On osseointegration in response to controlled surface nanotopography

Knowledge about the biological responses provoked by the surface modification of titanium implants on the nanoscale is still in its infancy. Although in vitro studies claim superior effects considering higher adhesion and proliferation of osteoblasts in the short term and even differentiation towards the osteogenic cell lineage in the long term, these responses do not necessarily reflect the actual outcome in the complex in vivo environment. Therefore, the main aim of this thesis was to evaluate the biological responses at the bone interface to titanium implants with controlled surface nanotopography. Both very early and late healing events were considered, and the phases of acute inflammation, bone regeneration and bone remodeling were evaluated, first in the rat tibia and thereafter in human maxillary bone. This was performed by screening and quantification of genes of interest, representing the different healing phases, by quantitative polymerase chain reaction (qPCR), and correlating these molecular events to morphological (histology and histomorphometry) and biomechanical (removal torque) outcomes of osseointegration. The first study used a specially designed implant with nanopatterns only at the cylindrical part facing the bone marrow and not the threads that were engaging the cortical bone. Analyses showed that the gene expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) and osteoclast marker cathepsin K (CatK) was downregulated at the nanopatterned implants at 3 and 6 days, respectively. This finding was consistent with fewer CD163-positive macrophages in the peri-implant tissue. Due to improved methodology, the nanopatterns could be applied to complex screw-shaped implants resembling clinical dental implants and used in the second, third and fourth studies. In the second study, evaluating the very early tissue-implant interactions, nanotopography downregulated the expression of monocyte chemoattractant protein-1 (MCP-1) at 12 hours and triggered the expression of osteocalcin (OC) at 3 days. This was in parallel with a relatively lower number of CD68-positive monocytes and a higher proportion of early-formed bone. In the third study, it was demonstrated that the nanotopography could downregulate the expression of the proinflammatory cytokine TNF-α even after 21 days. Osteoclastogenesis molecular activity was down-regulated at implants with combined nano- and microtopography at 6 days. A synergistic effect was disclosed, with the combination of micro- and nanotopography further attenuating the inflammatory response via TNF-α downregulation and resulting in an increased biomechanical stability, as judged by higher removal torque values. A human study showed that implants with nanotopography significantly increased the expression of all the targeted osteoblastic markers, namely, runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and OC, suggesting the promotion of bone formation. In conclusion, nanotopography per se, attenuates the initial inflammatory response and increases bone formation while down-regulating osteoclastogenesis and bone resorption molecular activities. Furthermore, the combined effect of micro- and nanotopography can further attenuate the inflammatory response and enhance the mechanical stability of the implants

Molecular Response to Nanopatterned Implants in the Human Jaw Bone

Implant surface modification by nanopatterning is an interesting route for enhancing osseointegration in humans. Herein, the molecular response to an intentional, controlled nanotopography pattern superimposed on screw-shaped titanium implants is investigated in human bone. When clinical implants are installed, additional two mini-implants, one with a machined surface (M) and one with a machined surface superimposed with a hemispherical nanopattern (MN), are installed in the posterior maxilla. In the second-stage surgery, after 6-8 weeks, the mini-implants are retrieved by unscrewing, and the implant-adherent cells are subjected to gene expression analysis using quantitative polymerase chain reaction (qPCR). Compared to those adherent to the machined (M) implants, the cells adherent to the nanopatterned (MN) implants demonstrate significant upregulation (1.8- to 2-fold) of bone-related genes (RUNX2, ALP, and OC). No significant differences are observed in the expression of the analyzed inflammatory and remodeling genes. Correlation analysis reveals that older patient age is associated with increased expression of proinflammatory cytokines (TNF-alpha and MCP-1) on the machined implants and decreased expression of proosteogenic factor (BMP-2) on the nanopatterned implants. Controlled nanotopography, in the form of hemispherical 60 nm protrusions, promotes gene expressions related to early osteogenic differentiation and osteoblastic activity in implant-adherent cells in the human jaw bone.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-02891]; BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy; Vastra Gotaland Region; Swedish government [ALFGBG-725641]; Swedish county councils, the ALF agreement [ALFGBG-725641]; TUA/Region Vastra Gotaland; Stiftelsen Handlanden Hjalmar Svensson; IngaBritt and Arne Lundberg Foundation; Eivind o Elsa K: son Sylvan Foundation; Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative</p

The role of well-defined nanotopography of titanium implants on osseointegration: cellular and&nbsp;molecular events in vivo

Dimitrios Karazisis,1&ndash;3 Ahmed M Ballo,1,2,4 Sarunas Petronis,2,5 Hossein Agheli,1,2 Lena Emanuelsson,1,2 Peter Thomsen,1,2 Omar Omar1,2 1Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 2BIOMATCELL, VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden; 3Department of Oral and Maxillofacial Surgery, Sahlgrenska Academy, University of Gothenburg, Sweden; 4Department of Oral Health Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada; 5Department of Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden, Bor&aring;s, Sweden Purpose: Mechanisms governing the cellular interactions with well-defined nanotopography are not well described in vivo. This is partly due to the difficulty in isolating a particular effect of nanotopography from other surface properties. This study employed colloidal lithography for nanofabrication on titanium implants in combination with an in vivo sampling procedure and different analytical techniques. The aim was to elucidate the effect of well-defined nanotopography on the molecular, cellular, and structural events of osseointegration.Materials and methods: Titanium implants were nanopatterned (Nano) with semispherical protrusions using colloidal lithography. Implants, with and without nanotopography, were implanted in rat tibia and retrieved after 3, 6, and 28 days. Retrieved implants were evaluated using quantitative polymerase chain reaction, histology, immunohistochemistry, and energy dispersive X-ray spectroscopy (EDS).Results: Surface characterization showed that the nanotopography was well defined in terms of shape (semispherical), size (79&plusmn;6 nm), and distribution (31&plusmn;2 particles/&micro;m2). EDS showed similar levels of titanium, oxygen, and carbon for test and control implants, confirming similar chemistry. The molecular analysis of the retrieved implants revealed that the expression levels of the inflammatory cytokine, TNF-&alpha;, and the osteoclastic marker, CatK, were reduced in cells adherent to the Nano implants. This was consistent with the observation of less CD163-positive macrophages in the tissue surrounding the Nano implant. Furthermore, periostin immunostaining was frequently detected around the Nano implant, indicating higher osteogenic activity. This was supported by the EDS analysis of the retrieved implants showing higher content of calcium and phosphate on the Nano implants.Conclusion: The results show that Nano implants elicit less periimplant macrophage infiltration and downregulate the early expression of inflammatory (TNF-&alpha;) and osteoclastic (CatK) genes. Immunostaining and elemental analyses show higher osteogenic activity at the Nano implant. It is concluded that an implant with the present range of well-defined nanocues attenuates the inflammatory response while enhancing mineralization during osseointegration. Keywords: nanofabrication, gene expression, immunohistochemistry, energy dispersive X-ray spectroscopy, inflammatory cytokines, bone formatio

The role of well-defined nanotopography of titanium implants on osseointegration: cellular and molecular events in vivo

Purpose: Mechanisms governing the cellular interactions with well-defined nanotopography are not well described in vivo. This is partly due to the difficulty in isolating a particular effect of nanotopography from other surface properties. This study employed colloidal lithography for nanofabrication on titanium implants in combination with an in vivo sampling procedure and different analytical techniques. The aim was to elucidate the effect of well-defined nanotopography on the molecular, cellular, and structural events of osseointegration. Materials and methods: Titanium implants were nanopatterned (Nano) with semispherical protrusions using colloidal lithography. Implants, with and without nanotopography, were implanted in rat tibia and retrieved after 3, 6, and 28 days. Retrieved implants were evaluated using quantitative polymerase chain reaction, histology, immunohistochemistry, and energy dispersive X-ray spectroscopy (EDS). Results: Surface characterization showed that the nanotopography was well defined in terms of shape (semispherical), size (79 +/- 6 nm), and distribution (31 +/- 2 particles/mu m(2)). EDS showed similar levels of titanium, oxygen, and carbon for test and control implants, confirming similar chemistry. The molecular analysis of the retrieved implants revealed that the expression levels of the inflammatory cytokine, TNF-alpha, and the osteoclastic marker, CatK, were reduced in cells adherent to the Nano implants. This was consistent with the observation of less CD163-positive macrophages in the tissue surrounding the Nano implant. Furthermore, periostin immunostaining was frequently detected around the Nano implant, indicating higher osteogenic activity. This was supported by the EDS analysis of the retrieved implants showing higher content of calcium and phosphate on the Nano implants. Conclusion: The results show that Nano implants elicit less periimplant macrophage infiltration and downregulate the early expression of inflammatory (TNF-alpha) and osteoclastic (CatK) genes. Immunostaining and elemental analyses show higher osteogenic activity at the Nano implant. It is concluded that an implant with the present range of well-defined nanocues attenuates the inflammatory response while enhancing mineralization during osseointegration

Influence of electrical parameters on morphology of nanostructured TiO 2

Ti6Al4V alloy micro rough surfaces with TiO2 self-organized nanostructured layers were synthesized using electrochemical anodization in phosphate/fluoride electrolyte, at different end potentials (5V, 10V, 15V, and 20 V). The current – time characteristics were recorded, and the link between current evolution and the morphology of developing oxide layers was investigated. On flat surfaces of Ti6Al4V alloy we developed TiO2 layers with different morphologies (random pores, nanopores of 25…50 nm, and highly organized nanotubes of 50…100 nm in diameter) depending on electrical parameters of anodization process. In our anodization cell, in optimized conditions, we are able to superimpose nanostructured oxide layers (nanotubular or nanoporous) over micro structured surfaces of titanium based materials used for biomedical implants

The effects of controlled nanotopography, machined topography and their combination on molecular activities, bone formation and biomechanical stability during osseointegration

The initial cellular and molecular activities at the bone interface of implants with controlled nanoscale topography and microscale roughness have previously been reported. However, the effects of such surface modifications on the development of osseointegration have not yet been determined. This study investigated the molecular events and the histological and biomechanical development of the bone interface in implants with nanoscale topography, microscale roughness or a combination of both. Polished and machined titanium implants with and without controlled nanopatterning (75 nm protrusions) were produced using colloidal lithography and coated with a thin titanium layer to unify the chemistry. The implants were inserted in rat tibiae and subjected to removal torque (RTQ) measurements, molecular analyses and histological analyses after 6, 21 and 28 days. The results showed that nanotopography superimposed on microrough, machined, surfaces promoted an early increase in RTQ and hence produced greater implant stability at 6 and 21 days. Two-way MANOVA revealed that the increased RTQ was influenced by microscale roughness and the combination of nanoscale and microscale topographies. Furthermore, increased bone-implant contact (BIC) was observed with the combined nanopatterned machined surface, although MANOVA results implied that the increased BIC was mainly dependent on microscale roughness. At the molecular level, the nanotopography, per se, and in synergy with microscale roughness, downregulated the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). In conclusion, controlled nanotopography superimposed on microrough machined implants promoted implant stability during osseointegration. Nanoscale-driven mechanisms may involve attenuation of the inflammatory response at the titanium implant site. Statement of Significance The role of combined implant microscale and nanotopography features for osseointegration is incompletely understood. Using colloidal lithography technique, we created an ordered nanotopography pattern superimposed on screwshaped implants with microscale topography. The midterm and late molecular, bone-implant contact and removal torque responses were analysed in vivo. Nanotopography superimposed on microrough, machined, surfaces promoted the implant stability, influenced by microscale topography and the combination of nanoscale and microscale topographies. Increased bone-implant contact was mainly dependent on microscale roughness whereas the nanotopography, per se, and in synergy with microscale roughness, attenuated the proinflammatory tumor necrosis factor alpha (TNF-alpha) expression. It is concluded that microscale and nanopatterns provide individual as well as synergistic effects on molecular, morphological and biomechanical implant-tissue processes in vivo. (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-02891]; BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy; Vastra Gotaland Region; Swedish government [ALFGBG725641]; Swedish county councils, the ALF agreement [ALFGBG725641]; TUA/Region Vastra Gotaland research grant; Stiftelsen Handlanden Hjalmar Svensson; IngaBritt and Arne Lundberg Foundation; Eivind o Elsa K: son Sylvan Foundation; Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative; Svenska Sallskapet for Medicinsk Forskning (SSMF)</p