Due to the bio-inert nature of titanium (Ti) and subsequent accompanying chronic inflammatory response, an implant's stability and function can be significantly affected, which is why various surface modifications have been employed, including the deposition of titanium oxide (TiO2) nanotubes (TNTs) onto the native surface through the anodic oxidation method. While the influence of nanotube diameter on cell behaviour and osteogenesis is very well documented, information regarding the effects of nanotube lateral spacing on the in vivo new bone formation process is insufficient and hard to find. Considering this, the present study's aim was to evaluate the mechanical properties and the osteogenic ability of two types of TNTs-based pins with different lateral spacing, e.g., 25 nm (TNTs) and 92 nm (spTNTs). The mechanical properties of the TNT-coated implants were characterised from a morphological point of view (tube diameter, spacing, and tube length) using scanning electron microscopy (SEM). In addition, the chemical composition of the implants was evaluated using X-ray photoelectron spectroscopy, while surface roughness and topography were characterised using atomic force microscopy (AFM). Finally, the implants' hardness and elastic modulus were investigated using nanoindentation measurements. The in vivo new bone formation was histologically evaluated (haematoxylin and eosin-HE staining) at 6 and 30 days post-implantation in a rat model. Mechanical characterisation revealed that the two morphologies presented a similar chemical composition and mechanical strength, but, in terms of surface roughness, the spTNTs exhibited a higher average roughness. The microscopic examination at 1 month post-implantation revealed that spTNTs pins (57.21 ± 34.93) were capable of promoting early new bone tissue formation to a greater extent than the TNTs-coated implants (24.37 ± 6.5), with a difference in the average thickness of the newly formed bone tissue of ~32.84 µm, thus highlighting the importance of this parameter when designing future dental/orthopaedic implants.

Department of Biochemistry and Molecular Biology Faculty of Biology University of Bucharest 91 95 Spl Independentei 050657 Bucharest Romania

Department of Materials Science and Engineering Chair of General Materials Properties Friedrich Alexander University of Erlangen Nürnberg Martensstraße 5 91058 Erlangen Germany

Department of Materials Science and Engineering WW4 LKO Friedrich Alexander University of Erlangen Nürnberg Martensstrasse 7 91058 Erlangen Germany

Faculty of Veterinary Medicine University of Agronomic Sciences and Veterinary Medicine 105 Spl Independentei 050097 Bucharest Romania

Regional Centre of Advanced Technologies and Materials Šlechtitelů 27 78371 Olomouc Czech Republic

Research Institute of the University of Bucharest University of Bucharest 050657 Bucharest Romania

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Tao B., Lan H., Zhou X., Lin C., Qin X., Wu M., Zhang Y., Chen S., Guo A., Li K., et al. Regulation of TiO2 nanotubes on titanium implants to orchestrate osteo/angio-genesis and osteoimmunomodulation for boosted osseointegration. Mater. Des. 2023;233:112268. doi: 10.1016/j.matdes.2023.112268. DOI

Messias A., Nicolau P., Guerra F. Titanium dental implants with different collar design and surface modifications: A systemic review on survival rates and marginal bone levels. Clin. Oral Implant. Res. 2019;30:20–48. doi: 10.1111/clr.13389. PubMed DOI

Ma A., Shang H., Song Y., Chen B., You Y., Han W., Zhang X., Zhang W., Li Y., Li C. Icariin-Functionalised Coating on TiO2 Nanotubes Surface to Improve Osteoblast activity In Vitro and Osteogenesis Ability In Vivo. Coatings. 2019;9:327. doi: 10.3390/coatings9050327. DOI

Lu X., Wu Z., Xu K., Wang X., Wang S., Qiu H., Li X., Chen J. Multifunctional Coatings of Titanium Implants Toward Promoting Ossoeintegration and Preventing Infection: Recent Developments. Front. Bioeng. Biotechnol. 2021;9:783816. doi: 10.3389/fbioe.2021.783816. PubMed DOI PMC

Liu Y., Rath B., Tingart M., Eschweiler J. Role of implant surface modification in ossoeintegration: A systemic review. J. Biomed. Mater. Res. Part A. 2020;108:470–484. doi: 10.1002/jbm.a.36829. PubMed DOI

Khaw J.S., Bowen C.R., Cartmell S.H. Effect of TiO2 Nanotube Pore Diameter on human Mesenchymal Stem Cells and Human Osteoblasts. Nanomaterials. 2020;10:2117. doi: 10.3390/nano10112117. PubMed DOI PMC

Hou C., An J., Zhao D., Ma X., Zhang W., Zhao W., Wu M., Zhang Z., Yuan F. Surface Modification Techniques to Pro-duce Micro/Nano-scale Topographies on Ti-based Implant Surfaces for Improved Osseointegration. Front. Bioeng. Biotechnol. 2022;10:835008. doi: 10.3389/fbioe.2022.835008. PubMed DOI PMC

Izmir M., Ercan B. Anodization of titanium alloys for orthopedic applications. Front. Chem. Sci. Eng. 2019;13:28–45. doi: 10.1007/s11705-018-1759-y. DOI

Lee K., Mazare A., Schmuki P. One-Dimensional Titanium Dioxide Nanomaterials: Nanotubes. Chem. Rev. 2014;114:9385–9454. doi: 10.1021/cr500061m. PubMed DOI

Batool S.A., Maqbool M.S., Javed M.A., Niaz A., Rehman M.A.U. A review on the Fabrication and Characterization of Titania Nanotubes Obtained via Electrochemical Anodization. Surfaces. 2022;5:456–480. doi: 10.3390/surfaces5040033. DOI

Park J., Tesler A.B., Gongadze E., Iglic A., Schmuki P., Mazare A. Nanoscale Topography of Anodic TiO2 Nanostructures Is Crucial for Cell-Surface Interactions. ACS Appl. Mater. Interfaces. 2024;16:4430–4438. doi: 10.1021/acsami.3c16033. PubMed DOI

Necula M.G., Mazare A., Ion R.N., Ozkan S., Park J., Schmuki P., Cimpean A. Lateral Spacing of TiO2 Nanotubes Modu-lates Osteoblast Behavior. Materials. 2019;12:2956. doi: 10.3390/ma12182956. PubMed DOI PMC

Bjursten L.M., Rasmusson L., Oh S., Smith G.C., Brammer K.S., Jin S. Titanium dioxide nanotubes enhance bone bonding in vivo. J. Biomed. Mater. Res. A. 2010;92A:1218–1224. doi: 10.1002/jbm.a.32463. PubMed DOI

von Wilmowsky C., Bauer S., Lutz R., Meisel M., Neukam F.W., Toyoshima T., Schmuki P., Nkenke E., Schlegel K.A. In Vivo Evaluation of Anodic TiO2 Nanotubes: An experimental Study in the Pig. J. Biomed. Mater. Res. Part B Appl. Biomater. 2009;89:165–171. doi: 10.1002/jbm.b.31201. PubMed DOI

Kang C.-G., Park Y.-B., Choi H., Oh S., Lee K.-W., Choi S.-H., Shim J.-S. Osseointegration of Implants Surface-treated with Various Diameters of TiO2 Nanotubes in Rabbit. J. Nanomater. 2015;2015:634650. doi: 10.1155/2015/634650. DOI

Wang N., Li H., Lu W., Li J., Wang J., Zhang Z., Liu Y. Effects of TiO2 nanotubes with different diameters on gene ex-pression and osseointegration of implants in minipigs. Biomaterials. 2011;32:6900–6911. doi: 10.1016/j.biomaterials.2011.06.023. PubMed DOI

Jang I., Shim S.-C., Choi D.-S., Cha B.-K., Lee J.-K., Choe B.-H., Choi W.-Y. Effect of TiO2 nanotubes arrays on osseointe-gration of orthodontic miniscrew. Biomed. Microdevices. 2015;17:76. doi: 10.1007/s10544-015-9986-1. PubMed DOI

Park J., Bauer S., Schmuki P., der Mark K. Narrow Window in Nanoscale Dependent Activation of Endothelial Cell Growth and Differentiation on TiO2 Nanotube Surfaces. Nano Lett. 2009;9:3157–3164. doi: 10.1021/nl9013502. PubMed DOI

Park J., Bauer S., Schlegel K.A., Neukam F.M., Von Der Mark K., Schmuki P. TiO2 nanotube surfaces: A 15 nm-an optimal length scale of surface topography for cell adhesion and differentiation. Small. 2009;5:666–671. doi: 10.1002/smll.200801476. PubMed DOI

Xu L., Yu Q., Jiang X.Q., Zhan F.Q., Yu W., Jiang X., Zhang F. The effect of anatase TiO2 nanotube layers on MC3T3-E1 preosteoblasts adhesion, proliferation and differentiation. J. Biomed. Mater. Res. Part A. 2010;94:1012–1022. doi: 10.1002/jbm.a.32687. PubMed DOI

Che C., Wang J., Guo W. Effect of TiO2 Nanotubes on Biological Activity of Osteoblasts and Focal Adhesion Kinase/Osteopontin Level. J. Biomed. Technol. 2024;20:793–799. doi: 10.1166/jbn.2024.3877. DOI

Brammer K.S., Oh S., Cobb C.J., Bjursten L.M., van der Heyde H., Jin S. Improved bone-forming functionality of diameter-controlled TiO2 nanotube surface. Acta Biomater. 2009;5:3215–3223. doi: 10.1016/j.actbio.2009.05.008. PubMed DOI

Yi Y., Park Y., Choi H., Lee K., Kim S., Kim K., Oh S., Shim J. The Evaluation of Ossoeintegration of Dental Implant Surface with Different Size of TiO2 Nanotube in Rats. J. Nanomater. 2015;2015:581713. doi: 10.1155/2015/581713. DOI

von Wilmowsky C., Bauer S., Roedl S., Neukam F.W., Schmuki P., Schlegel K.A. The diameter of anodic TiO2 nanotubes affects bone formation and correlates with the bone morphogenic protein-2 expression in vivo. Clin. Oral Implant. Res. 2011;23:359–366. doi: 10.1111/j.1600-0501.2010.02139.x. PubMed DOI

Yu M., Yang H., Li B., Wang R., Han Y. Molecular mechanisms of interrod spacing-mediated osseointegration via modulating inflammatory response and osteogenic differentiation. Chem. Eng. J. 2024;454:140141. doi: 10.1016/j.cej.2022.140141. DOI

Zhou J., Li B., Lu S., Zhang L., Han Y. Regulation of osteoblast proliferation and differentiation by interrod spacing of Sr-HA nanorods on microporous titania coatings. ACS Appl. Mater. Interfaces. 2013;5:5358–5365. doi: 10.1021/am401339n. PubMed DOI

Zhou J., Han Y., Lu S. Direct role of interrod spacing in mediating cell adhesion on Sr-HA nanorod-patterned coatings. Int. J. Nanomed. 2014;9:1243–1260. PubMed PMC

Kim H.S., Yoo H.S. Differentiation and focal adhesion of adipose-derived stem cells on nano-pillars arrays with different spacing. RSC Adv. 2015;5:49508–49512. doi: 10.1039/C5RA07608K. DOI

Chezzi B., Langonegro P., Fukuta N., Parisi L., Calestani D., Galli C., Salviati G., Macaluso G.M., Rossi F. Sub-Micropillar Spacing Modulates the Spatial Arrangement of Mouse MC3T3-E1 Osteoblastic Cells. Nanomaterials. 2019;9:1701. doi: 10.3390/nano9121701. PubMed DOI PMC

Kelvin-Agwu M.T.C., Adelodun M.O., Igwama G.T., Anyanwu E.C. Advancements in biomedical device implants: A comprehensive review of current technologies. Int. J. Front. Med. Surg. Res. 2024;6:19–28. doi: 10.53294/ijfmsr.2024.6.1.0037. DOI

Concalves A.D., Balestri W., Reinwald Y. Biomaterials. IntechOpen; London, UK: 2024. Biomedical implants for regenerative therapies; pp. 1–36.

Necula M.G., Mazare A., Negrescu A.M., Mitran V., Ozkan S., Trusca R., Park J., Schmuki P., Cimpean A. Macrophage-like Cells Are Responsive to Titania Nanotube Intertube Spacing—An In Vitro Study. Int. J. Mol. Sci. 2022;23:3558. doi: 10.3390/ijms23073558. PubMed DOI PMC

Ozkan S., Mazare A., Schmuki P. Critical parameters and factors in the formation of spaced TiO2 nanotubes by self-organizing anodization. Electrochim. Acta. 2018;268:435–447. doi: 10.1016/j.electacta.2018.02.120. DOI

Xu Y.N., Liu M.N., Wang M.C., Oloyede A., Bell J.M., Yan C. Nnaoindentation study of the mechanical behavior of the TiO2 nanotube arrays. J. Appl. Phys. 2015;118:145301. doi: 10.1063/1.4932213. DOI

Alfaraj T.A., Al-Madani S., Algahtani N.S., Almohammadi A.A., Alqahtani A.M., AlQabbani H.S., Bajunaid M.K., Alharthy B.A., Aljalfan N. Optimizing Osseointegration in Dental Implantology: A Cross-Disciplinary Review of Current and Emerging Strategies. Cures. 2023;15:e47943. doi: 10.7759/cureus.47943. PubMed DOI PMC

Li X., Wu J., Li D., Zou Q., Man Y., Zou L., Li W. Pro-Osteogenesis and in vivo tracking investigation of dental implantation system comprising novel mTi implant and HYH-Fe particles. Bioact. Mater. 2021;6:2658–2666. doi: 10.1016/j.bioactmat.2021.01.038. PubMed DOI PMC

Cruz M.B., Silva N., Marques J.F., Mata A., Silva F.S., Carames J. Biomimetic Implant Surfaces and Their Role in Biological Integration—A Concise Review. Biomimetics. 2022;7:74. doi: 10.3390/biomimetics7020074. PubMed DOI PMC

Lu X., Zhao Y., Peng X., Lu C., Wu Z., Xu H., Qin Y., Xu Y., Wang Q., Hao Y., et al. Comprehensive Overview of Interface Strategies in Implant Osseointegration. Adv. Funct. Mater. 2024. early view . DOI

Alves-Rezende M.C., Capalbo L.C., De Oliveira Limirio J.P.J., Capalbo B.C., Limirio P.H.J.O., Rosa J.L. The role of TiO2 nanotube surface on osseointegration of titanium implants: Biomechanical and histological study in rats. Micros. Res. Tech. 2020;83:817–823. doi: 10.1002/jemt.23473. PubMed DOI

Wang C., Gao S., Lu R., Wang X., Chen S. In Vitro and In Vivo Studies of Hydrogenated Titanium Dioxide Nanotubes with Superhydrophilic Surfaces during Early Osseointegration. Cells. 2022;11:3417. doi: 10.3390/cells11213417. PubMed DOI PMC

Li J., Zheng Y., Yu Z., Kankala R.K., Lin Q., Shi J., Chen C., Luo K., Chen A., Zhong Q. Surface-modified titanium and titanium-based alloys for improved osteogenesis: A critical review. Heliyon. 2024;10:e23779. doi: 10.1016/j.heliyon.2023.e23779. PubMed DOI PMC

Zhao X., You L., Wang T., Zhang X., Li Z., Ding L., Li J., Xiao C., Han F., Li B. Enhanced Ossoeintegration of Titanium Implants by Surface Modification with Silicon-doped Titania Nanotubes. Int. J. Nanomed. 2020;15:8583–8594. doi: 10.2147/IJN.S270311. PubMed DOI PMC

Yao L., Al-Bishari A.M., Shen J., Wang Z., Liu T., Sheng L., Wu G., Lu L., Xu L., Liu J. Ossoeintegration and an-ti-infection of dental implant under osteoporotic conditions promoted by gallium oxide nano-layer coated titanium di-oxide nanotube arrays. Ceram. Int. 2023;49:22961–22969. doi: 10.1016/j.ceramint.2023.04.121. DOI

Moon K.-S., Bae J.-M., Park Y.-B., Choi E.-J., Oh S.-H. Photobiomodulation-Based Synergic Effects of Pt-Coated TiO2 Nanotubes and 850nm Near-Infrared Irradiation on the Ossoeintegration Enhancement: In Vitro and In Vivo Evaluation. Nanomaterials. 2023;13:1377. doi: 10.3390/nano13081377. PubMed DOI PMC

Li Y., Tang L., Shen M., Wang Z., Huang X. A comparative study of Sr-loaded nano-textured Ti and TiO2 nanotube implants on osseointegration immediately after tooth extraction in Beagle dogs. Front. Mater. 2023;10:1213163. doi: 10.3389/fmats.2023.1213163. DOI

Hamlekhan A., Takoudis C., Sukotjo C., Mathew M.T., Virdi A., Shahbazian-Yassar R., Shokuhfar T. Recent progress toward surface modification of bone/Dental implants with titanium and zirconia dioxide nanotubes fabrication of TiO2 nanotubes. J. Nanotechnol. Smart Mater. 2014;1:301–314.

Miron R.J., Bohner M., Zhang Y., Bosshardth D.D. Osteoinduction and osteoimmunology: Emerging concepts. Periodontology 2000. 2024;94:9–26. doi: 10.1111/prd.12519. PubMed DOI

Amani H., Alipour M., Shahrirari E., Taboas J.M. Immunomodulatory Biomaterials: Tailoring Surface Properties to Mitigate Foreign Body Reaction and Enhance Tissue Regeneration. Adv. Healthc. Mater. 2024;13:2401253. doi: 10.1002/adhm.202401253. PubMed DOI

Mendonca G., Mendonca D.B., Simoes L.G., Araujo A.L., Leite E.R., Duarte W.R., Aragao F.J.L., Cooper L.F. The effects of implant surface nanoscale features of osteoblast specific gene expression. Biomaterials. 2009;30:4053–4062. doi: 10.1016/j.biomaterials.2009.04.010. PubMed DOI

Shekaran A., Garcia A.J. Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tis-sue engineering. Biochem. Biophys. Acta. 2011;1810:350–360. doi: 10.1016/j.bbagen.2010.04.006. PubMed DOI PMC

Wu B., Tang Y., Wang K., Zhou X., Xiang L. Nanostructured Titanium Implant Surface Facilitating Osseointegration from Protein Adsorption to Osteogenesis: The Example of TiO2 NATs. Int. J. Nanomed. 2023;17:1865–1879. doi: 10.2147/IJN.S362720. PubMed DOI PMC

Berger M.B., Slosar P., Schwartz Z., Cohen D.J., Goodman S.B., Anderson P.A., Boyan B.D. A Review of Biomimetic Topographies and Their Role in Promoting Bone Formation and Osseointegration: Implications for Clinical Use. Biomimetics. 2022;7:46. doi: 10.3390/biomimetics7020046. PubMed DOI PMC

Skoog S.A., Kumar G., Narayan R.J., Goering P.L. Biological responses to immobilized microscale and nanoscale surface topographies. Pharmacol. Ther. 2018;182:33–55. doi: 10.1016/j.pharmthera.2017.07.009. PubMed DOI

Fadzil A.F.A., Pramanik A., Basak A.K., Prakash C., Shankar S. Role of surface quality on biocompatibility of implants—A review. Ann. 3D Print. Med. 2022;8:100082. doi: 10.1016/j.stlm.2022.100082. DOI

Soliman A.M., Barreda D.R. Acute Inflammation in Tissue Healing. Int. J. Mol. Sci. 2023;24:641. doi: 10.3390/ijms24010641. PubMed DOI PMC

Pang X., He X., Qiu Z., Zhang H., Xie R., Liu Z., Gu Y., Zhao N., Xiang Q., Cui Y. Targeting integrin pathways: Mechanism and advances in therapy. Signal Transduct. Target. Ther. 2023;8:1. doi: 10.1038/s41392-022-01259-6. PubMed DOI PMC

do Nascimento M., Brito T.O., Lima A.M., Elias C.N. Protein interactions with osseointegrable titanium implants. Braz. J. Oral Sci. 2023;22:e238749. doi: 10.20396/bjos.v22i00.8668749. DOI

Sun Z., Costell M., Fässler R. Integrin activation by talin, kindlin and mechanical forces. Nat. Cell Biol. 2019;21:25–31. doi: 10.1038/s41556-018-0234-9. PubMed DOI

Mu P., Li Y., Zhang Y. High-throughput screening of rat mesenchymal stem cell behavior on gradient TiO2 nanotubes. ACS Biomater. Sci. Eng. 2018;4:2804–2814. doi: 10.1021/acsbiomaterials.8b00488. PubMed DOI

Oh S., Brammer K.S., Li Y.S. Stem cell fate dictated solely by altered nanotube dimension. Proc. Natl. Acad. Sci. USA. 2009;106:2130–2135. doi: 10.1073/pnas.0813200106. PubMed DOI PMC

Biggs M.J., Richards R.G., Gadegarrd N., McMurray R.J., Affrossman S., Wilkinson C.D. Interactions with the nanoscale topography: Adhesion quantification and signal transduction in cells of osteogenic and multipotent lineage. J. Biomed. Mater. Res. A. 2009;91:195–208. doi: 10.1002/jbm.a.32196. PubMed DOI

Ocampo R.A., Echeverry-Rendon M., Robledo S., Echeverria F. Effect of TiO2 nanotubes size, heat treatment, and UV radiation on osteoblast behaviour. Mater. Chem. Phys. 2022;275:125137. doi: 10.1016/j.matchemphys.2021.125137. DOI

Zhang H., Cooper L.F., Zhang X., Zhang Y., Deng F., Song J., Yang S. Titanium nano-tubes induce osteogenic differenti-ation through the FAK/RhoA/YAP cascade. RSC Adv. 2016;6:44062–44069. doi: 10.1039/C6RA04002K. DOI

Kong K., Chang Y., Hu Y., Qiao H., Zhao C., Rong K., Zhang P., Zhang J., Zhai Z., Li H. TiO2 Nanotubes Promote Osteogenic Differentiation Through Regulation of Yap and Piezo1. Front. Bioeng. Biotechnol. 2022;10:872088. doi: 10.3389/fbioe.2022.872088. PubMed DOI PMC