An in vitro analysis and physicochemical characterization of a nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite

An in vitro analysis and physicochemical characterization of a nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite | 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 Research Article An in vitro analysis and physicochemical characterization of a nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite Igor Silva Brum, Carlos Nelson Elias, Bianca Torres Ciambarella, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7140491/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Materials science has contributed to developing new nano-biomaterials for specific dentistry applications. The present work aims to characterize the physicochemical properties of a composite nanomaterial scaffold in the form of blocks for maxillofacial bone regeneration application. The scaffold had block shapes and was a mixture of nano-hydroxyapatite, β-Tricalcium phosphate, and type I collagen of bovine origin. The biomaterial was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), chemical composition microanalysis, and X-ray diffractometry (XRD). The wettability was measured using a distilled water contact angle with the surface. The phase transformation temperatures were measured using differential scanning calorimetry (DSC). In the SEM and TEM analyses, it was possible to identify the layers of the materials and, with microanalysis, quantify the chemical composition. The XRD spectra showed the presence of nano-hydroxyapatite and nano-ß-TCP. The wettability testing showed that the material is super hydrophilic. The results of the DSC testing showed that the analyzed sample undergoes endothermic transitions and transformation between 25 and 150°C. The results showed that the composite does not contain contamination from manufacturing procedures. It can be concluded that the n-HA/β-TCP and type 1 collagen composite are free of manufacturing contaminants, have higher hydrophilicity, and can be suited for clinical application. nano-hydroxyapatite ß-TCP bone nano-biomaterial collagen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Mixtures of nano-hydroxyapatite with collagen fibers are used to design materials for bone regeneration procedures [ 1 ]. The biocompatible mixture of collagen fiber and nano-hydroxyapatite promotes ideal conditions to create an efficient biomaterial for bone formation [ 2 ]. The physicochemical characteristics of nano-hydroxyapatite (n-HA) are highly efficient in bone remodeling. The n-HA induces a higher percentage of new matrix formation and blood vessel proliferation than micro-hydroxyapatite (micro-HA) [ 3 ]. Type 1 collagen from bovine or other natural sources, such as porcine pericardium, is widely used as a hemostatic barrier or protective barrier in cases of guided bone regeneration. However, its association with biomaterials that serve as pillars for bone remodeling surgery, such as hydroxyapatite and nano-hydroxyapatite, is being widely developed. Combining both promotes material stability during surgery and post-surgery [ 4 ]. Transmission electron microscopy (TEM) is an excellent tool for identifying collagen bundles in materials used in guided bone regeneration. Collagen fibrils are very characteristic, and the more organized they are, the more organic the sample will be and the better its biocompatibility [ 5 ]. Characterization methods using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are necessary to develop new biomaterials. These methods provide high-resolution images to identify the biomaterials in the samples [ 6 ]. Chemical composition analysis using a microprobe coupled to the SEM determines the semiquantitative percentages of each element in the sample. This technique is suited to identify the possible contaminants from manufacturing procedures that potentially can spoil the material's performance [ 7 ]. X-ray diffraction can identify the crystalline phases and chemical elements present in the material. The Rietveld technique quantifies each phase and calculates the chemical percentages of the material [ 8 ]. Currently, in the science of biomaterials, the diversity of technical analyses makes it easier to improve the development of biomaterials. It accelerates the emergence of new potential products. Combining several materials to form a single product becomes an interesting strategy to improve the final product applications in several areas, including bone formation [ 9 ]. The objective of the present work was to characterize the surface morphology and roughness, identify the crystalline phases, quantify the chemical composition, and analyze the energy variation during the heating of a composite made with a mixture of nano-hydroxyapatite/β-Tricalcium phosphate and type 1 collagen for use as a biomaterial. Materials and methods The analyzed material was synthesized by an inorganic reaction of two different salts, calcium nitrate and dibasic ammonium phosphate. The synthesis was very controlled, keeping the perfect mass balance and the necessary stoichiometry of the reaction, 10:6 calcium/phosphorus. The alloplastic biomaterial mixture was synthesized at the facilities of Regener Biomaterials Co (Curitiba, Brazil) and named Blue Bone®. The biomaterial had nanometric particles of hydroxyapatite and ß-TCP. The collagen type 1 was prepared at Regener® Biomaterials facilities by performing three simple steps: cleaning the raw material using two chemical baths (sodium hydroxide and acetone) for fat removal; extracting the type 1 collagen using acetic acid from the cleaned bovine tendons; and purifying the extracted material by lyophilization process. The collagen type 1 had an expected reabsorption time of up to 30 days. The chemical composition of the composite was determined using semiquantitative chemical analysis with a microprobe coupled to the SEM and TEM. Scanning Electron Microscopy and Transmission Electron Microscopy Gold-coated collagen type 1 surfaces were analyzed using a Field Emission GUN Quanta 250 FEG (FEI Company, Oregon, USA). A 5000 magnification was used to analyze the homogeneity, a 15,000 magnification to observe cell clusters, and a 20,000 magnification to identify specific cell types. For the SEM analysis, the fixation procedure started with osmium tetroxide and potassium ferrocyanide (1.0 wt%, 0.8 wt%, respectively) with a cacodylate buffer (0.1 M, pH 7.4) incubation for one h in the dark, followed by three sodium cacodylate buffer rinses in distilled water (0.2 M, pH 7.4) for one h. After this step, the sample was immersed in a sequential ethanol grade (25–100 vol%) rinse for specimen dehydration and slicing. The slices were immersed in hexamethyl silazane for 10 min before being placed in an evaporation chamber for drying. Specimen mount on aluminum stubs was achieved using colloidal silver adhesive (Electron Microscopy Sciences, Peabody, MA, USA). The specimens were coated with gold film by sputtering (Cool Sputter Coater—SCD 005, Bal-Tec, Berlin, Germany). The results of SEM analysis were complemented with roughness measurements using a Zygo NewView 7100 optical roughness meter (Zygo Corporation, Connecticut, United States). The implants' surface roughness parameters Ra, Rsk, Rms, Rku, PV, Rpk, Rk, and R3z were measured. The analyzed regions presented homogeneous surface roughness. Thin collagenous type 1 sections were analyzed using a JEOL JEM-1011 transmission electron microscope (JEOL, Ltd., Akishima, Tokyo, Japan), operating at 60 kV. Digital micrographs were captured using an ORIUS CCD digital camera (Gatan, Inc., Pleasanton, CA, USA) at 8000×, 10,000× and 25,000× magnification. The morphology of the samples was characterized using scanning electron microscopy Field Emission Gun (Quanta FEG 250; Hillsboro, Oregon 97124 - USA). The samples were prepared for TEM analysis: fixation in 2.5 wt% glutaraldehyde diluted in 0.1 M cacodylate buffer solution (overnight). Wash in 3 baths in cacodylate buffer solution (0.1 M) for 15 min each bath. Dehydration in 30 vol% acetone bath (15 min), 50 vol% acetone, 70 vol% acetone (15 min), 90 vol% acetone (15 min), 100 vol% acetone (15 min), and 100 vol% acetone (15 min) Infiltration in acetone + epon mixture (2:1) for two h; acetone + epon (1:1) for two h; acetone + epon mixture (1:2) for two h, infiltration in pure Epon (overnight) Inclusion in Epon and polymerization between 48 and 72 h at 60°C. Plate cuts with a thickness of 1 micrometer and staining with toluidine blue. Cutting with ultramicrotome to obtain 70 nm slides collected on 300 mesh copper grids. The slides were contrasted with uranyl acetate (for 20–30 min), and TEM observation was performed. The X-ray diffraction (XRD) The biomaterial was characterized by X-ray diffraction (XRD), porosimetry, and pycnometer tests. The X-ray diffraction (XRD) was performed using a Panalytical (Almelo, Netherlands) Empyrean diffractometer, with Cu-Kα radiation, 2θ range of 20–80°, a step width of 0.02°, and an exposure time of 5 s. The diffraction peaks were identified by comparing them with standard ICDD (International Centre for Diffraction Data) diffraction files and COD-Jan2012 (Crystallography Open Database) PDF2-2004 databases. The X-ray diffractograms were recorded on a Siemens diffractor (Bruker AXS; Durham—UK), model D-5000 (θ-θ), equipped with a graphite curved monochromator, secondary beam, and Cu tube. The quantitative analysis of the phases was determined by the mathematical refinement method proposed by Rietveld. Rietveld analysis of XRD data was used to identify and quantify the percentages of the phases. The Rietveld Method involves adjusting the theoretical diffraction peaks calculated from crystallographic information to the experimentally measured diffraction pattern. The criterion for this adjustment is to minimize the sum of the squares of the differences. Wettability The surface wettability was determined by measuring the contact angle with a goniometer First Ten Angstroms FTA-100 (First Ten Angstroms Co., Portsmouth, VA, USA). The contact angles were determined by averaging the values obtained at five different areas on the three sample surfaces using NaCl 0.9% solution. The wettability was determined by the contact angle measurement method. This methodology is the most used. For the measurement, a drop of 0.9% sodium chloride was placed on the surface of the membrane, and an image of the drop was obtained. The static contact angle was defined by fitting the Young-Laplace equation around the drop. Measurement of Thermodynamic Properties Using DSC The thermal stability of the mixture of nano-hydroxyapatite, β-Tricalcium phosphate, and type I collagen was determined by differential scanning calorimetry (DSC). The thermal behavior of the samples was analyzed by differential scanning calorimetry (DSC) using a Shimadzu DSC60 DSC calorimeter (Shimadzu, Kyoto, Japan). Two samples are used to perform DSC tests. The first sample is used to determine the thermal properties. The second sample must have known thermal properties to be used as a reference. All energy variations of the mixture of nano-hydroxyapatite, β-Tricalcium phosphate, and type I collagen, during heating were calculated based on the properties of the reference sample. The mixture samples with 8.0 ± 0.5 mg each were placed in aluminum pans. The pans were closed and weighed. Scans were performed between 25°C and 150°C at a 5°C/min rate. The tests were conducted without atmospheric control. The reference sample was an empty aluminum pan. The mixture and reference samples were kept at the same temperatures, and the difference in energy required to increase the temperature between them was determined. The reference sample has a defined thermal capacity throughout the temperature range scanned. The test determined the variation in energy with the temperature of the phase transformations and transitions. The thermal measurements yielded the temperature and energy for membrane denaturation. The phenomenon of denaturation is distinct from degradation. Denaturation is the rupture of interchain hydrogen bonds that leads to the formation of an amorphous material. The temperature at the beginning of denaturation ( T onset ), the temperature at the end of denaturation ( T endset ), the peak temperature of denaturation at maximum heat absorption ( T p ), the change of enthalpy (Δ H ), and the width at half-peak height (Δ T 1/2 ) were determined using the DSC curve. The peak denaturation temperature is the temperature at which collagen structure unfolding occurs. The thermal denaturation of the collagen membrane was characterized by its enthalpy (Δ H d ) and denaturation temperature ( T d ). The change of enthalpy (Δ H ) corresponds to the energy absorbed by the tissue during the helix-coil transformation of the collagen. Results Aleatory regions were selected for higher magnification and semiquantitative chemical analysis. Figures 1 , 2 , and 3 show the SEM photomicrographs. It is possible to observe different gray shades of material bordered by a light clear region. Figure 2 : The analyzed composite. Photomicrographs detailing the points where the semiquantitative chemical analyses were carried out (2a to 2f). Figure a (50x); Figures b, c (220x) and Figures d, e, f (1000x) magnification. The SEM-analyzed images showed sheets and plates of collagen in diverse planes of orientations and highly angular and porous surfaces of the calcium phosphate granules (Fig. 3 a, 3 b). Transmission Electron Microscopy (TEM) Transmission electron microscopy showed the type 1 collagen fibers organically arranged in straight bundles. It was also possible to see that there was no external agent in the type 1 collagen, indicating the purity of the sample. (Fig. 4 A, 4 B) Microanalysis of calcium phosphate In the microanalysis of the sample containing calcium phosphate, it was possible to identify the chemical elements forming the n-HA/β-TCP and type 1 collagen composite. (Fig. 5 a to 5 e) The chemical percentages of type 1 collagen fibers were recorded, and no impurities were identified. Figure 6 shows images of carbon, nitrogen, and oxygen distribution mapping on the samples' surfaces. Roughness Figure 7 shows the collagenous type 1 surface morphology image obtained by interferometry during roughness measurements. The surface was homogeneous. The roughness surface parameter is Ra 12.9 µm, Rms 13.1 µm, and Rku 1.1 µm. The X-ray diffraction (XRD) Figure 8 shows the diffraction spectra and peaks of the crystalline phases. The peaks of the diffractograms were identified based on information from the database using the Diffrac software. EVE. V4.22. (A) (B) Figure 8: The diffractogram of the sample (A), the peaks characterize only nano-hydroxyapatite. The diffractogram (B) peaks of the phases characterize nano-hydroxyapatite (in red) and nano-ß-TCP (in blue). Wettability In wettability testing, it was impossible to measure the droplet's angle of incidence due to the surface of the material being extremely hydrophilic, Fig. 9 . (Supplementary Material). DSC The material's thermal energy variation during heating characterized the thermal properties of the sample. The thermal measurement curve (Fig. 10 ) shows the variation in energy during the heating. The energy variation was identified at temperatures 61.1, 121.4, 128.7, and 133.6 o C. Discussion The morphological and chemical biomaterial analysis indicates that nano-hydroxyapatite/β-Tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite are suitable for further in vivo testing. Developing new graft biomaterials is an excellent alternative to improve the performance of the regenerative properties for patient treatments. Adding other types of components to hydroxyapatite has been proven to do so. Literature data showed that adding chitosan to nano-hydroxyapatite improved osteogenesis and reduced bacterial adhesion [ 10 ]. The collagen molecule contributes to key aspects of bone regeneration, such as cell migration, attachment, migration, cell division, and differentiation. Collagen participates in osteoinduction and osteoconduction in bone healing. In addition, collagen is the main scaffold that sustains mineralization in the human body, notably in the intrafibrillar mineralization pattern [ 11 , 12 ]. Previous work showed that the mixture of hydroxyapatite with collagen had characteristics such as microstructure, absorption kinetics, and mechanical properties suitable as bone substitutes [ 11 ]. The results of the present work corroborate those cited in the literature [ 13 – 16 ]. Figure 3 shows the interaction between calcium phosphate and collagen. Literature results using rabbits showed that the calcium concentration in the graft material used in surgery can form different types of hydroxyapatites [ 17 ]. In the tests carried out in the present work, it was possible to verify that the percentages of calcium and other chemical elements were similar to those of hydroxyapatite. Figure 3 b shows n-HA granules. The identification of material phases is mainly determined by XRD testing [ 18 ]. Data from the literature showed that the percentage of nano-hydroxyapatite was similar to that of dentin. The nano-hydroxyapatite induced more collagen cross-links, increased the rate of the organic matrix formation, and promoted better mineralization [ 19 ]. The association of nano-hydroxyapatite with collagen was analyzed in tests, and the results were compared with materials containing only hydroxyapatite. The results showed that the structure and performance can be improved by mixing appropriate percentages of collagen and hydroxyapatite [ 20 ]. When nano-hydroxyapatite is mixed with n-HA, the cell viability, cell integration, and differentiation processes are improved [ 21 ]. Moreover, it was demonstrated that n-HA and collagen scaffolds acted synergistically in the osteoconduction process and that bone morphogenetic protein-7 (BMP7) and BMP2 participated in the process [ 22 ]. The results of the DSC test showed that the analyzed sample undergoes endothermic transitions between 25 and 150°C (Fig. 10 ). The energy variation identified at a temperature of 61.1°C is related to the glass transition of the amorphous phase of collagen (Fig. 10 ). The second variation identified near 133°C is related to internal water loss. Results available in the literature show an energy variation that occurs at 230°C, which is associated with the denaturation of the collagen molecule chains. This third energy variation was not identified in the present study; the DSC test was interrupted at 150°C. Denaturation is different from degradation. During denaturation, the hydrogen bonds between the polymer chains are broken. Based on Fig. 10 , it is possible to identify the temperature at which denaturation began (Tonset), the temperature at the end of denaturation (Tendset), and the peak denaturation temperature at maximum heat absorption (Tp). The peak denaturation temperature is the temperature at which the collagen structure unfolds. The denaturation of type I collagen began (Tonset) at 47.6°C, with the transformation peak (Tp) occurring at 61.1°C, and the final denaturation temperature (Tendset) was 87.9°C. The denaturation temperature measured in this study differed from those reported in other studies [ 23 ]. The differences between the temperatures (Tonset, Tp, and Tendset) are attributed to collagens of different origins and compositions. Natural and synthetic collagens have various types and amino acid contents, as well as sample preparation for analysis, genetic lineage, animal age, fibrillation, mineralization, etc. The denaturation temperature of lyophilized type I collagen differs from that of hydrated collagen. The arrangement of the crosslinks influenced the denaturation temperature. Literature results show that collagen thermograms present parameters differences due to collagen type and origin [ 23 ]. The literature indicates that the first energy variation occurs between 30 and 150°C, and the endothermic peak (Td') is between 80°C and 104°C. The differences in the attributed properties are associated with the dehydration process and preparation of fibrillar collagen. Some researchers [ 23 ] show the DSC thermogram of the collagen sample, and the peak temperatures occur near 61.5 and 221.8°C. Literature results suggest that the peak energy variation near 61.5°C is associated with changing the triple helix structures of collagen molecules to randomly coiled structures [ 24 ]. During the structure change, intra, and intermolecular hydrogen bonds break by releasing weakly bound water. The stability of the triple helix structures of collagen molecules and the binding of water in the structure of the molecules depend on intra- and intermolecular hydrogen bonds. The literature also suggests that the energy variation near 221.8°C is due to the degradation of the polypeptide chains and the evaporation of residual and/or strongly bound water. The results obtained in the present work extended the previous works. Notably, the percentages of nano-hydroxyapatite and collagen influence the biomaterial's performance in bone repair. Conclusion Based on the results obtained in the present work, it can be concluded that: The material has a homogeneous surface morphology of collagen fibrils as bundles with different thicknesses and directions. Transmission electron microscopy showed the type 1 collagen fibers organically arranged in straight bundles. The surface roughness parameters measured using interferometry were Ra 12.9 µm, Rms 13.1 µm, and Rku 1.1 µm. The material has thermal energy variation during heating at temperatures 61.1, 121.4, 128.7, and 133.6 o C. The material is highly hydrophilic. The analyzed nano-biomaterial made with the mixture of nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen does not contain manufacturing processing impurities and is suitable for in vivo surgery applications. Declarations Author Contributions: Igor da Silva Brum: Conceptualization and co-wrote the manuscript. Carlos Nelson Elias: analysis of results, writing a review, Marco Antonio Alencar de Carvalho, Guilherme Aparecido Monteiro Duque da Fonseca, Bianca Torres Ciambarella: editing and analysis of results, Lucio Frigo co-wrote the manuscript. Jorge José de Carvalho: Analyze the experimental results of the materials. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Informed Consent Statement: The author confirms that consent has been obtained from the patient(s) for publishing this paper. Acknowledgments: The authors are grateful to the Brazilian Agencies CAPES, CNPq, FAPERJ, and FINEP. Conflicts of Interest: The authors declare no conflicts of interest. Ethics, Consent to Participate : Ethics, Consent to Participate, and Consent to Publish declarations: not applicable References Yu L, Wei M. Biomineralization of Collagen-Based Materials for Hard Tissue Repair. Int J Mol Sci. 2021;22(2):944. 10.3390/ijms22020944. PMID: 33477897; PMCID: PMC7833386. Lazarevic M, Petrovic S, Pierfelice TV, Ignjatovic N, Piattelli A, Vlajic Tovilovic T, Radunovic M. Antimicrobial and Osteogenic Effects of Collagen Membrane Decorated with Chitosan-Nano-Hydroxyapatite. Biomolecules. 2023;13(4):579. 10.3390/biom13040579. PMID: 37189328; PMCID: PMC10135971. Min KH, Kim DH, Kim KH, Seo JH, Pack SP. Biomimetic Scaffolds of Calcium-Based Materials for Bone Regeneration. Biomimetics (Basel). 2024;9(9):511. 10.3390/biomimetics9090511. PMID: 39329533; PMCID: PMC11430767. 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Regen Biomater. 2016;3(1):33–40. 10.1093/rb/rbv025. Epub 2015 Dec 31. PMID: 26816654; PMCID: PMC472327. Chen ZG, Mo XM, He CL, Wang HS Carbohydr. Polym., Martins AAS, Plepis VCA. A. M. G. J. Therm. Anal. Calorim. 2002, 67, 491. Yeh Jen-taut, Chang Haw-jer, Xiao Li-fang, Yang, Li, Zhu P, Huang Guo-xian, Yao W-H. (2010). Physicochemical properties and molecular weight characterization of porcine dermal collagen digested under varying conditions with clostridium histolytic collagenase. e-Polymers October 2010 10(1). 10.1515/epoly.2010.10.1.1226 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7140491","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491286623,"identity":"a16aeb92-5558-4f36-930a-3d9d71226f59","order_by":0,"name":"Igor Silva Brum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACNhDB2AAkmJmPQUTYidbCzpbGwJAAFGEmxiqwFn4eM7AWBkJa+PgXH/vAuONe4tpmnm8PPv7YJs/HzMD44WMOHodJPEuewXimOHHbYd7thjMSbhu2MTMwS87chk/LGWMGxrYEkJZt0jwJtxmBWtiYeYnTwvMMpMWesBb+HrgWNpCWRCJsYUtmYDyTYLztMJuZ5Iy028ltzIzNeP0i33/4MAPjjgTZbecPP5P4YHPbdn5788EPH/FoYZBIYGD+gyoEjiY8gP8AfvlRMApGwSgYBQwAQXZJnGI4pBgAAAAASUVORK5CYII=","orcid":"","institution":"State University of Rio de Janeiro, Rio de Janeiro","correspondingAuthor":true,"prefix":"","firstName":"Igor","middleName":"Silva","lastName":"Brum","suffix":""},{"id":491286628,"identity":"16f10bcc-c88b-4e24-aeb1-7349e7c6d943","order_by":1,"name":"Carlos Nelson Elias","email":"","orcid":"","institution":"IME Military Engineering Institute, Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Nelson","lastName":"Elias","suffix":""},{"id":491286629,"identity":"9283de68-f5bd-464f-8ad7-09d3b66d4df0","order_by":2,"name":"Bianca Torres Ciambarella","email":"","orcid":"","institution":"State University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"Torres","lastName":"Ciambarella","suffix":""},{"id":491286630,"identity":"cf52bf8a-c244-4cf0-90f8-c11897baa976","order_by":3,"name":"Guilherme Aparecido","email":"","orcid":"","institution":"State University of Rio de Janeiro, Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Guilherme","middleName":"","lastName":"Aparecido","suffix":""},{"id":491286632,"identity":"c4bf34ed-f5ac-43cb-87f2-4dc6499d99d0","order_by":4,"name":"Lucio Frigo","email":"","orcid":"","institution":"APCD School of Dentistry, São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Lucio","middleName":"","lastName":"Frigo","suffix":""},{"id":491286635,"identity":"280157d5-fa7f-4d62-aeb4-52d83a6e1a37","order_by":5,"name":"Marco Antônio Alencar Carvalho","email":"","orcid":"","institution":"State University of Rio de Janeiro, Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"Antônio Alencar","lastName":"Carvalho","suffix":""},{"id":491286638,"identity":"5471ea0f-707f-49be-a2aa-fa2bdd28ee22","order_by":6,"name":"Jorge José Carvalho","email":"","orcid":"","institution":"State University of Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"José","lastName":"Carvalho","suffix":""}],"badges":[],"createdAt":"2025-07-16 13:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7140491/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7140491/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87723335,"identity":"873134f9-d5cc-48e4-b232-93503bcad19d","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68269,"visible":true,"origin":"","legend":"\u003cp\u003eMicrograph of the sample indicating the regions of chemical analysis in the square boxes (A). The collagen type I membrane is made with a mixture of nano-hydroxyapatite/β-tricalcium phosphate. The round particle is a nano-hydroxyapatite/β-tricalcium phosphate (B).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/7d03f6324d02efc9996f5b03.jpg"},{"id":87723334,"identity":"61d63796-c1b7-46de-9aa2-d69ece8a3a3e","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75530,"visible":true,"origin":"","legend":"\u003cp\u003eThe analyzed composite. Photomicrographs detailing the points where the semiquantitative chemical analyses were carried out (2a to 2f). Figure a (50x); Figures b, c (220x) and Figures d, e, f (1000x) magnification.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/f9fd563f59f24cdee8e0a820.jpg"},{"id":87723856,"identity":"2dc9c431-eb78-484a-8682-85bd6363717d","added_by":"auto","created_at":"2025-07-28 10:23:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18929,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM photomicrographs of the surface morphology of the analyzed biomaterial. (a) Image obtained with secondary electrons. (b) image obtained with backscattered electrons. The area of ​​collagen fibers stands out in the red circle, and the calcium phosphate granule is highlighted in the yellow circle. Figure A (99x) and Figure B (100x) magnification.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/278f59ac1577ce34aa598b9b.jpg"},{"id":87723336,"identity":"425ccc63-efcf-441d-9a7b-996137e583bb","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30595,"visible":true,"origin":"","legend":"\u003cp\u003eThe TEM photomicrograph showed a well-organized type 1 collagen bundles (A) (800x magnification), which is characteristic of this model of bovine type 1 collagen fibrillar pattern (B) (12000x magnification)\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/0051338caad458a076164bd2.jpg"},{"id":87723340,"identity":"2c419525-9895-4db0-a72f-6d7b2354fdf1","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27112,"visible":true,"origin":"","legend":"\u003cp\u003eSpectrum of calcium phosphate microanalysis. Regions analyzed with 1000x magnification.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/84318b946f51a0b6e88bd3dc.jpg"},{"id":87723857,"identity":"328cc7a5-8f84-42af-9fd0-617b748fe37e","added_by":"auto","created_at":"2025-07-28 10:23:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79378,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of the distribution of chemical elements on the surface of one of the analyzed samples. (a) Sample surface, (b) Carbon, (c) Oxygen, and (d) Nitrogen.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/78e164dc4603619268c172b2.jpg"},{"id":87723338,"identity":"81c974f6-191e-4087-a2e3-1635559e8ea5","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":131657,"visible":true,"origin":"","legend":"\u003cp\u003ecollagenous type 1 surface morphology. Images obtained by interferometry.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/21bcc6d33ff7541ab663fb15.jpg"},{"id":87724847,"identity":"a7f95a7a-48a8-4374-a008-1b8ca8a6886b","added_by":"auto","created_at":"2025-07-28 10:31:16","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":60359,"visible":true,"origin":"","legend":"\u003cp\u003eThe diffractogram of the sample (A), the peaks characterize only nano-hydroxyapatite. The diffractogram (B) peaks of the phases characterize nano-hydroxyapatite (in red) and nano-ß-TCP (in blue).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/148af78454283efa9420b275.jpg"},{"id":87723350,"identity":"eae15759-ddde-43ea-a1ab-dfe6d78f0378","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":29434,"visible":true,"origin":"","legend":"\u003cp\u003eThe picture shows \u0026nbsp;the drop of 0.9% sodium chloride coming into contact with the surface of the hydroxyapatite and collagen sample.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/7e149283bac18b31ae24b0f2.jpg"},{"id":87723349,"identity":"fde70b70-4ce1-4ce1-8b8e-a40ab3268148","added_by":"auto","created_at":"2025-07-28 10:15:16","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":27890,"visible":true,"origin":"","legend":"\u003cp\u003eThermogram showing the material thermal properties measured using DSC.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/b3224e6a89feb61ecad61e21.jpg"},{"id":89807829,"identity":"f541e6b3-2b50-42c3-9411-09f500d0e806","added_by":"auto","created_at":"2025-08-25 09:24:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1145564,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7140491/v1/f75a519f-f4e9-437d-9707-f3b30a3f4dfe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An in vitro analysis and physicochemical characterization of a nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMixtures of nano-hydroxyapatite with collagen fibers are used to design materials for bone regeneration procedures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The biocompatible mixture of collagen fiber and nano-hydroxyapatite promotes ideal conditions to create an efficient biomaterial for bone formation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe physicochemical characteristics of nano-hydroxyapatite (n-HA) are highly efficient in bone remodeling. The n-HA induces a higher percentage of new matrix formation and blood vessel proliferation than micro-hydroxyapatite (micro-HA) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Type 1 collagen from bovine or other natural sources, such as porcine pericardium, is widely used as a hemostatic barrier or protective barrier in cases of guided bone regeneration. However, its association with biomaterials that serve as pillars for bone remodeling surgery, such as hydroxyapatite and nano-hydroxyapatite, is being widely developed. Combining both promotes material stability during surgery and post-surgery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTransmission electron microscopy (TEM) is an excellent tool for identifying collagen bundles in materials used in guided bone regeneration. Collagen fibrils are very characteristic, and the more organized they are, the more organic the sample will be and the better its biocompatibility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCharacterization methods using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are necessary to develop new biomaterials. These methods provide high-resolution images to identify the biomaterials in the samples [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChemical composition analysis using a microprobe coupled to the SEM determines the semiquantitative percentages of each element in the sample. This technique is suited to identify the possible contaminants from manufacturing procedures that potentially can spoil the material's performance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eX-ray diffraction can identify the crystalline phases and chemical elements present in the material. The Rietveld technique quantifies each phase and calculates the chemical percentages of the material [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrently, in the science of biomaterials, the diversity of technical analyses makes it easier to improve the development of biomaterials. It accelerates the emergence of new potential products. Combining several materials to form a single product becomes an interesting strategy to improve the final product applications in several areas, including bone formation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe objective of the present work was to characterize the surface morphology and roughness, identify the crystalline phases, quantify the chemical composition, and analyze the energy variation during the heating of a composite made with a mixture of nano-hydroxyapatite/β-Tricalcium phosphate and type 1 collagen for use as a biomaterial.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe analyzed material was synthesized by an inorganic reaction of two different salts, calcium nitrate and dibasic ammonium phosphate. The synthesis was very controlled, keeping the perfect mass balance and the necessary stoichiometry of the reaction, 10:6 calcium/phosphorus. The alloplastic biomaterial mixture was synthesized at the facilities of Regener Biomaterials Co (Curitiba, Brazil) and named Blue Bone\u0026reg;. The biomaterial had nanometric particles of hydroxyapatite and \u0026szlig;-TCP.\u003c/p\u003e\u003cp\u003eThe collagen type 1 was prepared at Regener\u0026reg; Biomaterials facilities by performing three simple steps: cleaning the raw material using two chemical baths (sodium hydroxide and acetone) for fat removal; extracting the type 1 collagen using acetic acid from the cleaned bovine tendons; and purifying the extracted material by lyophilization process. The collagen type 1 had an expected reabsorption time of up to 30 days.\u003c/p\u003e\u003cp\u003eThe chemical composition of the composite was determined using semiquantitative chemical analysis with a microprobe coupled to the SEM and TEM.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScanning Electron Microscopy and Transmission Electron Microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGold-coated collagen type 1 surfaces were analyzed using a Field Emission GUN Quanta 250 FEG (FEI Company, Oregon, USA). A 5000 magnification was used to analyze the homogeneity, a 15,000 magnification to observe cell clusters, and a 20,000 magnification to identify specific cell types. For the SEM analysis, the fixation procedure started with osmium tetroxide and potassium ferrocyanide (1.0 wt%, 0.8 wt%, respectively) with a cacodylate buffer (0.1 M, pH 7.4) incubation for one h in the dark, followed by three sodium cacodylate buffer rinses in distilled water (0.2 M, pH 7.4) for one h. After this step, the sample was immersed in a sequential ethanol grade (25\u0026ndash;100 vol%) rinse for specimen dehydration and slicing. The slices were immersed in hexamethyl silazane for 10 min before being placed in an evaporation chamber for drying. Specimen mount on aluminum stubs was achieved using colloidal silver adhesive (Electron Microscopy Sciences, Peabody, MA, USA). The specimens were coated with gold film by sputtering (Cool Sputter Coater\u0026mdash;SCD 005, Bal-Tec, Berlin, Germany). The results of SEM analysis were complemented with roughness measurements using a Zygo NewView 7100 optical roughness meter (Zygo Corporation, Connecticut, United States). The implants' surface roughness parameters Ra, Rsk, Rms, Rku, PV, Rpk, Rk, and R3z were measured. The analyzed regions presented homogeneous surface roughness.\u003c/p\u003e\u003cp\u003eThin collagenous type 1 sections were analyzed using a JEOL JEM-1011 transmission electron microscope (JEOL, Ltd., Akishima, Tokyo, Japan), operating at 60 kV. Digital micrographs were captured using an ORIUS CCD digital camera (Gatan, Inc., Pleasanton, CA, USA) at 8000\u0026times;, 10,000\u0026times; and 25,000\u0026times; magnification. The morphology of the samples was characterized using scanning electron microscopy Field Emission Gun (Quanta FEG 250; Hillsboro, Oregon 97124 - USA).\u003c/p\u003e\u003cp\u003eThe samples were prepared for TEM analysis: fixation in 2.5 wt% glutaraldehyde diluted in 0.1 M cacodylate buffer solution (overnight). Wash in 3 baths in cacodylate buffer solution (0.1 M) for 15 min each bath. Dehydration in 30 vol% acetone bath (15 min), 50 vol% acetone, 70 vol% acetone (15 min), 90 vol% acetone (15 min), 100 vol% acetone (15 min), and 100 vol% acetone (15 min) Infiltration in acetone\u0026thinsp;+\u0026thinsp;epon mixture (2:1) for two h; acetone\u0026thinsp;+\u0026thinsp;epon (1:1) for two h; acetone\u0026thinsp;+\u0026thinsp;epon mixture (1:2) for two h, infiltration in pure Epon (overnight) Inclusion in Epon and polymerization between 48 and 72 h at 60\u0026deg;C. Plate cuts with a thickness of 1 micrometer and staining with toluidine blue. Cutting with ultramicrotome to obtain 70 nm slides collected on 300 mesh copper grids. The slides were contrasted with uranyl acetate (for 20\u0026ndash;30 min), and TEM observation was performed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe X-ray diffraction (XRD)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe biomaterial was characterized by X-ray diffraction (XRD), porosimetry, and pycnometer tests. The X-ray diffraction (XRD) was performed using a Panalytical (Almelo, Netherlands) Empyrean diffractometer, with Cu-Kα radiation, 2θ range of 20\u0026ndash;80\u0026deg;, a step width of 0.02\u0026deg;, and an exposure time of 5 s.\u003c/p\u003e\u003cp\u003eThe diffraction peaks were identified by comparing them with standard ICDD (International Centre for Diffraction Data) diffraction files and COD-Jan2012 (Crystallography Open Database) PDF2-2004 databases. The X-ray diffractograms were recorded on a Siemens diffractor (Bruker AXS; Durham\u0026mdash;UK), model D-5000 (θ-θ), equipped with a graphite curved monochromator, secondary beam, and Cu tube. The quantitative analysis of the phases was determined by the mathematical refinement method proposed by Rietveld.\u003c/p\u003e\u003cp\u003eRietveld analysis of XRD data was used to identify and quantify the percentages of the phases. The Rietveld Method involves adjusting the theoretical diffraction peaks calculated from crystallographic information to the experimentally measured diffraction pattern. The criterion for this adjustment is to minimize the sum of the squares of the differences.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWettability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe surface wettability was determined by measuring the contact angle with a goniometer First Ten Angstroms FTA-100 (First Ten Angstroms Co., Portsmouth, VA, USA). The contact angles were determined by averaging the values obtained at five different areas on the three sample surfaces using NaCl 0.9% solution.\u003c/p\u003e\u003cp\u003eThe wettability was determined by the contact angle measurement method. This methodology is the most used. For the measurement, a drop of 0.9% sodium chloride was placed on the surface of the membrane, and an image of the drop was obtained. The static contact angle was defined by fitting the Young-Laplace equation around the drop.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of Thermodynamic Properties Using DSC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe thermal stability of the mixture of nano-hydroxyapatite, β-Tricalcium phosphate, and type I collagen was determined by differential scanning calorimetry (DSC). The thermal behavior of the samples was analyzed by differential scanning calorimetry (DSC) using a Shimadzu DSC60 DSC calorimeter (Shimadzu, Kyoto, Japan). Two samples are used to perform DSC tests. The first sample is used to determine the thermal properties. The second sample must have known thermal properties to be used as a reference. All energy variations of the mixture of nano-hydroxyapatite, β-Tricalcium phosphate, and type I collagen, during heating were calculated based on the properties of the reference sample.\u003c/p\u003e\u003cp\u003eThe mixture samples with 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg each were placed in aluminum pans. The pans were closed and weighed. Scans were performed between 25\u0026deg;C and 150\u0026deg;C at a 5\u0026deg;C/min rate. The tests were conducted without atmospheric control. The reference sample was an empty aluminum pan. The mixture and reference samples were kept at the same temperatures, and the difference in energy required to increase the temperature between them was determined. The reference sample has a defined thermal capacity throughout the temperature range scanned. The test determined the variation in energy with the temperature of the phase transformations and transitions.\u003c/p\u003e\u003cp\u003eThe thermal measurements yielded the temperature and energy for membrane denaturation. The phenomenon of denaturation is distinct from degradation. Denaturation is the rupture of interchain hydrogen bonds that leads to the formation of an amorphous material. The temperature at the beginning of denaturation (\u003cem\u003eT\u003c/em\u003e\u003csub\u003eonset\u003c/sub\u003e), the temperature at the end of denaturation (\u003cem\u003eT\u003c/em\u003e\u003csub\u003eendset\u003c/sub\u003e), the peak temperature of denaturation at maximum heat absorption (\u003cem\u003eT\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e), the change of enthalpy (Δ\u003cem\u003eH\u003c/em\u003e), and the width at half-peak height (Δ\u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) were determined using the DSC curve. The peak denaturation temperature is the temperature at which collagen structure unfolding occurs. The thermal denaturation of the collagen membrane was characterized by its enthalpy (Δ\u003cem\u003eH\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) and denaturation temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e). The change of enthalpy (Δ\u003cem\u003eH\u003c/em\u003e) corresponds to the energy absorbed by the tissue during the helix-coil transformation of the collagen.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAleatory regions were selected for higher magnification and semiquantitative chemical analysis. Figures\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show the SEM photomicrographs. It is possible to observe different gray shades of material bordered by a light clear region.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: The analyzed composite. Photomicrographs detailing the points where the semiquantitative chemical analyses were carried out (2a to 2f). Figure a (50x); Figures b, c (220x) and Figures d, e, f (1000x) magnification.\u003c/p\u003e\u003cp\u003eThe SEM-analyzed images showed sheets and plates of collagen in diverse planes of orientations and highly angular and porous surfaces of the calcium phosphate granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransmission Electron Microscopy (TEM)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTransmission electron microscopy showed the type 1 collagen fibers organically arranged in straight bundles. It was also possible to see that there was no external agent in the type 1 collagen, indicating the purity of the sample. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroanalysis of calcium phosphate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the microanalysis of the sample containing calcium phosphate, it was possible to identify the chemical elements forming the n-HA/β-TCP and type 1 collagen composite. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe chemical percentages of type 1 collagen fibers were recorded, and no impurities were identified. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows images of carbon, nitrogen, and oxygen distribution mapping on the samples' surfaces.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRoughness\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the collagenous type 1 surface morphology image obtained by interferometry during roughness measurements. The surface was homogeneous. The roughness surface parameter is Ra 12.9 \u0026micro;m, Rms 13.1 \u0026micro;m, and Rku 1.1 \u0026micro;m.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe X-ray diffraction (XRD)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure 8 shows the diffraction spectra and peaks of the crystalline phases. The peaks of the diffractograms were identified based on information from the database using the Diffrac software. EVE. V4.22.\u003c/p\u003e\u003cp\u003e(A) (B) \u003c/p\u003e\u003cp\u003eFigure 8: The diffractogram of the sample (A), the peaks characterize only nano-hydroxyapatite. The diffractogram (B) peaks of the phases characterize nano-hydroxyapatite (in red) and nano-\u0026szlig;-TCP (in blue).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWettability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn wettability testing, it was impossible to measure the droplet's angle of incidence due to the surface of the material being extremely hydrophilic, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e. (Supplementary Material).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDSC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe material's thermal energy variation during heating characterized the thermal properties of the sample. The thermal measurement curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e) shows the variation in energy during the heating. The energy variation was identified at temperatures 61.1, 121.4, 128.7, and 133.6 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe morphological and chemical biomaterial analysis indicates that nano-hydroxyapatite/β-Tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite are suitable for further \u003cem\u003ein vivo\u003c/em\u003e testing.\u003c/p\u003e\u003cp\u003eDeveloping new graft biomaterials is an excellent alternative to improve the performance of the regenerative properties for patient treatments. Adding other types of components to hydroxyapatite has been proven to do so. Literature data showed that adding chitosan to nano-hydroxyapatite improved osteogenesis and reduced bacterial adhesion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe collagen molecule contributes to key aspects of bone regeneration, such as cell migration, attachment, migration, cell division, and differentiation. Collagen participates in osteoinduction and osteoconduction in bone healing. In addition, collagen is the main scaffold that sustains mineralization in the human body, notably in the intrafibrillar mineralization pattern [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious work showed that the mixture of hydroxyapatite with collagen had characteristics such as microstructure, absorption kinetics, and mechanical properties suitable as bone substitutes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The results of the present work corroborate those cited in the literature [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the interaction between calcium phosphate and collagen.\u003c/p\u003e\u003cp\u003eLiterature results using rabbits showed that the calcium concentration in the graft material used in surgery can form different types of hydroxyapatites [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the tests carried out in the present work, it was possible to verify that the percentages of calcium and other chemical elements were similar to those of hydroxyapatite. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb shows n-HA granules.\u003c/p\u003e\u003cp\u003eThe identification of material phases is mainly determined by XRD testing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Data from the literature showed that the percentage of nano-hydroxyapatite was similar to that of dentin. The nano-hydroxyapatite induced more collagen cross-links, increased the rate of the organic matrix formation, and promoted better mineralization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe association of nano-hydroxyapatite with collagen was analyzed in tests, and the results were compared with materials containing only hydroxyapatite. The results showed that the structure and performance can be improved by mixing appropriate percentages of collagen and hydroxyapatite [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhen nano-hydroxyapatite is mixed with n-HA, the cell viability, cell integration, and differentiation processes are improved [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, it was demonstrated that n-HA and collagen scaffolds acted synergistically in the osteoconduction process and that bone morphogenetic protein-7 (BMP7) and BMP2 participated in the process [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe results of the DSC test showed that the analyzed sample undergoes endothermic transitions between 25 and 150\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The energy variation identified at a temperature of 61.1\u0026deg;C is related to the glass transition of the amorphous phase of collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The second variation identified near 133\u0026deg;C is related to internal water loss. Results available in the literature show an energy variation that occurs at 230\u0026deg;C, which is associated with the denaturation of the collagen molecule chains. This third energy variation was not identified in the present study; the DSC test was interrupted at 150\u0026deg;C. Denaturation is different from degradation. During denaturation, the hydrogen bonds between the polymer chains are broken.\u003c/p\u003e\u003cp\u003eBased on Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e, it is possible to identify the temperature at which denaturation began (Tonset), the temperature at the end of denaturation (Tendset), and the peak denaturation temperature at maximum heat absorption (Tp). The peak denaturation temperature is the temperature at which the collagen structure unfolds. The denaturation of type I collagen began (Tonset) at 47.6\u0026deg;C, with the transformation peak (Tp) occurring at 61.1\u0026deg;C, and the final denaturation temperature (Tendset) was 87.9\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe denaturation temperature measured in this study differed from those reported in other studies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The differences between the temperatures (Tonset, Tp, and Tendset) are attributed to collagens of different origins and compositions. Natural and synthetic collagens have various types and amino acid contents, as well as sample preparation for analysis, genetic lineage, animal age, fibrillation, mineralization, etc. The denaturation temperature of lyophilized type I collagen differs from that of hydrated collagen. The arrangement of the crosslinks influenced the denaturation temperature.\u003c/p\u003e\u003cp\u003eLiterature results show that collagen thermograms present parameters differences due to collagen type and origin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The literature indicates that the first energy variation occurs between 30 and 150\u0026deg;C, and the endothermic peak (Td') is between 80\u0026deg;C and 104\u0026deg;C. The differences in the attributed properties are associated with the dehydration process and preparation of fibrillar collagen. Some researchers [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] show the DSC thermogram of the collagen sample, and the peak temperatures occur near 61.5 and 221.8\u0026deg;C. Literature results suggest that the peak energy variation near 61.5\u0026deg;C is associated with changing the triple helix structures of collagen molecules to randomly coiled structures [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. During the structure change, intra, and intermolecular hydrogen bonds break by releasing weakly bound water. The stability of the triple helix structures of collagen molecules and the binding of water in the structure of the molecules depend on intra- and intermolecular hydrogen bonds. The literature also suggests that the energy variation near 221.8\u0026deg;C is due to the degradation of the polypeptide chains and the evaporation of residual and/or strongly bound water.\u003c/p\u003e\u003cp\u003eThe results obtained in the present work extended the previous works. Notably, the percentages of nano-hydroxyapatite and collagen influence the biomaterial's performance in bone repair.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on the results obtained in the present work, it can be concluded that:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe material has a homogeneous surface morphology of collagen fibrils as bundles with different thicknesses and directions.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTransmission electron microscopy showed the type 1 collagen fibers organically arranged in straight bundles.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe surface roughness parameters measured using interferometry were Ra 12.9 \u0026micro;m, Rms 13.1 \u0026micro;m, and Rku 1.1 \u0026micro;m.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe material has thermal energy variation during heating at temperatures 61.1, 121.4, 128.7, and 133.6 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe material is highly hydrophilic.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe analyzed nano-biomaterial made with the mixture of nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen does not contain manufacturing processing impurities and is suitable for \u003cem\u003ein vivo\u003c/em\u003e surgery applications.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eIgor da Silva Brum: Conceptualization and co-wrote the manuscript. Carlos Nelson Elias: analysis of results, writing a review, Marco Antonio Alencar de Carvalho,\u0026nbsp;Guilherme Aparecido Monteiro Duque da Fonseca,\u0026nbsp;Bianca Torres Ciambarella: editing and analysis of results, Lucio Frigo co-wrote the manuscript.\u0026nbsp;Jorge José de Carvalho: Analyze the experimental results of the materials. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eThe author confirms that consent has been obtained from the patient(s) for publishing this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors are grateful to the Brazilian Agencies CAPES, CNPq, FAPERJ, and FINEP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate\u003c/strong\u003e: Ethics, Consent to Participate, and Consent to Publish declarations: not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYu L, Wei M. Biomineralization of Collagen-Based Materials for Hard Tissue Repair. Int J Mol Sci. 2021;22(2):944. 10.3390/ijms22020944. PMID: 33477897; PMCID: PMC7833386.\u003c/li\u003e\n\u003cli\u003eLazarevic M, Petrovic S, Pierfelice TV, Ignjatovic N, Piattelli A, Vlajic Tovilovic T, Radunovic M. Antimicrobial and Osteogenic Effects of Collagen Membrane Decorated with Chitosan-Nano-Hydroxyapatite. Biomolecules. 2023;13(4):579. 10.3390/biom13040579. PMID: 37189328; PMCID: PMC10135971.\u003c/li\u003e\n\u003cli\u003eMin KH, Kim DH, Kim KH, Seo JH, Pack SP. Biomimetic Scaffolds of Calcium-Based Materials for Bone Regeneration. Biomimetics (Basel). 2024;9(9):511. 10.3390/biomimetics9090511. PMID: 39329533; PMCID: PMC11430767.\u003c/li\u003e\n\u003cli\u003eCarolina DN, Satari MH, Priosoeryanto BP, Susanto A, Sukotjo C, Kartasasmita RE. 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PMID: 28183611.\u003c/li\u003e\n\u003cli\u003eEnrich-Essvein T, Rodríguez-Navarro AB, Álvarez-Lloret P, Cifuentes-Jiménez C, Bolaños-Carmona MV, González-López S. Proanthocyanidin-functionalized hydroxyapatite nanoparticles as dentin biomodifier. Dent Mater. 2021;37(9):1437–1445. 10.1016/j.dental.2021.07.002. Epub 2021 Aug 3. PMID: 34353622.\u003c/li\u003e\n\u003cli\u003eWang YF, Wang CY, Wan P, Wang SG, Wang XM. Comparison of bone regeneration in alveolar bone of dogs on mineralized collagen grafts with two composition ratios of nano-hydroxyapatite and collagen. Regen Biomater. 2016;3(1):33–40. 10.1093/rb/rbv025. Epub 2015 Dec 31. PMID: 26816654; PMCID: PMC472327.\u003c/li\u003e\n\u003cli\u003eChen ZG, Mo XM, He CL, Wang HS Carbohydr. Polym., Martins AAS, Plepis VCA. A. M. G. J. Therm. Anal. Calorim. 2002, 67, 491.\u003c/li\u003e\n\u003cli\u003eYeh Jen-taut, Chang Haw-jer, Xiao Li-fang, Yang, Li, Zhu P, Huang Guo-xian, Yao W-H. (2010). Physicochemical properties and molecular weight characterization of porcine dermal collagen digested under varying conditions with clostridium histolytic collagenase. e-Polymers October 2010 10(1). 10.1515/epoly.2010.10.1.1226\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"nano-hydroxyapatite, ß-TCP, bone, nano-biomaterial, collagen","lastPublishedDoi":"10.21203/rs.3.rs-7140491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7140491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaterials science has contributed to developing new nano-biomaterials for specific dentistry applications. The present work aims to characterize the physicochemical properties of a composite nanomaterial scaffold in the form of blocks for maxillofacial bone regeneration application. The scaffold had block shapes and was a mixture of nano-hydroxyapatite, β-Tricalcium phosphate, and type I collagen of bovine origin. The biomaterial was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), chemical composition microanalysis, and X-ray diffractometry (XRD). The wettability was measured using a distilled water contact angle with the surface. The phase transformation temperatures were measured using differential scanning calorimetry (DSC). In the SEM and TEM analyses, it was possible to identify the layers of the materials and, with microanalysis, quantify the chemical composition. The XRD spectra showed the presence of nano-hydroxyapatite and nano-\u0026szlig;-TCP. The wettability testing showed that the material is super hydrophilic. The results of the DSC testing showed that the analyzed sample undergoes endothermic transitions and transformation between 25 and 150\u0026deg;C. The results showed that the composite does not contain contamination from manufacturing procedures. It can be concluded that the n-HA/β-TCP and type 1 collagen composite are free of manufacturing contaminants, have higher hydrophilicity, and can be suited for clinical application.\u003c/p\u003e","manuscriptTitle":"An in vitro analysis and physicochemical characterization of a nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) and type 1 collagen composite","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 10:15:11","doi":"10.21203/rs.3.rs-7140491/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3eb85d44-ca86-4303-98cb-cacb1937c570","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T09:23:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 10:15:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7140491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7140491","identity":"rs-7140491","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}