Chitosan-collagen scaffolds cross-linked with genipin demonstrated cytocompatibility at different levels of bioanalysis with human adipose-derived mesenchymal stem cells for regenerative medicine: a research article

Chitosan-collagen scaffolds cross-linked with genipin demonstrated cytocompatibility at different levels of bioanalysis with human adipose-derived mesenchymal stem cells for regenerative medicine: a research article | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chitosan-collagen scaffolds cross-linked with genipin demonstrated cytocompatibility at different levels of bioanalysis with human adipose-derived mesenchymal stem cells for regenerative medicine: a research article Idiberto José Zotarelli-Filho, Durval Ribas Filho, Nizar Abu Hamdeh, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9106198/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: One in five people over 65 years of age benefiting from in vitro-generated tissues and organs. This study aimed to analyze the cytocompatibility of Adipose-Derived Stem Cells (ADSC) at the chromosomal/chromatid level in the presence of the chitosan-collagen-genipin biomatrix. Methods: ADSC was obtained from human lipoaspirate by enzymatic method and centrifugation and then cultured in Petakas (control) and the chitosan-collagen-genipin biomatrices, with and without cryopreservation. ADSC were characterized for adhesion and proliferation by conventional optical microscopy, phase contrast and scanning electron microscopy, immunophenotyping, cell viability with Trypan blue test, chromosomal and chromatid stability by traditional cytogenetic techniques, neoplastic potential by Papanicolau test and cell differentiation capacity in three types of tissues. Results: The biomatrix intercrossed with genipin 0.75% v/v was selected for studies in this article since it presented good physicochemical stability. After cultivation in Petakas and biomatrices, ADSC maintained their intrinsic functions of adhesion, proliferation, and differentiation. Cytogenetic tests of ADSC after different culture conditions in biomatrices revealed no statistically significant differences (p<0.05) to control cells (cultured in Petakas). Discussion: Biomatrix intercrossed with genipin 0.75% v/v can stimulate the expression of ADSC activities. The presence of cations and anions on individual polymer chains can stimulate the cellular activities of ADSC without causing cellular stress or loss of quiescence in these cells. Conclusion: The chitosan-collagen-genipin biomatrix with genipin at 0.75% v/v was shown to be biocompatible both at the cellular level and at the chromosomal/chromatid level of adipose mesenchymal stem cells. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Stem cells Adipose-derived mesenchymal stem cells Biomatrices Scaffolds Chromosomal stability Chromatid stability Cytostability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION A significant number of people worldwide have seen their life expectancy increase due to regenerative medicine and tissue engineering [ 1 – 3 ]. In this context, it is worth noting that the main cause of hospitalization in Brazil is due to injuries caused by accidents in general. In 2022 alone, the Brazilian Ministry of Health invested more than 154.9 million in the manufacture of 3,298,667 orthoses, prostheses, and mobility devices, including wheelchairs [ 4 ]. In addition, the American Heart Association estimates that more than 6.5 million Americans suffer from congestive heart failure, and in Brazil, approximately 350,000 people have acute myocardial infarction per year [ 5 , 6 ]. Thus, tissue engineering is crucial to meet this demand through the development of biomaterials (scaffolds) to promote tissue regeneration [ 7 ]. In this regard, regenerative medicine and tissue engineering must comply with the entire cellular machinery to chemical, physical, and biological processes to achieve the appropriate biological niche, that is, to create a microenvironment favorable to tissue regeneration through signaling cascades and neovascularization [ 8 – 12 ]. Given this, in the present study, chitosan-collagen-genipin biomatrices were used, with the degree of deacetylation (DD) of chitosan being above 0.4 to achieve good solubility with collagen and to bind the Schiff base with genipin, conferring greater stability to the biomatrix to favor cell growth [ 13 , 14 ]. The mixture of the two biopolymers reconciles the cytocompatibility of collagen with the adhesion forces of chitosan. Collagen is bioadhesive through specific arginine-glycine-aspartate (RGD) sites. The RGD group promotes cell adhesion by binding to integrin receptors, thus promoting cell growth and differentiation [ 15 ]. Genipin is a hydrophilic organic compound whose structure was discovered in 1960 and is extracted from geniposide (origin: gardenia fruit) and has low cytotoxicity [ 16 ]. Sung et al. (1999) [ 17 ] found that genipin was 10,000 times less cytotoxic than glutaraldehyde.In this context of the biomatrices, the adult stem cells, prominent to adipose-derived stem cells (ADSC), appeared as an alternative for the cellular therapy, since they were found to have some degree of plasticity, with a capacity of self-renewal and differentiation in specialized progenitors. The most widely studied stem cells are those derived from bone marrow, which is divided into two main cell types: hematopoietic stem cells, which originate from blood cells, and the adipose-derived mesenchymal stem cells (ADSC), which are in the medullary stroma involving the hematopoietic cells and have the potential for differentiation in multiple tissues [ 1 , 2 , 18 , 19 ]. In addition, ADSC also has the advantages of self-renewal, immunomodulatory character, multipotentiality, ease of isolation, purification, and cryopreservation [ 1 ]. According to the recommendations of the International Society of Cell Therapy, to be used in studies, they must present the minimum characteristics as adherent and proliferative cells, differentiate into at least three cell lines (adipocytes, chondrocytes, and osteocytes), and present the mesenchymal panel with typical cell surface markers, in addition to cytogenetic stability. Some studies also show that there is an important link between fat cells and the vascular network. These cells represent a heterogeneous population of microvascular endothelial cells, constitute a convenient source of multipotent cells, and are non-restrictive [ 20 ]. ADSC, when in intense activity, can express the exosomes (extracellular vesicles-EV) according to the environment in which they are found. EVs can promote the activation of the immune system through the cytokines that carry on its membrane, such as tumor necrosis factor (TFN). An example of the role of exosomes in the immune system is demonstrated by the ability to act as 17 antigen presenters for T cells due to the presence of Major Histocompatibility Class (MHC) I and II molecules [ 10 , 21 , 22 ]. With the evolution of cell culture, Celartia® was formed to develop better quality instruments for in vitro cell growth, such as Petakas, to transform the ancient art of cell culture engineering, to increase ease of management reduce costs, and minimize the environmental impact of cell culture. Petaka is a device designed for the culture of adherent cells and the culture of non-adherent cells [ 23 ]. Therefore, in this scenario of technological advances in regenerative medicine and human tissue engineering, the present study aimed to respond to several information gaps by analyzing the cytocompatibility of chitosan-collagen-genipin biomatrices in the presence of mesenchymal stem cells of human adipose tissue, analyzing the frequency of chromatid and chromosome breaks and failures, and/or numerical chromosomal alterations in ADSC in biological samples from the two patients under different culture conditions (after the 3rd passage and after cryopreservation for 12 months), in the presence and absence of chitosan-collagen-genipin biomatrix, and analyze the presence of multinucleation and nuclear polymorphisms of ADSC after contact with the biomatrix. Also, this study investigated the intrinsic characteristics of these cells as the ability to differentiate the cultured cells in osteoblasts, chondroblasts, and adipocytes, immunophenotyping, and proliferation in the biomatrices. METHODS AND MATERIALS Study Design This study followed an experimental study model (research article), involving the analysis of human biological material at the in vitro level. Ethical Approval The present study was evaluated and approved by the research ethics committee of the Beneficência Portuguesa Hospital of São José do Rio Preto, São Paulo, Brazil, under number of approval process 030427. All procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2024. Informed Consent I nformed consent was obtained from all participants involved in the study, with all procedures explained in detail before participation. Clinical Trial Number Not applicable. Participants For the development of the present study, two patients aged between 20 and 30 years and candidates for an aesthetic procedure of liposuction were selected and accepted to donate the biological material (liposuction - adipose tissue) for the sole use of in vitro studies. Both patients signed the informed consent form. Obtaining and Selecting Adipose-Derived Mesenchymal Stem Cells Adipose-Derived Stem Cells (ADSC) from two young women (aged 20-30 years) were studied. The biological material was donated by plastic surgeon Dr. Luiz Fernando Frascino of São José do Rio Preto. The technique for extracting adipose tissue used manual liposuction with a 2.5 mm cannula. According to the protocol of Zuk et al. [24], adipose tissue was treated with Clostridium histolyticum collagenase type IV (Sigma-Aldrich) for two hours for extraction of vascular stromal fraction (Figure 1). The fat tissue suspension and cells were then taken to the Sepax system to obtain and wash the cell pellet (pellet) which was then resuspended in a culture medium to a concentration of about 2.5 x 10 5 cells mL -1 for culture in chitosan-collagen-genipin matrices, Petakas and cryopreservation for one year. The two samples of 2.0 mL of adipose tissue (01 sample per patient) were obtained from the abdomen region of two women who passed the selection criteria. Smoking or drug-dependent women with a history of autoimmune diseases, hematological diseases, previous chemo or radiotherapy, heart disease, renal, hepatic, and psychiatric diseases were excluded. Extraction of Stromal Vascular Fraction (SVF) from Adipose Tissue According to Coleman et.al. (2006) [25], the adipose tissue samples will be extracted by the manual liposuction method (with a 2.5 mm diameter Cannula) and taken to the laboratory for ADSC extraction procedures. Fatty tissue samples will be homogenized and an initial volume of 20.0 mL will be divided into 2.0 mL samples. Subsequently, to these volumes, 6.0 mL of collagenase type IV ( Clostridium hystoliticum - Sigma Aldrich) will be added in a ratio of 1:3 (fat: collagenase), and the mixture will be brought into a 37.0 ° C incubator, 5.0 % CO 2 , for 1.0 hour and a half. After this time, the mixture will be centrifuged at 800 xg for 10 minutes at 23.0 ° C and then the pellet will be resuspended in 10.0 mL culture medium, and 1.0 mL will be used for the assays. Cryopreservation According to Nardi and Meirelles (2006) [26], the cells will be frozen in RPMI 1640 culture medium containing 10.0% FBS (Gibco) and 10.0 % DMSO (Life Group). The cells will be trypsinized and resuspended in RPMI medium and transferred to a falcon tube (± 5.0 mL), centrifuged at 900 xg for 5.0 minutes, the supernatant removed and the pellet resuspended with the chosen freezing solution. The amount depends on the pellet or number of cells (concentration approximately 106 to 107 cells Ml-1) 1.0 mL of the prepared solution was placed in a screw-capped freezing tube, which will remain for about one hour in the freezer at -20.0 °C, about 24 hours -80.0 °C and will be transferred to the tank with liquid nitrogen (± - 196.0 °C), where it will remain for one year passage in the Petakas, cell growth in the matrices and genetic stability assays. Cellular Defrost The cryotubes with the cryopreserved cells were removed from the liquid nitrogen reservoir where they were stored and thawed in a 37.0 °C thermostatic bath. After, the cells were suspended in an Alfa MEM medium supplemented with 10.0 % FBS, and centrifuged at 400 xg for 10.0 min. The thawing process had an average duration of 3 minutes. Viability of ADSC by trypan blue According to Nardi and Meirelles (2006) [26], 10.0 μL of cell solution will be placed in the Neubauer chamber for quantification of live and dead cells, with replicates of n=3.0. The Neubauer chamber has its central band in a position determined millimetrically below the sides, so that the placement of a coverslip, well adhered to the edges of the depression, gives rise to a chamber with a very determined volume. After placing a sample of the cell suspension in this chamber so that it is filled up, but without extravasation, the viable (uncolored) cells are counted under the microscope. The chamber has four quadrants (Q1, Q2, Q3 and Q4) with an area of 0.1mm² each. When integrating a glass cover plate into the Neubauer Chamber, you get a height of 0.1mm. The volume capacity is then 0.1mm x 0.1mm² = 0.1mm³. Cultivation of ADSC in Petakas According to the modified Celartia® [23] methodology, ADSC will be cultured in Petakas in the approximate amount of 2.5 x 10 5 cells mL -1 , which will be submitted to the tenth passage in alpha-MEM medium with 10.0 % fetal bovine serum and antibiotics. The cells will be infused into the Petakas using a waterproof and sterile membrane. After infusion, the set (cells and Petaka) will be held horizontally on one side for 1.0 hours and soon thereafter will be turned on the other side and held for at least 12 hours. After this time, the set will be placed vertically for the cell culture. Culture of ADSC in the Chitosan-Collagen-Genipin Scaffolds For ADSC culture, according to Nardi and Meirelles (2006) [26], these cells were expanded in alpha MEM culture medium supplemented with 10.0% v/v fetal bovine serum containing 1.0 % v/v ampicillin and streptomycin. After reaching about 85.0% confluence, the cells revolved from the surface of the plate with trypsin-EDTA, and then the cells were counted in the Neubauer chamber to know the number of live cells to be seeded on the biomatrices. After sowing, the Biomatrices were transferred to an incubator at 37.0 °C with 5.0% carbon dioxide, a time of 72.0 h for further analysis. The matrices were sterilized in a solution of 70.0 % ethanol (overnight) and then in the presence of ultraviolet light (15.0 min). After washing the biomatrices six times with sterile water, about 1.0 x 10 5 ADSC were seeded into the biomatrices. Preparation of the Chitosan-Collagen-Genipin Scaffolds Based on the method described by Baldwin and Kiick [27], the previously characterized chitosan (Mw = 115 kDa and degree of deacetylation of 85.25 %) was dissolved in 10.00 mL of 2.50 % v/v acetic acid solution for 24 hours at room temperature. The collagen was dissolved. The chitosan-collagen scaffold was prepared by mixing the two solutions under stirring for 96 h. The solutions were transferred to a 96-well plate, with a volume of 170 μL, in a ratio of 1: 1 v/v. Finally, the scaffold was crosslinked at 0.75% v/v genipin. The mixture was then frozen in liquid nitrogen (-196.0 °C) and then lyophilized. Scanning Optical Microscopy (OM) and Scanning Electron Microscopy (SEM) In optical microscopy, the ADSCs in the Petakas were photographed in the third passage with 85.0 % confluence and also after the 48h time in the third passage, in which phase the largest number of cells in the mitotic phase can be found, with 2.5x10 6 ADSC. In SEM, images of the ADSCs in the Petakas and the Biomatrices were also captured in the third passage with 85.0 % confluence and also after the time of 48h in the third passage, phase in which one can find the largest number of cells in mitotic phase, with 2.1x10 6 ADSC. In addition, the presence of exosomal production on the surface of ADSCs was also analyzed. Immunophenotyping Assay After removal of the cells from the culture flasks, performed with the aid of the trypsin solution, they were suspended in 1.0 mL of PBS supplemented with 3.0 % human albumin (Grifols® 20.0 % Human Albumin, PBS/A). The resulting suspension was subjected to the determination of the number and proportion of non-viable cells by the Trypan blue staining method as described in section Cell Viability, followed by adjustment of the viable cell density to 106 mL-1 cells. The immunophenotypic evaluation of these cells was carried out using the immunophenotyping technique by flow cytometry on a FACSCanto II® (BD Biosciences) cytometer, using the monoclonal antibodies conjugated with fluorochromes, and their respective isotypic controls. Also, ADSCs from the last pass of all cultures, homogenized in PBS/A, were separated into 100.0 μL aliquots and dispensed into 5.0 mL tubes for flow cytometry. After, 5.0 μL of each antibody was added to the tubes and homogenized by vortex stirring. The suspensions were then incubated for 15.0 min at room temperature in the dark. Then 400.0 μL of PBS/A was added to each tube, followed by homogenization by shaking and centrifugation at 400 × g for 10.0 min. The supernatant was discarded and the pellet homogenized in 500.0 μL of PBS / A. To tube 3 of each sample was added 5.0 μL of 7-AAD moments before cytometer acquisition. The ADSCs were then acquired on a cytometer coupled to the BD FACSDiva II® analysis software (BD Biosciences) and subsequently analyzed by Infinicyt® software (Cytognos) for the binding/labeling of each antibody (marker expression) and 7-AAD (cells). In all, eighteen anti-CDs were analyzed, involving the CD. ADSC differentiation analysis (Trileneage) - Monitoring Osteogenic Differentiation Osteogenic differentiation was induced in ADSC cultures in 6-well plates exhibiting 85.0 % confluency. In these, three wells had the MEM / SFB alpha media supplemented with 10.0 nM dexamethasone, 10.0 mM β-glycerol phosphate, and 50.0 μg mL-1 ascorbate-2-phosphate (Sigma-Aldrich). The osteogenic differentiation medium (alpha MEM / DIFO) was prepared by mixing 5.0 mL of ascorbate-2-phosphate / β-glycerol phosphate solution in alpha MEM / SFB medium (0.05 % ascorbate-2- phosphate and 2.16 % β-glycerol phosphate in MEM alpha medium, m/v); 98.0 μL dexamethasone solution (0.1 % in ethanol, m/v, diluted in alpha MEM, 1:49, v/v); 44.02 mL of MEM / SFB alpha medium; After homogenization, the solution was membrane filtered with 0.22 μm micropores. The ADSCs were incubated with MEM / DIFO alpha medium at 37 ° C and 5.0 % CO 2 for 14 days, with medium replacement every 72 h. The same procedure was performed in parallel with the negative control wells, with MEM / SFB alpha culture medium. After the differentiation period, the plates were submitted to staining to reveal secretion of mineralized extracellular matrix (differentiation in osteocytes), according to the following procedures: removal of the culture medium from all wells (differentiation and control); rinsing well with PBS (2x); fixation of cells with 2.5 % aqueous solution of glutaraldehyde (m/v) for 1 h 30 min; rinse with distilled water (2x); dehydration with isopropanol 60.0% (v/v) and incubation at room temperature for 5 min; (1.37 % in ultrapure water, m / v, pH 4.1-4.3, Sigma-Aldrich) and incubation at room temperature for 45 min; discard the dye solution and rinse with PBS (3x); photo-documentation and inverted microscope analysis [26]. Chondrogenic Differentiation Chondrogenic differentiation was induced through the commercial StemPro® Chondrogenesis Differentiation Kit (Gibco®). For this, a micro mass culture was performed where the cells were resuspended in a concentration of 1.6 x 10 7 viable cells mL -1 , of this total cell was aliquoted 1.0 mL of cell suspension that was transferred to another Petaka, where it remained for 2 hours in an oven with 5.0 % CO 2 at 37.0 °C. After the incubation period, 500.0 μL of differentiation medium (StemPro Chondrogenesis Differentiation Kit - Gibco) was added, and in control cells standard culture medium. The media was replacedtwice a week. After 14 days the medium was removed and the cells were fixed with the cells fixed with 2.5 % glutaraldehyde (Merck / Millipore, USA) at room temperature for 90.0 minutes. Following fixation, the Alcian Blue dye (Sigma®, USA), (1.0 % prepared in 0.1N HCl (F.Maia® / Brazil)) was added for 30.0 minutes, then the cells were washed in 0.1 N of HCL by 3 times, and the staining was analyzed under inverted optical microscope. Where staining was observed indicates the synthesis of proteoglycans produced by the chondrocytes [26]. Adipogenic Differentiation For the induction of adipogenic differentiation, ADSC cultures were used in Petakas. Upon reaching 85.0 % confluency, three wells had the MEM / SFB alpha medium supplemented with 0.5 mM isobutylmethylxanthine (3-isobutyl-1-methylxanthine), 50.0 μM indomethacin and 0.5 μM dexamethasone (Sigma- Aldrich). The adipogenic differentiation medium (MEM/DIFA alpha) was prepared by mixing 3.0 mL of IBMX solution (0.18% in water); 90.0 μL of 1.0 % indomethacin solution in ethanol; 490.0 μL dexamethasone solution (0.1% in ethanol; diluted in alpha MEM, 1:49, v/v); and 46.4 mL of MEM / SFB alpha medium. After homogenization, the solution was membrane filtered with 0.22 μm (polyvinylidene fluoride membrane) micropores. ADSCs were incubated with MEM/ DIFA alpha medium at 37 °C and 5.0 % CO 2 for 14 days, with medium replacement every 72 h. The same procedure was performed in parallel, with negative control Petakas, with alpha MEM/SFB culture medium. After the period of differentiation, the plates were submitted to staining to reveal cytoplasmic accumulation of lipid droplets (differentiation in adipocytes), according to the following procedures: removal of culture medium from all wells (differentiation and control); rinsing well with PBS (2x); fixation of cells with 2.5 % aqueous solution of glutaraldehyde (m/v, Sigma-Aldrich) for 1 h 30 min; rinse well with distilled water (2x); dehydration with isopropanol 60.0 % (v/v) and incubation at room temperature for 5 min; (0.3 % in isopropanol 99.0 %, m / v, diluted in water 3: 2 v/v) and incubation at room temperature environment for 5.0 min; discard the dye solution and rinse with distilled water (2x); photo-documentation and inverted microscope analysis [26]. Cytogenetic Analysis of ADSC After cell expansion in the 3rd passage, after 48 h of culture in Petakas (control) and chitosan-collagen-genipin biomatrices, the cells were subjected to the usual staining with Giemsa, according to the protocol routinely used in the Genetics Laboratory of the Hospital de Base de São José do Rio Preto/SP. The study of chromosome stability was performed based on the presence of chromatids chromosome breaks and gaps, and numerical chromosome alterations. The findings were interpreted and described according to information and standardization contained in the International System for Human Cytogenetic Nomenclature (ISCN, 2024) [28]. Cytogenetic analysis was performed on 480 cells in total (240 from each patient), considering 60 metaphase cells from four samples from each participant. For each patient, two samples with metaphase cells were considered (60 control cells and 60 cells in the biomatrix). Thus, for each sample, from each patient, 60 metaphases of cells cultured in the biomatrix and 60 cells cultured in the Petakas were evaluated. After 12 months of cryopreservation, 60 metaphases cultured in the biomatrices and 60 cultured in the Petakas of each patient were also evaluated. Protocol for Obtaining Metaphase Chromosomes by Usual Staining Dividing adipose mesenchymal stem cells were blocked in metaphase with colcemid at 0.1μg mL -1 (Gibco), detached from the growth surface with trypsin at 0.25% (Gibco), and subsequently hypotonized by exposure to a 0.057M KCl solution (Merck). The cells were fixed in a 3:1 solution of methanol and acetic acid. Cytogenetic analysis of metaphase chromosomes was performed by usual staining [29] and the images were captured on a BX60 microscope (Olympus) using the ImagePro Plus program. The findings were checked by a cytogeneticist and the results were characterized according to the ISCN standards [28]. Adapted Pap Smear Test After the cultures reached ≅85.0% confluence, the culture medium was removed from the Petakas and discarded, and the wells were washed with PBS at 37.0 °C (3x). Afterward, the cells in the Petakas were stained with Harris hematoxylin, Orange G6, and EA36, which are included in the Papanicolaou® kit for exfoliative cytology (Newprov) [30]. The procedures were repeated for each petaka, at room temperature. This staining was performed by removing and discarding the PBS; incubation with 80.0%, 70.0%, 50.0% ethanol, and distilled water, for 2.0 min each. After removing and discarding the water, 1.0 mL of Harris hematoxylin stain was added, incubating for 2.0 min. The Petakas were washed several times (10x) with distilled water. The excess water was removed and incubation was carried out with 50.0%, 70.0%, 80.0%, and 96.0% ethanol, for 2.0 min each. After discarding the 96.0% ethanol, 1.0 mL of Orange G6 stain was added, incubating for 2.0 min. This dye was discarded and 1.0 mL of 96.0% ethanol was added, incubating for 2.0 min (2x). Then, 1.0 mL of EA36 dye was added, incubating for 2.0 min. The Petakas were then incubated three times with 96.0% ethanol, for 2.0 min each. After that, the Petakas were subjected to microscopy analysis, initially performed under an inverted microscope, with subsequent image capture performed on an Olympus BX51 microscope, coupled to a DP70 digital camera and DP70-BSW software. For each sample (each patient), images of at least ten visual fields were captured with 200x magnification, analyzing 100 cells after 72 hours of cultivation in Petakas, 100 cells in biomatrices, 100 cells after 12 months of cryopreservation of the same cells in Petakas and 100 cells in biomatrices, with a total of 800 cells for the two participants in the present study. Statistical Analysis For data analysis, a database was built in the Microsoft Excel spreadsheet which was exported to the Stata 18 and Minitab 21 statistical program. Common descriptive statistical analysis and the Anderson-Darling normality test were performed for all variables, controls, and treatments. Values of mean and standard deviation were also evaluated. After that, a parametric regression test was performed. As there was the presence of the predictors and the continuous and categorical predictors, the linear regression and residual analysis of Durbin-Watson. For all linear regression tests, an alpha level lower than 0.05 was adopted as significant. For the Durbin-Watson residue analysis, the reference significance level was 0.05, adopting an acceptable independence interval of 1.75 <dw <2.60 (according to the Durbin-Watson standard table, dU <dw <4-dU). RESULTS Physicochemical Properties of the Chitosan-Collagen-Genipin Biomatrix Due to the increased stiffness of these biomaterials with increasing genipin concentration, the cross-linked biomatrix with 0.75% v/v genipin exhibited moderate stiffness and a swelling rate of approximately 80.0% (Figure 1). The average pore size of the biomatrix was 40 µm (p<0.05). Regarding the degradation rate, the cross-linked biomatrix with 0.75% v/v genipin exhibited the lowest mass loss rate during immersion in human blood solution. Therefore, the biomatrix with 0.75% v/v genipin exhibited the best physicochemical characteristics in this study and was therefore the selected biomatrix. Cell Counting and Viability According to Figure 2, triplicate cell viability tests with trypan blue showed higher cell viability in the biomatrices with lower percentages of free amino groups, to the cells of the two patients of the present study. As an example, the chitosan (Ch) scaffolds presented the lowest cell viability (86.5 %) and the chitosan-collagen scaffolds (with 0.75 % and 1.00 % v / v genipin) had the highest cell viability (99.8 %) to the reference of 99.5 % as the minimum value recommended by the International Society for Cellular Therapy (ISCT). There was no statistical significance between the chitosan-collagen sample with 0.75% (v / v) genipin with the chitosan, collagen, and chitosan-collagen control groups, with p> 0.05. The residual analysis of Durbin-Watson confirmed that there was no similarity between these samples, with the result of dw between 1.75 <dw <2.60. Immunophenotyping Assay The culture of ADSC derived from human adipose tissue is shown with a phenotypically heterogeneous population. These cells were located in the upper right region of the dot-plot for FSC x SSC, corresponding to cells of larger size and granularity. Analysis of the described region revealed a high frequency (greater than 70.0 %) of antigen expression characteristic of ADSC (CD166, CD105, CD90, CD73, and CD54) in the third passage, both in the presence of the chitosan-collagen scaffolds with 0.75% of genipin and Petakas (control). The R1 region contains the cells of larger size and granularity. In M2 the percentage of cells positive for CD105, CD166, CD34, CD133, CD45, CD31, CD73, CD44, CD90, CD117, and CD54 were presented in the total population and the region of larger and granular cells. The histograms of expression analysis of CD31, CD90, CD34, CD117, CD54, CD73, CD44, CD133, CD45, CD105, and CD166 in cells of the region of larger size and granularity (Table 1 and Figure 3). Table 1. Representative results of ADSC immunophenotyping after Cultivation in Biomatrices and Petakas. CDs / Results (%) SVF 3 a Expansion (Petakas) 3 a Expansion Scaffolds After Cryopreservation (Petakas) After Cryopreservation (Scaffolds) CD 45 35.5 1.13 1.1 1.02 0.99 CD 14 46.2 0.14 0.13 0.12 0.14 CD 51,61 64.0 1.85 1.90 1.87 1.74 CD 54 10.3 47.28 48.2 45.6 46.7 CD 44 19.5 66.93 68.5 66.3 66.8 CD 49e 20.2 64.42 67.4 65.5 67.3 CD 34 55.4 0.71 0.51 0.43 0.32 CD 13 15.5 84.92 85.3 83.1 84.2 CD 31 34.5 0.21 0.23 0.19 0.18 CD 166 2.4 18.73 17.4 16.6 17.2 HLA-DR 10.3 0.45 0.43 0.41 0.44 HLA-ABC 3.5 19.14 20.1 18.4 20.2 CD 29 7.7 77.95 78.2 76.5 78.3 CD 146 14.3 0.49 0.32 0.29 0.30 CD 90 14.7 82.25 81.2 80.02 82.1 CD 105 4.4 31.06 32 30.3 31.3 CD 106 9.8 0.68 0.89 0.69 0.58 CD 73 18.8 52.04 54.41 49.4 53.4 Source: Own Authorship. Optical and Electronic Microscopy According to Figure 4, the ADSC adhered and proliferated in the Petakas, and the C and D images, can be observed in images A and B, the same cells can be analyzed in the mitotic process, with 2.5x10 6 ADSC mL -1 . In SEM images, the biopolymers of chitosan, collagen, and genipin were observed individually, as well as in their combined chitosan-collagen-genipin (scaffolds) form. It also analyzed the adhesion and proliferation of ADSC in the scaffolds, showing images with ADSC in the mitotic phase (with 2.1x10 6 ADSC mL - 1) and also images in the proliferative phase and with cytoplasmic projections, according to Figure 5. Cell Differentiation To prove the differentiation capacity of mesenchymal stem cells, we standardized an efficient protocol for differentiation in the osteogenic, chondrogenic, and adipogenic lines. After cell expansion and flow cytometry characterization, differentiation was started in the three lines described. After the adipogenic differentiation, the mesenchymal stem cells presented cytoplasmic red staining in small vesicles when submitted to the staining with Oil Red. The property of this dye to interact with lipids and neutral triglycerides shows a red color that shows the accumulation of these substances in cytoplasmic vesicles of the differentiated cells. As can be seen in Figure 5, mesenchymal stem cells lost their fibroblast morphology and acquired a spherical morphology with numerous vesicles, characterizing the accumulation of lipids. The process of differentiation of the mesenchymal stem cells into the osteogenic lineage was evidenced by the Alzarin Red S coloration that stains red deposits of calcium and other minerals. As can be seen in Figure 05, around the differentiated cells are reddish regions, which are supposed to be deposits of minerals from the formation of a mineralized extracellular matrix, similar to that of the bone tissue. Another striking feature is the change in morphology, as the fibroblast cells alter their shape to elongated and aligned cells, covering the entire culture plate and in some places, forming cellular clusters. After three weeks the mesenchymal stem cells induced for chondrogenic differentiation were stained with Alcian blue. Cells showed expansion in cell size with a significant increase in cytoplasm, becoming rounded and flattened. A bluish cell coloration can be observed due to the interaction of the dye with the glycosaminoglycan expressed during differentiation. Figure 6 shows the ADSCs in the third passage presenting fibroblastic morphology. Differentiated mesenchymal stem cells in adipogenic lineage stained with Oil Red, showing accumulation of lipid vesicles in cells. D differentiated mesenchymal stem cells in the osteogenic lineage stained with Alzarin Red S showing accumulation of calcium formed, and mesenchymal stem cells under optical microscopy during differentiation culture, showing the formation of colonies and alignment of cells with their elongated structure. Differentiated mesenchymal stem cells in chondrogenic lineage stained with Alcian blue, changes in cell morphology (rounded), and matrix production with blue-stained proteoglycans (n=6) were observed. Cytogenetic Analysis – Chromosomal and Chromatid Stability The study of the stability of the genetic material at the chromosomal level was performed based on the presence of chromosomal and chromatid breaks and gaps, in addition to numerical alterations. The cells were subjected to the usual staining with Giemsa, according to protocols routinely used in the Genetics Laboratory of the Hospital de Base (Hemocentro/São José do Rio Preto/SP). The findings were interpreted and described according to information and standardization contained in the ISCN [28]. The cytogenetic tests were performed after cell expansion in the 3rd passage, after 48 h of culture in Petakas (control) and chitosan-collagen scaffolds with genipin at 0.75% v/v. The same was repeated after 12 months of cryopreservation. A total of 480 cells were analyzed (samples from both participants), as previously described. To better understand the most precise time in days to obtain a greater number of ADSC in metaphase, for the interruption and stabilization of chromosomes, before and after cryopreservation, an analysis of cell growth kinetics was performed. Through this analysis, it can be understood that the best time to apply colcemid was after 24 hours (one day) of the second change of culture medium. The best time was from the eighth (8) to the ninth (9) day. Chromosomal analysis of the samples did not reveal any statistically significant difference between the frequencies of numerical alterations and/or chromatid and/or chromosome breaks or gaps, as shown in Table 2 and Figure 7, both in the metaphase chromosomes obtained after 48 h of culture in Petakas (control) and those obtained from cells cultured in the chitosan-collagen scaffolds with genipin at 0.75% v/v, before and after 12 months of cryopreservation. Table 2. Chromosomal and chromatid analysis of breaks/gaps and/or numerical alterations of ADSC in the presence or absence of biomatrices and before and after cryopreservation. N total = 960 metaphases Petakas_before (control) Breaks/Failures /Numerical Changes Biomatrices_ before Breaks/Failures /Numerical Changes Petakas_After (control) Breaks/Failures /Numerical Changes Biomatrices_ after Breaks/Failures /Numerical Changes p-value (α<0,05) Patient 1 Sample 1 Sample 2 01/00/01 01/00/00 03/01/02 03/00/00 03/02/00 00/02/03 03/01/02 03/01/02 p=4,23 p=3,45 Patient 2 Sample 1 Sample 2 01/01/00 02/01/01 02/00/00 01/01/00 03/02/00 03/02/02 02/01/02 03/01/02 p=2,23 p=1,45 Source: Own Authorship. Adapted Pap Smear Test All cell samples from the control and scaffolds were evaluated. For each sample and passage, having established the number of 400 cells per sample and passage, totaling 800 cells analyzed with the two patients, no cells with an altered number of nuclei and/or atypical nuclear morphology were identified (Figure 8). Based on the Bethesda classification system [31-33], the possible morphological alterations of the nucleus that could be identified correspond to atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells - cannot exclude high-grade intraepithelial lesion (HSIL, ASC-H). Multinucleation is characteristic of low-grade squamous intraepithelial lesions (LSIL). However, these events were not found in the ADSC samples of the present study. DISCUSSION Significance of Biomatrices in the Tissue Engineering In the context of tissue engineering and regenerative medicine, it is crucial to establish biomatrices that will enable the development of the appropriate biological niche in order to precisely achieve paracrine, autocrine, and endocrine effects through mesenchymal stem cells, exosomes, microRNAs, telocytes, and other cells and molecules. In this sense, biomaterials such as chitosan, collagen, and genipin present physicochemical and biological properties of cytocompatibility for regenerative processes, naturally maintaining tissue homeostasis and the functional stereochemical organization of the generated tissues and their systemic integration [1,34-38]. In the present study, the degradation of chitosan-collagen scaffolds with 0.75% v/v genipin was the most resilient in the presence of human blood in culture medium, as shown in Figure 1. This is explained by the higher energy required for the depolymerization of the crosslinking of the polymer chains (covalent crosslinking), mainly through the NH 2 groups of chitosan. The combination of chitosan, collagen, and genipin through covalent bonds enables balanced degradation kinetics. This occurs because chitosan reacts with carbonyl compounds through nucleophilic attack of its amino groups, forming covalent bonds with some groups of the collagen and genipin [13,14]. The solubility of the scaffolds increases through the protonation of the amino groups with increasing crosslinking with genipin [39-41]. These results of the present study are similar to the results published by Chiono et al. (2008) [39]. This revealed that the best concentration of genipin for biomatrices fabrication was 0.75% v/v, similar to the results reported by Baldwin and Kiick (2010) [27]. Also, chitosan, as a polyelectrolyte, stimulated the expression of ADSC activities by paracrine and autocrine factors. Type I collagen, through the site-specific arginine-glycine-aspartate (RGD), promoted the adhesion of these cells by binding to integrin receptors, thus promoting cell growth and anchoring of proteins and cells to the extracellular matrix. Studies reported by Bet, Goissis, and Plepis (1997) [42] reported that the physical unit of type I collagen, designated as the "D-period," is involved in controlling the biological activities that maintain the biological microenvironment in tissues. It is known that electrostatic ion-ion, ion-dipole, and dipole-dipole interactions, van der Waals interactions, π-electron interactions, and charge transfer complexes occur, forming ionic, hydrogen, and covalent bonds between the polymeric components of chitosan (-OH and -NH 2 groups) and collagen (OH-, -COOH, and -NH 2 groups), with negative Gibbs free energy (ΔG < 0), according to Domard and Taravel (1995) [15] and Israelachvili (2011) [41]. The mixing of polycations (chitosan) and polyanions (collagen) can occur by spontaneous aggregation, with the consequent release of counterions, increasing entropy (ΔS > 0) [41,43]. Thus, the presence of counterions, as well as the presence of cations and anions of the individual polymer chains, stimulated the cellular activities of ADSCs in the present study, without causing cellular stress (absence of exosomes on the cell surface) and loss of quiescence of these cells. Importance of Adipose-Derived Stem Cells ADSCs have the advantages of self-renewal, immunomodulatory character, multipotentiality, ease of isolation, purification, proliferation "in vitro" as well as cryopreservation [10]. ADSCs should exhibit minimal characteristics as adherent and proliferative cells, differentiate into at least three cell lines (adipocytes, chondrocytes, and osteocytes), and present the mesenchymal panel with the typical cell surface markers so that they can be used in studies, according to the recommendations of the International Society of Cell Therapy (ISCT) [44-47]. This differentiation translates the importance of these cells to be used with biomaterials as differentiation facilitators. Studies also show that there is an important link between fat cells and the vascular network [45]. These cells represent a heterogeneous population of microvascular endothelial cells, constitute a convenient source of multipotent cells, and are non-restrictive [47]. Under culture conditions, adipose stem cells grow easily in monolayers, maintain multipotentiality normally up to the tenth passage, and exhibit fibroblastroid morphology. CD4, CD14, CD166, and HLA-1 positive markers are required for the immunophenotyping of the ADSC, and the CD4, CD14, CD16, CD16, CD54, CD55, CD59, CD90, CD105, CD106, CD146, CD166 and HLA-1 positive markers for CD11b, CD14, CD19, CD31, CD34, CD45, CD79 alpha, CD80, CD117, CD133, CD144, HLA-DR and Stro-1, the abbreviation CD stands for the English Cluster Differentiation [10,44,45]. Also, ADSC secrete a cascade of cytokines and growth factors with paracrine, autocrine, and endocrine activities, such as macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), inflammatory protein macrophages (MIP-1α / CCL3) [46]. These factors when conjugated can produce some responses of the local immune system, stimulating angiogenesis and inducing proliferation and differentiation of mesenchymal stem cells in the desired tissue. In addition, ADSC induce the expression of junction proteins and increase microvascular integrity and nitric oxide (NO) production by macrophages [44-46]. Using the vacuum equipment, the amount of ADSC obtained with the 12 samples was significant when compared with results available in the literature [44,45]. The use of the 3.0 mm cannula in the liposuction process allowed to increase in the surface area of the extracted fat, facilitating the activity of the enzyme collagenase IV to extract the stromal vascular fraction (SVF). In addition to this, the extraction of adipose tissue with blood vessels in these cells was also obtained in a larger quantity, with similar work presented by Zuk et al. (2002) [24] and Nardi; Meirelles (2006) [26]. Cultivation in Petakas The study of adhesion and proliferation of ADSCs in Petakas has proved to be relevant with remarkable results against cell culture publications in traditional flasks as demonstrated by Zotarelli-Filho et al. (2013) [48], because it was possible to obtain a larger number of cells in less time, dispensed with the use of gaseous CO 2 , decreased the dehydration process of the cell culture medium and allowed more controlled gas exchange, thus ensuring longer pH stability of the cell culture medium. This also made it possible to reduce the consumption of the culture medium. Differentiation of ADSC According to Irioda et al. [49], maintaining the differentiation potential of the ADSC under cultivation is a sine qua non for achieving the desired therapeutic effect. Thus, even though it does not compose the proposed flowchart for the screening of the cultures of these cells, it is only through the induction of differentiation that its pluripotentiality can be attested. The International Society for Cellular Therapy (ISCT), after striving to standardize the name for ADSC, indicated among the minimum criteria for the demonstration of the pluripotency of ADSCs, the induction of their differentiation in adipocytes, osteoblasts, and chondroblasts [47]. The literary record on ADSCs however, contains a great diversity of differentiation reports. In these reports, even when restricted to ADSCs, they include cell types derived from the three embryonic leaflets [44-47]. In the present study, all the samples that maintained fibroblastoid growth patterns up to the third passage where in vitro cell differentiation analyses were started for the adipogenic, osteogenic, and chondrogenic lines. Differential staining allowed the detection of cytoplasmic lipid deposits typical of adipogenic differentiation, as well as the extracellular deposits of calcium crystals, typical of osteogenic differentiation, in all samples submitted to differentiation. Immunophenotyping Study Immunophenotypic characterization of ADSCs usually includes expression of the CD73, CD90, and CD105 markers; and absence of expression of CD11b or CD14, CD19 or CD79α, CD34, CD45, and major histocompatibility class II complexes, mainly HLA-DR [24]. Although the results of ADSC immunophenotyping have been representative and in agreement with the literature, mainly because of the CD13 +, CD29 +, CD44 +, CD54 +, CD90 +, CD166 +, CD146 +, CD 49e, CD73 +, and CD 105 +, the phenotype of these cells is not conclusive, since most of these antibodies are not only expressed in ADSC, appearing also in fibroblasts, according to studies by Zuk et al. (2002) [24]. Therefore, the alkaline phosphatase enzymatic activity assays were conclusive for the identification of ADSC, as reported also by Planat Bernard et al. [50,51]. Tumorigenic potential (Pap smear) and chromosomal stability Pap Smear Test Irioda et al. (2011) [30] were the first to report this type of application of the technique, including it as the first in the proposed algorithm. In the same publication, the authors also reported the detection of alterations in human ADSC. The Papanicolaou technique is a cost-effective tool used in the prevention and diagnosis of cervical cancer and has therefore been included in the routine screening of the adult female population in different countries [31-33]. Its efficiency and specificity are frequent targets of evaluation through different methodologies. Evaluations based on repeat testing, such as that of Soost et al. (1991) [52], for example, estimated the overall sensitivity and specificity of this technique at ≅80.0% and ≅99.4%, respectively. Studies using detection thresholds, such as that of Nanda et al. (2000) [53], despite identifying greater variation in results, also obtained high estimates for both sensitivity (≅70.0–80.0%) and specificity (≅95.0%). In population screening for cervical cancer prevention, more recent studies have revealed greater efficiency of other diagnostic techniques concerning the Papanicolaou test [54], however, corroborating what was proposed by Irioda et al. (2011) [30], we believe that this technique can contribute significantly to the evaluation of MSCs and other adherent cells in cultures for therapies, both due to its sensitivity/specificity and its ease of execution [32,33]. In the present study, no cells with altered numbers of nuclei and/or atypical nuclear morphology were identified in all samples and passages evaluated, with a total of 800 cells for the two participants. The Papanicolaou technique, when used in the analysis of desquamated cells from the uterine cervix, also allows the detection of other alterations, whose arrangement can characterize different types of lesions [31-33]. In the present study, it was decided to include only changes in the number and morphology of the nuclei. This decision was made because there was still no standardization for the use of this technique for mesenchymal stem cell cultures. In this way, errors in judgment were avoided, resulting from possible errors in sample preparation or even due to the evaluator's lack of training in the use of the technique, given that, even for the standardized use of the technique, differences in judgment are not uncommon [53]. Chromosome Stability In this study, a total of 480 cells from the two participants were analyzed. To better understand the most precise time in days of the metaphase phase of the ADSC for the interruption and stabilization of the chromosomes, before and after cryopreservation, an analysis of cell growth kinetics was performed. The best time was from the eighth (8) to the ninth (9) day, after the second change of culture medium. Thus, this analysis suggests an optimized guideline to better take advantage of the ideal time for metaphase interruption. After analysis, no numerical changes and/or breaks or chromosome or chromatid failures were evidenced, either in the metaphase chromosomes after 48 h of cultivation in Petakas (control) and chitosan-collagen biomatrices with genipin at 0.75% v/v, or after 12 months in the metaphase chromosomes that were extracted from the cryopreserved ADSC. Specific chromosomal anomalies are responsible for hundreds of syndromes that can be identified through karyotype analysis and study. They are responsible for a large proportion of reproductive losses, congenital malformations, and mental retardation, playing an important role in the pathogenesis of malignant hematological diseases, when acquired during life, associated with neoplasias. In these cases, studies of chromosomal disorders are important to aid in the diagnosis, prognosis, therapeutic conduct, and treatment monitoring of patients who present such alterations [30]. The normal functioning of the genetic system depends on the stability of the genetic material contained in the chromosomes [55]. The karyotype can change, leading to genetic alterations. Such alterations can be in the number of chromosomes or their structure. Changes in the number of chromosomes can lead to euploidy or aneuploidy. In the first form, there is an increase or decrease in the number of genomes in a cell, we have euploidy. In the second type of mutation, there is an increase or decrease in one or more chromosomes in the genome, altering the karyotype of those that present it [30]. Structural alterations modify the normal morphology of one or more chromosomes. Such chromosomal anomalies can affect entire chromosomes or chromosomal segments. Thus, there is a disorganization in the chromosomal structure that can be observed under the microscope in the following ways: - Deletion or deficiency: involves the loss of chromosomal material; - Duplication: a segment of the chromosome is represented twice or more. If the duplicated fragment includes the centromere, it can be incorporated into the karyotype as an extra chromosome; Inversion: involves the 180° inversion of a chromosome segment [55]. Therefore, in the present study, there were no significant chromatid and/or chromosomal alterations or anomalies, suggesting that the biomatrices are biocompatible and are strongly indicated for use in translational therapies. LIMITATIONS Due to the extensive laboratory analysis, financial constraints were a limiting factor, allowing analysis of samples from two patients. Nevertheless, all samples were performed in triplicate (replicates of n=30), and 480 cells (960 metaphases) were used for cytogenetic analyses. CONCLUSION The chitosan-collagen-genipin scaffolds, at the tested concentration of 0.75%, showed cytocompatibility at the cellular and chromosomal levels, since it allowed adhesion, proliferation, and biostimulation for the expression of normal cellular functions, and did not cause damage to adipose-derived stem cells. Abbreviations Adipose-Derived Stem Cells (ADSC) Tumor Necrosis Factor (TFN). Major Histocompatibility Class (MHC) Adipose-Derived Stem Cells (ADSC) International Society for Cellular Therapy (ISCT). Low-grade Squamous Intraepithelial Lesions (LSIL). Macrophage Colony Stimulating Factor (M-CSF) Declarations Author contributions: Conceptualization - IF, DF; Data curation – IF, HAK; Formal Analysis - IF, DF, MA; Investigation - IF, DF, NAH; Methodology – DF, NAH; Project administration - IF, DF, MA; Supervision – IF, NAH, HAK; Writing - original draft - IF, DF, MA, NAH, HAK; Writing-review & editing- IF, DF, MA, NAH, HAK. Ethical Approval The present study was evaluated and approved by the research ethics committee of the Beneficência Portuguesa Hospital of São José do Rio Preto, São Paulo, Brazil, under number of approval process 030427. All procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2024. Informed Consent I nformed consent was obtained from all participants involved in the study, with all procedures explained in detail before participation. Consent for Publication Informed consent was obtained from the patients. Availability of Data and Material All data generated or analyzed during this study are included in this published article. Funding National Council for Scientific and Technological Development (CNPq) from Brazil. Conflict of Interest The authors declare no conflict of interest, financial or otherwise. Acknowledgment First of all we thank the financial support of the National Council for Scientific and Technological Development (CNPq) from Brazil. Further, thank you very much Dr. Lílian Madi-Ravazzi for the excellent images obtained, responsible for the scanning electron microscope (SEM) from Department of microscopy and microanalysis and we also thank the laboratory of microscopy and microanalysis for the images of optical microscopy, both located at State University of São Paulo - IBILCE-UNESP, from São José do Rio Preto, Sao Paulo, Brazil. Clinical trial number Not applicable. References Biniazan F, Stoian A, Haykal S. Adipose-Derived Stem Cells: Angiogenetic Potential and Utility in Tissue Engineering. Int J Mol Sci. 2024 Feb 16;25(4):2356. doi: 10.3390/ijms25042356. Elias Ferreira Stricker P, Barth de Oliveira N, Felipe Mogharbel B, Lührs L, Irioda AC, Abdelwahid E, Regina Cavalli L, Zotarelli Filho IJ, Athayde Teixeira de Carvalho K. Meta-analysis of the Mesenchymal Stem Cells Immortalization Protocols: A Guideline for Regenerative Medicine. Curr Stem Cell Res Ther. 2023 Nov 27. doi: 10.2174/011574888X268464231016070900. Pacheco CMR, Ferreira PE, Saçaki CS, Tannous LA, Zotarelli-Filho IJ, Guarita-Souza LC, de Carvalho KAT. In vitro differentiation capacity of human breastmilk stem cells: A systematic review. World J Stem Cells. 2019 Nov 26;11(11):1005-1019. doi: 10.4252/wjsc.v11.i11.1005. Brazil. Ministry of Health. Available at: https://www.gov.br/saude/pt-br/assuntos/noticias/2020/outubro/sus-oferece-gratuitamente-orteses-e-proteses-sob-medida. Accessed at: 11/11/2024. Martin SS, Aday AW, Almarzooq ZI, Anderson CAM, Arora P, Avery CL, Baker-Smith CM, Barone Gibbs B, Beaton AZ, Boehme AK, Commodore-Mensah Y, Currie ME, Elkind MSV, Evenson KR, Generoso G, Heard DG, Hiremath S, Johansen MC, Kalani R, Kazi DS, Ko D, Liu J, Magnani JW, Michos ED, Mussolino ME, Navaneethan SD, Parikh NI, Perman SM, Poudel R, Rezk-Hanna M, Roth GA, Shah NS, St-Onge MP, Thacker EL, Tsao CW, Urbut SM, Van Spall HGC, Voeks JH, Wang NY, Wong ND, Wong SS, Yaffe K, Palaniappan LP; American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. 2024 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation. 2024 Feb 20;149(8):e347-e913. doi: 10.1161/CIR.0000000000001209. Epub 2024 Jan 24. Erratum in: Circulation. 2024 May 7;149(19):e1164. doi: 10.1161/CIR.0000000000001247. WHO-World Health Organization. WHO standards for prosthetics and orthotics. Available at: https://www.who.int/publications/i/item/9789241512480. Accessed at: 11/11/24. Boccafoschi F, Habermehl J, Vesentini S, Mantovani, D. Biological performances of collagen-based scaffolds for vascular tissue engineering. Biomaterials; 2005, 26 (35) : 7410-7417. Gou Y, Huang Y, Luo W, Li Y, Zhao P, Zhong J, Dong X, Guo M, Li A, Hao A, Zhao G, Wang Y, Zhu Y, Zhang H, Shi Y, Wagstaff W, Luu HH, Shi LL, Reid RR, He TC, Fan J. Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering. Bioact Mater. 2023 Dec 14;34:51-63. doi: 10.1016/j.bioactmat.2023.12.003. Greco OT, Filho IJZ, Bellini MF, Bilaqui A, Souza as junior et al. cardiomyopathy and cell therapy: ejection fraction improvement and cardiac muscle mass increasing, after a year of bone marrow stem cells transplantation by magnetic resonance image. j stem cell res ther. 2013, s6: 008. Zotarelli Filho IJ, Liedtke FS, Ferrari RS, Daher ID, Neiva EB, Moura MIP, Murad ACS. Protocol for obtaining and characterizing the stromal vascular fraction of human adipose tissue by mechanical dissociation: an original article. MedNEXT Journal of Medical and Health Sciences, 2022, 3(4). doi: https://doi.org/10.54448/mdnt22412. Daher ID, Moura MIP, Murad ACS, Ferrari RS, Neiva EB, Liedtke FS, Zotarelli Filho IJ. Major clinical findings of cellular therapy for intravitreal use in ischemic retinopathy and macular degeneration: a systematic review. MedNEXT Journal of Medical and Health Sciences, 2022, 3(3). https://doi.org/10.54448/mdnt22311. Kuo CK, Tuan RS. Mechanoactive tenogenic differentiation of human mesenchymal stem cells. Tissue Engineering, part a, 2008, v. 14, n. 10, p. 1615-1627. Muzzarelli RA, El Mehtedi M, Bottegoni C, Gigante A. Physical properties imparted by genipin to chitosan for tissue regeneration with human stem cells: a review. Int J Biol Macromol. 2016, dec;93(pt b):1366-1381. Alsarra IA. Chitosan topical gel formulation in the management of burn wounds. international journal of biological macromolecules, Guildford; 2009, 5 :16-21. Domard A, Taravel MN. Collagen and its interaction with chitosan: ii. influence of the physicochemical characteristics of collagen. Biomaterials; 1995, 16(11):865–71. Butler MF, Pudney PDA. Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin. Journal of Polymer Science Part A-Polymer Chemistry; 2003, 41(24) :3941-3953. Sung HW, Huang RN, Huang LL, Tsai CC. In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. Biomater sci polym ed.; 1999, 10:63–78. Abbott A. Cell rewind wins medicine nobel. nature; 2012, 490:151-152. Breyner NM, Hell RC, Carvalho LR. Effect of three dimensional chitosan porous scaffold on the differentiation of mesenchymal stem cells into chondrocytes. CTO; 191 : 119-28, 2010. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans R J, Keating A, Procko DJ, Horwitz EM. Minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement. Cytotherapy, 2006, v. 8, n. 4, p. 315-317. Roh YJ, Choi YH, Shin SH, Lee MK, Won YJ, Lee JH, Cho BS, Park KY, Seo SJ. Adipose tissue-derived exosomes alleviate particulate matter-induced inflammatory response and skin barrier damage in atopic dermatitis-like triple-cell model. PLoS One. 2024 Jan 19;19(1):e0292050. doi: 10.1371/journal.pone.0292050. Park N, Kim KS, Park CG, Jung HD, Park W, Na K. Adipose-derived stem cell-based anti-inflammatory paracrine factor regulation for the treatment of inflammatory bowel disease. J Control Release. 2024 Oct;374:384-399. doi: 10.1016/j.jconrel.2024.08.027. CELARTIA®, 2025. Available at: https://celartia.com/petaka-options/. Accessed at: 14/11/2024. Zuk PA, Zhu M, Ashjian P. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002, 13: 4279–4295. Coleman SR. Structural fat grafting: more than a permanent filler. Plast Reconstr Surg. 2006, sep;118(3 suppl):108s-120s. Nardi NB, Meirelles SL. Mesenchymal stem cells: isolation, in vitro expansion and characterization. Hep; 2006, 174: 249-82. Baldwin AD, Kiick KL. Polysaccharide-modified synthetic polymeric biomaterials. biopolymers; 2010, 94:128–140. ISCN 2024 - An International System for Human Cytogenomic Nomenclature (2024). Cytogenet Genome Res. 2024;164(Suppl 1):1-224. doi: 10.1159/000538512. Sanchez O, Escobar JI, Yunis JJ. A simple G banding technique. Lancet 1973;II:269. Irioda AC, Zocche L, Souza CMCO, Ferreira RJ, Aliprandini E, Cunha RC, Francisco JC, Guarita-souza LC, Malvezzi M, Beltrame MP, Mesquita LAF, Kuczera D, Chachques JC, Carvalho KAT. Pap test as the first step in sreening genetic stability in cell-based therapy. Journal of stem cell research and therapy, 2011, v. 1, n. 3, p. 1000106. National Cancer Institute. Bethesda System Website Atlas [Internet].1989. Available at: . Accessed at: 11/11/2024. Apgar BS, Zoschnick L, Wright JR TC. The 2001 Bethesda system terminology. American Family Physician, 2003, v. 68, p. 1992-1998. National Cancer Institute. NCI Cancer Atlas [Internet]. Available at: https://gis.cancer.gov/canceratlas/. Accessed at: 11/11/2024. National Institutes of Health Consensus Development Conference Statement on the Clinical Applications of Biomaterials. November 1-3, 1982. Artif Organs. 1983 May;7(2):260-5. PMID: 6688173. Luo W, Zhang H, Wan R, Cai Y, Liu Y, Wu Y, Yang Y, Chen J, Zhang D, Luo Z, Shang X. Biomaterials-Based Technologies in Skeletal Muscle Tissue Engineering. Adv Healthc Mater. 2024 Jul;13(18):e2304196. doi: 10.1002/adhm.202304196. Wang J, Liu M, Yang C, Pan Y, Ji S, Han N, Sun G. Biomaterials for bone defect repair: Types, mechanisms and effects. Int J Artif Organs. 2024 Feb;47(2):75-84. doi: 10.1177/03913988231218884. Xie Y, Ma C, Zhu Q, Fu T, Bai L, Lan X, Liu L, Xiao J. Facial nerve regeneration via body-brain crosstalk: The role of stem cells and biomaterials. Neurobiol Dis. 2024 Oct 1;200:106650. doi: 10.1016/j.nbd.2024.106650. Rahimi Darehbagh R, Seyedoshohadaei SA, Ramezani R, Rezaei N. Stem cell therapies for neurological disorders: current progress, challenges, and future perspectives. Eur J Med Res. 2024 Jul 25;29(1):386. doi: 10.1186/s40001-024-01987-1. Chiono V, Pulieri E, Vozzi G, Ciardelli G, Ahluwalia A, Giusti P. Genipin-crosslinked chitosan/gelatin blends for biomedical applications. J Mater Sci Mater Med; 2008,19: 889–898. Drago RS. Physical methods in chemistry. Saunders and Co., London, 1977. Israelachvili JN. Van der Waals forces in biological systems. Quarterly Reviews of Biophysics, 2011, 6(4), 341–387. Bet MR, Goissis G, Plepis AMG. Anionic collagen: calcium phosphate composites. preparation and characterization, Química Nova; 1997, 20 : 475-477. Hill TL. Thermodynamics of small systems. Benjamin, New York: Dover Pubns, 1963. Yuan C, Song W, Jiang X, Wang Y, Li C, Yu W, He Y. Adipose-derived stem cell-based optimization strategies for musculoskeletal regeneration: recent advances and perspectives. Stem Cell Res Ther. 2024 Mar 27;15(1):91. doi: 10.1186/s13287-024-03703-6. Wang K, Yang Z, Zhang B, Gong S, Wu Y. Adipose-Derived Stem Cell Exosomes Facilitate Diabetic Wound Healing: Mechanisms and Potential Applications. Int J Nanomedicine. 2024 Jun 17;19:6015-6033. doi: 10.2147/IJN.S466034. Zhou B, Chen Q, Zhang Q, Tian W, Chen T, Liu Z. Therapeutic potential of adipose-derived stem cell extracellular vesicles: from inflammation regulation to tissue repair. Stem Cell Res Ther. 2024 Aug 7;15(1):249. doi: 10.1186/s13287-024-03863-5. Sanchez-Guijo F, Vives J, Ruggeri A, Chabannon C, Corbacioglu S, Dolstra H, Farge D, Gagelmann N, Horgan C, Kuball J, Neven B, Rintala T, Rocha V, Sanchez-Ortega I, Snowden JA, Zwaginga JJ, Gnecchi M, Sureda A. Current challenges in cell and gene therapy: a joint view from the European Committee of the International Society for Cell & Gene Therapy (ISCT) and the European Society for Blood and Marrow Transplantation (EBMT). Cytotherapy. 2024 Jul;26(7):681-685. doi: 10.1016/j.jcyt.2024.02.007. Zotarelli Filho IJ, Frascino LF, Greco OT, Araujo JDD, Bilaqui A, Kassis EN, Ardito RV, Bonilla-Rodriguez GO. Chitosan-collagen scaffolds can regulate the biological activities of adipose mesenchymal stem cells for tissue engineering. J Regen Med Tissue Eng; 2013, 2:12. Irioda AC, Cassilha R, Zocche L, Francisco JC, Cunha RC, Ferreira PE, Guarita-Souza LC, Ferreira RJ, Mogharbel BF, Garikipati VN, Souza D, Beltrame MP, de Carvalho KA. Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression. Stem Cells Int. 2016;2016:2562718. doi: 10.1155/2016/2562718. Planat Bernard V, Silvestre JS, Cousin B. Plasticity of human adipose lineage cells towards endothelial cells: physiological and therapeutic perspectives. Circulation; 2004, 109: 656 -63. Planat Bernard V, Silvestre JS, Cousin B. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ. Res; 2003, 94: 223-9. Soost HJ, Lange HJ, Lehmacher W, Ruffing-Kullmann B. The validation of cervical cytology. Sensitivity, specificity and predictive values. Acta Cytologica, 1991, v. 35, n. 1, p. 8-14. Nanda K, McCrory DC, Myers RR, Bastian LA, Hasselblad V, Hickey JD, Matchar DB. Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review. Annals of Internal Medicine, 2000, v. 132, n. 10, p. 810-819. Karimi-Zarchi M, Peighmbari F, Karimi N, Rohi M, Chiti ZA Comparison of 3 ways of conventional pap smear, liquid-based cytology and colposcopy vs cervical biopsy for early diagnosis of premalignant lesions or cervical cancer in women with abnormal conventional Pap test. International Journal of Biomedical Science, 2013, v. 9, n. 4, p. 205-210. Sumner AT. Chromosomes – Organization and Function. Blackwell Publishing: United Kingdon-UK. 2003, 287p. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9106198","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":619885054,"identity":"efbcacfc-30f0-4bd2-8362-50e8f674f926","order_by":0,"name":"Idiberto José Zotarelli-Filho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYPACCxk2BsYHBx42MMhBRQ7gUc0MIiR42BiYDQ4kNjAYE68FyDBgAGoBIgJa5Nv7D36uqJDg4WNgZjyQuMMmfcPt5sOfCxju5OPSwthzmFnyzBmwwxgOJJ5Jy91w51ia9AyGZ5YNuJwlkcwg2dgG0sJ/4EBi2+HcDTdyzJh5GA4b4LKFTSKZ+WfjP5gtbYfTDW7kf/6MTwuPRDKbZGMDQkuCwY0cBml8WiR4DptZNhwDamEGa0kznHkjzUx6hsEznFrk2xsf32yosZGTb29m/vCxzUae70by488FFXdwakEKCBQ2ERpwax8Fo2AUjIJRAAC6O1GvpBEW4wAAAABJRU5ErkJggg==","orcid":"","institution":"UNESP - São Paulo State University. Institute of Biosciences, Humanities and Exact Sciences (Ibilce), Postgraduate Program in Food, Nutrition and Food Engineering","correspondingAuthor":true,"prefix":"","firstName":"Idiberto","middleName":"José","lastName":"Zotarelli-Filho","suffix":""},{"id":619885056,"identity":"255b817f-2b39-41fd-bde7-ebc6ff2c5592","order_by":1,"name":"Durval Ribas Filho","email":"","orcid":"","institution":"ABRAN - Brazilian Association of Nutrology","correspondingAuthor":false,"prefix":"","firstName":"Durval","middleName":"Ribas","lastName":"Filho","suffix":""},{"id":619885057,"identity":"ae97601c-e28a-4966-8969-afa915f05e32","order_by":2,"name":"Nizar Abu Hamdeh","email":"","orcid":"","institution":"Palestinian Clinical Research Center, Bethlehem, Palestine.","correspondingAuthor":false,"prefix":"","firstName":"Nizar","middleName":"Abu","lastName":"Hamdeh","suffix":""},{"id":619885060,"identity":"7695cddf-ba3e-4289-ab90-901673a58220","order_by":3,"name":"Haitham Abu Khadija","email":"","orcid":"","institution":"Hebrew University of Jerusalem, Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haitham","middleName":"Abu","lastName":"Khadija","suffix":""},{"id":619885061,"identity":"10b47ab2-7a73-4348-a5c7-8be731aa781a","order_by":4,"name":"Mohammad Alnees","email":"","orcid":"","institution":"Palestinian Clinical Research Center, Bethlehem, Palestine.","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Alnees","suffix":""}],"badges":[],"createdAt":"2026-03-12 14:54:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9106198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9106198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106724957,"identity":"24d997ed-6100-4002-a007-deb418d423af","added_by":"auto","created_at":"2026-04-12 18:30:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58408,"visible":true,"origin":"","legend":"\u003cp\u003eBox-Plot model graph showing statistical values of mean, standard deviation and decline of the crosslinking degree curve with increasing genipin concentration. Source: Own authorship.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/8e2a0320968e840e5038c3aa.jpg"},{"id":106521675,"identity":"261ff586-4748-47e3-9a9c-199b4ce865c2","added_by":"auto","created_at":"2026-04-09 13:03:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108991,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability results in the third passage. Biological sample representative of a patient. Source: Own Authorship\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/3e8539692d2ef1cf807ae619.jpg"},{"id":106724615,"identity":"ecd24334-be30-422b-9a8e-7f63b933ef41","added_by":"auto","created_at":"2026-04-12 18:28:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175960,"visible":true,"origin":"","legend":"\u003cp\u003eHistograms for the analysis of the expression of CD31, CD90, CD34, CD117, CD54, CD73, CD44, CD133, CD45, CD105 and CD166 in cells of the region of larger size and granularity. Representative result of ADSC immunophenotyping after culture in the biomatrices with 0.75% v/v genipin. Source: Own Authorship.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/9425cd487139c61e34dd5cfd.jpg"},{"id":106724614,"identity":"08ac5a03-891c-497b-8fa2-cd24ed43c5b8","added_by":"auto","created_at":"2026-04-12 18:28:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148618,"visible":true,"origin":"","legend":"\u003cp\u003eOptical microscopy images showing in Figures A and B the ADSC adhered in the Petakas and in the Figures C and D the ADSC in mitotic spindle. Source: Own Authorship.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/a68a8504fd1a80e171a0a876.jpg"},{"id":106521677,"identity":"f4b67ad5-6887-4696-bf63-268f70698eeb","added_by":"auto","created_at":"2026-04-09 13:03:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325694,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of individual biopolymers and together (chitosan-collagen-genipin-scaffolds) and scaffolds with ADSC adhered in mitotic phase and also proliferated. Source: Own Authorship.\u003c/p\u003e\n\u003cp\u003eSEM images of individual biopolymers and together (chitosan-collagen-genipin-scaffolds) and scaffolds with ADSC adhered in mitotic phase and also proliferated (with 2.1x10\u003csup\u003e6\u003c/sup\u003e ADSC mL\u003csup\u003e-1\u003c/sup\u003e). Source: Own Authorship.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/afb57af38ef88dd1ba08e4a8.jpg"},{"id":106521680,"identity":"8d7f23de-94d8-4ea2-9537-75716ee98b85","added_by":"auto","created_at":"2026-04-09 13:03:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":188830,"visible":true,"origin":"","legend":"\u003cp\u003eImages showing the differentiation of ADSCs in three different cell lines, such as adipocytes, osteocytes and chondrocytes. Source: Own Authorship.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/c413750149b15403176325b4.jpg"},{"id":106724782,"identity":"b6a41fc9-4b18-42c1-ba0d-d3ef29e5b451","added_by":"auto","created_at":"2026-04-12 18:29:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":57784,"visible":true,"origin":"","legend":"\u003cp\u003eOptical microscopy image representing the total sample of the present study, showing metaphase chromosomes of ADSC with their representative karyotype, with usual Giemsa staining (without trypsin). Image obtained before cryopreservation. Source: Own Authorship.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/80b5fa160c841dc057c162c7.jpg"},{"id":106725036,"identity":"eaaf91ae-d3a1-489a-96ac-cb97b6c43436","added_by":"auto","created_at":"2026-04-12 18:31:08","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":157381,"visible":true,"origin":"","legend":"\u003cp\u003eImages showing the nuclear quantification and morphology of the control and test ADSC samples. Source: Own Authorship.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/b149735cf78e6e0444c3711a.jpg"},{"id":107697769,"identity":"ffc65163-a040-4ea6-b9d0-21dba79e49fe","added_by":"auto","created_at":"2026-04-24 07:27:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1684857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/61b51aab-0d9b-44b8-bcc8-377c50df60c0.pdf"},{"id":106521673,"identity":"ffbd72bd-d153-4534-a10a-da06294cd0d2","added_by":"auto","created_at":"2026-04-09 13:03:32","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":180477,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9106198/v1/ce08e1bded0d72d875ea341e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan-collagen scaffolds cross-linked with genipin demonstrated cytocompatibility at different levels of bioanalysis with human adipose-derived mesenchymal stem cells for regenerative medicine: a research article","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eA significant number of people worldwide have seen their life expectancy increase due to regenerative medicine and tissue engineering [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In this context, it is worth noting that the main cause of hospitalization in Brazil is due to injuries caused by accidents in general. In 2022 alone, the Brazilian Ministry of Health invested more than 154.9\u0026nbsp;million in the manufacture of 3,298,667 orthoses, prostheses, and mobility devices, including wheelchairs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, the American Heart Association estimates that more than 6.5\u0026nbsp;million Americans suffer from congestive heart failure, and in Brazil, approximately 350,000 people have acute myocardial infarction per year [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, tissue engineering is crucial to meet this demand through the development of biomaterials (scaffolds) to promote tissue regeneration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this regard, regenerative medicine and tissue engineering must comply with the entire cellular machinery to chemical, physical, and biological processes to achieve the appropriate biological niche, that is, to create a microenvironment favorable to tissue regeneration through signaling cascades and neovascularization [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven this, in the present study, chitosan-collagen-genipin biomatrices were used, with the degree of deacetylation (DD) of chitosan being above 0.4 to achieve good solubility with collagen and to bind the Schiff base with genipin, conferring greater stability to the biomatrix to favor cell growth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mixture of the two biopolymers reconciles the cytocompatibility of collagen with the adhesion forces of chitosan. Collagen is bioadhesive through specific arginine-glycine-aspartate (RGD) sites. The RGD group promotes cell adhesion by binding to integrin receptors, thus promoting cell growth and differentiation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenipin is a hydrophilic organic compound whose structure was discovered in 1960 and is extracted from geniposide (origin: gardenia fruit) and has low cytotoxicity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Sung et al. (1999) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] found that genipin was 10,000 times less cytotoxic than glutaraldehyde.In this context of the biomatrices, the adult stem cells, prominent to adipose-derived stem cells (ADSC), appeared as an alternative for the cellular therapy, since they were found to have some degree of plasticity, with a capacity of self-renewal and differentiation in specialized progenitors. The most widely studied stem cells are those derived from bone marrow, which is divided into two main cell types: hematopoietic stem cells, which originate from blood cells, and the adipose-derived mesenchymal stem cells (ADSC), which are in the medullary stroma involving the hematopoietic cells and have the potential for differentiation in multiple tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, ADSC also has the advantages of self-renewal, immunomodulatory character, multipotentiality, ease of isolation, purification, and cryopreservation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to the recommendations of the International Society of Cell Therapy, to be used in studies, they must present the minimum characteristics as adherent and proliferative cells, differentiate into at least three cell lines (adipocytes, chondrocytes, and osteocytes), and present the mesenchymal panel with typical cell surface markers, in addition to cytogenetic stability. Some studies also show that there is an important link between fat cells and the vascular network. These cells represent a heterogeneous population of microvascular endothelial cells, constitute a convenient source of multipotent cells, and are non-restrictive [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eADSC, when in intense activity, can express the exosomes (extracellular vesicles-EV) according to the environment in which they are found. EVs can promote the activation of the immune system through the cytokines that carry on its membrane, such as tumor necrosis factor (TFN). An example of the role of exosomes in the immune system is demonstrated by the ability to act as 17 antigen presenters for T cells due to the presence of Major Histocompatibility Class (MHC) I and II molecules [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the evolution of cell culture, Celartia\u0026reg; was formed to develop better quality instruments for in vitro cell growth, such as Petakas, to transform the ancient art of cell culture engineering, to increase ease of management reduce costs, and minimize the environmental impact of cell culture. Petaka is a device designed for the culture of adherent cells and the culture of non-adherent cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, in this scenario of technological advances in regenerative medicine and human tissue engineering, the present study aimed to respond to several information gaps by analyzing the cytocompatibility of chitosan-collagen-genipin biomatrices in the presence of mesenchymal stem cells of human adipose tissue, analyzing the frequency of chromatid and chromosome breaks and failures, and/or numerical chromosomal alterations in ADSC in biological samples from the two patients under different culture conditions (after the 3rd passage and after cryopreservation for 12 months), in the presence and absence of chitosan-collagen-genipin biomatrix, and analyze the presence of multinucleation and nuclear polymorphisms of ADSC after contact with the biomatrix. Also, this study investigated the intrinsic characteristics of these cells as the ability to differentiate the cultured cells in osteoblasts, chondroblasts, and adipocytes, immunophenotyping, and proliferation in the biomatrices.\u003c/p\u003e"},{"header":"METHODS AND MATERIALS","content":"\u003cp\u003e\u003cstrong\u003eStudy Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study followed an experimental study model (research article), involving the analysis of human biological material at the \u003cem\u003ein vitro\u003c/em\u003e level.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was evaluated and approved by the research ethics committee of the Beneficência Portuguesa Hospital of São José do Rio Preto, São Paulo, Brazil, under number of approval process 030427.\u0026nbsp;All procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003enformed consent was obtained from all participants involved in the study, with all procedures explained in detail before participation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the development of the present study, two patients aged between 20 and 30 years and candidates for an aesthetic procedure of liposuction were selected and accepted to donate the biological material (liposuction - adipose tissue) for the sole use of in vitro studies. Both patients signed the informed consent form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObtaining and Selecting Adipose-Derived Mesenchymal Stem Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdipose-Derived Stem Cells (ADSC) from two young women (aged 20-30 years) were studied. The biological material was donated by plastic surgeon Dr. Luiz Fernando Frascino of São José do Rio Preto. The technique for extracting adipose tissue used manual liposuction with a 2.5 mm cannula. According to the protocol of Zuk et al. [24], adipose tissue was treated with Clostridium histolyticum collagenase type IV (Sigma-Aldrich) for two hours for extraction of vascular stromal fraction (Figure 1). The fat tissue suspension and cells were then taken to the Sepax system to obtain and wash the cell pellet (pellet) which was then resuspended in a culture medium to a concentration of about 2.5 x 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e for culture in chitosan-collagen-genipin matrices, Petakas and cryopreservation for one year. The two samples of 2.0 mL of adipose tissue (01 sample per patient) were obtained from the abdomen region of two women who passed the selection criteria. Smoking or drug-dependent women with a history of autoimmune diseases, hematological diseases, previous chemo or radiotherapy, heart disease, renal, hepatic, and psychiatric diseases were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of Stromal Vascular Fraction (SVF) from Adipose Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Coleman et.al. (2006) [25], the adipose tissue samples will be extracted by the manual liposuction method (with a 2.5 mm diameter Cannula) and taken to the laboratory for ADSC extraction procedures. Fatty tissue samples will be homogenized and an initial volume of 20.0 mL will be divided into 2.0 mL samples. Subsequently, to these volumes, 6.0 mL of collagenase type IV (\u003cem\u003eClostridium hystoliticum\u003c/em\u003e - Sigma Aldrich) will be added in a ratio of 1:3 (fat: collagenase), and the mixture will be brought into a 37.0 ° C incubator, 5.0 % CO\u003csub\u003e2\u003c/sub\u003e, for 1.0 hour and a half. After this time, the mixture will be centrifuged at 800 xg for 10 minutes at 23.0 ° C and then the pellet will be resuspended in 10.0 mL culture medium, and 1.0 mL will be used for the assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryopreservation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Nardi and Meirelles (2006) [26], the cells will be frozen in RPMI 1640 culture medium containing 10.0% FBS (Gibco) and 10.0 % DMSO (Life Group). The cells will be trypsinized and resuspended in RPMI medium and transferred to a falcon tube (± 5.0 mL), centrifuged at 900 xg for 5.0 minutes, the supernatant removed and the pellet resuspended with the chosen freezing solution. The amount depends on the pellet or number of cells (concentration approximately 106 to 107 cells Ml-1) 1.0 mL of the prepared solution was placed in a screw-capped freezing tube, which will remain for about one hour in the freezer at -20.0 °C, about 24 hours -80.0 °C and will be transferred to the tank with liquid nitrogen (± - 196.0 °C), where it will remain for one year passage in the Petakas, cell growth in the matrices and genetic stability assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular Defrost\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cryotubes with the cryopreserved cells were removed from the liquid nitrogen reservoir where they were stored and thawed in a 37.0 °C thermostatic bath. After, the cells were suspended in an Alfa MEM medium supplemented with 10.0 % FBS, and centrifuged at 400 xg for 10.0 min. The thawing process had an average duration of 3 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViability of ADSC by trypan blue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Nardi and Meirelles (2006) [26], 10.0 μL of cell solution will be placed in the Neubauer chamber for quantification of live and dead cells, with replicates of n=3.0. The Neubauer chamber has its central band in a position determined millimetrically below the sides, so that the placement of a coverslip, well adhered to the edges of the depression, gives rise to a chamber with a very determined volume. After placing a sample of the cell suspension in this chamber so that it is filled up, but without extravasation, the viable (uncolored) cells are counted under the microscope. The chamber has four quadrants (Q1, Q2, Q3 and Q4) with an area of 0.1mm² each. When integrating a glass cover plate into the Neubauer Chamber, you get a height of 0.1mm. The volume capacity is then 0.1mm x 0.1mm² = 0.1mm³.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultivation of ADSC in Petakas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the modified Celartia® [23] methodology, ADSC will be cultured in Petakas in the approximate amount of 2.5 x 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e, which will be submitted to the tenth passage in alpha-MEM medium with 10.0 % fetal bovine serum and antibiotics. The cells will be infused into the Petakas using a waterproof and sterile membrane. After infusion, the set (cells and Petaka) will be held horizontally on one side for 1.0 hours and soon thereafter will be turned on the other side and held for at least 12 hours. After this time, the set will be placed vertically for the cell culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture of ADSC in the Chitosan-Collagen-Genipin Scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor ADSC culture, according to Nardi and Meirelles (2006) [26], these cells were expanded in alpha MEM culture medium supplemented with 10.0% v/v fetal bovine serum containing 1.0 % v/v ampicillin and streptomycin. After reaching about 85.0% confluence, the cells revolved from the surface of the plate with trypsin-EDTA, and then the cells were counted in the Neubauer chamber to know the number of live cells to be seeded on the biomatrices. After sowing, the Biomatrices were transferred to an incubator at 37.0 °C with 5.0% carbon dioxide, a time of 72.0 h for further analysis. The matrices were sterilized in a solution of 70.0 % ethanol (overnight) and then in the presence of ultraviolet light (15.0 min). After washing the biomatrices six times with sterile water, about 1.0 x 10\u003csup\u003e5\u003c/sup\u003e ADSC were seeded into the biomatrices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of the Chitosan-Collagen-Genipin Scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the method described by Baldwin and Kiick [27], the previously characterized chitosan (Mw = 115 kDa and degree of deacetylation of 85.25 %) was dissolved in 10.00 mL of 2.50 % v/v acetic acid solution for 24 hours at room temperature. The collagen was dissolved. The chitosan-collagen scaffold was prepared by mixing the two solutions under stirring for 96 h. The solutions were transferred to a 96-well plate, with a volume of 170 μL, in a ratio of 1: 1 v/v. Finally, the scaffold was crosslinked at 0.75% v/v genipin. The mixture was then frozen in liquid nitrogen (-196.0 °C) and then lyophilized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning Optical Microscopy (OM) and Scanning Electron Microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn optical microscopy, the ADSCs in the Petakas were photographed in the third passage with 85.0 % confluence and also after the 48h time in the third passage, in which phase the largest number of cells in the mitotic phase can be found, with 2.5x10\u003csup\u003e6\u003c/sup\u003e ADSC. In SEM, images of the ADSCs in the Petakas and the Biomatrices were also captured in the third passage with 85.0 % confluence and also after the time of 48h in the third passage, phase in which one can find the largest number of cells in mitotic phase, with 2.1x10\u003csup\u003e6\u003c/sup\u003e ADSC. In addition, the presence of exosomal production on the surface of ADSCs was also analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunophenotyping Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter removal of the cells from the culture flasks, performed with the aid of the trypsin solution, they were suspended in 1.0 mL of PBS supplemented with 3.0 % human albumin (Grifols® 20.0 % Human Albumin, PBS/A). The resulting suspension was subjected to the determination of the number and proportion of non-viable cells by the Trypan blue staining method as described in section Cell Viability, followed by adjustment of the viable cell density to 106 mL-1 cells. The immunophenotypic evaluation of these cells was carried out using the immunophenotyping technique by flow cytometry on a FACSCanto II® (BD Biosciences) cytometer, using the monoclonal antibodies conjugated with fluorochromes, and their respective isotypic controls.\u003c/p\u003e\n\u003cp\u003eAlso, ADSCs from the last pass of all cultures, homogenized in PBS/A, were separated into 100.0 μL aliquots and dispensed into 5.0 mL tubes for flow cytometry. After, 5.0 μL of each antibody was added to the tubes and homogenized by vortex stirring. The suspensions were then incubated for 15.0 min at room temperature in the dark. Then 400.0 μL of PBS/A was added to each tube, followed by homogenization by shaking and centrifugation at 400 × g for 10.0 min. The supernatant was discarded and the pellet homogenized in 500.0 μL of PBS / A. To tube 3 of each sample was added 5.0 μL of 7-AAD moments before cytometer acquisition. The ADSCs were then acquired on a cytometer coupled to the BD FACSDiva II® analysis software (BD Biosciences) and subsequently analyzed by Infinicyt® software (Cytognos) for the binding/labeling of each antibody (marker expression) and 7-AAD (cells). In all, eighteen anti-CDs were analyzed, involving the CD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADSC differentiation analysis (Trileneage) - Monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsteogenic Differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOsteogenic differentiation was induced in ADSC cultures in 6-well plates exhibiting 85.0 % confluency. In these, three wells had the MEM / SFB alpha media supplemented with 10.0 nM dexamethasone, 10.0 mM β-glycerol phosphate, and 50.0 μg mL-1 ascorbate-2-phosphate (Sigma-Aldrich). The osteogenic differentiation medium (alpha MEM / DIFO) was prepared by mixing 5.0 mL of ascorbate-2-phosphate / β-glycerol phosphate solution in alpha MEM / SFB medium (0.05 % ascorbate-2- phosphate and 2.16 % β-glycerol phosphate in MEM alpha medium, m/v); 98.0 μL dexamethasone solution (0.1 % in ethanol, m/v, diluted in alpha MEM, 1:49, v/v); 44.02 mL of MEM / SFB alpha medium; After homogenization, the solution was membrane filtered with 0.22 μm micropores.\u003c/p\u003e\n\u003cp\u003eThe ADSCs were incubated with MEM / DIFO alpha medium at 37 ° C and 5.0 % CO\u003csub\u003e2\u003c/sub\u003e for 14 days, with medium replacement every 72 h. The same procedure was performed in parallel with the negative control wells, with MEM / SFB alpha culture medium. After the differentiation period, the plates were submitted to staining to reveal secretion of mineralized extracellular matrix (differentiation in osteocytes), according to the following procedures: removal of the culture medium from all wells (differentiation and control); rinsing well with PBS (2x); fixation of cells with 2.5 % aqueous solution of glutaraldehyde (m/v) for 1 h 30 min; rinse with distilled water (2x); dehydration with isopropanol 60.0% (v/v) and incubation at room temperature for 5 min; (1.37 % in ultrapure water, m / v, pH 4.1-4.3, Sigma-Aldrich) and incubation at room temperature for 45 min; discard the dye solution and rinse with PBS (3x); photo-documentation and inverted microscope analysis [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChondrogenic Differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChondrogenic differentiation was induced through the commercial StemPro® Chondrogenesis Differentiation Kit (Gibco®). For this, a micro mass culture was performed where the cells were resuspended in a concentration of 1.6 x 10\u003csup\u003e7\u003c/sup\u003e viable cells mL\u003csup\u003e-1\u003c/sup\u003e, of this total cell was aliquoted 1.0 mL of cell suspension that was transferred to another Petaka, where it remained for 2 hours in an oven with 5.0 % CO\u003csub\u003e2\u003c/sub\u003e at 37.0 °C. After the incubation period, 500.0 μL of differentiation medium (StemPro Chondrogenesis Differentiation Kit - Gibco) was added, and in control cells standard culture medium. The media was replacedtwice a week. After 14 days the medium was removed and the cells were fixed with the cells fixed with 2.5 % glutaraldehyde (Merck / Millipore, USA) at room temperature for 90.0 minutes. Following fixation, the Alcian Blue dye (Sigma®, USA), (1.0 % prepared in 0.1N HCl (F.Maia® / Brazil)) was added for 30.0 minutes, then the cells were washed in 0.1 N of HCL by 3 times, and the staining was analyzed under inverted optical microscope. Where staining was observed indicates the synthesis of proteoglycans produced by the chondrocytes [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdipogenic Differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the induction of adipogenic differentiation, ADSC cultures were used in Petakas. Upon reaching 85.0 % confluency, three wells had the MEM / SFB alpha medium supplemented with 0.5 mM isobutylmethylxanthine (3-isobutyl-1-methylxanthine), 50.0 μM indomethacin and 0.5 μM dexamethasone (Sigma- Aldrich). The adipogenic differentiation medium (MEM/DIFA alpha) was prepared by mixing 3.0 mL of IBMX solution (0.18% in water); 90.0 μL of 1.0 % indomethacin solution in ethanol; 490.0 μL dexamethasone solution (0.1% in ethanol; diluted in alpha MEM, 1:49, v/v); and 46.4 mL of MEM / SFB alpha medium. After homogenization, the solution was membrane filtered with 0.22 μm (polyvinylidene fluoride membrane) micropores.\u003c/p\u003e\n\u003cp\u003eADSCs were incubated with MEM/ DIFA alpha medium at 37 °C and 5.0 % CO\u003csub\u003e2\u003c/sub\u003e for 14 days, with medium replacement every 72 h. The same procedure was performed in parallel, with negative control Petakas, with alpha MEM/SFB culture medium. After the period of differentiation, the plates were submitted to staining to reveal cytoplasmic accumulation of lipid droplets (differentiation in adipocytes), according to the following procedures: removal of culture medium from all wells (differentiation and control); rinsing well with PBS (2x); fixation of cells with 2.5 % aqueous solution of glutaraldehyde (m/v, Sigma-Aldrich) for 1 h 30 min; rinse well with distilled water (2x); dehydration with isopropanol 60.0 % (v/v) and incubation at room temperature for 5 min; (0.3 % in isopropanol 99.0 %, m / v, diluted in water 3: 2 v/v) and incubation at room temperature environment for 5.0 min; discard the dye solution and rinse with distilled water (2x); photo-documentation and inverted microscope analysis [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytogenetic Analysis of ADSC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter cell expansion in the 3rd passage, after 48 h of culture in Petakas (control) and chitosan-collagen-genipin biomatrices, the cells were subjected to the usual staining with Giemsa, according to the protocol routinely used in the Genetics Laboratory of the Hospital de Base de São José do Rio Preto/SP. The study of chromosome stability was performed based on the presence of chromatids chromosome breaks and gaps, and numerical chromosome alterations. The findings were interpreted and described according to information and standardization contained in the International System for Human Cytogenetic Nomenclature (ISCN, 2024) [28]. Cytogenetic analysis was performed on 480 cells in total (240 from each patient), considering 60 metaphase cells from four samples from each participant. For each patient, two samples with metaphase cells were considered (60 control cells and 60 cells in the biomatrix). Thus, for each sample, from each patient, 60 metaphases of cells cultured in the biomatrix and 60 cells cultured in the Petakas were evaluated. After 12 months of cryopreservation, 60 metaphases cultured in the biomatrices and 60 cultured in the Petakas of each patient were also evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtocol for Obtaining Metaphase Chromosomes by Usual Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDividing adipose mesenchymal stem cells were blocked in metaphase with colcemid at 0.1μg mL\u003csup\u003e-1\u003c/sup\u003e (Gibco), detached from the growth surface with trypsin at 0.25% (Gibco), and subsequently hypotonized by exposure to a 0.057M KCl solution (Merck). The cells were fixed in a 3:1 solution of methanol and acetic acid. Cytogenetic analysis of metaphase chromosomes was performed by usual staining [29] and the images were captured on a BX60 microscope (Olympus) using the ImagePro Plus program. The findings were checked by a cytogeneticist and the results were characterized according to the ISCN standards [28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdapted Pap Smear Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the cultures reached\u0026nbsp;≅85.0% confluence, the culture medium was removed from the Petakas and discarded, and the wells were washed with PBS at 37.0 °C (3x). Afterward, the cells in the Petakas were stained with Harris hematoxylin, Orange G6, and EA36, which are included in the Papanicolaou® kit for exfoliative cytology (Newprov) [30]. The procedures were repeated for each petaka, at room temperature. This staining was performed by removing and discarding the PBS; incubation with 80.0%, 70.0%, 50.0% ethanol, and distilled water, for 2.0 min each. After removing and discarding the water, 1.0 mL of Harris hematoxylin stain was added, incubating for 2.0 min. The Petakas were washed several times (10x) with distilled water. The excess water was removed and incubation was carried out with 50.0%, 70.0%, 80.0%, and 96.0% ethanol, for 2.0 min each. After discarding the 96.0% ethanol, 1.0 mL of Orange G6 stain was added, incubating for 2.0 min. This dye was discarded and 1.0 mL of 96.0% ethanol was added, incubating for 2.0 min (2x). Then, 1.0 mL of EA36 dye was added, incubating for 2.0 min. The Petakas were then incubated three times with 96.0% ethanol, for 2.0 min each. After that, the Petakas were subjected to microscopy analysis, initially performed under an inverted microscope, with subsequent image capture performed on an Olympus BX51 microscope, coupled to a DP70 digital camera and DP70-BSW software. For each sample (each patient), images of at least ten visual fields were captured with 200x magnification, analyzing 100 cells after 72 hours of cultivation in Petakas, 100 cells in biomatrices, 100 cells after 12 months of cryopreservation of the same cells in Petakas and 100 cells in biomatrices, with a total of 800 cells for the two participants in the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor data analysis, a database was built in the Microsoft Excel spreadsheet which was exported to the Stata 18 and Minitab 21 statistical program. Common descriptive statistical analysis and the Anderson-Darling normality test were performed for all variables, controls, and treatments. Values of mean and standard deviation were also evaluated. After that, a parametric regression test was performed. As there was the presence of the predictors and the continuous and categorical predictors, the linear regression and residual analysis of Durbin-Watson. For all linear regression tests, an alpha level lower than 0.05 was adopted as significant. For the Durbin-Watson residue analysis, the reference significance level was 0.05, adopting an acceptable independence interval of 1.75 \u0026lt;dw \u0026lt;2.60 (according to the Durbin-Watson standard table, dU \u0026lt;dw \u0026lt;4-dU).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePhysicochemical Properties of the Chitosan-Collagen-Genipin Biomatrix\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDue to the increased stiffness of these biomaterials with increasing genipin concentration, the cross-linked biomatrix with 0.75% v/v genipin exhibited moderate stiffness and a swelling rate of approximately 80.0% (Figure 1). The average pore size of the biomatrix was 40 \u0026micro;m (p\u0026lt;0.05). Regarding the degradation rate, the cross-linked biomatrix with 0.75% v/v genipin exhibited the lowest mass loss rate during immersion in human blood solution. Therefore, the biomatrix with 0.75% v/v genipin exhibited the best physicochemical characteristics in this study and was therefore the selected biomatrix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Counting and Viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Figure 2, triplicate cell viability tests with trypan blue showed higher cell viability in the biomatrices with lower percentages of free amino groups, to the cells of the two patients of the present study. As an example, the chitosan (Ch) scaffolds presented the lowest cell viability (86.5 %) and the chitosan-collagen scaffolds (with 0.75 % and 1.00 % v / v genipin) had the highest cell viability (99.8 %) to the reference of 99.5 % as the minimum value recommended by the International Society for Cellular Therapy (ISCT).\u003c/p\u003e\n\u003cp\u003eThere was no statistical significance between the chitosan-collagen sample with 0.75% (v / v) genipin with the chitosan, collagen, and chitosan-collagen control groups, with p\u0026gt; 0.05. The residual analysis of Durbin-Watson confirmed that there was no similarity between these samples, with the result of dw between 1.75 \u0026lt;dw \u0026lt;2.60.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunophenotyping Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe culture of ADSC derived from human adipose tissue is shown with a phenotypically heterogeneous population. These cells were located in the upper right region of the dot-plot for FSC x SSC, corresponding to cells of larger size and granularity. Analysis of the described region revealed a high frequency (greater than 70.0 %) of antigen expression characteristic of ADSC (CD166, CD105, CD90, CD73, and CD54) in the third passage, both in the presence of the chitosan-collagen scaffolds with 0.75% of genipin and Petakas (control).\u003c/p\u003e\n\u003cp\u003eThe R1 region contains the cells of larger size and granularity. In M2 the percentage of cells positive for CD105, CD166, CD34, CD133, CD45, CD31, CD73, CD44, CD90, CD117, and CD54 were presented in the total population and the region of larger and granular cells. The histograms of expression analysis of CD31, CD90, CD34, CD117, CD54, CD73, CD44, CD133, CD45, CD105, and CD166 in cells of the region of larger size and granularity (Table 1 and Figure 3).\u003c/p\u003e\n\u003cp\u003eTable 1.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRepresentative results of ADSC immunophenotyping after Cultivation in Biomatrices and Petakas.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCDs / Results (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; SVF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eExpansion (Petakas)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e Expansion\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eScaffolds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter Cryopreservation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Petakas)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter Cryopreservation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Scaffolds)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e46.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 51,61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n 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valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e47.28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e48.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e45.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e46.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 44\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e19.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n 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\u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e67.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e65.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e67.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e55.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.71\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n 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valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e83.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e84.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e34.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n 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valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e17.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHLA-DR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.43\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n 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\u003cp\u003e\u003cstrong\u003eCD 146\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e82.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e81.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e80.02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e82.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 105\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.06\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 106\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.69\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 73\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e52.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e54.41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e49.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e53.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSource: Own Authorship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptical and Electronic Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Figure 4, the ADSC adhered and proliferated in the Petakas, and the C and D images, can be observed in images A and B, the same cells can be analyzed in the mitotic process, with 2.5x10\u003csup\u003e6\u003c/sup\u003e ADSC mL\u003csup\u003e-1\u003c/sup\u003e. In SEM images, the biopolymers of chitosan, collagen, and genipin were observed individually, as well as in their combined chitosan-collagen-genipin (scaffolds) form. It also analyzed the adhesion and proliferation of ADSC in the scaffolds, showing images with ADSC in the mitotic phase (with 2.1x10\u003csup\u003e6\u003c/sup\u003e ADSC mL\u003csup\u003e-\u003c/sup\u003e1) and also images in the proliferative phase and with cytoplasmic projections, according to Figure 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prove the differentiation capacity of mesenchymal stem cells, we standardized an efficient protocol for differentiation in the osteogenic, chondrogenic, and adipogenic lines. After cell expansion and flow cytometry characterization, differentiation was started in the three lines described.\u003c/p\u003e\n\u003cp\u003eAfter the adipogenic differentiation, the mesenchymal stem cells presented cytoplasmic red staining in small vesicles when submitted to the staining with Oil Red. The property of this dye to interact with lipids and neutral triglycerides shows a red color that shows the accumulation of these substances in cytoplasmic vesicles of the differentiated cells. As can be seen in Figure 5, mesenchymal stem cells lost their fibroblast morphology and acquired a spherical morphology with numerous vesicles, characterizing the accumulation of lipids.\u003c/p\u003e\n\u003cp\u003eThe process of differentiation of the mesenchymal stem cells into the osteogenic lineage was evidenced by the Alzarin Red S coloration that stains red deposits of calcium and other minerals. As can be seen in Figure 05, around the differentiated cells are reddish regions, which are supposed to be deposits of minerals from the formation of a mineralized extracellular matrix, similar to that of the bone tissue. Another striking feature is the change in morphology, as the fibroblast cells alter their shape to elongated and aligned cells, covering the entire culture plate and in some places, forming cellular clusters.\u003c/p\u003e\n\u003cp\u003eAfter three weeks the mesenchymal stem cells induced for chondrogenic differentiation were stained with Alcian blue. Cells showed expansion in cell size with a significant increase in cytoplasm, becoming rounded and flattened. A bluish cell coloration can be observed due to the interaction of the dye with the glycosaminoglycan expressed during differentiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Figure 6 shows the ADSCs in the third passage presenting fibroblastic morphology. Differentiated mesenchymal stem cells in adipogenic lineage stained with Oil Red, showing accumulation of lipid vesicles in cells. D differentiated mesenchymal stem cells in the osteogenic lineage stained with Alzarin Red S showing accumulation of calcium formed, and mesenchymal stem cells under optical microscopy during differentiation culture, showing the formation of colonies and alignment of cells with their elongated structure. Differentiated mesenchymal stem cells in chondrogenic lineage stained with Alcian blue, changes in cell morphology (rounded), and matrix production with blue-stained proteoglycans (n=6) were observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytogenetic Analysis \u0026ndash; Chromosomal and Chromatid Stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study of the stability of the genetic material at the chromosomal level was performed based on the presence of chromosomal and chromatid breaks and gaps, in addition to numerical alterations. The cells were subjected to the usual staining with Giemsa, according to protocols routinely used in the Genetics Laboratory of the Hospital de Base (Hemocentro/S\u0026atilde;o Jos\u0026eacute; do Rio Preto/SP). The findings were interpreted and described according to information and standardization contained in the ISCN [28].\u003c/p\u003e\n\u003cp\u003eThe cytogenetic tests were performed after cell expansion in the 3rd passage, after 48 h of culture in Petakas (control) and chitosan-collagen scaffolds with genipin at 0.75% v/v. The same was repeated after 12 months of cryopreservation. A total of 480 cells were analyzed (samples from both participants), as previously described.\u003c/p\u003e\n\u003cp\u003eTo better understand the most precise time in days to obtain a greater number of ADSC in metaphase, for the interruption and stabilization of chromosomes, before and after cryopreservation, an analysis of cell growth kinetics was performed. Through this analysis, it can be understood that the best time to apply colcemid was after 24 hours (one day) of the second change of culture medium. The best time was from the eighth (8) to the ninth (9) day. Chromosomal analysis of the samples did not reveal any statistically significant difference between the frequencies of numerical alterations and/or chromatid and/or chromosome breaks or gaps, as shown in Table 2 and Figure 7, both in the metaphase chromosomes obtained after 48 h of culture in Petakas (control) and those obtained from cells cultured in the chitosan-collagen scaffolds with genipin at 0.75% v/v, before and after 12 months of cryopreservation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. Chromosomal and chromatid analysis of breaks/gaps and/or numerical alterations of ADSC in the presence or absence of biomatrices and before and after cryopreservation.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"654\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eN total = 960 metaphases\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1958%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePetakas_before (control)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBreaks/Failures\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e/Numerical Changes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4189%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiomatrices_\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebefore\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBreaks/Failures\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e/Numerical Changes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePetakas_After\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(control)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBreaks/Failures\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e/Numerical Changes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiomatrices_\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eafter\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBreaks/Failures\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e/Numerical Changes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026alpha;\u0026lt;0,05)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePatient 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample 2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1958%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e01/00/01\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e01/00/00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4189%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/01/02\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/00/00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/02/00\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e00/02/03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/01/02\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/01/02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep=4,23\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep=3,45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePatient 2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSample 2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1958%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e01/01/00\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e02/01/01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4189%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e02/00/00\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e01/01/00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/02/00\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/02/02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e02/01/02\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e03/01/02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep=2,23\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep=1,45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSource: Own Authorship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdapted Pap Smear Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll cell samples from the control and scaffolds were evaluated. For each sample and passage, having established the number of 400 cells per sample and passage, totaling 800 cells analyzed with the two patients, no cells with an altered number of nuclei and/or atypical nuclear morphology were identified (Figure 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBased on the Bethesda classification system [31-33], the possible morphological alterations of the nucleus that could be identified correspond to atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells - cannot exclude high-grade intraepithelial lesion (HSIL, ASC-H). Multinucleation is characteristic of low-grade squamous intraepithelial lesions (LSIL). However, these events were not found in the ADSC samples of the present study.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eSignificance of Biomatrices in the Tissue Engineering\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the context of tissue engineering and regenerative medicine, it is crucial to establish biomatrices that will enable the development of the appropriate biological niche in order to precisely achieve paracrine, autocrine, and endocrine effects through mesenchymal stem cells, exosomes, microRNAs, telocytes, and other cells and molecules. In this sense, biomaterials such as chitosan, collagen, and genipin present physicochemical and biological properties of cytocompatibility for regenerative processes, naturally maintaining tissue homeostasis and the functional stereochemical organization of the generated tissues and their systemic integration [1,34-38]. \u003c/p\u003e\n\u003cp\u003eIn the present study, the degradation of chitosan-collagen scaffolds with 0.75% v/v genipin was the most resilient in the presence of human blood in culture medium, as shown in Figure 1. This is explained by the higher energy required for the depolymerization of the crosslinking of the polymer chains (covalent crosslinking), mainly through the NH\u003csub\u003e2\u003c/sub\u003e groups of chitosan. The combination of chitosan, collagen, and genipin through covalent bonds enables balanced degradation kinetics. This occurs because chitosan reacts with carbonyl compounds through nucleophilic attack of its amino groups, forming covalent bonds with some groups of the collagen and genipin [13,14].\u003c/p\u003e\n\u003cp\u003eThe solubility of the scaffolds increases through the protonation of the amino groups with increasing crosslinking with genipin [39-41]. These results of the present study are similar to the results published by Chiono et al. (2008) [39]. This revealed that the best concentration of genipin for biomatrices fabrication was 0.75% v/v, similar to the results reported by Baldwin and Kiick (2010) [27]. \u003c/p\u003e\n\u003cp\u003eAlso, chitosan, as a polyelectrolyte, stimulated the expression of ADSC activities by paracrine and autocrine factors. Type I collagen, through the site-specific arginine-glycine-aspartate (RGD), promoted the adhesion of these cells by binding to integrin receptors, thus promoting cell growth and anchoring of proteins and cells to the extracellular matrix. Studies reported by Bet, Goissis, and Plepis (1997) [42] reported that the physical unit of type I collagen, designated as the \"D-period,\" is involved in controlling the biological activities that maintain the biological microenvironment in tissues. \u003c/p\u003e\n\u003cp\u003eIt is known that electrostatic ion-ion, ion-dipole, and dipole-dipole interactions, van der Waals interactions, π-electron interactions, and charge transfer complexes occur, forming ionic, hydrogen, and covalent bonds between the polymeric components of chitosan (-OH and -NH\u003csub\u003e2\u003c/sub\u003e groups) and collagen (OH-, -COOH, and -NH\u003csub\u003e2\u003c/sub\u003e groups), with negative Gibbs free energy (ΔG \u0026lt; 0), according to Domard and Taravel (1995) [15] and Israelachvili (2011) [41].\u003c/p\u003e\n\u003cp\u003eThe mixing of polycations (chitosan) and polyanions (collagen) can occur by spontaneous aggregation, with the consequent release of counterions, increasing entropy (ΔS \u0026gt; 0) [41,43]. Thus, the presence of counterions, as well as the presence of cations and anions of the individual polymer chains, stimulated the cellular activities of ADSCs in the present study, without causing cellular stress (absence of exosomes on the cell surface) and loss of quiescence of these cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImportance of Adipose-Derived Stem Cells \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eADSCs have the advantages of self-renewal, immunomodulatory character, multipotentiality, ease of isolation, purification, proliferation \"in vitro\" as well as cryopreservation [10]. ADSCs should exhibit minimal characteristics as adherent and proliferative cells, differentiate into at least three cell lines (adipocytes, chondrocytes, and osteocytes), and present the mesenchymal panel with the typical cell surface markers so that they can be used in studies, according to the recommendations of the International Society of Cell Therapy (ISCT) [44-47]. This differentiation translates the importance of these cells to be used with biomaterials as differentiation facilitators. Studies also show that there is an important link between fat cells and the vascular network [45].\u003c/p\u003e\n\u003cp\u003eThese cells represent a heterogeneous population of microvascular endothelial cells, constitute a convenient source of multipotent cells, and are non-restrictive [47]. Under culture conditions, adipose stem cells grow easily in monolayers, maintain multipotentiality normally up to the tenth passage, and exhibit fibroblastroid morphology. CD4, CD14, CD166, and HLA-1 positive markers are required for the immunophenotyping of the ADSC, and the CD4, CD14, CD16, CD16, CD54, CD55, CD59, CD90, CD105, CD106, CD146, CD166 and HLA-1 positive markers for CD11b, CD14, CD19, CD31, CD34, CD45, CD79 alpha, CD80, CD117, CD133, CD144, HLA-DR and Stro-1, the abbreviation CD stands for the English Cluster Differentiation [10,44,45].\u003c/p\u003e\n\u003cp\u003eAlso, ADSC secrete a cascade of cytokines and growth factors with paracrine, autocrine, and endocrine activities, such as macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), inflammatory protein macrophages (MIP-1α / CCL3) [46]. These factors when conjugated can produce some responses of the local immune system, stimulating angiogenesis and inducing proliferation and differentiation of mesenchymal stem cells in the desired tissue. In addition, ADSC induce the expression of junction proteins and increase microvascular integrity and nitric oxide (NO) production by macrophages [44-46].\u003c/p\u003e\n\u003cp\u003eUsing the vacuum equipment, the amount of ADSC obtained with the 12 samples was significant when compared with results available in the literature [44,45]. The use of the 3.0 mm cannula in the liposuction process allowed to increase in the surface area of the extracted fat, facilitating the activity of the enzyme collagenase IV to extract the stromal vascular fraction (SVF). In addition to this, the extraction of adipose tissue with blood vessels in these cells was also obtained in a larger quantity, with similar work presented by Zuk et al. (2002) [24] and Nardi; Meirelles (2006) [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultivation in Petakas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study of adhesion and proliferation of ADSCs in Petakas has proved to be relevant with remarkable results against cell culture publications in traditional flasks as demonstrated by Zotarelli-Filho et al. (2013) [48], because it was possible to obtain a larger number of cells in less time, dispensed with the use of gaseous CO\u003csub\u003e2\u003c/sub\u003e, decreased the dehydration process of the cell culture medium and allowed more controlled gas exchange, thus ensuring longer pH stability of the cell culture medium. This also made it possible to reduce the consumption of the culture medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentiation of ADSC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Irioda et al. [49], maintaining the differentiation potential of the ADSC under cultivation is a sine qua non for achieving the desired therapeutic effect. Thus, even though it does not compose the proposed flowchart for the screening of the cultures of these cells, it is only through the induction of differentiation that its pluripotentiality can be attested.\u003c/p\u003e\n\u003cp\u003eThe International Society for Cellular Therapy (ISCT), after striving to standardize the name for ADSC, indicated among the minimum criteria for the demonstration of the pluripotency of ADSCs, the induction of their differentiation in adipocytes, osteoblasts, and chondroblasts [47]. The literary record on ADSCs however, contains a great diversity of differentiation reports. In these reports, even when restricted to ADSCs, they include cell types derived from the three embryonic leaflets [44-47].\u003c/p\u003e\n\u003cp\u003eIn the present study, all the samples that maintained fibroblastoid growth patterns up to the third passage where in vitro cell differentiation analyses were started for the adipogenic, osteogenic, and chondrogenic lines. Differential staining allowed the detection of cytoplasmic lipid deposits typical of adipogenic differentiation, as well as the extracellular deposits of calcium crystals, typical of osteogenic differentiation, in all samples submitted to differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunophenotyping Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunophenotypic characterization of ADSCs usually includes expression of the CD73, CD90, and CD105 markers; and absence of expression of CD11b or CD14, CD19 or CD79α, CD34, CD45, and major histocompatibility class II complexes, mainly HLA-DR [24]. Although the results of ADSC immunophenotyping have been representative and in agreement with the literature, mainly because of the CD13 +, CD29 +, CD44 +, CD54 +, CD90 +, CD166 +, CD146 +, CD 49e, CD73 +, and CD 105 +, the phenotype of these cells is not conclusive, since most of these antibodies are not only expressed in ADSC, appearing also in fibroblasts, according to studies by Zuk et al. (2002) [24]. Therefore, the alkaline phosphatase enzymatic activity assays were conclusive for the identification of ADSC, as reported also by Planat Bernard et al. [50,51].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumorigenic potential (Pap smear) and chromosomal stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePap Smear Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIrioda et al. (2011) [30] were the first to report this type of application of the technique, including it as the first in the proposed algorithm. In the same publication, the authors also reported the detection of alterations in human ADSC. The Papanicolaou technique is a cost-effective tool used in the prevention and diagnosis of cervical cancer and has therefore been included in the routine screening of the adult female population in different countries [31-33]. Its efficiency and specificity are frequent targets of evaluation through different methodologies.\u003c/p\u003e\n\u003cp\u003eEvaluations based on repeat testing, such as that of Soost et al. (1991) [52], for example, estimated the overall sensitivity and specificity of this technique at ≅80.0% and ≅99.4%, respectively. Studies using detection thresholds, such as that of Nanda et al. (2000) [53], despite identifying greater variation in results, also obtained high estimates for both sensitivity (≅70.0–80.0%) and specificity (≅95.0%).\u003c/p\u003e\n\u003cp\u003eIn population screening for cervical cancer prevention, more recent studies have revealed greater efficiency of other diagnostic techniques concerning the Papanicolaou test [54], however, corroborating what was proposed by Irioda et al. (2011) [30], we believe that this technique can contribute significantly to the evaluation of MSCs and other adherent cells in cultures for therapies, both due to its sensitivity/specificity and its ease of execution [32,33].\u003c/p\u003e\n\u003cp\u003eIn the present study, no cells with altered numbers of nuclei and/or atypical nuclear morphology were identified in all samples and passages evaluated, with a total of 800 cells for the two participants. The Papanicolaou technique, when used in the analysis of desquamated cells from the uterine cervix, also allows the detection of other alterations, whose arrangement can characterize different types of lesions [31-33]. In the present study, it was decided to include only changes in the number and morphology of the nuclei. This decision was made because there was still no standardization for the use of this technique for mesenchymal stem cell cultures. In this way, errors in judgment were avoided, resulting from possible errors in sample preparation or even due to the evaluator's lack of training in the use of the technique, given that, even for the standardized use of the technique, differences in judgment are not uncommon [53].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosome Stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, a total of 480 cells from the two participants were analyzed. To better understand the most precise time in days of the metaphase phase of the ADSC for the interruption and stabilization of the chromosomes, before and after cryopreservation, an analysis of cell growth kinetics was performed. The best time was from the eighth (8) to the ninth (9) day, after the second change of culture medium. \u003c/p\u003e\n\u003cp\u003eThus, this analysis suggests an optimized guideline to better take advantage of the ideal time for metaphase interruption. After analysis, no numerical changes and/or breaks or chromosome or chromatid failures were evidenced, either in the metaphase chromosomes after 48 h of cultivation in Petakas (control) and chitosan-collagen biomatrices with genipin at 0.75% v/v, or after 12 months in the metaphase chromosomes that were extracted from the cryopreserved ADSC. Specific chromosomal anomalies are responsible for hundreds of syndromes that can be identified through karyotype analysis and study. \u003c/p\u003e\n\u003cp\u003eThey are responsible for a large proportion of reproductive losses, congenital malformations, and mental retardation, playing an important role in the pathogenesis of malignant hematological diseases, when acquired during life, associated with neoplasias. In these cases, studies of chromosomal disorders are important to aid in the diagnosis, prognosis, therapeutic conduct, and treatment monitoring of patients who present such alterations [30].\u003c/p\u003e\n\u003cp\u003eThe normal functioning of the genetic system depends on the stability of the genetic material contained in the chromosomes [55]. The karyotype can change, leading to genetic alterations. Such alterations can be in the number of chromosomes or their structure. Changes in the number of chromosomes can lead to euploidy or aneuploidy. In the first form, there is an increase or decrease in the number of genomes in a cell, we have euploidy. In the second type of mutation, there is an increase or decrease in one or more chromosomes in the genome, altering the karyotype of those that present it [30].\u003c/p\u003e\n\u003cp\u003eStructural alterations modify the normal morphology of one or more chromosomes. Such chromosomal anomalies can affect entire chromosomes or chromosomal segments. Thus, there is a disorganization in the chromosomal structure that can be observed under the microscope in the following ways: - Deletion or deficiency: involves the loss of chromosomal material; - Duplication: a segment of the chromosome is represented twice or more. If the duplicated fragment includes the centromere, it can be incorporated into the karyotype as an extra chromosome; Inversion: involves the 180° inversion of a chromosome segment [55].\u003c/p\u003e\n\u003cp\u003eTherefore, in the present study, there were no significant chromatid and/or chromosomal alterations or anomalies, suggesting that the biomatrices are biocompatible and are strongly indicated for use in translational therapies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLIMITATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the extensive laboratory analysis, financial constraints were a limiting factor, allowing analysis of samples from two patients. Nevertheless, all samples were performed in triplicate (replicates of n=30), and 480 cells (960 metaphases) were used for cytogenetic analyses.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe chitosan-collagen-genipin scaffolds, at the tested concentration of 0.75%, showed cytocompatibility at the cellular and chromosomal levels, since it allowed adhesion, proliferation, and biostimulation for the expression of normal cellular functions, and did not cause damage to adipose-derived stem cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAdipose-Derived Stem Cells (ADSC)\u003c/p\u003e\n\u003cp\u003eTumor Necrosis Factor (TFN).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMajor Histocompatibility Class (MHC)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdipose-Derived Stem Cells (ADSC)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInternational Society for Cellular Therapy (ISCT).\u003c/p\u003e\n\u003cp\u003eLow-grade Squamous Intraepithelial Lesions (LSIL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMacrophage Colony Stimulating Factor (M-CSF)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization -\u003c/strong\u003e IF, DF; \u003cstrong\u003eData curation –\u003c/strong\u003e IF, HAK; \u003cstrong\u003eFormal Analysis -\u003c/strong\u003e IF, DF, MA; \u003cstrong\u003eInvestigation -\u003c/strong\u003e IF, DF, NAH; \u003cstrong\u003eMethodology –\u003c/strong\u003e DF, NAH; \u003cstrong\u003eProject administration -\u003c/strong\u003e IF, DF, MA; \u003cstrong\u003eSupervision –\u003c/strong\u003e IF, NAH, HAK; \u003cstrong\u003eWriting - original draft -\u003c/strong\u003e IF, DF, MA, NAH, HAK; \u003cstrong\u003eWriting-review \u0026amp; editing-\u003c/strong\u003e IF, DF, MA, NAH, HAK.\u003c/p\u003e\n\u003cp\u003eEthical Approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study was evaluated and approved by the research ethics committee of the Beneficência Portuguesa Hospital of São José do Rio Preto, São Paulo, Brazil, under number of approval process 030427.\u0026nbsp;All procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003enformed consent was obtained from all participants involved in the study, with all procedures explained in detail before participation.\u003c/p\u003e\n\u003cp\u003eConsent for Publication\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from the patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAvailability of Data and Material\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Council for Scientific and Technological Development (CNPq) from Brazil.\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst of all we thank the financial support of the National Council for Scientific and Technological Development (CNPq) from Brazil. Further, thank you very much Dr. Lílian Madi-Ravazzi for the excellent images obtained, responsible for the scanning electron microscope (SEM) from Department of microscopy and microanalysis and we also thank the laboratory of microscopy and microanalysis for the images of optical microscopy, both located at State University of São Paulo - IBILCE-UNESP, from São José do Rio Preto, Sao Paulo, Brazil.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBiniazan F, Stoian A, Haykal S. Adipose-Derived Stem Cells: Angiogenetic Potential and Utility in Tissue Engineering. Int J Mol Sci. 2024 Feb 16;25(4):2356. doi: 10.3390/ijms25042356. \u003c/li\u003e\n\u003cli\u003eElias Ferreira Stricker P, Barth de Oliveira N, Felipe Mogharbel B, L\u0026uuml;hrs L, Irioda AC, Abdelwahid E, Regina Cavalli L, Zotarelli Filho IJ, Athayde Teixeira de Carvalho K. Meta-analysis of the Mesenchymal Stem Cells Immortalization Protocols: A Guideline for Regenerative Medicine. Curr Stem Cell Res Ther. 2023 Nov 27. doi: 10.2174/011574888X268464231016070900. \u003c/li\u003e\n\u003cli\u003ePacheco CMR, Ferreira PE, Sa\u0026ccedil;aki CS, Tannous LA, Zotarelli-Filho IJ, Guarita-Souza LC, de Carvalho KAT. In vitro differentiation capacity of human breastmilk stem cells: A systematic review. World J Stem Cells. 2019 Nov 26;11(11):1005-1019. doi: 10.4252/wjsc.v11.i11.1005. \u003c/li\u003e\n\u003cli\u003eBrazil. Ministry of Health. Available at: https://www.gov.br/saude/pt-br/assuntos/noticias/2020/outubro/sus-oferece-gratuitamente-orteses-e-proteses-sob-medida. Accessed at: 11/11/2024.\u003c/li\u003e\n\u003cli\u003eMartin SS, Aday AW, Almarzooq ZI, Anderson CAM, Arora P, Avery CL, Baker-Smith CM, Barone Gibbs B, Beaton AZ, Boehme AK, Commodore-Mensah Y, Currie ME, Elkind MSV, Evenson KR, Generoso G, Heard DG, Hiremath S, Johansen MC, Kalani R, Kazi DS, Ko D, Liu J, Magnani JW, Michos ED, Mussolino ME, Navaneethan SD, Parikh NI, Perman SM, Poudel R, Rezk-Hanna M, Roth GA, Shah NS, St-Onge MP, Thacker EL, Tsao CW, Urbut SM, Van Spall HGC, Voeks JH, Wang NY, Wong ND, Wong SS, Yaffe K, Palaniappan LP; American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. 2024 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation. 2024 Feb 20;149(8):e347-e913. doi: 10.1161/CIR.0000000000001209. Epub 2024 Jan 24. Erratum in: Circulation. 2024 May 7;149(19):e1164. doi: 10.1161/CIR.0000000000001247. \u003c/li\u003e\n\u003cli\u003eWHO-World Health Organization. WHO standards for prosthetics and orthotics. Available at: https://www.who.int/publications/i/item/9789241512480. Accessed at: 11/11/24.\u003c/li\u003e\n\u003cli\u003eBoccafoschi F, Habermehl J, Vesentini S, Mantovani, D. Biological performances of collagen-based scaffolds for vascular tissue engineering. Biomaterials; 2005, 26 (35) : 7410-7417.\u003c/li\u003e\n\u003cli\u003eGou Y, Huang Y, Luo W, Li Y, Zhao P, Zhong J, Dong X, Guo M, Li A, Hao A, Zhao G, Wang Y, Zhu Y, Zhang H, Shi Y, Wagstaff W, Luu HH, Shi LL, Reid RR, He TC, Fan J. Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering. Bioact Mater. 2023 Dec 14;34:51-63. doi: 10.1016/j.bioactmat.2023.12.003. \u003c/li\u003e\n\u003cli\u003eGreco OT, Filho IJZ, Bellini MF, Bilaqui A, Souza as junior et al. cardiomyopathy and cell therapy: ejection fraction improvement and cardiac muscle mass increasing, after a year of bone marrow stem cells transplantation by magnetic resonance image. j stem cell res ther. 2013, s6: 008. \u003c/li\u003e\n\u003cli\u003eZotarelli Filho IJ, Liedtke FS, Ferrari RS, Daher ID, Neiva EB, Moura MIP, Murad ACS. Protocol for obtaining and characterizing the stromal vascular fraction of human adipose tissue by mechanical dissociation: an original article. MedNEXT Journal of Medical and Health Sciences, 2022, 3(4). doi: https://doi.org/10.54448/mdnt22412.\u003c/li\u003e\n\u003cli\u003eDaher ID, Moura MIP, Murad ACS, Ferrari RS, Neiva EB, Liedtke FS, Zotarelli Filho IJ. Major clinical findings of cellular therapy for intravitreal use in ischemic retinopathy and macular degeneration: a systematic review. MedNEXT Journal of Medical and Health Sciences, 2022, 3(3). https://doi.org/10.54448/mdnt22311.\u003c/li\u003e\n\u003cli\u003eKuo CK, Tuan RS. Mechanoactive tenogenic differentiation of human mesenchymal stem cells. Tissue Engineering, part a, 2008, v. 14, n. 10, p. 1615-1627.\u003c/li\u003e\n\u003cli\u003eMuzzarelli RA, El Mehtedi M, Bottegoni C, Gigante A. Physical properties imparted by genipin to chitosan for tissue regeneration with human stem cells: a review. Int J Biol Macromol. 2016, dec;93(pt b):1366-1381.\u003c/li\u003e\n\u003cli\u003eAlsarra IA. Chitosan topical gel formulation in the management of burn wounds. international journal of biological macromolecules, Guildford; 2009, 5 :16-21.\u003c/li\u003e\n\u003cli\u003eDomard A, Taravel MN. Collagen and its interaction with chitosan: ii. influence of the physicochemical characteristics of collagen. Biomaterials; 1995, 16(11):865\u0026ndash;71.\u003c/li\u003e\n\u003cli\u003eButler MF, Pudney PDA. Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin. Journal of Polymer Science Part A-Polymer Chemistry; 2003, 41(24) :3941-3953.\u003c/li\u003e\n\u003cli\u003eSung HW, Huang RN, Huang LL, Tsai CC. In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. Biomater sci polym ed.; 1999, 10:63\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eAbbott A. Cell rewind wins medicine nobel. nature; 2012, 490:151-152.\u003c/li\u003e\n\u003cli\u003eBreyner NM, Hell RC, Carvalho LR. Effect of three dimensional chitosan porous scaffold on the differentiation of mesenchymal stem cells into chondrocytes. CTO; 191 : 119-28, 2010.\u003c/li\u003e\n\u003cli\u003eDominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans R J, Keating A, Procko DJ, Horwitz EM. Minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement. Cytotherapy, 2006, v. 8, n. 4, p. 315-317.\u003c/li\u003e\n\u003cli\u003eRoh YJ, Choi YH, Shin SH, Lee MK, Won YJ, Lee JH, Cho BS, Park KY, Seo SJ. Adipose tissue-derived exosomes alleviate particulate matter-induced inflammatory response and skin barrier damage in atopic dermatitis-like triple-cell model. PLoS One. 2024 Jan 19;19(1):e0292050. doi: 10.1371/journal.pone.0292050.\u003c/li\u003e\n\u003cli\u003ePark N, Kim KS, Park CG, Jung HD, Park W, Na K. Adipose-derived stem cell-based anti-inflammatory paracrine factor regulation for the treatment of inflammatory bowel disease. J Control Release. 2024 Oct;374:384-399. doi: 10.1016/j.jconrel.2024.08.027. \u003c/li\u003e\n\u003cli\u003eCELARTIA\u0026reg;, 2025. Available at: https://celartia.com/petaka-options/. Accessed at: 14/11/2024.\u003c/li\u003e\n\u003cli\u003eZuk PA, Zhu M, Ashjian P. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002, 13: 4279\u0026ndash;4295.\u003c/li\u003e\n\u003cli\u003eColeman SR. Structural fat grafting: more than a permanent filler. Plast Reconstr Surg. 2006, sep;118(3 suppl):108s-120s.\u003c/li\u003e\n\u003cli\u003eNardi NB, Meirelles SL. Mesenchymal stem cells: isolation, in vitro expansion and characterization. Hep; 2006, 174: 249-82.\u003c/li\u003e\n\u003cli\u003eBaldwin AD, Kiick KL. Polysaccharide-modified synthetic polymeric biomaterials. biopolymers; 2010, 94:128\u0026ndash;140.\u003c/li\u003e\n\u003cli\u003eISCN 2024 - An International System for Human Cytogenomic Nomenclature (2024). Cytogenet Genome Res. 2024;164(Suppl 1):1-224. doi: 10.1159/000538512.\u003c/li\u003e\n\u003cli\u003eSanchez O, Escobar JI, Yunis JJ. A simple G banding technique. Lancet 1973;II:269.\u003c/li\u003e\n\u003cli\u003eIrioda AC, Zocche L, Souza CMCO, Ferreira RJ, Aliprandini E, Cunha RC, Francisco JC, Guarita-souza LC, Malvezzi M, Beltrame MP, Mesquita LAF, Kuczera D, Chachques JC, Carvalho KAT. Pap test as the first step in sreening genetic stability in cell-based therapy. Journal of stem cell research and therapy, 2011, v. 1, n. 3, p. 1000106.\u003c/li\u003e\n\u003cli\u003eNational Cancer Institute. Bethesda System Website Atlas [Internet].1989. Available at: \u0026lt;http://nih.techriver.net/index.php\u0026gt;. Accessed at: 11/11/2024. \u003c/li\u003e\n\u003cli\u003eApgar BS, Zoschnick L, Wright JR TC. The 2001 Bethesda system terminology. American Family Physician, 2003, v. 68, p. 1992-1998.\u003c/li\u003e\n\u003cli\u003eNational Cancer Institute. NCI Cancer Atlas [Internet]. Available at: https://gis.cancer.gov/canceratlas/. Accessed at: 11/11/2024.\u003c/li\u003e\n\u003cli\u003eNational Institutes of Health Consensus Development Conference Statement on the Clinical Applications of Biomaterials. November 1-3, 1982. Artif Organs. 1983 May;7(2):260-5. PMID: 6688173.\u003c/li\u003e\n\u003cli\u003eLuo W, Zhang H, Wan R, Cai Y, Liu Y, Wu Y, Yang Y, Chen J, Zhang D, Luo Z, Shang X. Biomaterials-Based Technologies in Skeletal Muscle Tissue Engineering. Adv Healthc Mater. 2024 Jul;13(18):e2304196. doi: 10.1002/adhm.202304196. \u003c/li\u003e\n\u003cli\u003eWang J, Liu M, Yang C, Pan Y, Ji S, Han N, Sun G. Biomaterials for bone defect repair: Types, mechanisms and effects. Int J Artif Organs. 2024 Feb;47(2):75-84. doi: 10.1177/03913988231218884. \u003c/li\u003e\n\u003cli\u003eXie Y, Ma C, Zhu Q, Fu T, Bai L, Lan X, Liu L, Xiao J. Facial nerve regeneration via body-brain crosstalk: The role of stem cells and biomaterials. Neurobiol Dis. 2024 Oct 1;200:106650. doi: 10.1016/j.nbd.2024.106650. \u003c/li\u003e\n\u003cli\u003eRahimi Darehbagh R, Seyedoshohadaei SA, Ramezani R, Rezaei N. Stem cell therapies for neurological disorders: current progress, challenges, and future perspectives. Eur J Med Res. 2024 Jul 25;29(1):386. doi: 10.1186/s40001-024-01987-1.\u003c/li\u003e\n\u003cli\u003eChiono V, Pulieri E, Vozzi G, Ciardelli G, Ahluwalia A, Giusti P. Genipin-crosslinked chitosan/gelatin blends for biomedical applications. J Mater Sci Mater Med; 2008,19: 889\u0026ndash;898.\u003c/li\u003e\n\u003cli\u003eDrago RS. Physical methods in chemistry. Saunders and Co., London, 1977.\u003c/li\u003e\n\u003cli\u003eIsraelachvili JN. Van der Waals forces in biological systems. Quarterly Reviews of Biophysics, 2011, 6(4), 341\u0026ndash;387.\u003c/li\u003e\n\u003cli\u003eBet MR, Goissis G, Plepis AMG. Anionic collagen: calcium phosphate composites. preparation and characterization, Qu\u0026iacute;mica Nova; 1997, 20 : 475-477.\u003c/li\u003e\n\u003cli\u003eHill TL. Thermodynamics of small systems. Benjamin, New York: Dover Pubns, 1963.\u003c/li\u003e\n\u003cli\u003eYuan C, Song W, Jiang X, Wang Y, Li C, Yu W, He Y. Adipose-derived stem cell-based optimization strategies for musculoskeletal regeneration: recent advances and perspectives. Stem Cell Res Ther. 2024 Mar 27;15(1):91. doi: 10.1186/s13287-024-03703-6. \u003c/li\u003e\n\u003cli\u003eWang K, Yang Z, Zhang B, Gong S, Wu Y. Adipose-Derived Stem Cell Exosomes Facilitate Diabetic Wound Healing: Mechanisms and Potential Applications. Int J Nanomedicine. 2024 Jun 17;19:6015-6033. doi: 10.2147/IJN.S466034. \u003c/li\u003e\n\u003cli\u003eZhou B, Chen Q, Zhang Q, Tian W, Chen T, Liu Z. Therapeutic potential of adipose-derived stem cell extracellular vesicles: from inflammation regulation to tissue repair. Stem Cell Res Ther. 2024 Aug 7;15(1):249. doi: 10.1186/s13287-024-03863-5. \u003c/li\u003e\n\u003cli\u003eSanchez-Guijo F, Vives J, Ruggeri A, Chabannon C, Corbacioglu S, Dolstra H, Farge D, Gagelmann N, Horgan C, Kuball J, Neven B, Rintala T, Rocha V, Sanchez-Ortega I, Snowden JA, Zwaginga JJ, Gnecchi M, Sureda A. Current challenges in cell and gene therapy: a joint view from the European Committee of the International Society for Cell \u0026amp; Gene Therapy (ISCT) and the European Society for Blood and Marrow Transplantation (EBMT). Cytotherapy. 2024 Jul;26(7):681-685. doi: 10.1016/j.jcyt.2024.02.007. \u003c/li\u003e\n\u003cli\u003eZotarelli Filho IJ, Frascino LF, Greco OT, Araujo JDD, Bilaqui A, Kassis EN, Ardito RV, Bonilla-Rodriguez GO. Chitosan-collagen scaffolds can regulate the biological activities of adipose mesenchymal stem cells for tissue engineering. J Regen Med Tissue Eng; 2013, 2:12.\u003c/li\u003e\n\u003cli\u003eIrioda AC, Cassilha R, Zocche L, Francisco JC, Cunha RC, Ferreira PE, Guarita-Souza LC, Ferreira RJ, Mogharbel BF, Garikipati VN, Souza D, Beltrame MP, de Carvalho KA. Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease \u0026alpha;4-Integrin Expression. Stem Cells Int. 2016;2016:2562718. doi: 10.1155/2016/2562718.\u003c/li\u003e\n\u003cli\u003ePlanat Bernard V, Silvestre JS, Cousin B. Plasticity of human adipose lineage cells towards endothelial cells: physiological and therapeutic perspectives. Circulation; 2004, 109: 656 -63.\u003c/li\u003e\n\u003cli\u003ePlanat Bernard V, Silvestre JS, Cousin B. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ. Res; 2003, 94: 223-9.\u003c/li\u003e\n\u003cli\u003eSoost HJ, Lange HJ, Lehmacher W, Ruffing-Kullmann B. The validation of cervical cytology. Sensitivity, specificity and predictive values. Acta Cytologica, 1991, v. 35, n. 1, p. 8-14.\u003c/li\u003e\n\u003cli\u003eNanda K, McCrory DC, Myers RR, Bastian LA, Hasselblad V, Hickey JD, Matchar DB. Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review. Annals of Internal Medicine, 2000, v. 132, n. 10, p. 810-819. \u003c/li\u003e\n\u003cli\u003eKarimi-Zarchi M, Peighmbari F, Karimi N, Rohi M, Chiti ZA Comparison of 3 ways of conventional pap smear, liquid-based cytology and colposcopy vs cervical biopsy for early diagnosis of premalignant lesions or cervical cancer in women with abnormal conventional Pap test. International Journal of Biomedical Science, 2013, v. 9, n. 4, p. 205-210.\u003c/li\u003e\n\u003cli\u003eSumner AT. Chromosomes \u0026ndash; Organization and Function. Blackwell Publishing: United Kingdon-UK. 2003, 287p.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adipose-derived mesenchymal stem cells, Biomatrices, Scaffolds, Chromosomal stability, Chromatid stability, Cytostability","lastPublishedDoi":"10.21203/rs.3.rs-9106198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9106198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e One in five people over 65 years of age benefiting from in vitro-generated tissues and organs. This study aimed to analyze the cytocompatibility of Adipose-Derived Stem Cells (ADSC) at the chromosomal/chromatid level in the presence of the chitosan-collagen-genipin biomatrix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e ADSC was obtained from human lipoaspirate by enzymatic method and centrifugation and then cultured in Petakas (control) and the chitosan-collagen-genipin biomatrices, with and without cryopreservation. ADSC were characterized for adhesion and proliferation by conventional optical microscopy, phase contrast and scanning electron microscopy, immunophenotyping, cell viability with Trypan blue test, chromosomal and chromatid stability by traditional cytogenetic techniques, neoplastic potential by Papanicolau test and cell differentiation capacity in three types of tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The biomatrix intercrossed with genipin 0.75% v/v was selected for studies in this article since it presented good physicochemical stability. After cultivation in Petakas and biomatrices, ADSC maintained their intrinsic functions of adhesion, proliferation, and differentiation. Cytogenetic tests of ADSC after different culture conditions in biomatrices revealed no statistically significant differences (p\u0026lt;0.05) to control cells (cultured in Petakas).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion: \u003c/strong\u003eBiomatrix intercrossed with genipin 0.75% v/v can stimulate the expression of ADSC activities. The presence of cations and anions on individual polymer chains can stimulate the cellular activities of ADSC without causing cellular stress or loss of quiescence in these cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The chitosan-collagen-genipin biomatrix with genipin at 0.75% v/v was shown to be biocompatible both at the cellular level and at the chromosomal/chromatid level of adipose mesenchymal stem cells.\u003c/p\u003e","manuscriptTitle":"Chitosan-collagen scaffolds cross-linked with genipin demonstrated cytocompatibility at different levels of bioanalysis with human adipose-derived mesenchymal stem cells for regenerative medicine: a research article","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 13:03:28","doi":"10.21203/rs.3.rs-9106198/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc3c3502-de56-4c8a-9608-1c8714e8c142","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65969022,"name":"Biological sciences/Biological techniques"},{"id":65969023,"name":"Biological sciences/Biotechnology"},{"id":65969024,"name":"Biological sciences/Cancer"},{"id":65969025,"name":"Biological sciences/Cell biology"},{"id":65969026,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-24T07:25:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 13:03:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9106198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9106198","identity":"rs-9106198","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}