Onion membrane based composite scaffolds incorporated with N-Boc L-cysteine methyl ester enhances mineralization for bone tissue engineering applications

Onion membrane based composite scaffolds incorporated with N-Boc L-cysteine methyl ester enhances mineralization for bone tissue engineering applications | 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 Onion membrane based composite scaffolds incorporated with N-Boc L-cysteine methyl ester enhances mineralization for bone tissue engineering applications Sivasankar MV, Sreenivasa Rao Parcha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4849833/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Composite scaffolds S1(C-MFC-PCL), S2 (C-MFC-PCL-H), and S3 (C-MFC-PCL-Zr) containing micro-fibrillated cellulose (MFC), chitosan (C), polycaprolactone (PCL), zirconium oxide (Zr), and hydroxyapatite (H) were synthesized by freeze-drying process. N-Boc-L-cysteine methyl ester (NBLCME) was synthesized and incorporated into the composite scaffolds S1, S2, and S3 at different concentrations (20–100µg/ml). FTIR analysis confirmed the interactions between S1, S2, S3, and NBLCME. SEM analysis showed that the S1, S2, and S3 had 70–85% porosity with a pore diameter range of 100–450µm. The scaffolds S1, S2, and S3 scaffolds achieved sustained drug delivery following Fickian diffusion behavior (n ≤ 0.45). The cytotoxic effects of NBLCME treated scaffolds (S1, S2, and S3) on MG63 cell line were studied by examining cell viability, alkaline phosphatase activity (ALP), Alizarin red S activity (ARS), and cell adhesion. The cytotoxicity of the treated scaffolds on MG63 cell line was dose-dependent, with no cytotoxic effects at concentrations below 60µg/ml. However, higher concentrations of NBLCME (> 60µg/ml) significantly reduced ALP and ARS activity of MG63 cells due to lactate dehydrogenase leakage. Composite scaffolds S1, S2, and S3 showed significant results in mechanical properties, swelling behavior, sustainable drug release, slow degradation rate, cell adhesion, growth, and proliferation. S3 composite scaffold exhibit excellent properties than other composite scaffolds S2 and S3. Therefore, S3 can be used as promising biomaterial for bone tissue engineering. Biological sciences/Stem cells Physical sciences/Materials science/Biomaterials Chitosan Microfibrillated cellulose Zirconium NBLCME MG63 cells cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tissue engineering has emerged as a promising approach for tissue regeneration, with a focus on the development of biomaterials for scaffold production ( 1 ). Biomaterials used in bone tissue engineering should possess both bioresorbability and bioactivity, allowing for bone regeneration and degradation over time ( 2 – 3 ). The establishment of stable direct contact between bone and scaffold surface is crucial for optimal scaffold development, requiring structural, mechanical, and functional integration (osteointegration) ( 4 ). Scaffolds made from biomaterials with these properties have the potential to support cell attachment, growth, and proliferation, facilitating tissue regeneration ( 5 – 7 ). The combination of bioresorbable and bioactive biomaterials in scaffold design holds promise for inducing bone regeneration and promoting successful tissue integration ( 2 – 3 ). Microfibrillated cellulose (MFC) is a cellulose material obtained through mechanical disintegration without hydrolysis, consisting of long cellulose microfibrils with diameters ranging from 20nm to 60nm ( 8 – 10 ). MFC are abundant natural materials found in plants and bacteria and exhibits a web-like structure and contains both amorphous and crystalline parts. It has a low percolation threshold and a strong ability to form stable networks, making it a valuable filler material in the production of biocomposites ( 11 ). Biocomposites made with MFC have been manufactured using various polymers such as polyvinyl alcohol, poly lactic acid, polycaprolactone, pectin, starch, and chitosan ( 12 – 16 ). The use of MFC in these biocomposites enhances their properties and expands their potential applications in areas like tissue engineering ( 17 ). They possess positive properties for biomedical applications and cell culture, including biocompatibility, protein binding surface, and mechanical strength ( 18 – 19 ). The high density of reactive hydroxyl groups on the surface of cellulose fibers enables the immobilization of cell adhesive proteins, such as fibronectin, facilitating cell adhesion ( 20 – 22 ). The glucan chain structure in cellulose fibers is densely packed which provides mechanical strength to support cell aggregate structures, but there have been limited studies due to the absence of an intrinsic 3D architecture ( 23 – 24 ). To address the lack of 3D architecture, the use of chitosan, a linear polysaccharide derived from chitin, can provide an additional 3D structure for cellulose-based cell scaffolds ( 25 – 26 ). Chitosan, derived from chitin, is a linear polysaccharide and the second most abundant natural polymer after cellulose. Chitosan is a biopolymer composed of β-( 1 , 4 ) linked 2-deoxy-2-amino-D-glucopyranose and is obtained by alkaline de-acetylation of chitin. It is widely investigated for its low toxicity, biodegradability, and cytocompatibility properties, making it a potential biomaterial for various biomedical and bioengineering applications ( 27 – 30 ). One of the main concerns with chitosan materials has the low mechanical properties ( 31 ). Chemical modifications have gained attention from researchers as a means to enhance the mechanical properties of chitosan due to the variety of available modifications using the numerous amino and hydroxyl side groups ( 32 – 33 ). Poly-caprolactone (PCL) is a semi-crystalline polyester widely used as biomaterials in medical applications. PCL has a low melting point (55°C) and suitable properties (porosity, degradation time, and bioreabsorption) for bone tissue regeneration. PCL has a poor wetting surface and establishes weak interactions with biological fluids, preventing cell adhesion and proliferation ( 34 ). Hydroxyapatite (HA) is a ceramic material characterized by a hexagonal network structure, constituting the primary mineral component of both teeth and bones. It exhibits biocompatibility and does not elicit an inflammatory response ( 22 – 23 ). HA has slow resorption, maintaining its initial state for 2–3 years after implantation, allowing for gradual bone tissue growth and cell proliferation within the material ( 24 – 26 ). Additionally, HA demonstrates excellent mechanical properties, with a compressive strength of up to 160MPa, making it suitable for applications in low load conditions and small bone areas ( 21 ). But recent decades, Zirconia has been extensively used in bone tissue engineering and dental restorations due to its superior mechanical strength and biocompatibility, which enhance the cell differentiation, adhesion, and proliferation ( 35 – 36 ). Zirconia and nano-crystals exhibit unique properties, such as heightened chemical reactivity and an augmented surface area. Modifications to surface characteristics exert a considerable impact on cellular reactions, protein absorption, and mineralization, all of which are pivotal aspects in bone tissue engineering ( 37 – 41 ). Due to its remarkable strength and ideal biocompatibility, zirconia has found extensive use in dental restorations and medical implants ( 42 ). The MG-63 human osteoblastic cell line is widely used in osteogenesis research. Cell stability across multiple passages in culture is crucial for effective biological investigations. MG-63 cells offer valuable insights into the interaction between cells and materials ( 38 ). Hence, the aim of the research is to enhance the mechanical and chemical properties, as well as to evaluate its biological ability by incorporating the bioceramic materials such as hydroxyapatite and zirconium oxide in a novel microfibrillated cellulose reinforced natural polymer-based sponge composed of chitosan was fabricated via freeze-drying method, for repairing bone defect. The prepared composite scaffolds displayed significant impact on drug release was unveiled, contributing to the development of an effective sustained release system. The composite scaffolds S1, S2, and S3 achieved sustained drug delivery following Fickian diffusion behavior (n ≤ 0.45). The biocompatibility of S1, S2, and S3 was systematically assessed through MTT assay, cell adhesion, and cell proliferation, affirming their suitability for bone applications. It is suggested that the S3 scaffold were superior to S1 and S2, S3 is quite promising biomaterial for application in bone tissue engineering and drug delivery. Materials and Methods 2.1.1. Materials Onions ( Allium cepa L. probably originate from central Asia) used in this study was purchased from the market (Warangal, India), chitosan (Mw 100–150 kDa with deacetylation degree of 75–85%) were procured from Sigma-Aldrich (India), Zirconium Oxide, hydroxyapatite, PCL (Mw = 80,000) were purchased from Merck (India). dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), 3-(4,5-dimethyhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton-X100, fluorescein isothiocyanate isomer I (FITC), 4,6-diamindino-2-phenylindole (DAPI), Bovine Serum Albumin (BSA), phosphate buffer saline (PBS), 4% paraformaldehyde, 25% Ammonia solution, p-Nitrophenyl phosphate (pNP), Alizarin red S staining, Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution were acquired from Hi-media (India). The human osteoblast cells (MG63 cell lines) were obtained from National Centre for Cell Science (India). Acetic acid (≥ 99.7%) and Ethanol analytical grade were purchased from Spectrochem (India), Double distilled water was used throughout the experiments. 2.1.2. Extraction of Microfibrillated cellulose (MFC): The onion membrane was thoroughly washed with dd.water to remove dirt and then dried using the freeze drying method for 24 hours. The dried samples were ground into a fine powder using a cutting blender for further analysis. MFC was extracted from the powdered sample of onion skin using an alkaline extraction method. To remove lignin and amorphous hemicellulose, the samples were treated with a 17.5% sodium hydroxide solution for 1 hour at 23ºC (1:25w/v ratio). The onion powder was then thoroughly washed with dd.water and treated with a 10% acetic acid solution. Finally, the treated powder was dried in a hot air oven at 100ºC for 24 hours to obtain the resulting dried MFC for further characterization. The resulting dried MFC were used for further characterization. 2.1.3. Preparation of scaffolds S1 (C-MFC-PCL), S2 (C-MFC-PCL-H), and S3 (C-MFC-PCL-Z): Composite scaffolds of chitosan (C) and microfibrillated cellulose (MFC) were fabricated by varying percentage of composite in the final mixture. A 2% (w/v) chitosan (C) solution was prepared in a 0.5M acetic acid. To this solution, 0.5% (w/v) microfibrillated cellulose (MFC) was added under stirring condition to obtain a uniform mixture (C-MFC). 1% (w/v) PCL solution was prepared in an acetic acid and it was slowly added into C-MFC solution kept for overnight to obtain S1, followed by addition of 1% (w/v) hydroxyapatite (S2) and zirconium (S3) and left stirring for 4-5hr. The solution was introduced into 24-well cell culture plates and maintained in a freezer at -80°C for 24h. The samples S1, S2, and S3 were then lyophilized in a freeze dryer until dried. The resulting microporous scaffolds were neutralized with 1N NaOH, washed with dd.water and immersed in DI water overnight to achieve a neutral pH. The washed scaffolds S1, S2, and S3 were freeze dried again and stored in a desiccator under vacuum. The synthesized molecule NBLCME was dissolved in methanol at different concentrations (20, 40, 60, 80, and 100µg/ml). Composite scaffolds S1, S2, and S3 were immersed in the methanol solution containing NBLCME overnight. The scaffolds S1, S2, and S3 were then dried in a hot air oven for 2h. Thorough washing of the scaffolds with dd.water was performed, followed by freeze drying for further analysis. 2.1.4. Scanning electron microscopy (SEM) analysis The surface of the scaffolds S1, S2, and S3 was coated with a thin layer of gold using a sputter-coating technique. Scanning electron microscopy (SEM) was employed to study the ultra-structural analysis of the scaffolds. The samples were examined at an accelerating voltage of 15–20 kV during the SEM analysis. 2.1.5. Porosity studies: The porosity of fabricated composite scaffolds S1, S2, and S3 was estimated by immersing them in absolute ethanol and measuring the weight changes ( 43 ). The weights before and after immersion were recorded, along with the volume before immersion. These values, along with the constant density of ethanol, were used to calculate the porosity. The experiment was conducted in triplicate to ensure the consistency of the results. The porosity was calculated using the Eq. 1, $$\:P=\frac{W2-W1}{\rho\:V1}X100$$ Where the weight before immersion (W 1 ), weight after immersion (W 2 ), constant density of ethanol (ρ) and the volume before immersion (V 1 ) were used in the calculation. 2.1.6. Mechanical Testing: The composite scaffolds S1, S2, and S3 were molded into a cylindrical shape with 10 mm in diameter and 8 mm in height. The constructs were placed under compressive load using a universal testing machine (INSTRON, MA, USA) at a crosshead speed 0.5 mm/min up to failure or until the sample reached a 70% reduction in height. The test was performed by following ASTM F2027. The reported values were the average values determined from three specimens. 2.1.7. Fourier Transform Infrared Spectroscopy (FT-IR) analysis: FTIR spectroscopy was employed to investigate intermolecular interactions between functional groups of different materials. The scaffolds were first milled into powder and then mixed with potassium bromide (KBr) salt. The mixture was compressed into potassium bromide disks, which were used for FTIR spectral analysis. FTIR spectra were recorded using a PERKIN ELMER 2DTGS instrument, covering a range between 4000 and 400 cm − 1 . 2.1.8. Swelling Studies: Composite scaffolds were cut into equal-weight pieces and immersed in PBS (pH 7.4, 37ºC). The scaffolds were removed at different time intervals, and the water on the surface was gently blotted on filter paper and weighed (W d ). S (%) \(\:=\frac{{w}_{w-}{w}_{d}}{{w}_{d}}\times\:100\:\) Where S, W w and W d are the swelling percentage, wet, and dry weight of the scaffolds, respectively. 2.1.9. In vitro biodegradation studies: The degradation study was conducted by immersing the scaffolds in lysozyme-containing buffer (PBS, pH̴ 7.4) at 37ºC for 28 days. The scaffolds were initially weighed (W i ) and then immersed in the lysozyme buffer for the specified duration. At 7, 14, 21, and 28 days, the scaffolds were removed from the buffer, washed in deionized water to remove surface ions, and freeze-dried. The final weight of the scaffolds (W f ) was recorded after freeze-drying. This experimental setup allowed for the assessment of scaffold degradation over time and the determination of the weight loss experienced by the scaffolds. The degradation was calculated using the formula Degradation (%) = \(\:\frac{{W}_{i\:-\:{W}_{f}}}{{W}_{f}}\:\times\:100\) 2.2. Protein Adsorption study: Protein adsorption capacity of scaffolds was analyzed using ( 44 ) with slight modifications. Untreated composite scaffolds as control and treated scaffolds S1, S2, and S3 with NBLCME in different concentration (20, 40, 60, 80, and 100 µg/ml) having equal weights were taken and pretreated with 70% ethanol for 1h followed by hydration with PBS (pH, 7.4) for 10–30 min. Hydrated scaffolds were incubate in 0.5ml 10% FBS for 2h at 37ºC to ensure completion of adsorption process. Gently rinsed the scaffolds with PBS thrice, to remove excess unbound protein and ions which are not adsorbed on the surface. Finally, add 1ml of 1% (W/V) SDS solution to separate the adsorbed protein. Afterward, shake the solution for 1h and centrifuge at 5000g for 15min at 4ºC and collect the supernatant. Total protein concentration was quantified using dye adding assay. BSA as standard and evaluate protein concentration were recorded the absorbance at a wavelength of 720nm. 2.2.1. In vitro drug release study: In vitro release studies were carried out in PBS buffer at pH-7.4. N-Boc-L-cysteine methyl ester solution (1mg/ml in methanol) was prepared first. Treated scaffolds S1, S2, and S3 were put into 10ml of release medium (PBS buffer) in an incubator shaker (100 rpm) at 37°C. The supernatant from drug containing solutions (1ml) were taken at constant time intervals from the release medium and were replaced with same amount fresh buffer. The concentration of N-Boc-L-cysteine methyl ester drug in the samples was measured by using micro plate reader, at 254 nm. 2.2.2. Cell viability assay: Cells were seeded on the scaffolds at a density of 3x10 5 cells/well on treated scaffolds (S1, S2, and S3) with different concentrations of NBLCME (20, 40, 60, 80, and 100µg/ml) for 1, 3, 5, and 7 days. MTT assay was performed using 0.50mg/ml medium concentration of MTT dye, which was added to each well and incubated at 37°C for 4 hours. Living cells converted the MTT dye to a formazan product with mitochondria. The solubilization reagent, dimethyl sulfoxide (DMSO), 150µl was added to the cell cultures and left for 2 hours. The absorbance of each cultured solution was measured at 540 nm using a microplate reader. 2.2.3. Determination of ALP activity: Cells were seeded on the scaffolds at a density of 3x10 5 cells/well and incubated in a CO 2 incubator for 72 hours. The media from the cell culture was aspirated, and the cells were rinsed twice with PBS. Cells in each well were fixed with 4% formaldehyde for 10 minutes. After washing, the cells were treated with 30µl of p-Nitrophenyl phosphate containing 5mM MgCl 2 at 37ºC for 30 minutes. The reaction was terminated by adding 30µl of 0.5N NaOH. The ALP activity was assayed by measuring the conversion of p-nitrophenyl phosphate to p-nitrophenol, which resulted in a color change. The color change was measured using a microplate reader at 405 nm. 2.2.4. ARS staining: Cells were seeded on scaffolds (3x10 5 cells/well) and placed in CO 2 incubator for 72h. Aspirate the media from cell culture, and cells-matrix were gently rinsed with PBS twice. Cells of each well were fixed with 4% formaldehyde for 10 min. After washing, cells were treated with 40mM or 2% Alizarin red S (pH 4.1–4.3) for 20min at room temperature with gentle shaking. Later, cell-matrix were washed 3–4 times with dd.water while shaking for 5min. For quantification of staining, 200µl of 10%v/v acetic acid was added to each sample and incubate for 30min with shaking. The cell layer on the substrate was collected with acetic acid (10%v/v) and transferred to 1.5ml centrifuge tube. After that tubes were heated to 85ºC for 10min and transfer to ice for 5min then tubes were centrifuge at 20,000g for 15min. Supernatant were transferred to new tube and neutralized with 50µl of 10% v/v ammonium hydroxide. The ARS activity was measured using microplate reader (Thermo-scientific) at 405 nm. 2.2.5. Fluorescent labelling of microfilament: Cell morphology was qualitatively analyzed after 4 days of culture using fluorescein isothiocyanate isomer I (FITC) fluorescent dye to stain the cell membrane and cytoplasmic proteins, and 4,6-diamindino-2-phenylindole (DAPI) to counterstain the cell nuclei. The cell-matrix were rinsed with PBS for 3 times/5mins each, fixing them in 4% formaldehyde, permeabilizing the cells with 0.5% Triton X-100, blocking the reaction with 5% BSA, staining with FITC isomer-I solution (5µg/ml), and counterstaining with DAPI solution (0.08µg/ml). After each step, the samples were washed thoroughly with PBS for 5 times/5min each. The cell-matrix was observed under a fluorescence microscope to analyze the cell morphology. Results And Discussion Fabricated composite scaffolds S1, S2, and S3 for bone tissue engineering requires an interconnected pore and highly porous structure to support cell attachment, proliferation, and tissue growth, as well as nutrient flow. The freeze-drying technique was employed to achieve the desired porous structure in the scaffolds. The resulting composite material S1, S2, and S3 exhibited adjustable morphology, consisting of a fibrous material integrated with a sponge component. SEM images in Fig. 1 (a-g) showcased the top view and cross sections of the S1, S2, and S3 scaffolds, revealing their porous nature. Fabricated composite scaffolds S1, S2, and S3 showed highly porous structure, as observed at higher magnification, consisted favorable interconnected pores, which facilitate cell attachment and new bone tissue ingrowth. Porosity is a crucial factor for ideal scaffolds in tissue engineering applications, as it allows cells to passes through the pores and attach at suitable area for further proliferation. Figure 2 a show the porosity of the synthesized composite scaffolds S1, S2, and S3 showing approximately 73%, 76%, and 80% respectively. The presence of NBLCME throughout the scaffolds contributed to the reduced porosity, as NBLCME binds to the polymer chains and helps maintain the integrity of the polymer chains. The obtained porosity in the prepared composite scaffolds was deemed sufficient, as it facilitates the supply of nutrients and oxygen to the interior regions of the scaffolds. Scaffolds for bone tissue engineering need sufficient mechanical strength to support tissue regeneration and maintain integrity during cell growth ( 27 – 28 ). However, there is a tradeoff between material porosity and mechanical strength in porous scaffolds ( 26 ). Compression modulus tests were conducted on S1, S2, and S3 scaffolds to evaluate their stress-strain relations. The compressive modulus of chitosan-MFC scaffolds was determined to be 0.83 ± 0.21MPa, while pure chitosan exhibited an extremely low compression modulus of approximately 0.28 ± 0.11MPa [29]. The addition of PCL, PCL-HA, and PCL-Zr to chitosan-microfibrillated cellulose increased the compression modulus, resulting in a value of 1.2 (S1), 2.23 (S2), and 2.7 MPa (S3) respectively were shown in Fig. 2 b. The addition of microfibrillated cellulose, PCL, and bio-ceramics materials to chitosan scaffolds resulted in an increased compression modulus, indicating improved mechanical strength. This increase in compressive modulus can be attributed to the strong ionic interactions between chitosan (C) and all copolymers content leading to the formation of a S1, S2, and S3 complex. Although all three scaffolds S1, S2, and S3 show good mechanical properties, S3 was higher than other scaffolds due to toughness of zirconium oxide (Zr) compare to hydroxyapatite. FTIR analysis confirmed the presence of these strong ionic interactions in the S1, S2, and S3 scaffolds, further supporting that they contribute to the increased compressive modulus. The FTIR spectra of treated and untreated S1, S2, and S3 scaffolds, along with Chitosan, MFC, polycaprolactone, hydroxyapatite, zirconium, and NBLCME were compared. Untreated and treated scaffolds (S1, S2, and S3) with the drug showed a broad absorption band between 3300–3500 cm − 1 and 3291–3361 cm − 1 , corresponding to the stretching vibrations of -OH and -NH groups. Additional absorption bands at 2880–2900 cm − 1 , 1430 cm − 1 , and 1050 cm − 1 indicated the stretching vibrations of C-H, -CH 2 scissoring, and CH 2 -O-CH 2 groups, respectively. Chitosan exhibited a distinct absorption band at 1170 cm − 1 , attributed to the free primary NH 2 group at the C2 position. The peak at 1655 cm − 1 indicated the presence of acetylated amino groups in chitosan, suggesting incomplete de-acetylation. Peaks at 1326 cm − 1 and 1030 cm − 1 corresponded to the C-N stretching of the NH 2 group and C-O-C stretching vibration, respectively. In addition, the integration of ZrO 2 in S3 was confirmed by the characteristic’s peaks observed at 503 cm − 1 , which attributed to Zr-O stretching vibration of ZrO 2 . Broad peaks in the range of 1155 − 980 cm-1 correspond to the stretching vibration of phosphate (PO 4 3− ), indicating the presence of phosphate groups. The strong peak at 1740 cm − 1 in the cross-linked samples indicated successful cross-linking shown in Fig. 3 , which prevents swelling, impacting the integrity and strength of the polymer network. Increased cross-linking resulted in a denser macromolecular network, limiting hydration and swelling capacity. Swelling property of scaffolds are important for regulating cell infiltration, adhesion, and nutrient transportation. The addition of PCL and bio-ceramics contents in the C-MFC composite scaffolds reduces the degree of water absorption due to intermolecular crosslinking and weakening of hydrogen bonds. As shown in Fig. 4 a, the swelling ratio of the scaffolds (S1, S2, and S3) remained the same after 6000 seconds, indicating a high degree of swelling (420, 580, and 650% respectively). Chitosan swells in aqueous medium due to protonation and ionization of amino and carboxyl groups, while MFC, being hydrophilic, swells rapidly in the presence of PBS, resulting in increased scaffolds weight (S2 and S3). In particular, S1 scaffold water absorbtion was around 420% which is significantly less compared to other scaffolds S2 and S3 containing HA and Zr, due to hydrophobic characteristics of PCL. Figure 4 b shows the in vitro degradation profile of composite scaffolds S1, S2, and S3. At 7 days, the scaffolds S1, S2, and S3 had degraded to 9, 15, and 28% respectively, with the rate of degradation increasing over time. By day 14, approximately 11, 17, and 19% degradation had occurred, and at the end of 28 days, the scaffolds S1, S2, and S3 had degraded to 15, 30, and 32% respectively. The degradation of the scaffold is attributed to the presence of lysozyme in the human body, which degrades chitosan by hydrolyzing the β-1,4 glycosidic bond between N-acetylglucosamine units. The interaction between chitosan and cellulose microfiber was found to be good, as supported by the study ( 45 ). MFC, being hydrophilic, swells rapidly in the presence of PBS, resulting in a decrease in the interactions between chitosan and cellulose microfiber due to the loosening of physical cross-linking between them. The degradation products of the scaffold can attract more cells towards it, thereby improving the bioactivity of the scaffolds. Controlled degradation rate is essential for an ideal scaffold in bone tissue engineering applications. Protein adsorption on scaffolds can influence cell adhesion and is affected by factors such as surface properties and material composition ( 46 ). The NBLCME treated scaffolds S1, S2, and S3 with different dose concentrations (20, 40, 60, 80, and 100µg/ml) showed higher protein adsorption compared to the untreated scaffolds S1, S2, and S3 (2.1, 2.8, and 3.4µg/mg) were shown in Fig. 4 c. This increase in protein adsorption can be attributed to the distribution of NBLCME particles on the scaffold surfaces, which increases binding sites for proteins and promotes electrostatic interactions ( 47 ). Increasing the concentration of the drug in S1, S2, and S3 scaffolds resulted in a reduction in protein adsorption. This can be attributed to the decrease in scaffold porosity with increasing drug concentration, which directly affects the surface area available for protein adsorption. The scaffolds exhibited a considerable amount of protein adsorption, which is reported to be sufficient for promoting cell adhesion and proliferation. The in vitro drug release from S1, S2, and S3 scaffolds follows a sustained and gradual pattern, with a high release rate observed over a longer period in 72 hours. Moreover, the burst release completely disappears in composite scaffolds, and the cumulative releases are 15, 17, and 18% for S1, S2, and S3 respectively, during the first 72 h. Even though the absolute value of drug release is low, it gradually releases with increasing time as is evident in the case of S3. A comparative measurement shows that 18% of drug release occurs in time 1, 5, 10, and 72 h in S1, S2, and S3 respectively, exhibiting sustained release in S3 as compared to S1 and S2. If we look into the release mechanism involves three distinct steps: liquid penetration into the matrix, dissolution of the drug, and diffusion of the drug out of the matrix ( 48 ). The rate-determining step for drug release is considered to be the slower diffusion process, which is influenced by the sluggish swelling ability of C-MFC scaffolds. The increased cross-linking of C-MFC systems results in a denser network structure, restricting swelling and preventing the diffusion of drug molecules from the network to the release medium. The release kinetics of the drug are best described by the Korsmeyer-Peppas model leading to the exponent “n” values of 0.23, 0.25, and 0.26 for S1, S2, and S3 respectively, indicating Fickian diffusion (n < 0.45) as the dominant mechanism. Other models such as zero order, first order, and Higuchi models were verified but correlation r 2 values are not satisfactory in fitting the release data, while the Korsmeyer-Peppas model is a perfect fit shown in Fig. 4 d. Figure 5 (a-c) demonstrated the viability of MG63 cells which is expressed as the relative absorbance percentage of the control group. MG63 cells cultured with treated scaffolds S1, S2, and S3 showed varying responses to different dose concentrations of NBLCME (20, 40, 60, 80, and 100µg/ml). At a concentration of 80 and 100µg/ml, the proliferation rates of MG63 cells were lower after 1, 3, 5, and 7 days of culture. However, at lower concentrations of 20, 40, and 60µg/ml, the cell proliferation rates were higher. The critical concentration of NBLCME can be considered as 80µg/ml, as it showed lower proliferation rates. The treated scaffolds S1, S2, and S3 exhibited dose and time-dependent cytotoxicity at concentrations higher than 60µg/ml, while showing little sensitivity to increased dose and extended time at concentrations lower than 60µg/ml. It has been suggested that the higher concentration of NBLCME treated scaffolds inhibits the cell proliferation. ALP activity in MG63 cells incubated with treated scaffolds S1, S2, and S3 for 7 days was assessed to evaluate the bioactivity of NBLCME in the context of bone-related implants. In our study Fig. 6 (a-c) shows ALP activity of MG63 cells cultured with low concentrations of NBLCME (60, 40 and 20µg/ml) exhibited higher ALP activity compared to the control group, consistent with the results of the MTT assay. However, at a critical concentration of 60µg/ml, NBLCME led to significantly lower ALP activity, indicating that osteogenesis was inhibited by NBLCME-treated scaffolds at concentrations exceeding 60µg/ml. Although fabricated scaffolds S1, S2, and S3 showed good activity on alkaline phosphatase assay, but S3 exhibits the superior in ALP activity than other scaffolds S1 and S2. Alizarin red S staining was used to evaluate the relative value of calcium deposition on cells cultured on NBLCME-treated scaffolds (S1, S2, and S3) and untreated scaffolds as a control for 7 days. Alizarin red S solution selectively bound to calcium salts, allowing for the quantification of calcium deposition were shown in Fig. 7 (A-C) . Cells cultured with NBLCME-treated scaffolds at concentrations of 20, 40, and 60µg/ml exhibited higher levels of Alizarin red S staining compared to the 80, 100 µg/ml, and control group. Notably, the 60 µg/ml concentration of NBLCME-treated scaffolds S3 showed the highest amount of calcium deposition than the other scaffolds S1 and S2 were shown in Fig. 7 . Immunofluorescence staining was performed to observe the change in microfilament distribution in MG63 cells cultured on treated scaffolds S1, S2, and S3, and control for 4 days. As shown in Fig. 8 (a-i) , control cells showed well-developed actin fibers, while no differences were observed at low NBLCME concentrations (60µg/ml). Severe damage to actin fibers was observed at higher NBLCME concentrations (100µg/ml) were shown in Fig. 8 (c, f, and i) , possibly due to damage in the cell membrane, which induced cytotoxicity and lactate dehydrogenase (LDH) release. The release of LDH was influenced by the contact surface area between NBLCME and the cell membrane, with higher concentrations impeding LDH release. The cytotoxicity of treated scaffolds S1, S2, and S3 to MG63 cells is likely caused by intracellular activity without physical damage to the plasma membrane. Conclusion The composite scaffolds S1, S2, and S3 was fabricated using the freeze-drying method, and its bioactivity was confirmed through enhanced bio-mineralization and protein adsorption. The solubility and swelling properties decrease due to cross-linking of C-MFC systems with an increase in the degree of substitution on the chitosan backbone compared to S1 and S2. Significant improvement in mechanical property in S2 and S3 than S1, but S3 showed higher compression modulus due to higher strength and toughness of zirconium oxide. Although the hydroxyapatite is incorporated in S2 shows less compression modulus than S3 due to brittle nature of HA. The treated scaffold S3 exhibited sustained release of a drug, with a high release rate over a longer period, reaching a total drug release of 18% in 72 hours. In vitro studies using osteoblast-like MG-63 cells showed that the treated composite scaffold S3 significantly improved cell proliferation and calcium deposition, with lower concentrations showing better results. Cyto-compatibility studies using MG63 cells demonstrated an increase in cell viability over time, indicating the biocompatibility of the composite scaffold S3. However, further investigation is required to study the release of LDH and its effect on cell apoptosis, as well as in vivo models. Overall, the S3 composite scaffold are suitable candidate for controlled drug delivery and ideal for bone tissue engineering applications, but further research is needed to fully understand it’s potential. Declarations Declaration of competing interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution 1. Sivasankar MVConceptualization; Data curation; Formal analysis; Investigation; Resources; Roles/Writing – original draft; Writing & editing.2. Sreenivasa Rao ParchaProject Adminstration, Conceptualization; Supervision; Investigation; Roles/Writing – original draft. Acknowledgement: The authors thank the National Institute of Technology, for providing research facility, Ministry of Human Resources and Development, Government of India, New Delhi. Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Vacanti, J. P., Vacanti, C. A., Lanza, R. P., Langer, R. & Vacanti, J. Principles of Tissue Engineering, 2nd ed., Academic Press , CA, pp. 3–9. (2000). Hench, L. L. & Polak, J. M. Third-Gen Biomed. Mater. Sci. 295 , 1014–1023. (2002). Temenoff, J. S., Lu, L. & Mikos, A. G. in: J.E. Davies (Ed.), Bone Engineering, em squared incorporated, Toronto , pp 454–459. (2000). 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Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Nov, 2024 Reviews received at journal 12 Oct, 2024 Reviews received at journal 07 Oct, 2024 Reviews received at journal 29 Sep, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviewers agreed at journal 21 Sep, 2024 Reviewers invited by journal 21 Sep, 2024 Editor assigned by journal 15 Sep, 2024 Editor invited by journal 09 Sep, 2024 Submission checks completed at journal 06 Sep, 2024 First submitted to journal 02 Aug, 2024 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-4849833","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":363358918,"identity":"ddc23d85-5516-41b8-8f5f-5f4ad24fd6e1","order_by":0,"name":"Sivasankar MV","email":"","orcid":"","institution":"National Institute of Technology Warangal","correspondingAuthor":false,"prefix":"","firstName":"Sivasankar","middleName":"","lastName":"MV","suffix":""},{"id":363358919,"identity":"0406b2f3-baba-451c-adf5-a29cbc06a0de","order_by":1,"name":"Sreenivasa Rao Parcha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYLCChAobHn4wo4BYLQ/OpMlJNoC0GBCpg/Fh22FjgwMgJjFa5PtPp0kkth1O3Hx+deKHBwYM8vxiB/BrMbiRu00i4Vx64rYbbzdLAB1mOHN2AgEtErybDRLKrIFazm4AaUkwuE1Ai3z/WaAWNubEzTPObv5BlBaGA7kbHyS0ORsb8PduI84WoF+AWoCBLHGDd5tFgoEEYb8AHbbh4A9QVAJdeBPIkOeXJuQwOJAAq5QgVjkI8B8gRfUoGAWjYBSMJAAAy5RKGc5nBuMAAAAASUVORK5CYII=","orcid":"","institution":"National Institute of Technology Warangal","correspondingAuthor":true,"prefix":"","firstName":"Sreenivasa","middleName":"Rao","lastName":"Parcha","suffix":""}],"badges":[],"createdAt":"2024-08-02 17:02:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4849833/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4849833/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66321027,"identity":"808d82ab-9308-46f0-9472-2c29483c5065","added_by":"auto","created_at":"2024-10-10 11:41:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":693887,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs shows highly porous and interconnected pore of composite scaffolds in form of sponge-fiber. Top and cross sections of S1 \u003cstrong\u003e(a \u0026amp; b)\u003c/strong\u003e, S2 \u003cstrong\u003e(c \u0026amp; d)\u003c/strong\u003e, and S3\u003cstrong\u003e (e \u0026amp; f)\u003c/strong\u003e respectively, and \u003cstrong\u003eg)\u003c/strong\u003e fiber diameter of MFC is 6.14 and 11.07µm. \u003cstrong\u003eScale - 100µm\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/4922ca4a98a5d3b32396bc97.png"},{"id":66320273,"identity":"4ba4da5a-2b37-4bdf-9d5d-b7c43057f6be","added_by":"auto","created_at":"2024-10-10 11:33:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea and b)\u003c/strong\u003e Porosity and compression modulus of the different composite scaffolds S1, S2, and S3. Data\u003cstrong\u003e \u003c/strong\u003eare the means ± standard deviations from three specimens.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/7708f2e2ae35aad375796332.png"},{"id":66321135,"identity":"ce7550cb-dd8e-4e06-a4dd-e5f453dbc14e","added_by":"auto","created_at":"2024-10-10 11:49:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":489882,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of composite scaffolds a) S3, b) S2, c) S1, d) C-MFC, e) NBLCME, f) PCL, g) MFC, h) Zr, i) HAP, and j) Chitosan.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/bb2e1363a8ba360e11bf4b8d.png"},{"id":66320276,"identity":"736a4a80-a487-4b8a-837e-f547b44b2a3a","added_by":"auto","created_at":"2024-10-10 11:33:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":331260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Swelling behavior of composite scaffolds, \u003cstrong\u003eb)\u003c/strong\u003e in vitro degradtion of composite scaffolds, \u003cstrong\u003ec)\u003c/strong\u003eaverage amount of adsorped protein after 2h of incubation on different composite scaffolds of S1, S2, and S3. \u003cstrong\u003ed)\u003c/strong\u003e Cumulative drug release profile for S1, S2, and S3 scaffolds. Data\u003cstrong\u003e \u003c/strong\u003eare the means ± standard deviations from three specimens.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/b7ec5d2548bff06b9798fd18.png"},{"id":66321031,"identity":"3669d4fd-1906-4cce-86ee-285715dc72f1","added_by":"auto","created_at":"2024-10-10 11:41:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":289702,"visible":true,"origin":"","legend":"\u003cp\u003eProliferation of MG63 cells cultured on various composite scaffolds \u003cstrong\u003e(a)\u003c/strong\u003e S1, \u003cstrong\u003e(b)\u003c/strong\u003e S2, and \u003cstrong\u003e(c)\u003c/strong\u003e S3 treated with drug in different concentration for different culture time points. Data\u003cstrong\u003e \u003c/strong\u003eare the means ± standard deviations from three specimens.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/62d48b047a54547d6249b234.png"},{"id":66321029,"identity":"296dba7b-76db-4705-967e-860fb185c3a2","added_by":"auto","created_at":"2024-10-10 11:41:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":266453,"visible":true,"origin":"","legend":"\u003cp\u003eALP assay on osteobalst-like cells (MG63 cells) cultured on various composite scaffolds \u003cstrong\u003e(a)\u003c/strong\u003e S1, \u003cstrong\u003e(b)\u003c/strong\u003eS2, and \u003cstrong\u003e(c)\u003c/strong\u003e S3 for 7 days. Data\u003cstrong\u003e \u003c/strong\u003eare the means ± standard deviations from three specimens.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/e9189c167e4e2819debd4062.png"},{"id":66321030,"identity":"ffb936c4-8778-4855-a0ef-170c1c3663c2","added_by":"auto","created_at":"2024-10-10 11:41:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":277800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a -f)\u003c/strong\u003e Alizarin red S staining of osteoblast like MG63 cells cultured on different composite scaffolds S1 \u003cstrong\u003e(A)\u003c/strong\u003e, S2 \u003cstrong\u003e(B)\u003c/strong\u003e, and S3 \u003cstrong\u003e(C)\u003c/strong\u003e. Effect of NBLCME treated composite scaffolds S1, S2, and S3 on calcium mineralization in osteoblast like MG63 cells. The supplementation of 20, 40, 60, 80, and 100µg/ml NBLCME appreciably improved the calcium mineralization, whereas the higher conc. decreases the calcium deposition in the osteoblast like cells MG63 cells in 7 days of treatment. \u003cstrong\u003eg)\u003c/strong\u003eQuantification of calcium mineralization deposits by Alizarin red S assay of MG63 cells cultured on various composite scaffolds S1, S2, and S3 for 7 days. Data\u003cstrong\u003e \u003c/strong\u003eare the means ± standard deviations from three specimens.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/55efc6fe31ab74649a9cd849.png"},{"id":66320278,"identity":"dbc88951-1912-4b5b-bf58-9ac3791cd18c","added_by":"auto","created_at":"2024-10-10 11:33:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":325433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-i) \u003c/strong\u003eOsteoblast like MG63 cell morphology by fluorescence staining of microfilament dyed green and cell nucleus dyed blue after cells were cultured on different scaffolds S1, S2, and S3 for 4 days. \u003cstrong\u003ea)\u003c/strong\u003e S1-control, \u003cstrong\u003eb)\u003c/strong\u003e S1-60µg/ml, \u003cstrong\u003ec)\u003c/strong\u003e S1-100µg/ml,\u003cstrong\u003e d)\u003c/strong\u003e S2-control, \u003cstrong\u003ee)\u003c/strong\u003e S2-60µg/ml, \u003cstrong\u003ef)\u003c/strong\u003e S2-100µg/ml, \u003cstrong\u003eg)\u003c/strong\u003e S3-control, \u003cstrong\u003eh)\u003c/strong\u003eS3-60µg/ml, and\u003cstrong\u003e i)\u003c/strong\u003e S3-100µg/ml respectively. The MG63 cells are well developed actin fibers of the control cells and lower dose of NBLCME at 60µg/ml were shown by the dashed ring (\u003cstrong\u003ea, d, g\u003c/strong\u003e- control, and \u003cstrong\u003eb, e, h\u003c/strong\u003e- S1, S2, S3). The severe damage and disappeared regions of microfilament actin fibers of the higher dose of NBLCME at 100µg/ml were shown by the dashed ring (\u003cstrong\u003ec, f, i\u003c/strong\u003e- S1, S2, S3).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/4024b1389699310bb10f66af.png"},{"id":66321783,"identity":"854f6f26-b054-4213-9ff1-26dca889aca8","added_by":"auto","created_at":"2024-10-10 11:57:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3570776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/83c36c47-4303-4be1-b9f6-b4f9c5e8f5b7.pdf"},{"id":66320281,"identity":"e27ffe4a-c9f1-4761-88b0-a774c0dfaac8","added_by":"auto","created_at":"2024-10-10 11:33:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":757634,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4849833/v1/0d2935e25e0e9af155da5d5e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Onion membrane based composite scaffolds incorporated with N-Boc L-cysteine methyl ester enhances mineralization for bone tissue engineering applications","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTissue engineering has emerged as a promising approach for tissue regeneration, with a focus on the development of biomaterials for scaffold production (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Biomaterials used in bone tissue engineering should possess both bioresorbability and bioactivity, allowing for bone regeneration and degradation over time (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The establishment of stable direct contact between bone and scaffold surface is crucial for optimal scaffold development, requiring structural, mechanical, and functional integration (osteointegration) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Scaffolds made from biomaterials with these properties have the potential to support cell attachment, growth, and proliferation, facilitating tissue regeneration (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The combination of bioresorbable and bioactive biomaterials in scaffold design holds promise for inducing bone regeneration and promoting successful tissue integration (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Microfibrillated cellulose (MFC) is a cellulose material obtained through mechanical disintegration without hydrolysis, consisting of long cellulose microfibrils with diameters ranging from 20nm to 60nm (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). MFC are abundant natural materials found in plants and bacteria and exhibits a web-like structure and contains both amorphous and crystalline parts. It has a low percolation threshold and a strong ability to form stable networks, making it a valuable filler material in the production of biocomposites (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Biocomposites made with MFC have been manufactured using various polymers such as polyvinyl alcohol, poly lactic acid, polycaprolactone, pectin, starch, and chitosan (\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The use of MFC in these biocomposites enhances their properties and expands their potential applications in areas like tissue engineering (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). They possess positive properties for biomedical applications and cell culture, including biocompatibility, protein binding surface, and mechanical strength (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The high density of reactive hydroxyl groups on the surface of cellulose fibers enables the immobilization of cell adhesive proteins, such as fibronectin, facilitating cell adhesion (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The glucan chain structure in cellulose fibers is densely packed which provides mechanical strength to support cell aggregate structures, but there have been limited studies due to the absence of an intrinsic 3D architecture (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). To address the lack of 3D architecture, the use of chitosan, a linear polysaccharide derived from chitin, can provide an additional 3D structure for cellulose-based cell scaffolds (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Chitosan, derived from chitin, is a linear polysaccharide and the second most abundant natural polymer after cellulose. Chitosan is a biopolymer composed of β-(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) linked 2-deoxy-2-amino-D-glucopyranose and is obtained by alkaline de-acetylation of chitin. It is widely investigated for its low toxicity, biodegradability, and cytocompatibility properties, making it a potential biomaterial for various biomedical and bioengineering applications (\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). One of the main concerns with chitosan materials has the low mechanical properties (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Chemical modifications have gained attention from researchers as a means to enhance the mechanical properties of chitosan due to the variety of available modifications using the numerous amino and hydroxyl side groups (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Poly-caprolactone (PCL) is a semi-crystalline polyester widely used as biomaterials in medical applications. PCL has a low melting point (55\u0026deg;C) and suitable properties (porosity, degradation time, and bioreabsorption) for bone tissue regeneration. PCL has a poor wetting surface and establishes weak interactions with biological fluids, preventing cell adhesion and proliferation (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Hydroxyapatite (HA) is a ceramic material characterized by a hexagonal network structure, constituting the primary mineral component of both teeth and bones. It exhibits biocompatibility and does not elicit an inflammatory response (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). HA has slow resorption, maintaining its initial state for 2\u0026ndash;3 years after implantation, allowing for gradual bone tissue growth and cell proliferation within the material (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Additionally, HA demonstrates excellent mechanical properties, with a compressive strength of up to 160MPa, making it suitable for applications in low load conditions and small bone areas (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). But recent decades, Zirconia has been extensively used in bone tissue engineering and dental restorations due to its superior mechanical strength and biocompatibility, which enhance the cell differentiation, adhesion, and proliferation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Zirconia and nano-crystals exhibit unique properties, such as heightened chemical reactivity and an augmented surface area. Modifications to surface characteristics exert a considerable impact on cellular reactions, protein absorption, and mineralization, all of which are pivotal aspects in bone tissue engineering (\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Due to its remarkable strength and ideal biocompatibility, zirconia has found extensive use in dental restorations and medical implants (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The MG-63 human osteoblastic cell line is widely used in osteogenesis research. Cell stability across multiple passages in culture is crucial for effective biological investigations. MG-63 cells offer valuable insights into the interaction between cells and materials (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Hence, the aim of the research is to enhance the mechanical and chemical properties, as well as to evaluate its biological ability by incorporating the bioceramic materials such as hydroxyapatite and zirconium oxide in a novel microfibrillated cellulose reinforced natural polymer-based sponge composed of chitosan was fabricated via freeze-drying method, for repairing bone defect. The prepared composite scaffolds displayed significant impact on drug release was unveiled, contributing to the development of an effective sustained release system. The composite scaffolds S1, S2, and S3 achieved sustained drug delivery following Fickian diffusion behavior (n\u0026thinsp;\u0026le;\u0026thinsp;0.45). The biocompatibility of S1, S2, and S3 was systematically assessed through MTT assay, cell adhesion, and cell proliferation, affirming their suitability for bone applications. It is suggested that the S3 scaffold were superior to S1 and S2, S3 is quite promising biomaterial for application in bone tissue engineering and drug delivery.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.1. Materials\u003c/h2\u003e \u003cp\u003eOnions (\u003cem\u003eAllium cepa L.\u003c/em\u003e probably originate from central Asia) used in this study was purchased from the market (Warangal, India), chitosan (Mw 100\u0026ndash;150 kDa with deacetylation degree of 75\u0026ndash;85%) were procured from Sigma-Aldrich (India), Zirconium Oxide, hydroxyapatite, PCL (Mw\u0026thinsp;=\u0026thinsp;80,000) were purchased from Merck (India). dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), 3-(4,5-dimethyhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton-X100, fluorescein isothiocyanate isomer I (FITC), 4,6-diamindino-2-phenylindole (DAPI), Bovine Serum Albumin (BSA), phosphate buffer saline (PBS), 4% paraformaldehyde, 25% Ammonia solution, p-Nitrophenyl phosphate (pNP), Alizarin red S staining, Dulbecco\u0026rsquo;s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution were acquired from Hi-media (India). The human osteoblast cells (MG63 cell lines) were obtained from National Centre for Cell Science (India). Acetic acid (\u0026ge;\u0026thinsp;99.7%) and Ethanol analytical grade were purchased from Spectrochem (India), Double distilled water was used throughout the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1.2. Extraction of Microfibrillated cellulose (MFC):\u003c/h2\u003e \u003cp\u003eThe onion membrane was thoroughly washed with dd.water to remove dirt and then dried using the freeze drying method for 24 hours. The dried samples were ground into a fine powder using a cutting blender for further analysis. MFC was extracted from the powdered sample of onion skin using an alkaline extraction method. To remove lignin and amorphous hemicellulose, the samples were treated with a 17.5% sodium hydroxide solution for 1 hour at 23\u0026ordm;C (1:25w/v ratio). The onion powder was then thoroughly washed with dd.water and treated with a 10% acetic acid solution. Finally, the treated powder was dried in a hot air oven at 100\u0026ordm;C for 24 hours to obtain the resulting dried MFC for further characterization. The resulting dried MFC were used for further characterization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.1.3. Preparation of scaffolds S1 (C-MFC-PCL), S2 (C-MFC-PCL-H), and S3 (C-MFC-PCL-Z):\u003c/h2\u003e \u003cp\u003eComposite scaffolds of chitosan (C) and microfibrillated cellulose (MFC) were fabricated by varying percentage of composite in the final mixture. A 2% (w/v) chitosan (C) solution was prepared in a 0.5M acetic acid. To this solution, 0.5% (w/v) microfibrillated cellulose (MFC) was added under stirring condition to obtain a uniform mixture (C-MFC). 1% (w/v) PCL solution was prepared in an acetic acid and it was slowly added into C-MFC solution kept for overnight to obtain S1, followed by addition of 1% (w/v) hydroxyapatite (S2) and zirconium (S3) and left stirring for 4-5hr. The solution was introduced into 24-well cell culture plates and maintained in a freezer at -80\u0026deg;C for 24h. The samples S1, S2, and S3 were then lyophilized in a freeze dryer until dried. The resulting microporous scaffolds were neutralized with 1N NaOH, washed with dd.water and immersed in DI water overnight to achieve a neutral pH. The washed scaffolds S1, S2, and S3 were freeze dried again and stored in a desiccator under vacuum. The synthesized molecule NBLCME was dissolved in methanol at different concentrations (20, 40, 60, 80, and 100\u0026micro;g/ml). Composite scaffolds S1, S2, and S3 were immersed in the methanol solution containing NBLCME overnight. The scaffolds S1, S2, and S3 were then dried in a hot air oven for 2h. Thorough washing of the scaffolds with dd.water was performed, followed by freeze drying for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.1.4. Scanning electron microscopy (SEM) analysis\u003c/h2\u003e \u003cp\u003eThe surface of the scaffolds S1, S2, and S3 was coated with a thin layer of gold using a sputter-coating technique. Scanning electron microscopy (SEM) was employed to study the ultra-structural analysis of the scaffolds. The samples were examined at an accelerating voltage of 15\u0026ndash;20 kV during the SEM analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.1.5. Porosity studies:\u003c/h2\u003e \u003cp\u003eThe porosity of fabricated composite scaffolds S1, S2, and S3 was estimated by immersing them in absolute ethanol and measuring the weight changes (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The weights before and after immersion were recorded, along with the volume before immersion. These values, along with the constant density of ethanol, were used to calculate the porosity. The experiment was conducted in triplicate to ensure the consistency of the results. The porosity was calculated using the Eq.\u0026nbsp;1,\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:P=\\frac{W2-W1}{\\rho\\:V1}X100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere the weight before immersion (W\u003csub\u003e1\u003c/sub\u003e), weight after immersion (W\u003csub\u003e2\u003c/sub\u003e), constant density of ethanol (ρ) and the volume before immersion (V\u003csub\u003e1\u003c/sub\u003e) were used in the calculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.1.6. Mechanical Testing:\u003c/h2\u003e \u003cp\u003eThe composite scaffolds S1, S2, and S3 were molded into a cylindrical shape with 10 mm in diameter and 8 mm in height. The constructs were placed under compressive load using a universal testing machine (INSTRON, MA, USA) at a crosshead speed 0.5 mm/min up to failure or until the sample reached a 70% reduction in height. The test was performed by following ASTM F2027. The reported values were the average values determined from three specimens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.1.7. Fourier Transform Infrared Spectroscopy (FT-IR) analysis:\u003c/h2\u003e \u003cp\u003eFTIR spectroscopy was employed to investigate intermolecular interactions between functional groups of different materials. The scaffolds were first milled into powder and then mixed with potassium bromide (KBr) salt. The mixture was compressed into potassium bromide disks, which were used for FTIR spectral analysis. FTIR spectra were recorded using a PERKIN ELMER 2DTGS instrument, covering a range between 4000 and 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.1.8. Swelling Studies:\u003c/h2\u003e \u003cp\u003eComposite scaffolds were cut into equal-weight pieces and immersed in PBS (pH 7.4, 37\u0026ordm;C). The scaffolds were removed at different time intervals, and the water on the surface was gently blotted on filter paper and weighed (W\u003csub\u003ed\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eS (%) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\frac{{w}_{w-}{w}_{d}}{{w}_{d}}\\times\\:100\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eWhere S, W\u003csub\u003ew\u003c/sub\u003e and W\u003csub\u003ed\u003c/sub\u003e are the swelling percentage, wet, and dry weight of the scaffolds, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.1.9. In vitro biodegradation studies:\u003c/h2\u003e \u003cp\u003eThe degradation study was conducted by immersing the scaffolds in lysozyme-containing buffer (PBS, pH̴ 7.4) at 37\u0026ordm;C for 28 days. The scaffolds were initially weighed (W\u003csub\u003ei\u003c/sub\u003e) and then immersed in the lysozyme buffer for the specified duration. At 7, 14, 21, and 28 days, the scaffolds were removed from the buffer, washed in deionized water to remove surface ions, and freeze-dried. The final weight of the scaffolds (W\u003csub\u003ef\u003c/sub\u003e) was recorded after freeze-drying. This experimental setup allowed for the assessment of scaffold degradation over time and the determination of the weight loss experienced by the scaffolds. The degradation was calculated using the formula\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eDegradation (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{W}_{i\\:-\\:{W}_{f}}}{{W}_{f}}\\:\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Protein Adsorption study:\u003c/h2\u003e \u003cp\u003eProtein adsorption capacity of scaffolds was analyzed using (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) with slight modifications. Untreated composite scaffolds as control and treated scaffolds S1, S2, and S3 with NBLCME in different concentration (20, 40, 60, 80, and 100 \u0026micro;g/ml) having equal weights were taken and pretreated with 70% ethanol for 1h followed by hydration with PBS (pH, 7.4) for 10\u0026ndash;30 min. Hydrated scaffolds were incubate in 0.5ml 10% FBS for 2h at 37\u0026ordm;C to ensure completion of adsorption process. Gently rinsed the scaffolds with PBS thrice, to remove excess unbound protein and ions which are not adsorbed on the surface. Finally, add 1ml of 1% (W/V) SDS solution to separate the adsorbed protein. Afterward, shake the solution for 1h and centrifuge at 5000g for 15min at 4\u0026ordm;C and collect the supernatant. Total protein concentration was quantified using dye adding assay. BSA as standard and evaluate protein concentration were recorded the absorbance at a wavelength of 720nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.2.1. In vitro drug release study:\u003c/h2\u003e \u003cp\u003eIn \u003cem\u003evitro\u003c/em\u003e release studies were carried out in PBS buffer at pH-7.4. N-Boc-L-cysteine methyl ester solution (1mg/ml in methanol) was prepared first. Treated scaffolds S1, S2, and S3 were put into 10ml of release medium (PBS buffer) in an incubator shaker (100 rpm) at 37\u0026deg;C. The supernatant from drug containing solutions (1ml) were taken at constant time intervals from the release medium and were replaced with same amount fresh buffer. The concentration of N-Boc-L-cysteine methyl ester drug in the samples was measured by using micro plate reader, at 254 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.2.2. Cell viability assay:\u003c/h2\u003e \u003cp\u003eCells were seeded on the scaffolds at a density of 3x10\u003csup\u003e5\u003c/sup\u003ecells/well on treated scaffolds (S1, S2, and S3) with different concentrations of NBLCME (20, 40, 60, 80, and 100\u0026micro;g/ml) for 1, 3, 5, and 7 days. MTT assay was performed using 0.50mg/ml medium concentration of MTT dye, which was added to each well and incubated at 37\u0026deg;C for 4 hours. Living cells converted the MTT dye to a formazan product with mitochondria. The solubilization reagent, dimethyl sulfoxide (DMSO), 150\u0026micro;l was added to the cell cultures and left for 2 hours. The absorbance of each cultured solution was measured at 540 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.2.3. Determination of ALP activity:\u003c/h2\u003e \u003cp\u003eCells were seeded on the scaffolds at a density of 3x10\u003csup\u003e5\u003c/sup\u003ecells/well and incubated in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 72 hours. The media from the cell culture was aspirated, and the cells were rinsed twice with PBS. Cells in each well were fixed with 4% formaldehyde for 10 minutes. After washing, the cells were treated with 30\u0026micro;l of p-Nitrophenyl phosphate containing 5mM MgCl\u003csub\u003e2\u003c/sub\u003e at 37\u0026ordm;C for 30 minutes. The reaction was terminated by adding 30\u0026micro;l of 0.5N NaOH. The ALP activity was assayed by measuring the conversion of p-nitrophenyl phosphate to p-nitrophenol, which resulted in a color change. The color change was measured using a microplate reader at 405 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.2.4. ARS staining:\u003c/h2\u003e \u003cp\u003eCells were seeded on scaffolds (3x10\u003csup\u003e5\u003c/sup\u003ecells/well) and placed in CO\u003csub\u003e2\u003c/sub\u003e incubator for 72h. Aspirate the media from cell culture, and cells-matrix were gently rinsed with PBS twice. Cells of each well were fixed with 4% formaldehyde for 10 min. After washing, cells were treated with 40mM or 2% Alizarin red S (pH 4.1\u0026ndash;4.3) for 20min at room temperature with gentle shaking. Later, cell-matrix were washed 3\u0026ndash;4 times with dd.water while shaking for 5min. For quantification of staining, 200\u0026micro;l of 10%v/v acetic acid was added to each sample and incubate for 30min with shaking. The cell layer on the substrate was collected with acetic acid (10%v/v) and transferred to 1.5ml centrifuge tube. After that tubes were heated to 85\u0026ordm;C for 10min and transfer to ice for 5min then tubes were centrifuge at 20,000g for 15min. Supernatant were transferred to new tube and neutralized with 50\u0026micro;l of 10% v/v ammonium hydroxide. The ARS activity was measured using microplate reader (Thermo-scientific) at 405 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.2.5. Fluorescent labelling of microfilament:\u003c/h2\u003e \u003cp\u003eCell morphology was qualitatively analyzed after 4 days of culture using fluorescein isothiocyanate isomer I (FITC) fluorescent dye to stain the cell membrane and cytoplasmic proteins, and 4,6-diamindino-2-phenylindole (DAPI) to counterstain the cell nuclei. The cell-matrix were rinsed with PBS for 3 times/5mins each, fixing them in 4% formaldehyde, permeabilizing the cells with 0.5% Triton X-100, blocking the reaction with 5% BSA, staining with FITC isomer-I solution (5\u0026micro;g/ml), and counterstaining with DAPI solution (0.08\u0026micro;g/ml). After each step, the samples were washed thoroughly with PBS for 5 times/5min each. The cell-matrix was observed under a fluorescence microscope to analyze the cell morphology.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results And Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003cp\u003eFabricated composite scaffolds S1, S2, and S3 for bone tissue engineering requires an interconnected pore and highly porous structure to support cell attachment, proliferation, and tissue growth, as well as nutrient flow. The freeze-drying technique was employed to achieve the desired porous structure in the scaffolds. The resulting composite material S1, S2, and S3 exhibited adjustable morphology, consisting of a fibrous material integrated with a sponge component. SEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003e(a-g)\u003c/b\u003e showcased the top view and cross sections of the S1, S2, and S3 scaffolds, revealing their porous nature. Fabricated composite scaffolds S1, S2, and S3 showed highly porous structure, as observed at higher magnification, consisted favorable interconnected pores, which facilitate cell attachment and new bone tissue ingrowth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePorosity is a crucial factor for ideal scaffolds in tissue engineering applications, as it allows cells to passes through the pores and attach at suitable area for further proliferation. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea show the porosity of the synthesized composite scaffolds S1, S2, and S3 showing approximately 73%, 76%, and 80% respectively. The presence of NBLCME throughout the scaffolds contributed to the reduced porosity, as NBLCME binds to the polymer chains and helps maintain the integrity of the polymer chains. The obtained porosity in the prepared composite scaffolds was deemed sufficient, as it facilitates the supply of nutrients and oxygen to the interior regions of the scaffolds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eScaffolds for bone tissue engineering need sufficient mechanical strength to support tissue regeneration and maintain integrity during cell growth (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, there is a tradeoff between material porosity and mechanical strength in porous scaffolds (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Compression modulus tests were conducted on S1, S2, and S3 scaffolds to evaluate their stress-strain relations. The compressive modulus of chitosan-MFC scaffolds was determined to be 0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21MPa, while pure chitosan exhibited an extremely low compression modulus of approximately 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11MPa [29]. The addition of PCL, PCL-HA, and PCL-Zr to chitosan-microfibrillated cellulose increased the compression modulus, resulting in a value of 1.2 (S1), 2.23 (S2), and 2.7 MPa (S3) respectively were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. The addition of microfibrillated cellulose, PCL, and bio-ceramics materials to chitosan scaffolds resulted in an increased compression modulus, indicating improved mechanical strength. This increase in compressive modulus can be attributed to the strong ionic interactions between chitosan (C) and all copolymers content leading to the formation of a S1, S2, and S3 complex. Although all three scaffolds S1, S2, and S3 show good mechanical properties, S3 was higher than other scaffolds due to toughness of zirconium oxide (Zr) compare to hydroxyapatite. FTIR analysis confirmed the presence of these strong ionic interactions in the S1, S2, and S3 scaffolds, further supporting that they contribute to the increased compressive modulus.\u003c/p\u003e \u003cp\u003eThe FTIR spectra of treated and untreated S1, S2, and S3 scaffolds, along with Chitosan, MFC, polycaprolactone, hydroxyapatite, zirconium, and NBLCME were compared. Untreated and treated scaffolds (S1, S2, and S3) with the drug showed a broad absorption band between 3300\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3291\u0026ndash;3361 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the stretching vibrations of -OH and -NH groups. Additional absorption bands at 2880\u0026ndash;2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1430 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1050 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated the stretching vibrations of C-H, -CH\u003csub\u003e2\u003c/sub\u003e scissoring, and CH\u003csub\u003e2\u003c/sub\u003e-O-CH\u003csub\u003e2\u003c/sub\u003e groups, respectively. Chitosan exhibited a distinct absorption band at 1170 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, attributed to the free primary NH\u003csub\u003e2\u003c/sub\u003e group at the C2 position. The peak at 1655 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated the presence of acetylated amino groups in chitosan, suggesting incomplete de-acetylation. Peaks at 1326 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1030 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to the C-N stretching of the NH\u003csub\u003e2\u003c/sub\u003e group and C-O-C stretching vibration, respectively. In addition, the integration of ZrO\u003csub\u003e2\u003c/sub\u003e in S3 was confirmed by the characteristic\u0026rsquo;s peaks observed at 503 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which attributed to Zr-O stretching vibration of ZrO\u003csub\u003e2\u003c/sub\u003e. Broad peaks in the range of 1155\u0026thinsp;\u0026minus;\u0026thinsp;980 cm-1 correspond to the stretching vibration of phosphate (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e), indicating the presence of phosphate groups. The strong peak at 1740 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the cross-linked samples indicated successful cross-linking shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, which prevents swelling, impacting the integrity and strength of the polymer network. Increased cross-linking resulted in a denser macromolecular network, limiting hydration and swelling capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSwelling property of scaffolds are important for regulating cell infiltration, adhesion, and nutrient transportation. The addition of PCL and bio-ceramics contents in the C-MFC composite scaffolds reduces the degree of water absorption due to intermolecular crosslinking and weakening of hydrogen bonds. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the swelling ratio of the scaffolds (S1, S2, and S3) remained the same after 6000 seconds, indicating a high degree of swelling (420, 580, and 650% respectively). Chitosan swells in aqueous medium due to protonation and ionization of amino and carboxyl groups, while MFC, being hydrophilic, swells rapidly in the presence of PBS, resulting in increased scaffolds weight (S2 and S3). In particular, S1 scaffold water absorbtion was around 420% which is significantly less compared to other scaffolds S2 and S3 containing HA and Zr, due to hydrophobic characteristics of PCL.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows the in vitro degradation profile of composite scaffolds S1, S2, and S3. At 7 days, the scaffolds S1, S2, and S3 had degraded to 9, 15, and 28% respectively, with the rate of degradation increasing over time. By day 14, approximately 11, 17, and 19% degradation had occurred, and at the end of 28 days, the scaffolds S1, S2, and S3 had degraded to 15, 30, and 32% respectively. The degradation of the scaffold is attributed to the presence of lysozyme in the human body, which degrades chitosan by hydrolyzing the β-1,4 glycosidic bond between N-acetylglucosamine units. The interaction between chitosan and cellulose microfiber was found to be good, as supported by the study (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). MFC, being hydrophilic, swells rapidly in the presence of PBS, resulting in a decrease in the interactions between chitosan and cellulose microfiber due to the loosening of physical cross-linking between them. The degradation products of the scaffold can attract more cells towards it, thereby improving the bioactivity of the scaffolds. Controlled degradation rate is essential for an ideal scaffold in bone tissue engineering applications.\u003c/p\u003e \u003cp\u003eProtein adsorption on scaffolds can influence cell adhesion and is affected by factors such as surface properties and material composition (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The NBLCME treated scaffolds S1, S2, and S3 with different dose concentrations (20, 40, 60, 80, and 100\u0026micro;g/ml) showed higher protein adsorption compared to the untreated scaffolds S1, S2, and S3 (2.1, 2.8, and 3.4\u0026micro;g/mg) were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. This increase in protein adsorption can be attributed to the distribution of NBLCME particles on the scaffold surfaces, which increases binding sites for proteins and promotes electrostatic interactions (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Increasing the concentration of the drug in S1, S2, and S3 scaffolds resulted in a reduction in protein adsorption. This can be attributed to the decrease in scaffold porosity with increasing drug concentration, which directly affects the surface area available for protein adsorption. The scaffolds exhibited a considerable amount of protein adsorption, which is reported to be sufficient for promoting cell adhesion and proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe in vitro drug release from S1, S2, and S3 scaffolds follows a sustained and gradual pattern, with a high release rate observed over a longer period in 72 hours. Moreover, the burst release completely disappears in composite scaffolds, and the cumulative releases are 15, 17, and 18% for S1, S2, and S3 respectively, during the first 72 h. Even though the absolute value of drug release is low, it gradually releases with increasing time as is evident in the case of S3. A comparative measurement shows that 18% of drug release occurs in time 1, 5, 10, and 72 h in S1, S2, and S3 respectively, exhibiting sustained release in S3 as compared to S1 and S2. If we look into the release mechanism involves three distinct steps: liquid penetration into the matrix, dissolution of the drug, and diffusion of the drug out of the matrix (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). The rate-determining step for drug release is considered to be the slower diffusion process, which is influenced by the sluggish swelling ability of C-MFC scaffolds. The increased cross-linking of C-MFC systems results in a denser network structure, restricting swelling and preventing the diffusion of drug molecules from the network to the release medium. The release kinetics of the drug are best described by the Korsmeyer-Peppas model leading to the exponent \u0026ldquo;n\u0026rdquo; values of 0.23, 0.25, and 0.26 for S1, S2, and S3 respectively, indicating Fickian diffusion (n\u0026thinsp;\u0026lt;\u0026thinsp;0.45) as the dominant mechanism. Other models such as zero order, first order, and Higuchi models were verified but correlation r\u003csup\u003e2\u003c/sup\u003e values are not satisfactory in fitting the release data, while the Korsmeyer-Peppas model is a perfect fit shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(a-c)\u003c/b\u003e demonstrated the viability of MG63 cells which is expressed as the relative absorbance percentage of the control group. MG63 cells cultured with treated scaffolds S1, S2, and S3 showed varying responses to different dose concentrations of NBLCME (20, 40, 60, 80, and 100\u0026micro;g/ml). At a concentration of 80 and 100\u0026micro;g/ml, the proliferation rates of MG63 cells were lower after 1, 3, 5, and 7 days of culture. However, at lower concentrations of 20, 40, and 60\u0026micro;g/ml, the cell proliferation rates were higher. The critical concentration of NBLCME can be considered as 80\u0026micro;g/ml, as it showed lower proliferation rates. The treated scaffolds S1, S2, and S3 exhibited dose and time-dependent cytotoxicity at concentrations higher than 60\u0026micro;g/ml, while showing little sensitivity to increased dose and extended time at concentrations lower than 60\u0026micro;g/ml. It has been suggested that the higher concentration of NBLCME treated scaffolds inhibits the cell proliferation.\u003c/p\u003e \u003cp\u003eALP activity in MG63 cells incubated with treated scaffolds S1, S2, and S3 for 7 days was assessed to evaluate the bioactivity of NBLCME in the context of bone-related implants. In our study Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e(a-c)\u003c/b\u003e shows ALP activity of MG63 cells cultured with low concentrations of NBLCME (60, 40 and 20\u0026micro;g/ml) exhibited higher ALP activity compared to the control group, consistent with the results of the MTT assay. However, at a critical concentration of 60\u0026micro;g/ml, NBLCME led to significantly lower ALP activity, indicating that osteogenesis was inhibited by NBLCME-treated scaffolds at concentrations exceeding 60\u0026micro;g/ml. Although fabricated scaffolds S1, S2, and S3 showed good activity on alkaline phosphatase assay, but S3 exhibits the superior in ALP activity than other scaffolds S1 and S2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlizarin red S staining was used to evaluate the relative value of calcium deposition on cells cultured on NBLCME-treated scaffolds (S1, S2, and S3) and untreated scaffolds as a control for 7 days. Alizarin red S solution selectively bound to calcium salts, allowing for the quantification of calcium deposition were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e(A-C)\u003c/b\u003e. Cells cultured with NBLCME-treated scaffolds at concentrations of 20, 40, and 60\u0026micro;g/ml exhibited higher levels of Alizarin red S staining compared to the 80, 100 \u0026micro;g/ml, and control group. Notably, the 60 \u0026micro;g/ml concentration of NBLCME-treated scaffolds S3 showed the highest amount of calcium deposition than the other scaffolds S1 and S2 were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining was performed to observe the change in microfilament distribution in MG63 cells cultured on treated scaffolds S1, S2, and S3, and control for 4 days. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e \u003cb\u003e(a-i)\u003c/b\u003e, control cells showed well-developed actin fibers, while no differences were observed at low NBLCME concentrations (60\u0026micro;g/ml). Severe damage to actin fibers was observed at higher NBLCME concentrations (100\u0026micro;g/ml) were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e(c, f, and i)\u003c/b\u003e, possibly due to damage in the cell membrane, which induced cytotoxicity and lactate dehydrogenase (LDH) release. The release of LDH was influenced by the contact surface area between NBLCME and the cell membrane, with higher concentrations impeding LDH release. The cytotoxicity of treated scaffolds S1, S2, and S3 to MG63 cells is likely caused by intracellular activity without physical damage to the plasma membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Conclusion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003cp\u003eThe composite scaffolds S1, S2, and S3 was fabricated using the freeze-drying method, and its bioactivity was confirmed through enhanced bio-mineralization and protein adsorption. The solubility and swelling properties decrease due to cross-linking of C-MFC systems with an increase in the degree of substitution on the chitosan backbone compared to S1 and S2. Significant improvement in mechanical property in S2 and S3 than S1, but S3 showed higher compression modulus due to higher strength and toughness of zirconium oxide. Although the hydroxyapatite is incorporated in S2 shows less compression modulus than S3 due to brittle nature of HA. The treated scaffold S3 exhibited sustained release of a drug, with a high release rate over a longer period, reaching a total drug release of 18% in 72 hours. In vitro studies using osteoblast-like MG-63 cells showed that the treated composite scaffold S3 significantly improved cell proliferation and calcium deposition, with lower concentrations showing better results. Cyto-compatibility studies using MG63 cells demonstrated an increase in cell viability over time, indicating the biocompatibility of the composite scaffold S3. However, further investigation is required to study the release of LDH and its effect on cell apoptosis, as well as in vivo models. Overall, the S3 composite scaffold are suitable candidate for controlled drug delivery and ideal for bone tissue engineering applications, but further research is needed to fully understand it\u0026rsquo;s potential.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e1. Sivasankar MVConceptualization; Data curation; Formal analysis; Investigation; Resources; Roles/Writing \u0026ndash; original draft; Writing \u0026amp; editing.2. Sreenivasa Rao ParchaProject Adminstration, Conceptualization; Supervision; Investigation; Roles/Writing \u0026ndash; original draft.\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e \u003cp\u003eThe authors thank the National Institute of Technology, for providing research facility, Ministry of Human Resources and Development, Government of India, New Delhi.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVacanti, J. P., Vacanti, C. A., Lanza, R. P., Langer, R. \u0026amp; Vacanti, J. Principles of Tissue Engineering, 2nd ed., \u003cem\u003eAcademic Press\u003c/em\u003e, CA, pp. 3\u0026ndash;9. (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHench, L. L. \u0026amp; Polak, J. 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Cell proliferation and controlled drug release studies of nanohybrids based on chitosan-glactic acid and montmorillonite. \u003cem\u003eActa Biomater.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 93\u0026ndash;100 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chitosan, Microfibrillated cellulose, Zirconium, NBLCME, MG63 cells, cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-4849833/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4849833/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eComposite scaffolds S1(C-MFC-PCL), S2 (C-MFC-PCL-H), and S3 (C-MFC-PCL-Zr) containing micro-fibrillated cellulose (MFC), chitosan (C), polycaprolactone (PCL), zirconium oxide (Zr), and hydroxyapatite (H) were synthesized by freeze-drying process. N-Boc-L-cysteine methyl ester (NBLCME) was synthesized and incorporated into the composite scaffolds S1, S2, and S3 at different concentrations (20\u0026ndash;100\u0026micro;g/ml). FTIR analysis confirmed the interactions between S1, S2, S3, and NBLCME. SEM analysis showed that the S1, S2, and S3 had 70\u0026ndash;85% porosity with a pore diameter range of 100\u0026ndash;450\u0026micro;m. The scaffolds S1, S2, and S3 scaffolds achieved sustained drug delivery following Fickian diffusion behavior (n\u0026thinsp;\u0026le;\u0026thinsp;0.45). The cytotoxic effects of NBLCME treated scaffolds (S1, S2, and S3) on MG63 cell line were studied by examining cell viability, alkaline phosphatase activity (ALP), Alizarin red S activity (ARS), and cell adhesion. The cytotoxicity of the treated scaffolds on MG63 cell line was dose-dependent, with no cytotoxic effects at concentrations below 60\u0026micro;g/ml. However, higher concentrations of NBLCME (\u0026gt;\u0026thinsp;60\u0026micro;g/ml) significantly reduced ALP and ARS activity of MG63 cells due to lactate dehydrogenase leakage. Composite scaffolds S1, S2, and S3 showed significant results in mechanical properties, swelling behavior, sustainable drug release, slow degradation rate, cell adhesion, growth, and proliferation. S3 composite scaffold exhibit excellent properties than other composite scaffolds S2 and S3. Therefore, S3 can be used as promising biomaterial for bone tissue engineering.\u003c/p\u003e","manuscriptTitle":"Onion membrane based composite scaffolds incorporated with N-Boc L-cysteine methyl ester enhances mineralization for bone tissue engineering applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-10 11:33:34","doi":"10.21203/rs.3.rs-4849833/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-06T08:57:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-12T12:21:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-07T23:23:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-30T02:13:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23820908913792539461424717142334618834","date":"2024-09-27T22:08:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261831903182180959377620763358460809891","date":"2024-09-24T15:36:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111184001501140393683511113575551064970","date":"2024-09-21T19:32:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-21T14:07:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-15T05:50:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-09T17:03:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-06T05:06:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-02T17:00:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fec5cd3c-c217-4574-b2ab-69aab300a4a1","owner":[],"postedDate":"October 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":38652780,"name":"Biological sciences/Stem cells"},{"id":38652781,"name":"Physical sciences/Materials science/Biomaterials"}],"tags":[],"updatedAt":"2024-12-11T10:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-10 11:33:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4849833","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4849833","identity":"rs-4849833","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}