In Vitro Biological Characterization of Titanium Alloys TNTM and TNHF as Potential Biomaterials for Bone Tissue Engineering

In Vitro Biological Characterization of Titanium Alloys TNTM and TNHF as Potential Biomaterials for Bone Tissue Engineering | 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 In Vitro Biological Characterization of Titanium Alloys TNTM and TNHF as Potential Biomaterials for Bone Tissue Engineering Andressa Francine Martins, Rocky Bruno Maschian, Anibal De Andrade Mendes Filho, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8492609/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Annually, worldwide, approximately 2.5 million biomaterials are used in orthopedics, with significant market growth. Orthopedic implants represent the largest share of this sector, accounting for approximately 80% of global use, with a major economic impact also in Brazil. The main metals used in orthopedic implants include titanium alloys. Titanium alloys have been used in the manufacture of medical devices and bone substitutes to improve the quality of life of the world’s population, given population aging and increased life expectancy. This work investigates the in vitro biological interaction related to the characterization of β-metastable titanium alloys TNTM (Ti-29Nb-13Ta-4Mo) and TNHF (Ti-16Nb-10Hf) for application in bone tissues, aiming at their use in medical applications. Biomaterials Bone tissue engineering Cellular adhesion Titanium alloys Figures Figure 1 Figure 2 Figure 3 Introduction and Objective Tissue engineering and regenerative medicine aim to improve life expectancy and quality of life through techniques to restore damaged organs and tissues [ 1 ]. Biomaterials enable the total or partial replacement of compromised structures and are designed to interact with biological systems in a safe and efficient manner [ 2 , 3 ]. These materials must exhibit biocompatibility and absence of toxicity, avoiding undesirable physiological responses [ 3 ]. In bone tissue engineering, biomaterials play a role in the regeneration and stabilization of defects caused by trauma, diseases, and developmental anomalies [ 4 ]. In general, alternatives include bone grafts and the use of natural or synthetic materials, such as ceramics, polymers, and metals [ 5 ]. Thus, advances in the development of these biomaterials expand the therapeutic options available for bone reconstruction and regeneration [ 6 ]. Annually, worldwide, approximately 2.5 million biomaterials are used in orthopedics, with significant market growth, increasing from US $ 25 billion in 2008 to US $ 88 billion in 2017. Orthopedic implants represent the largest share of this sector, accounting for approximately 80% of global use, with a major economic impact also in Brazil [ 3 , 7 ]. Metallic biomaterials stand out for their versatility, being widely employed in bone stabilization and joint replacement [ 8 ]. Their mechanical and physicochemical properties, such as corrosion resistance, fatigue resistance, and load-bearing capacity, justify their clinical application [ 9 ]. However, challenges such as the release of toxic ions, wear, corrosion, and mismatch of elastic modulus may compromise biocompatibility and bone integrity [ 2 , 7 , 9 ]. Currently, the main metals used in orthopedic implants include titanium alloys, such as commercially pure titanium (Ti CP) and Ti-6Al-4V [ 7 ]. Since the 1960s, titanium and its alloys have been used in medical implants due to their high mechanical strength, biocompatibility, low density, and excellent corrosion resistance provided by the passive TiO₂ layer [ 9 , 10 ]. These characteristics favor osseointegration and implant durability, although the effectiveness of the passive layer depends on its thickness and stability [ 10 ]. Ti CP is the most widely used; however, it exhibits low wear resistance, which limits its application in high-load conditions [ 7 , 9 ]. To overcome these limitations, alloys such as Ti-6Al-4V were developed, although they may present potential toxic effects associated with Al and V [ 8 , 10 ]. Therefore, this work investigated the in vitro biological interaction related to the characterization of the titanium alloys TNTM (Ti-29Nb-13Ta-4Mo) and TNHF (Ti-16Nb-10Hf) for application in bone tissues, aiming at their use in biomedical applications. Methodology The titanium samples were previously washed, sterilized by moist heat in an autoclave (121°C for 15 minutes), and handled in a biological safety cabinet, ensuring preservation of the material properties and sterility. Cytotoxicity was evaluated according to ISO 10993-5 using Vero cells, a fibroblast-like cell line widely employed in in vitro biological assays. Cells were cultured in a supplemented HAM-F10 medium, maintained at 37°C and 5% CO₂, and used when cell viability exceeded 90%. Cell viability was confirmed by morphological observation and trypan blue exclusion staining. Direct contact cytotoxicity assays were performed in triplicate, with positive (latex) and negative (ideal culture conditions) controls, allowing evaluation of the direct interaction between cells and titanium samples after 24 hours of incubation. In addition, samples were prepared for SEM analysis to observe cell adhesion and ultrastructure, being fixed in 2.5% glutaraldehyde and stored between 2 and 4°C. After washing with distilled water, samples were dehydrated in a graded ethanol series (50–100%), followed by critical point drying using liquid CO₂ to preserve structural integrity. Finally, a gold coating was applied by sputtering for SEM image acquisition. Results and discussion The initial assessment of cell viability indicated approximately 94% viable Vero cells, exhibiting morphology characteristic of the cell line, as shown in Fig. 1 . Fonte: autores Cytotoxicity assays are fundamental for the initial biological evaluation of biomaterials, as they allow the assessment of potential damage to cell viability and their safety for clinical applications [ 11 ]. Direct contact cytotoxicity evaluated the interaction of Vero cells with the TNHF and TNTM titanium alloys. Figure 2 shows the morphological pattern of cells in the negative and positive controls, with the negative control exhibiting viable, adherent cells with fibroblast-like morphology [ 11 , 12 ]. The positive control showed the expected cytotoxic response, with evident morphological alterations [ 13 , 14 ]. Also in Fig. 2 , cells in contact with the TNHF and TNTM alloys maintained the characteristic morphology of the Vero cell line, with no signs of cytotoxicity. The cells exhibited an elongated, mononuclear shape and adhesion to the substrate, similar to the negative control. These results indicate good cell–material interaction with the evaluated alloys. Therefore, the obtained data demonstrate the potential of these titanium alloys for use as biomaterials in biomedical applications. Fonte: autors Cell interaction with the TNHF and TNTM samples was analyzed by SEM, revealing cell adhesion on the surfaces, as shown in Fig. 3 . The TNHF sample exhibited good cell spreading, with flat, well-spread cells, centralized nuclei, and the presence of focal adhesion points, indicating a surface favorable to cell adhesion (Fig. 3 A) [ 15 ]. In contrast, the TNTM alloy displayed a distinct surface with a differentiated pattern of cell interaction (Fig. 3 B). Limited cell spreading and more rounded cells were observed, some indicating a division process. This behavior suggests a lower or slower cell–surface interaction compared to TNHF [ 16 ]. Fonte: autors Finals Considerations and Future Perspectives This study aimed to perform the in vitro biological characterization of titanium alloys using Vero cell cultures, targeting their application as biomaterials for the treatment of bone tissue injuries. In vitro assays are essential tools for the initial assessment of safety and cell–material interactions, allowing the prediction of biomaterial behavior in simulated environments. The TNTM and TNHF alloys showed good cellular responses, with no evidence of cytotoxic effects, indicating good biocompatibility. The evaluation of cell adhesion enabled the analysis of cell interactions with the biomaterial surfaces. It was observed that the TNHF alloy promoted greater cell spreading, whereas the TNTM alloy exhibited intermediate adhesion, associated with its surface characteristics. Overall, both alloys demonstrated satisfactory cell–material interactions. These results indicate the potential of TNTM and TNHF alloys for applications in bone tissue engineering and regenerative medicine. Declarations The authors declare that they have no personal, commercial, academic, political, financial, or any other type of conflict of interest regarding the evaluation and publication of this article. Author Contribution Todos os autores auxiliaram no trabalho Acknowledgments The authors would like to thank the Federal University of ABC (UFABC) for the institutional support and infrastructure provided for the development of this work. Special thanks are given to the Tissue Engineering and Biomolecules Laboratory and to the UFABC Multiuser Facilities for the technical and scientific support, equipment availability, and assistance during the experimental procedures and the analisis. Finally, the authors acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES) for the financial support, which was essential for the execution of this research. References Rodrigues LB. Aplicações de biomateriais em ortopedia. Universidade Estadual do Sudoeste da Bahia - Estudos Tecnológicos em Engenharia, v.9, n.2, pp. 63–76, 2013. Moreno MSMS. Engenharia de tecidos na substituição de tecido ósseo. Universidade Fernando Pessoa - Faculdade de Ciências da Saúde. Dissertação. Porto; 2014. Pires ALR, Bierhalz ACK, Moraes ÂM, Biomateriais. Tipos, aplicações e mercado. Química Nova, v. 38, n. 7, pp. 957–971, ago. 2015. Meijer GJ, Bruijn JD, Koole R, van Blitterswijk CA. Cell-Based bone tissue engineering. PLoS Med v. 2007;4(2):e9. Silva SBT, Andrade AF, Figueiredo BQ, Freitas FG, Barcelos LB, Peres MLA, Silvano RCND, Soares RS. Surgical reconstruction using biomaterials: an integrative literature review. Volume 10. Research, Society and Development; 2021. Pedroso R. Uso de biomateriais na regeneração e engenharia de tecido ósseo: uma revisão de literatura. Florianópolis: Centro de Ciências da Saúde - Universidade Federal de Santa Catarina; 2022. Festas A, Ramos A, Davim J. Medical devices biomaterials – A review. Proceedings of the Institution of Mechanical Engineers, Part L: Journal of Materials: Design and Applications. v. 234, n.1, pp. 218–228, 2020. Silva VV. Aplicação de biomateriais em ortopedia e engenharia de tecido ósseo. Revista Saúde e Meio Ambiente – RESMA, v. 5, n.2, pp. 14–27, 2017. Pilliar RM. Metallic Biomaterials. Biomedical materials, v. Volume 2. Cham: Springer; 2021. pp. 1–47. Girón J, Kerstner E, Medeiros T, Oliveira L, Machado GM, Malfatti CF, Pranke P. Biomaterials for bone regeneration: an orthopedic and dentistry overview. Brazilian J Med Biol Res v 54, n.9, 2021. Côrrea DOG. Citotoxicidade de novas ligas a base de titânio visando aplicações biomédicas. Bauru: Universidade Estadual Paulista. Faculdade de Ciências; 2022. Duarte CRA. Avaliação da citotoxicidade in vitro de composições de fosfato de cálcio para uso de recuperação óssea. Universidade do Estado de Santa Catarina, Centro de Ciências Agroveterinárias. Lages: Programa de Pós-Graduação em Ciência Animal; 2015. Ambrosio FN. Caracterização de filmes de quitosana e poli (álcool vinílico) para engenharia de tecidos. Universidade Federal do ABC, Programa de Pós-Graduação em Engenharia Biomédica. São Bernardo do Campo - SP; 2018. Masson AO, Lombello CB. Ensaios de citotoxicidade in vitro: comparação dos métodos de contato direto e por extrato. 25 Congresso Brasileiro de Engenharia Biomédica. Foz do Iguaçu, PR); 2016. Niinomi M, Liu Y, Masaki N, Liu H, Li H. Biomedical titanium alloys with Young’s moduli close to that of cortical bone. Regenerative Biomaterials, V. 3, n.3, pp. 173–85, 2016. Marin E, Lanzutti A. Biomedical Applications of Titanium Alloys: A Comprehensive Review. Materials, n.17, v. 1, p.114, 2023. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 19 Feb, 2026 Reviews received at journal 14 Feb, 2026 Reviews received at journal 11 Feb, 2026 Reviews received at journal 10 Feb, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers invited by journal 22 Jan, 2026 Editor assigned by journal 04 Jan, 2026 Submission checks completed at journal 04 Jan, 2026 First submitted to journal 31 Dec, 2025 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-8492609","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":579971617,"identity":"8e45ad25-493c-4ae0-9185-caf29d0a64ba","order_by":0,"name":"Andressa Francine Martins","email":"data:image/png;base64,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","orcid":"","institution":"Universidade Federal do ABC","correspondingAuthor":true,"prefix":"","firstName":"Andressa","middleName":"Francine","lastName":"Martins","suffix":""},{"id":579971618,"identity":"46307de2-7f0f-45bf-a071-a91d4c848ed6","order_by":1,"name":"Rocky Bruno Maschian","email":"","orcid":"","institution":"Universidade Federal do ABC","correspondingAuthor":false,"prefix":"","firstName":"Rocky","middleName":"Bruno","lastName":"Maschian","suffix":""},{"id":579971619,"identity":"abed9ce8-793d-48ea-aa32-507b47979840","order_by":2,"name":"Anibal De Andrade Mendes Filho","email":"","orcid":"","institution":"Universidade Federal do ABC","correspondingAuthor":false,"prefix":"","firstName":"Anibal","middleName":"De Andrade Mendes","lastName":"Filho","suffix":""},{"id":579971620,"identity":"0704c1e1-d568-42f2-a28e-a0786c459aca","order_by":3,"name":"Christiane Bertachini Lombello","email":"","orcid":"","institution":"Universidade Federal do ABC","correspondingAuthor":false,"prefix":"","firstName":"Christiane","middleName":"Bertachini","lastName":"Lombello","suffix":""}],"badges":[],"createdAt":"2026-01-01 00:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8492609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8492609/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101286624,"identity":"26176246-bdc7-4022-bbfe-e5e268aafad2","added_by":"auto","created_at":"2026-01-28 06:50:03","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative micrograph of the standard morphology of Vero cells. Cells cultured for inoculation in cytotoxicity experiments exhibiting the characteristic morphological pattern of the cell line, with elongated or polygonal cells forming a monolayer. Magnification: 400x.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFonte: autores\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8492609/v1/5ae2289743177642eb442bc0.jpeg"},{"id":101286625,"identity":"ddce6a98-7e09-43f9-90eb-d692c5f22627","added_by":"auto","created_at":"2026-01-28 06:50:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":737393,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative micrographs of Vero cell morphology after 24 hours of interaction with the controls and titanium alloy samples, obtained by phase-contrast light microscopy. [A] Negative control, [B] Positive control, [C] TNHF, and [D] TNTM. Magnification: 400×.\u003c/p\u003e\n\u003cp\u003eFonte: autores\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8492609/v1/4db2917a19d44a09268dcf1c.jpeg"},{"id":101286626,"identity":"47681726-48d9-46c9-88df-c0cd696598b5","added_by":"auto","created_at":"2026-01-28 06:50:03","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":981555,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of Vero cell adhesion on the surface of titanium alloy samples: [A] TNHF and [B] TNTM. Scanning Electron Microscopy.\u003c/p\u003e\n\u003cp\u003eFonte: autores\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8492609/v1/ba2e0d29f2adaffa8c3b2cfd.jpeg"},{"id":101286627,"identity":"a162897f-45a6-4912-bca4-466690a6f9dd","added_by":"auto","created_at":"2026-01-28 06:50:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2328449,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8492609/v1/7fad5dff-d6d4-4f1c-9687-9bb9ec84bd11.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Vitro Biological Characterization of Titanium Alloys TNTM and TNHF as Potential Biomaterials for Bone Tissue Engineering","fulltext":[{"header":"Introduction and Objective","content":"\u003cp\u003eTissue engineering and regenerative medicine aim to improve life expectancy and quality of life through techniques to restore damaged organs and tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Biomaterials enable the total or partial replacement of compromised structures and are designed to interact with biological systems in a safe and efficient manner [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These materials must exhibit biocompatibility and absence of toxicity, avoiding undesirable physiological responses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In bone tissue engineering, biomaterials play a role in the regeneration and stabilization of defects caused by trauma, diseases, and developmental anomalies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In general, alternatives include bone grafts and the use of natural or synthetic materials, such as ceramics, polymers, and metals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, advances in the development of these biomaterials expand the therapeutic options available for bone reconstruction and regeneration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Annually, worldwide, approximately 2.5\u0026nbsp;million biomaterials are used in orthopedics, with significant market growth, increasing from US\u003cspan\u003e$\u003c/span\u003e25\u0026nbsp;billion in 2008 to US\u003cspan\u003e$\u003c/span\u003e88\u0026nbsp;billion in 2017. Orthopedic implants represent the largest share of this sector, accounting for approximately 80% of global use, with a major economic impact also in Brazil [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Metallic biomaterials stand out for their versatility, being widely employed in bone stabilization and joint replacement [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Their mechanical and physicochemical properties, such as corrosion resistance, fatigue resistance, and load-bearing capacity, justify their clinical application [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, challenges such as the release of toxic ions, wear, corrosion, and mismatch of elastic modulus may compromise biocompatibility and bone integrity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Currently, the main metals used in orthopedic implants include titanium alloys, such as commercially pure titanium (Ti CP) and Ti-6Al-4V [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since the 1960s, titanium and its alloys have been used in medical implants due to their high mechanical strength, biocompatibility, low density, and excellent corrosion resistance provided by the passive TiO₂ layer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These characteristics favor osseointegration and implant durability, although the effectiveness of the passive layer depends on its thickness and stability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Ti CP is the most widely used; however, it exhibits low wear resistance, which limits its application in high-load conditions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To overcome these limitations, alloys such as Ti-6Al-4V were developed, although they may present potential toxic effects associated with Al and V [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, this work investigated the \u003cem\u003ein vitro\u003c/em\u003e biological interaction related to the characterization of the titanium alloys TNTM (Ti-29Nb-13Ta-4Mo) and TNHF (Ti-16Nb-10Hf) for application in bone tissues, aiming at their use in biomedical applications.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003eThe titanium samples were previously washed, sterilized by moist heat in an autoclave (121\u0026deg;C for 15 minutes), and handled in a biological safety cabinet, ensuring preservation of the material properties and sterility. Cytotoxicity was evaluated according to ISO 10993-5 using Vero cells, a fibroblast-like cell line widely employed in in vitro biological assays. Cells were cultured in a supplemented HAM-F10 medium, maintained at 37\u0026deg;C and 5% CO₂, and used when cell viability exceeded 90%. Cell viability was confirmed by morphological observation and trypan blue exclusion staining. Direct contact cytotoxicity assays were performed in triplicate, with positive (latex) and negative (ideal culture conditions) controls, allowing evaluation of the direct interaction between cells and titanium samples after 24 hours of incubation. In addition, samples were prepared for SEM analysis to observe cell adhesion and ultrastructure, being fixed in 2.5% glutaraldehyde and stored between 2 and 4\u0026deg;C. After washing with distilled water, samples were dehydrated in a graded ethanol series (50\u0026ndash;100%), followed by critical point drying using liquid CO₂ to preserve structural integrity. Finally, a gold coating was applied by sputtering for SEM image acquisition.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThe initial assessment of cell viability indicated approximately 94% viable Vero cells, exhibiting morphology characteristic of the cell line, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFonte: autores\u003c/p\u003e \u003cp\u003eCytotoxicity assays are fundamental for the initial biological evaluation of biomaterials, as they allow the assessment of potential damage to cell viability and their safety for clinical applications [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Direct contact cytotoxicity evaluated the interaction of Vero cells with the TNHF and TNTM titanium alloys. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the morphological pattern of cells in the negative and positive controls, with the negative control exhibiting viable, adherent cells with fibroblast-like morphology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The positive control showed the expected cytotoxic response, with evident morphological alterations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Also in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, cells in contact with the TNHF and TNTM alloys maintained the characteristic morphology of the Vero cell line, with no signs of cytotoxicity. The cells exhibited an elongated, mononuclear shape and adhesion to the substrate, similar to the negative control. These results indicate good cell\u0026ndash;material interaction with the evaluated alloys. Therefore, the obtained data demonstrate the potential of these titanium alloys for use as biomaterials in biomedical applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFonte: autors\u003c/p\u003e \u003cp\u003eCell interaction with the TNHF and TNTM samples was analyzed by SEM, revealing cell adhesion on the surfaces, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The TNHF sample exhibited good cell spreading, with flat, well-spread cells, centralized nuclei, and the presence of focal adhesion points, indicating a surface favorable to cell adhesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, the TNTM alloy displayed a distinct surface with a differentiated pattern of cell interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Limited cell spreading and more rounded cells were observed, some indicating a division process. This behavior suggests a lower or slower cell\u0026ndash;surface interaction compared to TNHF [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFonte: autors\u003c/p\u003e\n\u003ch3\u003eFinals Considerations and Future Perspectives\u003c/h3\u003e\n\u003cp\u003eThis study aimed to perform the in vitro biological characterization of titanium alloys using Vero cell cultures, targeting their application as biomaterials for the treatment of bone tissue injuries. In vitro assays are essential tools for the initial assessment of safety and cell\u0026ndash;material interactions, allowing the prediction of biomaterial behavior in simulated environments. The TNTM and TNHF alloys showed good cellular responses, with no evidence of cytotoxic effects, indicating good biocompatibility. The evaluation of cell adhesion enabled the analysis of cell interactions with the biomaterial surfaces. It was observed that the TNHF alloy promoted greater cell spreading, whereas the TNTM alloy exhibited intermediate adhesion, associated with its surface characteristics. Overall, both alloys demonstrated satisfactory cell\u0026ndash;material interactions. These results indicate the potential of TNTM and TNHF alloys for applications in bone tissue engineering and regenerative medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no personal, commercial, academic, political, financial, or any other type of conflict of interest regarding the evaluation and publication of this article.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTodos os autores auxiliaram no trabalho\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors would like to thank the Federal University of ABC (UFABC) for the institutional support and infrastructure provided for the development of this work. Special thanks are given to the Tissue Engineering and Biomolecules Laboratory and to the UFABC Multiuser Facilities for the technical and scientific support, equipment availability, and assistance during the experimental procedures and the analisis. Finally, the authors acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES) for the financial support, which was essential for the execution of this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRodrigues LB. Aplica\u0026ccedil;\u0026otilde;es de biomateriais em ortopedia. Universidade Estadual do Sudoeste da Bahia - Estudos Tecnol\u0026oacute;gicos em Engenharia, v.9, n.2, pp. 63\u0026ndash;76, 2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreno MSMS. Engenharia de tecidos na substitui\u0026ccedil;\u0026atilde;o de tecido \u0026oacute;sseo. Universidade Fernando Pessoa - Faculdade de Ci\u0026ecirc;ncias da Sa\u0026uacute;de. 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Regenerative Biomaterials, V. 3, n.3, pp. 173\u0026ndash;85, 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarin E, Lanzutti A. Biomedical Applications of Titanium Alloys: A Comprehensive Review. Materials, n.17, v. 1, p.114, 2023.\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"in-vitro-models","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [In vitro models](https://link.springer.com/journal/44164)","snPcode":"44164","submissionUrl":"https://submission.springernature.com/new-submission/44164/3","title":"In vitro models","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biomaterials, Bone tissue engineering, Cellular adhesion, Titanium alloys","lastPublishedDoi":"10.21203/rs.3.rs-8492609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8492609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnnually, worldwide, approximately 2.5\u0026nbsp;million biomaterials are used in orthopedics, with significant market growth. Orthopedic implants represent the largest share of this sector, accounting for approximately 80% of global use, with a major economic impact also in Brazil. The main metals used in orthopedic implants include titanium alloys. Titanium alloys have been used in the manufacture of medical devices and bone substitutes to improve the quality of life of the world\u0026rsquo;s population, given population aging and increased life expectancy. This work investigates the in vitro biological interaction related to the characterization of β-metastable titanium alloys TNTM (Ti-29Nb-13Ta-4Mo) and TNHF (Ti-16Nb-10Hf) for application in bone tissues, aiming at their use in medical applications.\u003c/p\u003e","manuscriptTitle":"In Vitro Biological Characterization of Titanium Alloys TNTM and TNHF as Potential Biomaterials for Bone Tissue Engineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 06:49:58","doi":"10.21203/rs.3.rs-8492609/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-19T14:02:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-14T08:17:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T14:35:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T07:57:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299477861490096104407452860240468590115","date":"2026-02-09T08:45:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165182461744848204192526596290816264904","date":"2026-02-06T04:27:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27576754794363308198811382929239553449","date":"2026-02-05T13:23:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246732054968628248722481712979848208606","date":"2026-02-03T14:12:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-23T00:35:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T04:45:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T03:27:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"In vitro models","date":"2026-01-01T00:26:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"in-vitro-models","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [In vitro models](https://link.springer.com/journal/44164)","snPcode":"44164","submissionUrl":"https://submission.springernature.com/new-submission/44164/3","title":"In vitro models","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"146ceb85-e2b7-4e69-98fe-bd48651347cb","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T14:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 06:49:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8492609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8492609","identity":"rs-8492609","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}