Biocompatibility and bio-corrosion behavior of medical grade titanium alloy developed through the direct energy deposition (DED) method

Biocompatibility and bio-corrosion behavior of medical grade titanium alloy developed through the direct energy deposition (DED) method | 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 Biocompatibility and bio-corrosion behavior of medical grade titanium alloy developed through the direct energy deposition (DED) method Soumyalata D, Rajesh Shanmugavel, Adam Khan M, Anish Nair, Andrej Jeromen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8659275/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 Additive manufacturing of titanium alloys has gained significant attention for biomedical applications due to its ability to fabricate complex geometries with controlled microstructures. In this study, a titanium alloy was fabricated using the direct energy deposition (DED) technique and evaluated in the as-built and double-aged conditionsfor biomedical suitability. Post-processing involved a two-stage aging treatment at 720°C and 620°Cto enhance microstructural stability and mechanical performance. Optical microscopy revealed a fine acicular lath structure in the as-built alloy resulting from rapid solidification, while the heat-treated alloy exhibited a coarser and thermodynamically stable α + βmicrostructure. The as-built DED Ti alloy showed a hardness of 360 Hv and density of 4.29 g/cc, which increased to 371 Hv and 4.49 g/cc after heat treatment due to martensite decomposition, secondary α precipitation, and porosity reduction. Cytocompatibility assessed using L929 fibroblast cellsfollowing ISO 10993-5 (MTT assay)confirmed cell viability above 70% for 72 hfor both conditions, with the as-built alloy exhibiting comparatively higher viability. Electrochemical corrosion studies conducted in simulated body fluid (SBF) demonstrated very low corrosion current densities (~10⁻³ mA/cm²) and corrosion rates (~0.009 mm/year), indicating excellent corrosion resistance. Notably, the as-built alloy exhibited superior passivation behavior due to the formation of a stable TiO₂-rich oxide layer. These results confirm the suitability of DED-fabricated titanium alloys for biomedical implant applications. titanium additive manufacturing cytotoxicity corrosion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The use of metals in biomedical engineering has evolved significantly over the past two decades. Metallic materials are widely preferred due to their excellent mechanical strength, which effectively supports biomedical applications [1]. Among these materials, titanium alloys are especially favoured for orthopaedic applications, as they are subjected to heavy cyclic loads and complex mechanical articulation under various physiological conditions [2,3]. However, in certain critical clinical populations such as elderly patients, individuals with diabetes, and those with metabolic disorders medical metal implants exhibit significantly higher failure rates, thereby increasing the likelihood of revision surgeries [4]. One of the primary contributing factors to this behavior is the altered biochemistry of body fluids in these patients. Consequently, titanium alloys have been strongly promoted by researchers and scientists for biomedical engineering applications due to their outstanding metallurgical and biocompatible properties [5]. One of the major challenges in the utilization of titanium alloys lies in their production and processing routes. For several decades, researchers have faced significant difficulties in achieving directionally solidified and single crystal titanium alloys. El Khalloufi (2021) reported the key challenges associated with the processing and commercialization of titanium-based alloys, highlighting limitations related to cost, scalability, and metallurgical control [6]. Among these challenges, the processing of titanium metal powders remains a critical issue. Powder based routes are commonly employed for the fabrication of titanium alloys due to their flexibility and near-net-shape capability [7]. Despite the availability of multiple processing techniques, the development of low-cost titanium alloys has played a vital role in expanding their industrial and biomedical applications [8]. However, a major constraint has been maintaining the metallurgical quality of the alloy without compromising its mechanical and functional properties [9]. Subsequently, the incorporation of nickel into titanium led to the development of nickel–titanium alloys, which gained significant attention as shape memory alloys due to their unique functional behavior. In recent years the titanium alloy with advanced manufacturing process such as laser base powder sintering took role to meet the industrial demand [10]. It is also coined as laser additive manufacturing process to build the complex and bottle neck shapes. There are different types of additive manufacturing process in current scenario to print metal parts. Especially the direct metal laser sintering (DMLS) process is most prominent and common process available to develop the additive-built titanium alloy and other superalloys [11]. The major constraint in the additive-built production process is delta – phase formation. Research on heat treatment and its biocompatibility of additive built titanium alloys are in tropical research to meet the readers. Important factors such as biocompatibility and its viability for the implementation of such novel process plays a vital role in advanced production process [12, 13]. The other techniques for additive manufacturing of titanium alloy open for research. The development of alloy through energy deposition and post processing are in demand for future implementation. In this research the novel technique such as direct energy deposition (DED) is proposed to print the titanium alloy for biomedical application. The alloy developed through the DED process will be post process to enhance the metallurgical quality and mechanical properties. Following this process, the material may be involved to investigate the bio compatibility via cytotoxicity and corrosion studies. From this analysis this research outcome will encourage the budding engineers to learn and feed with new data for bio medical applications. Materials and Method The titanium (Ti) alloy developed through the direct energy deposition (DED) is used to investigate the cytotoxicity and bio – compatibility for the further applications. It is one of the advanced and feasible method to develop metal component. Table 1 shows the optimal process parameter design for industrial standard to develop the titanium alloy through direct energy deposition method. The commercial titanium metal powder of particle size 30–160 µm with a medical standard is used for additive manufacturing. Figure 1 shows the schematic illustration and actual titanium alloy developed from the industrial standard metal powder. The spectroscopic analysis of the DED Ti alloy has Ti – 88.2%; Al – 6.85%, V – 4.6%, C – 0.008 and traces of iron. All the DED samples are in need of secondary process to compete the original product in terms of machining and post processing. Table 1 The standard process parameter used to develop the titanium alloy through direct energy deposition method Process Parameters Process condition Units Speed of Scan 5 mm/s Power 1250 Watts Volume rate 15 mm 3 /s Diameter of beam 3 mm As a base work the metal samples developed through the DED process is sliced using wire electrical discharge machining process. The dimensions factors of the test sample vary with respect the individual standards of experimental requirements. The test samples of DED Ti alloy are post processed to compare the metallurgical phase transformation, hardness and the density of the material. The post processing of the material was done with double aging process following the standard heat treatment plan. Figure 2 shows the heat treatment plan proposed for the DED Ti alloy. A tubular furnace which is designed to a maximum of 1400°C is used for the heat treatment plan. The samples are place inside the furnace with the help of 99.9% pure alumina boat and the cooling process are made through furnace cooling as the standards process conditions given in the plan. After heat treatment the samples are metallurgically polished to study the microstructural transformation developed due to the double ageing process. An optical microscope (Make: Vertimet) is used to reveal the microstructure of the material before and after post processing. From the optical image the inference on phase transformation will be inferred for discussion. Following, micro hardness and density values are calculated to discuss about the material quality for future analysis. As a core work, the samples are sliced to a dimension of 3mm x 3mm x 3mm for cell viability and cytotoxicity analysis. After slicing the samples are polished and cleaned with ultrasonicator. After metal cleaning process, the samples are sterilised for fibroblast cell culture analysis. The L929 fibroblast cell is the common cell used to study the cytotoxicity analysis following the standard MTT Assay protocol (ISO 10993). On the other way, to substantiate the materials quality the metal bio – compatibility analysis was done with simulated body fluid (SBF) using potentiostat (electrochemical workstation). The samples are maintained to a dimension of 10mm x 10mm x 2mm for electrochemical corrosion studies. Similar to the cell cytotoxicity analysis the sample preparation procedures are followed. In house lab reagents are used to prepare the simulated body fluid following the authentic procedure of Kokubo SBF medium for bio corrosion analysis [14]. The saturated calomel electrode is a reference electrode and the platinum is used as a standard electrode. The potential (± 250 mV) and sweep rate 1 mV/s is the common factor defined for electrochemical corrosion studies. ASTM standards for the electrochemical studies (ASTM F2129) is followed for the investigation. From the analysis, the outcomes are technical justified to recommend the experimentation to future work. Results and discussion The titanium alloy developed through the direct energy deposition (DED Ti alloy) method is evaluated to report on the microstructural transformation of the material. The structural morphology of the as – built and heat – treated DED Ti alloy observed through the microscope is as given in the Fig. 3 . For the as – built DED Ti alloy, the dense fine lath structure in a closed network is noticed due to the rapid cooling of metal pool. It is an effect of thermal gradient suppressed the metal pool to solidify at an average rate of 10 3 – 10 5 °C/s during the deposition. However, in the heat-treated DED Ti alloy the micro structures are coarse and more stable with clear grain boundary. Literature supports that the high cooling rate will suppress the β ◊ α transformation with high strain energy in the grain at 720°C. On second stage of ageing (620°C) secondary alpha phase precipitation nucleated within the beta phase (as matrix). Does the combination of alpha – beta phase has increased the density of the grains in the as – built DED Ti alloy. At 720°C the material decomposes the α′ → α + β, relieve residual stresses, stabilize β; and 620°C is the precipitate fine secondary α phase, refining the microstructure and also strengthen alloy. It is very important to note that the as – built DED Ti alloy has 360 Hv and 4.29 g/cc which is less than the heat treated DED Ti alloy which has 371 Hv and 4.49 g/cc. The higher hardness and density of the heat-treated DED Ti alloy compared to the as-built condition are attributed to diffusion-driven decomposition of metastable α′ martensite into equilibrium α + β phases, fine secondary α precipitation, and the reduction of process-induced porosity and lattice defects during double aging, resulting in a denser and mechanically more stable microstructure. MTT Assay test and evaluation for DED Ti alloy. The preparation of conditioned media was done following the ethylene oxide (EtO) sterilization procedure. The metal alloys were incubated in complete culture medium (DMEM high glucose supplemented with 10% fetal bovine serum and 1% antibiotic antimycotic solution) at an ISO-recommended extraction ratio of 0.2 g/mL. The incubation was carried out for 24, 48, and 72 hours to obtain time dependent conditioned media. In continuation to the control medium preparation, the cytotoxicity of the metals was assessed following the MTT assay procedure using L929 fibroblast cell line. The cells were seeded at a density of 1 × 10⁴ cells per well in 100 µL of complete medium in 96-well plates and allowed to attach for 24 hours. The cells were then treated with conditioned media obtained from the metal alloys at different time points. Control wells contained cells cultured in fresh complete medium. Figure 4 shows the optical image of the control samples of fibroblast cells. Each condition, including controls, was tested in five replicates to find average results and also for the results interpretations. After 24 hours of treatment, 5 mg/mL MTT solution, prepared in phosphate-buffered saline, was added to each well. The plates were gently shaken and incubated for 4 hours at 37°C in a humidified incubator with 5% CO₂. Subsequently, 100 µL of 10% SDS solution was added to each well to solubilize the formazan crystals, followed by overnight incubation. Absorbance was measured using a microplate reader (TECAN) at 570 nm with a reference wavelength of 630 nm. The evaluation procedure for the cell growth in proximity to metal alloys were performed to assess the ability of L929 cells to grow in the presence of metal alloys, the alloys were placed in 24-well culture plates, and L929 cells were seeded at a density of 1 × 10⁵ cells per well in complete medium. The cultures were incubated for 48 hours under standard conditions. After incubation, cell growth and morphology in the vicinity of the metal alloys were observed and documented using a Zeiss optical microscope. The phase contrast (microscopic) images of L929 fibroblast cells cultured on the 3D-printed material show good cell adhesion, typical spindle shaped morphology, and uniform spreading across the surface (Fig. 5 ). The cells appear viable, with no obvious signs of cytotoxicity such as rounding, detachment, or membrane damage in the as – built DED Ti alloy. The formation of interconnected cell networks and sustained cell density over time indicates that the as – built DED Ti alloy provides a favourable surface for fibroblast attachment and proliferation compared to the heat – treated DED Ti alloy. However, the confirmation of the cell count and its rate of behaviour in quantitative mode are further investigated. The quantitative data arrived from the cell viability results as shown in Figure, demonstrate that the DED Ti alloy maintains the acceptable cytocompatibility with the embed of L929 fibroblasts over 72 hours. Both as-built and heat-treated (HT) samples exhibit cell viability comparable to the control at 24 hours, indicating minimal acute cytotoxic effects and negligible release of harmful leachables during early exposure in accordance with ISO 10993-5 criteria (> 70% viability). A time-dependent reduction in viability is observed at 48 and 72 hours, more pronounced in the HT condition; however, viability values remain above the cytotoxic threshold, confirming that post-processing does not induce severe biological incompatibility (Fig. 6 ). The comparatively higher viability of the as-built samples suggests that heat treatment may alter surface chemistry or residual stress states, influencing protein adsorption and cell–material interactions. Overall, the results confirm that the 3D-printed material supports fibroblast survival over extended culture periods and is suitable for further evaluation through other bio chemical environment. Bio – corrosion with simulated body fluid (SBF) The bio corrosion studies on DED Ti alloy; as – built and heat – treated are exposed to simulated body fluid using electrochemical polarisation work station. The DED Ti alloy, before and after post processing are used to study about the bio – compatibility using simulated body fluid as given in the Table 2 . From the data it is clear that the both as-built and heat-treated DED Ti alloys exhibit very low corrosion current densities (~ 10⁻³ mA/cm²) and extremely low corrosion rates (~ 0.009 mm/year) under the SBF environment, confirming the inherently high corrosion resistance of titanium alloys in physiological environments. It is an indication of surface protection and passive layer formation (TiO x in this case) and suppressing the metal dissolution with respect to the alloying elements. There is a shift in corrosion potential for heat treated sample with reduction in corrosion current density. This implies that the corrosion process is predominantly controlled by passive film properties rather than bulk microstructural changes. However, for the as – built slightly negative trend in corrosion potential indicates positive sign highlighting its ability to rapidly form and regenerate a protective oxide layer despite higher initial electrochemical activity. From a biomedical perspective, this behavior is particularly significant, as it suggests that the as-built DED Ti alloy provides corrosion protection comparable to the heat-treated condition, while potentially minimizing ion release and associated cytotoxicity. Table 2 Data extracted from the TAFEL plot for DED Samples at two different cases exposed to simulated body fluid. DED Ti alloy Corrosion potential E corr (mV) Current density I corr (mA/cm 2 ) Corrosion rate CR (mm/year) As – built -120.35 0.001031 0.006125 Heat – treated -100.73 0.001986 0.009626 Figure 7 illustrates the electrochemical behaviour of the both as built and heat-treated DED Ti alloy metal samples exposed to simulated body fluid. The as – built DED Ti alloy reveals with asymmetry potential and current density curve under the simulated body fluid condition. The titanium alloys are stable to produce thick film over the adjunct surface and forms a passive layer to protect the material from further corrosion. Even though the presence of chloride ions in the simulated body fluid will induce the passive film to get break at the earliest. However, the as – built DED Ti alloy has sharp and perfect profile from the TAFEL plot to inform that the material is very much stable to protect the surface from aggressive body fluid environment (Fig. 7 a). This polarization behavior of the as-built titanium alloy in simulated body fluid exhibits a reversal characteristic of strong passivation. This response is attributed to the formation and rapid regeneration of a protective TiO₂ passive layer, influenced by the inherent surface roughness and microstructural heterogeneity of the as-built condition (Fig. 7 c). However, for the heat-treated samples, results from the electrochemical polarisation studies are little diverse from the analysis. It has been noticed that the reversal of anodic reaction observed in the polarization curve (Fig. 7 b). Which is attributed to the inherent electrochemical characteristics of the alloy elements; particularly the formation, breakdown, and depassivation of a surface passive film by the activation of alloy elements during the electrochemical reactions with simulated body fluid. The break of film and getting depassivation is due to the instability of the material exposed under the working medium. Technically speaking, the alteration of oxides charge transfer kinetics (for Ti and Al) at the interface induces such vulnerable reactions. It actually happens at the higher potential and the rate of conductivity simultaneously increased with ions fluctuations. At lower potentials, active electrochemical reactions were observed under relatively stabilized environmental conditions. Previous studies on commercial titanium implant materials have reported that the evolution of a TiOₓ passive layer during electrochemical exposure effectively protects the material from catastrophic or continuous corrosion (15). In the present study, the as-built DED titanium alloy demonstrated superior corrosion protection and reduced cell cytotoxicity compared to the heat-treated DED titanium alloy. Although the heat-treated DED titanium alloy exhibited a slightly higher overall oxide content, this was accompanied by a measurable reduction in aluminum and vanadium concentrations at the surface. This observation suggests preferential dissolution or outward diffusion of Al and V from the alloy matrix during electrochemical exposure, likely induced by thermal treatment–related microstructural changes. In contrast, the as-built DED titanium alloy retained a more stable surface chemistry, promoting the formation of a protective TiO₂-rich passive layer and thereby enhancing corrosion resistance and biocompatibility relative to the heat-treated condition. Conclusions The study confirms the feasibility of fabricating biomedical grade titanium alloys using the direct energy deposition (DED) process. From the investigations and detailed analysis, following points were derived as an conclusion. The as-built DED Ti alloy exhibited a fine acicular lath microstructure due to rapid solidification, while double aging promoted a stable α + β phase transformation. Heat treatment increased hardness from 360 Hv to 371 Hv and density from 4.29 g/cc to 4.49 g/cc, attributed to martensite decomposition, secondary α precipitation, and defect reduction. Cytotoxicity assessment (ISO 10993-5) using L929 fibroblast cells confirmed cell viability above 70% for both as-built and heat-treated conditions. Electrochemical studies in simulated body fluid (SBF) revealed very low corrosion rates (~ 0.009 mm/year), indicating excellent corrosion resistance. The as-built DED Ti alloy demonstrated superior passivation behavior and favourable biological response, highlighting its suitability for biomedical implant applications without mandatory post-processing. Declarations Author Contribution Author ContributionsSoumyalata D. (Affiliation 1): Conceptualization, methodology, experimental investigation, data curation, formal analysis, writing – original draft.Rajesh S. (Affiliation 1): Conceptualization, supervision, validation, resources, writing – review & editing, project administration.Adam Khan M. (Affiliation 2): Methodology, biocompatibility study design, cytotoxicity analysis guidance, writing – review & editing.Anish Nair (Affiliation 1,3): Experimental investigation, electrochemical corrosion studies, data analysis, visualization.Andrej Jeromen (Affiliation 3): Additive manufacturing process expertise, DED process validation, technical review.Edvard Govekar (Affiliation 3): Supervision, critical review, funding acquisition, overall technical guidance.All authors have read and agreed to the published version of the manuscript. Acknowledgement AcknowledgementsThe authors would like to express their sincere gratitude to the Department of Mechanical Engineering, Kalasalingam Academy of Research and Education, Krishnankoil, India, for providing the necessary laboratory facilities and institutional support to carry out this research work.The authors also acknowledge the support and cooperation extended by the Department of Mechanical Engineering, Al Shabaka Technical Institutional Academy (ASTI Academy), Dubai, UAE, particularly for technical discussions and academic collaboration related to the biocompatibility and materials characterization aspects of this study.Further, the authors gratefully acknowledge the Faculty of Mechanical Engineering, University of Ljubljana, Slovenia, for their support in additive manufacturing expertise, experimental guidance, and collaborative research contributions.The authors sincerely appreciate the valuable technical assistance and collaborative environment provided by all the participating institutions, which significantly contributed to the successful completion of this work. 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Adam Khan, J. T. Winowlin Jappes, Assessment on heat treatment and machinability of DMLS processed Ti64 alloy, Progress in Additive Manufacturing (2024). doi.org/10.1007/s40964-024-00848-x El-Bassyouni, G.T., Mouneir, S.M. & El-Shamy, A.M. Advances in surface modifications of titanium and its alloys: implications for biomedical and pharmaceutical applications. Multiscale and Multidiscip. Model. Exp. and Des. 8, 265 (2025). Ruben Raj Mathew, M. Adam Khan, J. T. Winowlin Jappes Investigation on surface science and cell viability of laser micro engraved DMLS Ti64 alloy, Journal of Bio- and Tribo-Corrosion (2025). (Accepted) M. Adam Khan, N. Ram Prasad, S. Navaneetha Krishnan, S. Karthic Raja, J. T. Winowlin Jappes & Muthukannan Duraiselvam (2017): Laser treated austenitic steel and nickel alloy for human implants, Materials and Manufacturing Processes, DOI: 10.1080/10426914.2017.1364746 S. Sivakumar, M. Adam Khan and Giftson J Senapathy (2020) Biocompatibility and surface studies on electro spark machined titanium based human implants, Journal of Bio- and Tribo-Corrosion 6: 1-11. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8659275","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":593006597,"identity":"71c61a1d-1c7f-4895-93c2-55af97aff565","order_by":0,"name":"Soumyalata D","email":"","orcid":"","institution":"Kalasalingam Ac","correspondingAuthor":false,"prefix":"","firstName":"Soumyalata","middleName":"","lastName":"D","suffix":""},{"id":593006600,"identity":"ebdcfbf7-954d-4a24-aa6e-1b1051761fee","order_by":1,"name":"Rajesh 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2","display":"","copyAsset":false,"role":"figure","size":138278,"visible":true,"origin":"","legend":"\u003cp\u003ePlan for DED Ti alloy heat treatment using tubular furnace\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/4a385c717cb6e660c7932f51.png"},{"id":103504544,"identity":"8e2ade39-4584-4e4d-a293-a48a3694b32e","added_by":"auto","created_at":"2026-02-26 13:20:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":917264,"visible":true,"origin":"","legend":"\u003cp\u003eThe optical image of as – built and heat – treated DED Ti alloy\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/65c2fd902cd1d318559e1946.png"},{"id":103070026,"identity":"d8c85057-40e4-46de-b818-8c63f6531b07","added_by":"auto","created_at":"2026-02-20 12:12:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":642784,"visible":true,"origin":"","legend":"\u003cp\u003eOptical image for controlled sample for comparison\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/d7e3909271b7885dd0bf5335.png"},{"id":103070024,"identity":"02f508cb-0ab3-48c6-b9be-7db209bb84cd","added_by":"auto","created_at":"2026-02-20 12:12:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1336410,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic image of the L929 Fibroblast Cell over as – built and heat – treated DED Ti alloy\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/b14f54f44393808ef528850e.png"},{"id":103070025,"identity":"46736238-ecb1-4e33-afe6-3e8819ac695c","added_by":"auto","created_at":"2026-02-20 12:12:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":272311,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative results of as – built and heat – treated DED Ti alloy cell viability with respect to the different durations\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/8b94ccd380a89b72501a392d.png"},{"id":103070023,"identity":"d2e848eb-ecf2-47e0-b5ff-891584c6628b","added_by":"auto","created_at":"2026-02-20 12:12:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":446493,"visible":true,"origin":"","legend":"\u003cp\u003eIndicates the TAFEL plot recorded from polarisation studies for (a) as – built DED Ti alloy and (b) heat treated DED Ti alloy; following the electron image and EDS value recorded from the SBF exposed area of (c, e) as – built DED Ti alloy and (d, f) heat treated DED Ti alloy.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/53c20c642b38cd50e97bf63f.png"},{"id":103509345,"identity":"b6b81b52-a901-4f95-8154-b3102c73b22f","added_by":"auto","created_at":"2026-02-26 13:58:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4923019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8659275/v1/fcc55837-9b0f-493c-892c-8cdb8d8fc984.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biocompatibility and bio-corrosion behavior of medical grade titanium alloy developed through the direct energy deposition (DED) method","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe use of metals in biomedical engineering has evolved significantly over the past two decades. Metallic materials are widely preferred due to their excellent mechanical strength, which effectively supports biomedical applications [1]. Among these materials, titanium alloys are especially favoured for orthopaedic applications, as they are subjected to heavy cyclic loads and complex mechanical articulation under various physiological conditions [2,3]. However, in certain critical clinical populations such as elderly patients, individuals with diabetes, and those with metabolic disorders medical metal implants exhibit significantly higher failure rates, thereby increasing the likelihood of revision surgeries [4]. One of the primary contributing factors to this behavior is the altered biochemistry of body fluids in these patients. Consequently, titanium alloys have been strongly promoted by researchers and scientists for biomedical engineering applications due to their outstanding metallurgical and biocompatible properties [5].\u003c/p\u003e \u003cp\u003eOne of the major challenges in the utilization of titanium alloys lies in their production and processing routes. For several decades, researchers have faced significant difficulties in achieving directionally solidified and single crystal titanium alloys. El Khalloufi (2021) reported the key challenges associated with the processing and commercialization of titanium-based alloys, highlighting limitations related to cost, scalability, and metallurgical control [6]. Among these challenges, the processing of titanium metal powders remains a critical issue. Powder based routes are commonly employed for the fabrication of titanium alloys due to their flexibility and near-net-shape capability [7]. Despite the availability of multiple processing techniques, the development of low-cost titanium alloys has played a vital role in expanding their industrial and biomedical applications [8]. However, a major constraint has been maintaining the metallurgical quality of the alloy without compromising its mechanical and functional properties [9]. Subsequently, the incorporation of nickel into titanium led to the development of nickel\u0026ndash;titanium alloys, which gained significant attention as shape memory alloys due to their unique functional behavior.\u003c/p\u003e \u003cp\u003eIn recent years the titanium alloy with advanced manufacturing process such as laser base powder sintering took role to meet the industrial demand [10]. It is also coined as laser additive manufacturing process to build the complex and bottle neck shapes. There are different types of additive manufacturing process in current scenario to print metal parts. Especially the direct metal laser sintering (DMLS) process is most prominent and common process available to develop the additive-built titanium alloy and other superalloys [11]. The major constraint in the additive-built production process is delta \u0026ndash; phase formation. Research on heat treatment and its biocompatibility of additive built titanium alloys are in tropical research to meet the readers. Important factors such as biocompatibility and its viability for the implementation of such novel process plays a vital role in advanced production process [12, 13]. The other techniques for additive manufacturing of titanium alloy open for research. The development of alloy through energy deposition and post processing are in demand for future implementation.\u003c/p\u003e \u003cp\u003eIn this research the novel technique such as direct energy deposition (DED) is proposed to print the titanium alloy for biomedical application. The alloy developed through the DED process will be post process to enhance the metallurgical quality and mechanical properties. Following this process, the material may be involved to investigate the bio compatibility via cytotoxicity and corrosion studies. From this analysis this research outcome will encourage the budding engineers to learn and feed with new data for bio medical applications.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003eThe titanium (Ti) alloy developed through the direct energy deposition (DED) is used to investigate the cytotoxicity and bio \u0026ndash; compatibility for the further applications. It is one of the advanced and feasible method to develop metal component. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the optimal process parameter design for industrial standard to develop the titanium alloy through direct energy deposition method. The commercial titanium metal powder of particle size 30\u0026ndash;160 \u0026micro;m with a medical standard is used for additive manufacturing. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the schematic illustration and actual titanium alloy developed from the industrial standard metal powder. The spectroscopic analysis of the DED Ti alloy has Ti \u0026ndash; 88.2%; Al \u0026ndash; 6.85%, V \u0026ndash; 4.6%, C \u0026ndash; 0.008 and traces of iron. All the DED samples are in need of secondary process to compete the original product in terms of machining and post processing.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe standard process parameter used to develop the titanium alloy through direct energy deposition method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProcess Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProcess condition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUnits\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpeed of Scan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emm/s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePower\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWatts\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolume rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emm\u003csup\u003e3\u003c/sup\u003e/s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiameter of beam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a base work the metal samples developed through the DED process is sliced using wire electrical discharge machining process. The dimensions factors of the test sample vary with respect the individual standards of experimental requirements. The test samples of DED Ti alloy are post processed to compare the metallurgical phase transformation, hardness and the density of the material. The post processing of the material was done with double aging process following the standard heat treatment plan. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the heat treatment plan proposed for the DED Ti alloy. A tubular furnace which is designed to a maximum of 1400\u0026deg;C is used for the heat treatment plan. The samples are place inside the furnace with the help of 99.9% pure alumina boat and the cooling process are made through furnace cooling as the standards process conditions given in the plan. After heat treatment the samples are metallurgically polished to study the microstructural transformation developed due to the double ageing process. An optical microscope (Make: Vertimet) is used to reveal the microstructure of the material before and after post processing. From the optical image the inference on phase transformation will be inferred for discussion. Following, micro hardness and density values are calculated to discuss about the material quality for future analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a core work, the samples are sliced to a dimension of 3mm x 3mm x 3mm for cell viability and cytotoxicity analysis. After slicing the samples are polished and cleaned with ultrasonicator. After metal cleaning process, the samples are sterilised for fibroblast cell culture analysis. The L929 fibroblast cell is the common cell used to study the cytotoxicity analysis following the standard MTT Assay protocol (ISO 10993). On the other way, to substantiate the materials quality the metal bio \u0026ndash; compatibility analysis was done with simulated body fluid (SBF) using potentiostat (electrochemical workstation). The samples are maintained to a dimension of 10mm x 10mm x 2mm for electrochemical corrosion studies. Similar to the cell cytotoxicity analysis the sample preparation procedures are followed. In house lab reagents are used to prepare the simulated body fluid following the authentic procedure of Kokubo SBF medium for bio corrosion analysis [14]. The saturated calomel electrode is a reference electrode and the platinum is used as a standard electrode. The potential (\u0026plusmn;\u0026thinsp;250 mV) and sweep rate 1 mV/s is the common factor defined for electrochemical corrosion studies. ASTM standards for the electrochemical studies (ASTM F2129) is followed for the investigation. From the analysis, the outcomes are technical justified to recommend the experimentation to future work.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThe titanium alloy developed through the direct energy deposition (DED Ti alloy) method is evaluated to report on the microstructural transformation of the material. The structural morphology of the as \u0026ndash; built and heat \u0026ndash; treated DED Ti alloy observed through the microscope is as given in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. For the as \u0026ndash; built DED Ti alloy, the dense fine lath structure in a closed network is noticed due to the rapid cooling of metal pool. It is an effect of thermal gradient suppressed the metal pool to solidify at an average rate of 10\u003csup\u003e3\u003c/sup\u003e \u0026ndash; 10\u003csup\u003e5\u003c/sup\u003e \u0026deg;C/s during the deposition. However, in the heat-treated DED Ti alloy the micro structures are coarse and more stable with clear grain boundary. Literature supports that the high cooling rate will suppress the β \u0026loz; α transformation with high strain energy in the grain at 720\u0026deg;C. On second stage of ageing (620\u0026deg;C) secondary alpha phase precipitation nucleated within the beta phase (as matrix). Does the combination of alpha \u0026ndash; beta phase has increased the density of the grains in the as \u0026ndash; built DED Ti alloy. At 720\u0026deg;C the material decomposes the α\u0026prime; \u0026rarr; α\u0026thinsp;+\u0026thinsp;β, relieve residual stresses, stabilize β; and 620\u0026deg;C is the precipitate fine secondary α phase, refining the microstructure and also strengthen alloy. It is very important to note that the as \u0026ndash; built DED Ti alloy has 360 Hv and 4.29 g/cc which is less than the heat treated DED Ti alloy which has 371 Hv and 4.49 g/cc. The higher hardness and density of the heat-treated DED Ti alloy compared to the as-built condition are attributed to diffusion-driven decomposition of metastable α\u0026prime; martensite into equilibrium α\u0026thinsp;+\u0026thinsp;β phases, fine secondary α precipitation, and the reduction of process-induced porosity and lattice defects during double aging, resulting in a denser and mechanically more stable microstructure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMTT Assay test and evaluation for DED Ti alloy.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe preparation of conditioned media was done following the ethylene oxide (EtO) sterilization procedure. The metal alloys were incubated in complete culture medium (DMEM high glucose supplemented with 10% fetal bovine serum and 1% antibiotic antimycotic solution) at an ISO-recommended extraction ratio of 0.2 g/mL. The incubation was carried out for 24, 48, and 72 hours to obtain time dependent conditioned media. In continuation to the control medium preparation, the cytotoxicity of the metals was assessed following the MTT assay procedure using L929 fibroblast cell line. The cells were seeded at a density of 1 \u0026times; 10⁴ cells per well in 100 \u0026micro;L of complete medium in 96-well plates and allowed to attach for 24 hours. The cells were then treated with conditioned media obtained from the metal alloys at different time points. Control wells contained cells cultured in fresh complete medium. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the optical image of the control samples of fibroblast cells. Each condition, including controls, was tested in five replicates to find average results and also for the results interpretations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 24 hours of treatment, 5 mg/mL MTT solution, prepared in phosphate-buffered saline, was added to each well. The plates were gently shaken and incubated for 4 hours at 37\u0026deg;C in a humidified incubator with 5% CO₂. Subsequently, 100 \u0026micro;L of 10% SDS solution was added to each well to solubilize the formazan crystals, followed by overnight incubation. Absorbance was measured using a microplate reader (TECAN) at 570 nm with a reference wavelength of 630 nm. The evaluation procedure for the cell growth in proximity to metal alloys were performed to assess the ability of L929 cells to grow in the presence of metal alloys, the alloys were placed in 24-well culture plates, and L929 cells were seeded at a density of 1 \u0026times; 10⁵ cells per well in complete medium. The cultures were incubated for 48 hours under standard conditions. After incubation, cell growth and morphology in the vicinity of the metal alloys were observed and documented using a Zeiss optical microscope.\u003c/p\u003e \u003cp\u003eThe phase contrast (microscopic) images of L929 fibroblast cells cultured on the 3D-printed material show good cell adhesion, typical spindle shaped morphology, and uniform spreading across the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The cells appear viable, with no obvious signs of cytotoxicity such as rounding, detachment, or membrane damage in the as \u0026ndash; built DED Ti alloy. The formation of interconnected cell networks and sustained cell density over time indicates that the as \u0026ndash; built DED Ti alloy provides a favourable surface for fibroblast attachment and proliferation compared to the heat \u0026ndash; treated DED Ti alloy. However, the confirmation of the cell count and its rate of behaviour in quantitative mode are further investigated. The quantitative data arrived from the cell viability results as shown in Figure, demonstrate that the DED Ti alloy maintains the acceptable cytocompatibility with the embed of L929 fibroblasts over 72 hours. Both as-built and heat-treated (HT) samples exhibit cell viability comparable to the control at 24 hours, indicating minimal acute cytotoxic effects and negligible release of harmful leachables during early exposure in accordance with ISO 10993-5 criteria (\u0026gt;\u0026thinsp;70% viability). A time-dependent reduction in viability is observed at 48 and 72 hours, more pronounced in the HT condition; however, viability values remain above the cytotoxic threshold, confirming that post-processing does not induce severe biological incompatibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The comparatively higher viability of the as-built samples suggests that heat treatment may alter surface chemistry or residual stress states, influencing protein adsorption and cell\u0026ndash;material interactions. Overall, the results confirm that the 3D-printed material supports fibroblast survival over extended culture periods and is suitable for further evaluation through other bio chemical environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eBio – corrosion with simulated body fluid (SBF)\u003c/h3\u003e\n\u003cp\u003eThe bio corrosion studies on DED Ti alloy; as \u0026ndash; built and heat \u0026ndash; treated are exposed to simulated body fluid using electrochemical polarisation work station.\u003c/p\u003e \u003cp\u003eThe DED Ti alloy, before and after post processing are used to study about the bio \u0026ndash; compatibility using simulated body fluid as given in the Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. From the data it is clear that the both as-built and heat-treated DED Ti alloys exhibit very low corrosion current densities (~\u0026thinsp;10⁻\u0026sup3; mA/cm\u0026sup2;) and extremely low corrosion rates (~\u0026thinsp;0.009 mm/year) under the SBF environment, confirming the inherently high corrosion resistance of titanium alloys in physiological environments. It is an indication of surface protection and passive layer formation (TiO\u003csub\u003ex\u003c/sub\u003e in this case) and suppressing the metal dissolution with respect to the alloying elements. There is a shift in corrosion potential for heat treated sample with reduction in corrosion current density. This implies that the corrosion process is predominantly controlled by passive film properties rather than bulk microstructural changes. However, for the as \u0026ndash; built slightly negative trend in corrosion potential indicates positive sign highlighting its ability to rapidly form and regenerate a protective oxide layer despite higher initial electrochemical activity. From a biomedical perspective, this behavior is particularly significant, as it suggests that the as-built DED Ti alloy provides corrosion protection comparable to the heat-treated condition, while potentially minimizing ion release and associated cytotoxicity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eData extracted from the TAFEL plot for DED Samples at two different cases exposed to simulated body fluid.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDED Ti alloy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrosion potential\u003c/p\u003e \u003cp\u003eE\u003csub\u003ecorr\u003c/sub\u003e (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCurrent density \u003c/p\u003e \u003cp\u003eI\u003csub\u003ecorr\u003c/sub\u003e (mA/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorrosion rate\u003c/p\u003e \u003cp\u003eCR (mm/year)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs \u0026ndash; built\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-120.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.001031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.006125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeat \u0026ndash; treated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-100.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.001986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009626\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e illustrates the electrochemical behaviour of the both as built and heat-treated DED Ti alloy metal samples exposed to simulated body fluid. The as \u0026ndash; built DED Ti alloy reveals with asymmetry potential and current density curve under the simulated body fluid condition. The titanium alloys are stable to produce thick film over the adjunct surface and forms a passive layer to protect the material from further corrosion. Even though the presence of chloride ions in the simulated body fluid will induce the passive film to get break at the earliest. However, the as \u0026ndash; built DED Ti alloy has sharp and perfect profile from the TAFEL plot to inform that the material is very much stable to protect the surface from aggressive body fluid environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). This polarization behavior of the as-built titanium alloy in simulated body fluid exhibits a reversal characteristic of strong passivation. This response is attributed to the formation and rapid regeneration of a protective TiO₂ passive layer, influenced by the inherent surface roughness and microstructural heterogeneity of the as-built condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, for the heat-treated samples, results from the electrochemical polarisation studies are little diverse from the analysis. It has been noticed that the reversal of anodic reaction observed in the polarization curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Which is attributed to the inherent electrochemical characteristics of the alloy elements; particularly the formation, breakdown, and depassivation of a surface passive film by the activation of alloy elements during the electrochemical reactions with simulated body fluid. The break of film and getting depassivation is due to the instability of the material exposed under the working medium. Technically speaking, the alteration of oxides charge transfer kinetics (for Ti and Al) at the interface induces such vulnerable reactions. It actually happens at the higher potential and the rate of conductivity simultaneously increased with ions fluctuations.\u003c/p\u003e \u003cp\u003eAt lower potentials, active electrochemical reactions were observed under relatively stabilized environmental conditions. Previous studies on commercial titanium implant materials have reported that the evolution of a TiOₓ passive layer during electrochemical exposure effectively protects the material from catastrophic or continuous corrosion (15). In the present study, the as-built DED titanium alloy demonstrated superior corrosion protection and reduced cell cytotoxicity compared to the heat-treated DED titanium alloy. Although the heat-treated DED titanium alloy exhibited a slightly higher overall oxide content, this was accompanied by a measurable reduction in aluminum and vanadium concentrations at the surface. This observation suggests preferential dissolution or outward diffusion of Al and V from the alloy matrix during electrochemical exposure, likely induced by thermal treatment\u0026ndash;related microstructural changes. In contrast, the as-built DED titanium alloy retained a more stable surface chemistry, promoting the formation of a protective TiO₂-rich passive layer and thereby enhancing corrosion resistance and biocompatibility relative to the heat-treated condition.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe study confirms the feasibility of fabricating biomedical grade titanium alloys using the direct energy deposition (DED) process. From the investigations and detailed analysis, following points were derived as an conclusion.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe as-built DED Ti alloy exhibited a fine acicular lath microstructure due to rapid solidification, while double aging promoted a stable α\u0026thinsp;+\u0026thinsp;β phase transformation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eHeat treatment increased hardness from 360 Hv to 371 Hv and density from 4.29 g/cc to 4.49 g/cc, attributed to martensite decomposition, secondary α precipitation, and defect reduction.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCytotoxicity assessment (ISO 10993-5) using L929 fibroblast cells confirmed cell viability above 70% for both as-built and heat-treated conditions.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eElectrochemical studies in simulated body fluid (SBF) revealed very low corrosion rates (~\u0026thinsp;0.009 mm/year), indicating excellent corrosion resistance. The as-built DED Ti alloy demonstrated superior passivation behavior and favourable biological response, highlighting its suitability for biomedical implant applications without mandatory post-processing.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor ContributionsSoumyalata D. (Affiliation 1): Conceptualization, methodology, experimental investigation, data curation, formal analysis, writing \u0026ndash; original draft.Rajesh S. (Affiliation 1): Conceptualization, supervision, validation, resources, writing \u0026ndash; review \u0026amp; editing, project administration.Adam Khan M. (Affiliation 2): Methodology, biocompatibility study design, cytotoxicity analysis guidance, writing \u0026ndash; review \u0026amp; editing.Anish Nair (Affiliation 1,3): Experimental investigation, electrochemical corrosion studies, data analysis, visualization.Andrej Jeromen (Affiliation 3): Additive manufacturing process expertise, DED process validation, technical review.Edvard Govekar (Affiliation 3): Supervision, critical review, funding acquisition, overall technical guidance.All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgementsThe authors would like to express their sincere gratitude to the Department of Mechanical Engineering, Kalasalingam Academy of Research and Education, Krishnankoil, India, for providing the necessary laboratory facilities and institutional support to carry out this research work.The authors also acknowledge the support and cooperation extended by the Department of Mechanical Engineering, Al Shabaka Technical Institutional Academy (ASTI Academy), Dubai, UAE, particularly for technical discussions and academic collaboration related to the biocompatibility and materials characterization aspects of this study.Further, the authors gratefully acknowledge the Faculty of Mechanical Engineering, University of Ljubljana, Slovenia, for their support in additive manufacturing expertise, experimental guidance, and collaborative research contributions.The authors sincerely appreciate the valuable technical assistance and collaborative environment provided by all the participating institutions, which significantly contributed to the successful completion of this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHyun-Do Jung , Titanium and Its Alloys for Biomedical Applications, MDPI Publisher, 2022.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSenopati G., Rahman Rashid R.A., Kartika I., Palanisamy S. Recent Development of Low-Cost \u0026beta;-Ti Alloys for Biomedical Applications: A Review. Metals. 2023;13:194.\u003c/li\u003e\n \u003cli\u003eH.J. Rack, J.I. Qazi, Titanium alloys for biomedical applications, Materials Science and Engineering: C, 26(8), 2006, 1269-1277.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYang J, Liu C, Sun H, Liu Y, Liu Z, Zhang D, Zhao G, Wang Q and Yang D (2022) The progress in titanium alloys used as biomedical implants: From the view of reactive oxygen species. Front. Bioeng. Biotechnol. 10:1092916.\u003c/li\u003e\n \u003cli\u003eMarin E, Lanzutti A. Biomedical Applications of Titanium Alloys: A Comprehensive Review. Materials (Basel). 2023 Dec 25;17(1):114. doi: 10.3390/ma17010114.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEl Khalloufi, Mohammed, Olivier Drevelle, and Gervais Soucy. 2021. \u0026quot;Titanium: An Overview of Resources and Production Methods\u0026quot; Minerals 11, no. 12: 1425.\u003c/li\u003e\n \u003cli\u003eStanley Abkowitz, Susan Abkowitz, Harvey Fisher, Titanium alloy components manufacture from blended elemental powder and the qualification process, Editor(s): Ma Qian, Francis H. (Sam) Froes, Titanium Powder Metallurgy, Butterworth-Heinemann, 2015, Pages 299-312.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhiyi Zou, Matthew K. Dunstan, Brandon McWilliams, Stuart Robertson, Richard Hague, Marco Simonelli, Development of low-cost Ti alloys with a balanced strength and ductility with generation of ultra-fine microstructures, Journal of Alloys and Compounds, 1030, 2025, 180786.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLaskowska D, Bałasz B, Żurawski Ł. Evolution of the Metallographic Structure of Additively Manufactured Ti-6Al-4V and Ti-6Al-7Nb Titanium Alloys. Materials. 2026; 19 (1): 80.\u003c/li\u003e\n \u003cli\u003eJinlong Su, Fulin Jiang, Jie Teng, Lequn Chen, Ming Yan, Guillermo Requena, Lai-Chang Zhang, Y Morris Wang, Ilya V Okulov, Hongmei Zhu and Chaolin Tan, Recent innovations in laser additive manufacturing of titanium alloys, 2024 Int. J. Extrem. Manuf. 6 032001\u003c/li\u003e\n \u003cli\u003eRuben Raj Mathew, M. Adam Khan, J. T. Winowlin Jappes, Assessment on heat treatment and machinability of DMLS processed Ti64 alloy, Progress in Additive Manufacturing (2024). doi.org/10.1007/s40964-024-00848-x\u003c/li\u003e\n \u003cli\u003eEl-Bassyouni, G.T., Mouneir, S.M. \u0026amp; El-Shamy, A.M. Advances in surface modifications of titanium and its alloys: implications for biomedical and pharmaceutical applications. Multiscale and Multidiscip. Model. Exp. and Des. 8, 265 (2025).\u003c/li\u003e\n \u003cli\u003eRuben Raj Mathew, M. Adam Khan, J. T. Winowlin Jappes Investigation on surface science and cell viability of laser micro engraved DMLS Ti64 alloy, Journal of Bio- and Tribo-Corrosion (2025). (Accepted)\u003c/li\u003e\n \u003cli\u003eM. Adam Khan, N. Ram Prasad, S. Navaneetha Krishnan, S. Karthic Raja, J. T. Winowlin Jappes \u0026amp; Muthukannan Duraiselvam (2017): Laser treated austenitic steel and nickel alloy for human implants, Materials and Manufacturing Processes, DOI: 10.1080/10426914.2017.1364746\u003c/li\u003e\n \u003cli\u003eS. Sivakumar, M. Adam Khan and Giftson J Senapathy (2020) Biocompatibility and surface studies on electro spark machined titanium based human implants, Journal of Bio- and Tribo-Corrosion 6: 1-11.\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":"titanium, additive manufacturing, cytotoxicity, corrosion","lastPublishedDoi":"10.21203/rs.3.rs-8659275/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8659275/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdditive manufacturing of titanium alloys has gained significant attention for biomedical applications due to its ability to fabricate complex geometries with controlled microstructures. In this study, a titanium alloy was fabricated using the direct energy deposition (DED) technique and evaluated in the as-built and double-aged conditionsfor biomedical suitability. Post-processing involved a two-stage aging treatment at 720°C and 620°Cto enhance microstructural stability and mechanical performance. Optical microscopy revealed a fine acicular lath structure in the as-built alloy resulting from rapid solidification, while the heat-treated alloy exhibited a coarser and thermodynamically stable α + βmicrostructure. The as-built DED Ti alloy showed a hardness of 360 Hv and density of 4.29 g/cc, which increased to 371 Hv and 4.49 g/cc after heat treatment due to martensite decomposition, secondary α precipitation, and porosity reduction. Cytocompatibility assessed using L929 fibroblast cellsfollowing ISO 10993-5 (MTT assay)confirmed cell viability above 70% for 72 hfor both conditions, with the as-built alloy exhibiting comparatively higher viability. Electrochemical corrosion studies conducted in simulated body fluid (SBF) demonstrated very low corrosion current densities (~10⁻³ mA/cm²) and corrosion rates (~0.009 mm/year), indicating excellent corrosion resistance. Notably, the as-built alloy exhibited superior passivation behavior due to the formation of a stable TiO₂-rich oxide layer. These results confirm the suitability of DED-fabricated titanium alloys for biomedical implant applications.\u003c/p\u003e","manuscriptTitle":"Biocompatibility and bio-corrosion behavior of medical grade titanium alloy developed through the direct energy deposition (DED) method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-20 12:12:22","doi":"10.21203/rs.3.rs-8659275/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":"fe753f0b-d53d-4ae2-9905-ec87efe4cf5a","owner":[],"postedDate":"February 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-20T12:12:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-20 12:12:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8659275","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8659275","identity":"rs-8659275","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}