Surface modification of ultrafine-grained titanium: Influence on mechanical properties, cytocompatibility, and osseointegration potential.

OBJECTIVE: The main objective of this study was to demonstrate that dental implants made from ultrafine-grain titanium (UFG-Ti) can be created that replicate state of the art surfaces of standard coarse-grain titanium (Ti), showing excellent cytocompatibility and osseointegration potential while also providing improved mechanical properties. MATERIAL AND METHODS: UFG-Ti was prepared by continuous equal channel angular processing (ECAP), and surfaces were treated by sandblasting and acid etching. Mechanical properties (tensile and fatigue strength), wettability, and roughness parameters were evaluated. Human trabecular bone-derived osteoblast precursor cells (HBCs) were cultured on all samples to examine cytocompatibility and mineralization after 4 and 28 days, respectively. Biomechanical pull-out measurements were performed in a rabbit in vivo model 4 weeks after implantation. RESULTS: Both yield and tensile strength as well as fatigue endurance were higher for UFG-Ti compared to Ti by 40%, 45%, and 34%, respectively. Fatigue endurance was slightly reduced following surface treatment. Existing surface treatment protocols could be applied to UFG-Ti and resulted in similar roughness and wettability as for standard Ti. Cell attachment and spreading were comparable on all samples, but mineralization was higher for the surfaces with hydrophilic treatment with no significant difference between UFG-Ti and Ti. Pull-out tests revealed that osseointegration of surface-treated UFG-Ti was found to be similar to that of surface-treated Ti. CONCLUSION: It could be demonstrated that existing surface treatments for Ti can be translated to UFG-Ti and, furthermore, that dental implants made from surface-treated UFG-Ti exhibit superior mechanical properties while maintaining cytocompatibility and osseointegration potential.

Chemically modified titanium-zirconium implants in comparison with commercially pure titanium controls stimulate the early molecular pathways of bone healing.

OBJECTIVES: Titanium-zirconium (TiZr) has been proposed as a mechanically stronger alternative to commercially pure titanium for oral and orthopaedic implants. However, not much is known on the osseointegration kinetics of TiZr surfaces. In this study, we aimed to identify the genetic response of bone around TiZr implants compared to pure Ti. MATERIAL AND METHODS: Microtextured and hydrophilic TiZr implants (tests) and cpTi implants grade IV (controls) were placed in the tibia of 30 New Zealand white rabbits. At 2, 4 and 12 weeks, the implants were subjected to removal torque test (RTQ). The expression of a panel of genes involved in the process of osseointegration was measured in the bone around the test and control implants by means of quantitative real-time polymerase chain reaction (PCR) and compared to the control samples. RESULTS: The controls yielded statistically significant higher RTQ at 4 weeks, but the RTQ of the tests had a larger increase between 4 and 12 weeks, when both groups reached similar values. The gene expression analysis showed that all selected markers for bone formation, bone remodelling and cytokines were significantly upregulated around TiZr implants after 2 weeks. After 4 weeks of healing, two bone formation markers were significantly more expressed in the test samples, while at 12 weeks, the expression of all genes was similar in the two groups. CONCLUSIONS: TiZr implants showed comparable biomechanical outcomes to cpTi up to 12 weeks of healing. However, at early healing stages, they showed a significant upregulation of osteogenesis and osteoclastogenesis markers.

Comparable responses of osteoblast lineage cells to microstructured hydrophilic titanium-zirconium and microstructured hydrophilic titanium.

OBJECTIVES: Although titanium (Ti) is commonly used for dental implants, Ti alloy materials are being developed to improve their physical material properties. Studies indicate that osteoblast differentiation and maturation of human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) respond to microstructured Ti and titanium-aluminum-vanadium (Ti6Al4V) surfaces in a similar manner. The goal of this study was to determine whether this is the case for osteoblast lineage cells grown on microstructured TiZr surfaces and whether their response is affected by surface nanotexture and hydrophilicity. MATERIALS AND METHODS: Grade 4 Ti and TiZr (13-17% Zr) disks were modified by large grit sand-blasting and acid-etching with storage in saline solution, resulting in a complex microstructured and hydrophilic surface corresponding to the commercially available implants SLActive® and Roxolid® SLActive® (Institut Straumann AG, Basel, Switzerland). The subsequent Ti modSLA and TiZr modSLA surfaces were characterized and osteogenic markers were measured. RESULTS: Evaluation of physical parameters revealed that the fabrication method was capable of inducing a microstructured and hydrophilic surface on both the Ti and TiZr disks. Overall, the surfaces were similar, but differences in nanostructure morphology/density and surface chemistry were detected. On Ti modSLA and TiZr modSLA, osteoblastic differentiation and maturation markers were enhanced in both MSCs and NHOsts, while inflammatory markers decreased compared with TCPS. CONCLUSIONS: These results indicate a similar positive cell response of MSCs and NHOsts when cultured on Ti modSLA and TiZr modSLA. Both surfaces were hydrophilic, indicating the importance of this property to osteoblast lineage cells.

Nanostructures and hydrophilicity influence osseointegration: a biomechanical study in the rabbit tibia.

OBJECTIVE: Implant surface properties have long been identified as an important factor to promote osseointegration. The importance of nanostructures and hydrophilicity has recently been discussed. The aim of this study was to investigate how nanostructures and wettability influence osseointegration and to identify whether the wettability, the nanostructure or both in combination play the key role in improved osseointegration. MATERIALS AND METHODS: Twenty-six adult rabbits each received two Ti grade 4 discs in each tibia. Four different types of surface modifications with different wettability and nanostructures were prepared: hydrophobic without nanostructures (SLA), with nanostructures (SLAnano); hydrophilic with two different nanostructure densities (low density: pmodSLA, high density: SLActive). All four groups were intended to have similar chemistry and microroughness. The surfaces were evaluated with contact angle measurements, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy and interferometry. After 4 and 8 weeks healing time, pull-out tests were performed. RESULTS: SLA and SLAnano were hydrophobic, whereas SLActive and pmodSLA were super-hydrophilic. No nanostructures were present on the SLA surface, but the three other surface modifications clearly showed the presence of nanostructures, although more sparsely distributed on pmodSLA. The hydrophobic samples showed higher carbon contamination levels compared with the hydrophilic samples. After 4 weeks healing time, SLActive implants showed the highest pull-out values, with significantly higher pull-out force than SLA and SLAnano. After 8 weeks, the SLActive implants had the highest pull-out force, significantly higher than SLAnano and SLA. CONCLUSIONS: The strongest bone response was achieved with a combination of wettability and the presence of nanostructures (SLActive).

Spontaneously formed nanostructures on titanium surfaces.

OBJECTIVES: The aim was to investigate the evolution of nanostructures on the SLActive surface, as a function of time, storage conditions, material dependence and to identify the step in which the reorganization of the outermost titanium oxide layer into well defined nanostructures takes place. MATERIAL AND METHODS: Titanium grade 2 discs were surface modified in seven different modes; (1) SLA (sand blasted, large grit, acid etched) protocol. (2) SLActive protocol (SLA stored in 0.9% NaCl solution), (3) SLActive, but stored in water instead of 0.9% NaCl solution, (4) pmod SLA: SLA discs subjected to oxygen plasma cleaning and stored in 0.9% NaCl solution, (5) SLAnano: SLActive discs aged for several months and then dried, (6) Mod A: same etching procedure and storage as for SLActive, but no sand blasting prior to etching, (7) pmod P: the discs were polished, oxygen plasma cleaned and stored in 0.9% NaCl solution. In addition TiZr alloy discs were prepared like the Ti SLActive samples. The surfaces were evaluated with SEM, interferometry, contact angle measurements and XPS. RESULTS: The samples stored dry were hydrophobic whereas the discs stored in liquid were hydrophilic. The evolution of nanostructures took 2 weeks, thereafter they were stable over time. The nanostructures occured after storage both in water and NaCl solution. Nanostructures were formed on Ti and TiZr although the morphology and distribution was quite different between the two materials. CONCLUSIONS: Acid etching in conjunction with storage in aqueous solution is responsible for the reorganization of the outermost titanium oxide layer into well defined nanostructures.

Assessing the osteogenic potential of zirconia and titanium surfaces with an advanced in vitro model.

OBJECTIVES: In recent years, zirconia dental implants have gained increased attention especially for patients with thin gingival biotypes or patients seeking metal-free restoration. While physical and chemical material surface properties govern the blood-material interaction and subsequent osseointegration processes, the organizational principles underlying the interplay of biochemical and biophysical cues are still not well understood. Therefore, this study investigated how the interaction of a microstructured zirconia surface with blood influences its osseointegration potential compared to microstructured titanium with or without additional nanostructures. METHODS: Microstructured zirconia and micro- (and nano)structured titanium surfaces were fabricated via sandblasting followed by acid etching and their topographical as well as physico-chemical features were thoroughly characterized. Following, an advanced in vitro approach mimicking the initial blood interaction of material surfaces upon implantation was applied. Fibrinogen adsorption, human blood coagulation as well as their influence on cell fate decisions of primary human bone and progenitor cells (HBC) were studied. RESULTS: Obtained surface micro- and nanostructures on titanium surfaces were sharp with rugged peaks whereas zirconia surfaces were less rough with structures being shallower, more round and granular. Compared to titanium surfaces, the zirconia surface showed increased fibrinogen adsorption, higher levels of total accessible fibrinogen γ-chain moieties yielding in increased platelet adhesion and activation and consequently thrombogenicity. Mineralization of HBC on microstructured surfaces was significantly higher on zirconia than on titanium, but was significantly lower compared to titanium surfaces with nanostructures. SIGNIFICANCE: This study provides insights into blood-material interaction and subsequent cellular events that are important for implant surface development.

Osteogenic response of human MSCs and osteoblasts to hydrophilic and hydrophobic nanostructured titanium implant surfaces.

Microstructured implant surfaces created by grit blasting and acid etching titanium (Ti) support osseointegration. This effect is further enhanced by storing in aqueous solution to retain hydrophilicity, but this also leads to surface nanostructure formation. The purpose of this study was to assess the contributions of nanostructures on the improved osteogenic response of osteoblast lineage cells to hydrophilic microstructured Ti. Human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) were cultured separately on non-nanostructured/hydrophobic (SLA), nanostructured/hydrophilic (modSLA), or nanostructured/hydrophobic (SLAnano) Ti surfaces. XPS showed elevated carbon levels on SLA and SLAnano compared to modSLA. Contact angle measurements indicated only modSLA was hydrophilic. Confocal laser microscopy revealed minor differences in mean surface roughness. SEM showed the presence of nanostructures on modSLA and SLAnano. MSCs and NHOst cells exhibited similar morphology on the substrates and osteoblastic differentiation and maturation were greatest on modSLA. These results suggest that when the appropriate microstructure is present, hydrophilicity may play a greater role in stimulating MSC and NHOst osteoblastic differentiation and maturation than the presence of nanostructures generated during storage in an aqueous environment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3137-3148, 2016.

Microstructure and mechanical properties of Ti-15Zr alloy used as dental implant material.

Ti-Zr alloys have recently started to receive a considerable amount of attention as promising materials for dental applications. This work compares mechanical properties of a new Ti-15Zr alloy to those of commercially pure titanium Grade4 in two surface conditions - machined and modified by sand-blasting and etching (SLA). As a result of significantly smaller grain size in the initial condition (1-2µm), the strength of Ti-15Zr alloy was found to be 10-15% higher than that of Grade4 titanium without reduction in the tensile elongation or compromising the fracture toughness. The fatigue endurance limit of the alloy was increased by around 30% (560MPa vs. 435MPa and 500MPa vs. 380MPa for machined and SLA-treated surfaces, respectively). Additional implant fatigue tests showed enhanced fatigue performance of Ti-15Zr over Ti-Grade4.

Proliferation, behavior, and differentiation of osteoblasts on surfaces of different microroughness.

OBJECTIVES: Titanium surface roughness is recognized as an important parameter influencing osseointegration. However, studies concerning the effect of well-defined surface topographies of titanium surfaces on osteoblasts have been limited in scope. In the present study we have investigated how Ti surfaces of different micrometer-scale roughness influence proliferation, migration, and differentiation of osteoblasts in-vitro. METHODS: Titanium replicas with surface roughnesses (Ra) of approximately 0, 1, 2, and 4µm were produced and MG-63 osteoblasts were cultured on these surfaces for up to 5 days. The effect of surface micrometer-scale roughness on proliferation, migration in time-lapse microscopy experiments, as well as the expression of alkaline phosphatase, osteocalcin, vascular-endothelial growth factor (VEGF), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated. RESULTS: Proliferation of MG-63 cells was found to decrease gradually with increasing surface roughness. However, the highest expression of alkaline phosphatase, osteocalcin and VEGF was observed on surfaces with Ra values of approximately 1 and 2µm. Further increase in surface roughness resulted in decreased expression of all investigated parameters. The cell migration speed measured in time-lapse microscopy experiments was significantly lower on surfaces with a Ra value of about 4µm, compared to those with lower roughness. No significant effect of surface roughness on the expression of OPG and RANKL was observed. SIGNIFICANCE: Thus, surfaces with intermediate Ra roughness values of 1-2µm seem to be optimal for osteoblast differentiation. Neither proliferation nor differentiation of osteoblasts appears to be supported by surfaces with higher or lower Ra values.

Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood.

Early and effective integration of a metal implant into bone tissue is of crucial importance for its long-term stability. While different material properties including surface roughness and wettability but also initial blood-implant surface interaction are known to influence this osseointegration, implications of the latter process are still poorly understood. In this study, early interaction between blood and the implant surface and how this affects the mechanism of osseointegration were investigated. For this, blood coagulation on a micro-roughened hydrophobic titanium (Ti) surface (SLA-H(phob)) and on a hydrophilic micro-roughened Ti surface with nanostructures (SLActive-H(phil)NS), as well as the effects of whole human blood pre-incubation of these two surfaces on the differentiation potential of primary human bone cells (HBC) was assessed. Interestingly, pre-incubation with blood resulted in a dense fibrin network over the entire surface on SLActive-H(phil)NS but only in single patches of fibrin and small isolated fibre complexes on SLA-H(phob). On SLActive-H(phil)NS, the number of HBCs attaching to the fibrin network was greatly increased and the cells displayed enhanced cell contact to the fibrin network. Notably, HBCs displayed increased expression of the osteogenic marker proteins alkaline phosphatase and collagen-I when cultivated on both surfaces upon blood pre-incubation. Additionally, blood pre-treatment promoted an earlier and enhanced mineralization of HBCs cultivated on SLActive-H(phil)NS compared to SLA-H(phob). The results presented in this study therefore suggest that blood pre-incubation of implant surfaces mimics a more physiological situation, eventually providing a more predictive in vitro model for the evaluation of novel bone implant surfaces.

Biomechanical and Histomorphometrical Evaluation of TiZr Alloy Implants: An in vivo Study in the Rabbit.

BACKGROUND: Clinically, there is a demand for mechanically stronger alloyed implants; however, not much evidence exists with regard to these materials. PURPOSE: To test the osseointegration property of TiZr1317 implants in a rabbit model. MATERIALS AND METHODS: Hydrophilic titanium-zirconium alloy (TiZr1317) implants with sand-blasted and acid-etched surface (test) and hydrophilic cpTi implants with the same treatment (control) were placed pairwise in the hind limbs (two in each tibia and one in each femur) of 36 Swedish lop-eared rabbits. After 2, 4, and 12 weeks (n = 12/time point), the bone samples were subjected to removal torque (RTQ, proximal tibia and femur) and histologic/histomorphometric (distal tibia) testings. RESULTS: The control presented significantly higher RTQ than the test at 2 weeks (55 vs 36 Ncm). No differences were observed for other time points. The test presented higher mean BIC than the control (19.25 vs 13.89 %) at 4 weeks; however, there were no statistical differences for the following time point tested in vivo.The new bone area was significantly higher for the test at 4 weeks in the marrow areas. CONCLUSION: The TiZr1317 implants presented comparable biologic outcomes to that of the cpTi implants through a 12-week evaluation period.

The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation.

Titanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.

Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC).

OBJECTIVES: Osteogenesis on titanium (Ti) surfaces is a complex process involving cell-substrate and cell-cell interaction of osteoblasts and endothelial cells. The aim of this study was to investigate the osteogenic properties of Ti surfaces on osteoblasts in the presence of endothelial cells (ECs). METHODS: Osteoblast-like cells (MG63 cells) and human umbilical vein endothelial cells (HUVECs) were grown in cocultures on four kinds of Ti surfaces: acid-etched (A), coarse-grit-blasted and acid-etched (SLA), hydrophilic A (modA) and hydrophilic SLA (modSLA) surfaces. MG63 cells in single cultures served as controls. Cell ratios and cell types in cocultures were determined and isolated using flow cytometry. Cell numbers were obtained by direct cell counting. In MG63 cells, alkaline phosphatase (ALP) activity was determined and protein levels of osteocalcin (OC) and osteoprotegerin (OPG) were detected with enzyme-linked immunosorbant assay (ELISA). The mRNA levels of ALP, OC and OPG of sorted MG63 cells were determined with real time polymerase chain reaction (PCR). RESULTS: MG63 cells proliferated in the presence of HUVECs, which showed higher cell numbers on Ti surfaces (A, SLA, modSLA) after 72h, and lower cell numbers on Ti surfaces (modA, SLA, modSLA) after 120h in comparison to single cultures. Protein and mRNA levels of ALP and OPG were higher in cocultures than in single cultures, while OC exhibited a lower expression. These three parameters were higher expressed on modA, SLA and modSLA surfaces compared to A surfaces. SIGNIFICANCE: Cocultures of osteoblasts and endothelial cells represent the most recently developed research model for investigating osteogenesis and angiogenesis which play both a major role in bone healing. This paper investigates for the first time the osteogenic properties of titanium surfaces used for dental implants with a coculture system with osteoblast-like cells and endothelial cells: (1) In cocultures with ECs (HUVECs) osteoblast-like cells (MG63 cells) show enhanced expression of early differentiation markers and osteogenic factors on Ti surfaces compared to single cultures of MG63 cells. (2) The differentiation and the expression of an osteogenic phenotype of osteoblast-like cells (MG63 cells) in coculture with ECs (HUVECs) is enhanced by both hydrophilicity and roughness of Ti surfaces.