Osteoblast differentiation on tissue culture polystyrene (TCPS) requires Wnt/beta-catenin signaling, regulating modulators of the Wnt pathway like Dickkopf-1 (Dkk1) and Dkk2. Osteoblast differentiation is increased on microstructured titanium (Ti) surfaces compared to TCPS; therefore, we hypothesized that surface topography and hydrophilicity affect Dkk1 and Dkk2 expression and that their roles in osteoblast differentiation on Ti differs depending on cell maturation state. Human osteoblast-like MG63 cells, normal human osteoblasts (HOBs), and human mesenchymal stem cells (MSCs), as well as MG63 cells stably silenced for Dkk1 or Dkk2 were grown for 6 days on TCPS and Ti surfaces (PT [Ra<0.2 μm], SLA [Ra = 4 μm], modSLA [hydrophilic-SLA]). Dkk1 and Dkk2 mRNA and protein increased on SLA and modSLA for all cell types, but exogenous rhDkk1 and rhDkk2 affected MSCs differently than MG63 cells and HOBs. Silencing Dkk1 reduced MG63 cell number on TCPS and PT, but increased differentiation on these substrates. Silencing Dkk2 reduced stimulatory effects of SLA and modSLA on osteoblast differentiation; Dkk2 but not Dkk1 restored these effects. Antibodies to Dkk1 or Dkk2 specifically blocked substrate-dependent changes caused by the proteins, demonstrating their autocrine action. This indicates major roles for Dkk1 and the canonical Wnt pathway in early-stage differentiation, and for Dkk2 and Wnt/Ca2+-dependent signaling in late-stage differentiation on microstructured and hydrophilic surfaces, during osseointegration.
Osseointegration; Titanium; Osteoblast; Mesenchymal stem cell; Surface roughness; Cell signaling
Multiple biomaterials are clinically available to spine surgeons for performing interbody fusion. Poly-ether-ether-ketone (PEEK) is used frequently for lumbar spine interbody fusion, but alternative materials are also used, including titanium (Ti) alloys. Previously, we showed that osteoblasts exhibit a more differentiated phenotype when grown on machined or grit-blasted titanium aluminum vanadium (Ti6Al4V) alloys with micron-scale roughened surfaces than when grown on smoother Ti6Al4V surfaces or on tissue culture polystyrene (TCPS). We hypothesized that osteoblasts cultured on rough Ti alloy substrates would present a more mature osteoblast phenotype than cells cultured on PEEK, suggesting that textured Ti6Al4V implants may provide a more osteogenic surface for interbody fusion devices.
The aim of the present study was to compare osteoblast response to smooth Ti6Al4V (sTiAlV) and roughened Ti6Al4V (rTiAlV) with their response to PEEK with respect to differentiation and production of factors associated with osteogenesis.
This in vitro study compared the phenotype of human MG63 osteoblast-like cells cultured on PEEK, sTiAlV, or rTiAlV surfaces and their production of bone morphogenetic proteins (BMPs).
Surface properties of PEEK, sTiAlV, and rTiAlV discs were determined. Human MG63 cells were grown on TCPS and the discs. Confluent cultures were harvested, and cell number, alkaline phosphatase–specific activity, and osteocalcin were measured as indicators of osteoblast maturation. Expression of messenger RNA (mRNA) for BMP2 and BMP4 was measured by real-time polymerase chain reaction. Levels of BMP2, BMP4, and BMP7 proteins were also measured in the conditioned media of the cell cultures.
Although roughness measurements for sTiAlV (Sa=0.09±0.01), PEEK (Sa=0.43±0.07), and rTiAlV (Sa= 1.81±0.51) varied, substrates had similar contact angles, indicating comparable wettability. Cell morphology differed depending on the surface. Cells cultured on Ti6Al4V had lower cell number and increased alkaline phosphatase specific activity, osteocalcin, BMP2, BMP4, and BMP7 levels in comparison to PEEK. In particular, roughness significantly increased the mRNA levels of BMP2 and BMP4 and secreted levels of BMP4.
These data demonstrate that rTiAlV substrates increase osteoblast maturation and produce an osteogenic environment that contains BMP2, BMP4, and BMP7. The results show that modifying surface structure is sufficient to create an osteogenic environment without addition of exogenous factors, which may induce better and faster bone during interbody fusion.
Ti6Al4V; PEEK; Osteoblast; BMP; Roughness
Surface roughness and surface free energy are two important factors that regulate cell responses to biomaterials. Previous studies established that titanium substrates with micron-scale and submicron scale topographies promote osteoblast differentiation and osteogenic local factor production and that there is a synergistic response to microrough Ti surfaces that have retained their high surface energy via processing that limits hydrocarbon contamination. This study tested the hypothesis that the synergistic response of osteoblasts to these modified surfaces depends on both surface microstructure and surface energy.
Ti disks were manufactured to present three different surface structures: smooth pretreatment surfaces (PT) with Ra of 0.2 µm; acid-etched surfaces (A) with a submicron roughness Ra of 0.83 µm; and sandblasted/acid-etched surfaces (SLA) with Ra of 3–4 µm. Modified acid-etched (modA) and modified sandblasted/acid-etched (modSLA) titanium substrates, which have low contamination and present a hydroxylated/hydrated surface layer to retain high surface energy, were compared with regular low surface energy A and SLA surfaces. Human osteoblast-like MG63 cells were cultured on these substrates and their responses, including cell shape, growth, differentiation (alkaline phosphatase, osteocalcin), and local factor production (TGF-β1, PGE2, osteoprotegerin [OPG]) were analyzed (N=6 per variable). Data were normalized to cell number.
There were no significant differences between smooth PT and A surfaces except for a small increase in OPG. Compared to A surfaces, MG63 cells produced 30% more osteocalcin on modA, and 70% more on SLA. However, growth on modSLA increased osteocalcin by more than 250%, which exceeded the sum of independent effects of surface energy and topography. Similar effects were noted when levels of latent TGF-β1, PGE2 and OPG were measured in the conditioned media.
The results demonstrate a synergistic effect between high surface energy and topography of Ti substrates and show that both micron scale and submicron scale structural features are necessary.
Titanium; Surface energy; Microstructure; Submicron roughness; Osteoblast differentiation
The microstructure and wettability of titanium (Ti) surfaces directly impact osteoblast differentiation in vitro and in vivo. These surface properties are important variables that control initial interactions of an implant with the physiological environment, potentially affecting osseointegration. The objective of this study was to use polyelectrolyte thin films to investigate how surface chemistry modulates response of human MG63 osteoblast-like cells to surface microstructure. Three polyelectrolytes, chitosan, poly(l-glutamic acid), and poly(l-lysine), were used to coat Ti substrates with two different microtopographies (PT, Sa = 0.37 µm and SLA, Sa = 2.54 µm). The polyelectrolyte coatings significantly increased wettability of PT and SLA without altering micron-scale roughness or morphology of the surface. Enhanced wettability of all coated PT surfaces was correlated with increased cell numbers whereas cell number was reduced on coated SLA surfaces. Alkaline phosphatase specific activity was increased on coated SLA surfaces than on uncoated SLA whereas no differences in enzyme activity were seen on coated PT compared to uncoated PT. Culture on chitosan-coated SLA enhanced osteocalcin and osteoprotegerin production. Integrin expression on smooth surfaces was sensitive to surface chemistry, but microtexture was the dominant variable in modulating integrin expression on SLA. These results suggest that surface wettability achieved using different thin films has a major role in regulating osteoblast response to Ti, but this is dependent on the microtexture of the substrate.
Wettability; Titanium; Surface roughness; Osteoblast
Interaction between implant surface and surrounding bone influences implant fixation. We attempted to improve the bone-implant interaction by 1) adding surface micro scale topography by acid etching, and 2) removing surface-adherent pro-inflammatory agents by plasma cleaning. Implant fixation was evaluated by implant osseointegration and biomechanical fixation.
The study consisted of two paired animal sub-studies where 10 skeletally mature Labrador dogs were used. Grit blasted titanium alloy implants were inserted press fit in each proximal tibia. In the first study grit blasted implants were compared with acid etched grit blasted implants. In the second study grit blasted implants were compared with acid etched grit blasted implants that were further treated with plasma sterilization. Implant performance was evaluated by histomorphometrical investigation (tissue-to-implant contact, peri-implant tissue density) and mechanical push-out testing after four weeks observation time.
Neither acid etching nor plasma sterilization of the grit blasted implants enhanced osseointegration or mechanical fixation in this press-fit canine implant model in a statistically significant manner.
Acid etching; biocompatibility; endotoxin; implant surgery; grit blasting; plasma sterilization; titanium.
Peri-implant bone formation depends on the ability of mesenchymal cells to colonize the implant surface and differentiate into osteoblasts. Human mesenchymal stem cells (HMSCs) undergo osteoblastic differentiation on microstructured titanium (Ti) surfaces in the absence of exogenous factors, but the mechanisms are unknown. Wnt proteins are associated with an osteoblast phenotype, but how Wnt signaling regulates HMSC differentiation on microstructured Ti surfaces is not known. HMSCs were cultured on tissue culture polystyrene or Ti (PT [Sa=0.33μm, θ=96°], SLA [Sa=2.5μm, θ=132°], modSLA [hydrophilic-SLA]). Expression of calcium-dependent Wnt ligand WNT5A increased and canonical Wnt pathway ligands decreased on microstructured Ti in a time-dependent manner. Treatment of HMSCs with canonical ligand Wnt3a preserved the mesenchymal phenotype on smooth surfaces. Treatment with Wnt5a increased osteoblastic differentiation. Expression of integrins ITGA1, ITGA2, and ITGAV increased over time and correlated with increased WNT5A expression. Treatment of HMSCs with Wnt5a, but not Wnt3a, increased integrin expression. Regulation of integrin expression due to surface roughness and energy was ablated in WNT5A-knockdown HMSCs. This indicates that surface properties regulate stem cell fate and induce osteoblast differentiation via the Wnt calcium-dependent pathway. Wnt5a enhances osteogenesis through a positive feedback with integrins and local factor regulation, particularly though BMP signaling.
Cell signaling; Surface roughness; Titanium; Stem cell; Growth factors
Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Previous researches proved that chemical surface modification of titanium implants could be used to improve Bone-to-implant contact. In this study, the surface topography, chemistry and biocompatibility of polished titanium surfaces treated with mixed solution of three acids containing HCl, HF and H3PO4 with different etched conditions for example concentration, time and addition of calcium chloride were studied. Osteoblast cells (MG-63) were cultured on different groups of titanium surfaces. In order to investigate titanium surfaces, SEM, AFM and EDS analyses were carried out. The results showed that surfaces treated with HCl–HF–H3PO4 had higher roughness, lower cytotoxicity level and better biocompatibility than controls. Moreover, addition of calcium chloride into mixed solution of three acids containing HCl, HF and H3PO4 is an important, predominant and new technique for obtaining biofunction in metals for biomedical use including dentistry.
The purpose of this study was to characterize the osseointegration of the fibronectin-coated implant surface.
Sand-blasted, large-grit, acid-etched (SLA) surface implants, with or without a thin calcium phosphate and fibronectin coating, were placed in edentulous mandibles of dogs 8 weeks after extraction. All dogs were sacrificed forhistological and histomorphometric evaluation after 4- and 8-week healing periods.
All types of implants were clinically stable without any mobility. Although the bone-to-implant contact and bone density of the SLA implants coated with calcium phosphate (CaP)/fibronectin were lower than the uncoated SLA implants, there were no significant differences between the uncoated SLA surface group and the SLA surface coated with CaP/fibronectin group.
Within the limits of this study, SLA surfaces coated with CaP/fibronectin were shown to have comparable bone-to-implant contact and bone density to uncoated SLA surfaces.
Biocompatible coated materials; Bone density; Calcium phosphate; Dental implants; Fibronectins
Microtexture and chemistry of implant surfaces are important variables for modulating cellular responses. Surface chemistry and wettability are connected directly. While each of these surface properties can influence cell response, it is difficult to decouple their specific contributions. To address this problem, the aims of this study were to develop a surface wettability gradient with a specific chemistry without altering micron scale roughness and to investigate the role of surface wettability on osteoblast response. Microtextured sandblasted/acid-etched (SLA, Sa = 3.1 μm) titanium disks were treated with oxygen plasma to increase reactive oxygen density on the surface. At 0, 2, 6, 10, and 24 h after removing them from the plasma, the surfaces were coated with chitosan for 30 min, rinsed and dried. Modified SLA surfaces are denoted as SLA/h in air prior to coating. Surface characterization demonstrated that this process yielded differing wettability (SLA0 < SLA2 < SLA10 < SLA24) without modifying the micron scale features of the surface. Cell number was reduced in a wettability-dependent manner, except for the most water-wettable surface, SLA24. There was no difference in alkaline phosphatase activity with differing wettability. Increased wettability yielded increased osteocalcin and osteoprotegerin production, except on the SLA24 surfaces. mRNA for integrins α1, α2, α5, β1, and β3 was sensitive to surface wettability. However, surface wettability did not affect mRNA levels for integrin α3. Silencing β1 increased cell number with reduced osteocalcin and osteoprotegerin in a wettability-dependent manner. Surface wettability as a primary regulator enhanced osteoblast differentiation, but integrin expression and silencing β1 results indicate that surface wettability regulates osteoblast through differential integrin expression profiles than microtexture does. The results may indicate that both microtexture and wettability with a specific chemistry have important regulatory effects on osseointegration. Each property had different effects, which were mediated by different integrin receptors.
Wettability; Oxygen plasma; Chitosan; Titanium; Osteoblast; Integrin
Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.2μm), microstructured sandblasted/acid etched surface (SLA, Ra=3-4μm), and a microstructured Ti plasma-sprayed surface (TPS, Ra=4μm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-β1, PKC, and α2β1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-β1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-β1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-β type II receptor or by reducing TGF-β1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of α2 or a double knockdown of α2β1 also reduced OPG, as well as production of TGF-β1. These results indicate that substrate dependent OPG production is regulated by TGF-β1, PKC, and α2β1 and suggest a mechanism by which α2β1-signaling increases PKC, resulting in TGF-β1 production and TGF-β1 then acts on its receptor to increase transcription of OPG.
Osteoblast; TGF-β1; Osteoprotegerin; Titanium; Microtopography
Titanium (Ti) osseointegration is critical for the success of dental and orthopaedic 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.
(4 to 6) nanotopography; titanium oxide; surface roughness; titanium; bone; implant; osteoblasts
Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces. Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA. Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.
Titanium; microstructure; surface energy; osteoblast; angiogenesis; VEGF
Microstructured and high surface energy titanium substrates increase osseointegration in vivo. In vitro, osteoblast differentiation is increased, but effects of the surface directly on multipotent mesenchymal stem cells (MSCs) and consequences for MSCs in the peri-implant environment are not known. We evaluated responses of human MSCs to substrate surface properties and examined the underlying mechanisms involved. MSCs exhibited osteoblast characteristics (alkaline phosphatase, RUNX2, and osteocalcin) when grown on microstructured Ti; this effect was more robust with increased hydrophilicity. Factors produced by osteoblasts grown on microstructured Ti were sufficient to induce co-cultured MSC differentiation to osteoblasts. Silencing studies showed that this was due to signaling via α2β1 integrins in osteoblasts on the substrate surface and paracrine action of secreted Dkk2. Thus, human MSCs are sensitive to substrate properties that induce osteoblastic differentiation; osteoblasts interact with these surface properties via α2β1 and secrete Dkk2, which acts on distal MSCs.
STATEMENT OF PROBLEM
Macroscopic and especially microscopic properties of implant surfaces play a major role in the osseous healing of dental implants. Dental implants with modified surfaces have shown stronger osseointegration than implants which are only turned (machined). Advanced surface modification techniques such as anodic oxidation and Ca-P application have been developed to achieve faster and stronger bonding between the host bone and the implant.
The purpose of this study was to investigate the effect of surface treatment of titanium dental implant on implant stability after insertion using the rabbit tibia model.
MATERIAL AND METHODS
Three test groups were prepared: sandblasted, large-grit and acid-etched (SLA) implants, anodic oxidized implants, and anodized implants with Ca-P immersion. The turned implants served as control. Twenty rabbits received 80 implants in the tibia. Resonance frequencies were measured at the time of implant insertion, 2 weeks and 4 weeks of healing. Removal torque values (RTV) were measured 2 and 4 weeks after insertion.
The implant stability quotient (ISQ) values of implants for resonance frequency analysis (RFA) increased significantly (P < .05) during 2 weeks of healing period although there were no significant differences among the test and control groups (P > .05). The test and control implants also showed significantly higher ISQ values during 4 weeks of healing period (P < .05). No significant differences, however, were found among all the groups. All the groups showed no significant differences in ISQ values between 2 and 4 weeks after implant insertion (P > .05). The SLA, anodized and Ca-P immersed implants showed higher RTVs at 2 and 4 weeks of healing than the machined one (P < .05). However, there was no significant difference among the experimental groups.
The surface-modified implants appear to provide superior implant stability to the turned one. Under the limitation of this study, however, we suggest that neither anodic oxidation nor Ca-P immersion techniques have any advantage over the conventional SLA technique with respect to implant stability.
surface treatment; bone to implant contact; removal torque; dental implant
Bioactivity and osteoconductivity of titanium degrade over time after surface processing. This time-dependent degradation is substantial and defined as the biological aging of titanium. UV treatment has shown to reactivate the aged surfaces, a process known as photofunctionalization. This study determined whether there is a difference in the behavior of biological aging for titanium with micro-nano-hybrid topography and titanium with microtopography alone, following functionalization. Titanium disks were acid etched to create micropits on the surface. Micro-nano-hybrid surfaces were created by depositioning 300-nm diameter TiO2 nodules onto the micropits using a previously established self-assembly protocol. These disks were stored for 8 weeks in the dark to allow sufficient aging, then treated with UV light for 48 hours. Rat bone marrow–derived osteoblasts were cultured on fresh disks (immediately after UV treatment), 3-day-old disks (disks stored for 3 days after UV treatment), and 7-day- old disks. The rates of cell attachment, spread, proliferation, and levels of alkaline phosphatase activity, and calcium deposition were reduced by 30%–50% on micropit surfaces, depending on the age of the titanium. In contrast, 7-day-old hybrid surfaces maintained equivalent levels of bioactivity compared with the fresh surfaces. Both micropit and micro-nano-hybrid surfaces were superhydrophilic immediately after UV treatment. However, after 7 days, the micro-nano- hybrid surfaces became hydrorepellent, while the micropit surfaces remained hydrophilic. The sustained bioactivity levels of the micro-nano-hybrid surfaces were nullified by treating these surfaces with Cl−anions. A thin TiO2 coating on the micropit surface without the formation of nanonodules did not result in the prevention or alleviation of the time-dependent decrease in biological activity. In conclusion, the micro-nano-hybrid titanium surfaces may slow the rate of time-dependent degradation of titanium bioactivity after UV photofunctionalization compared with titanium surfaces with microtopography alone. This antibiological aging effect was largely regulated by its sustained electropositivity uniquely conferred in TiO2 nanonodules, and was independent of the degree of hydrophilicity. These results demonstrate the potential usefulness of these hybrid surfaces to effectively utilize the benefits of UV photofunctionalization and provide a model to explore the mechanisms underlying antibiological aging properties.
bone–titanium integration; nanonodule; super osseointegration; dental and orthopedic implants; nanotechnology
The present study was performed to evaluate the effect of erbium-doped: yttrium, aluminium and garnet (Er:YAG) laser irradiation on sand-blasted, large grit, acid-etched (SLA) implant surface microstructure according to varying energy levels and application times of the laser.
The implant surface was irradiated by the Er:YAG laser under combined conditions of 100, 140, or 180 mJ/pulse and an application time of 1 minute, 1.5 minutes, or 2 minutes. Scanning electron microscopy (SEM) was used to examine the surface roughness of the specimens.
All experimental conditions of Er:YAG laser irradiation, except the power setting of 100 mJ/pulse for 1 minute and 1.5 minutes, led to an alteration in the implant surface. SEM evaluation showed a decrease in the surface roughness of the implants. However, the difference was not statistically significant. Alterations of implant surfaces included meltdown and flattening. More extensive alterations were present with increasing laser energy and application time.
To ensure no damage to their surfaces, it is recommended that SLA implants be irradiated with an Er:YAG laser below 100 mJ/pulse and 1.5 minutes for detoxifying the implant surfaces.
Dental implants; Lasers
Our study was designed to evaluate osseointegration among implants with three surface treatments: plasma-sprayed titanium (P), plasma-sprayed titanium with hydroxyapatite (PHA), and chemical-textured titanium with hydroxyapatite (CHA). Average surface roughness (Ra) was 27 microns for the P group, 17 microns for the PHA group, and 26 microns for the CHA group. Bilateral distal intramedullary implants were placed in the femora of thirty rabbits. Histomorphometry of scanning electron microscopy images was used to analyze the amount of bone around the implants at 6 and 12 weeks after implantation. Greater amounts of osseointegration were observed in the hydroxyapatite-coated groups than in the noncoated group. For all implant surfaces, osseointegration was greater at the diaphyseal level compared to the metaphyseal level. No significant differences were seen in osseointegration between the 6 and 12 week time points. Although the average surface roughness of the P and the CHA groups was similar, osseointegration of the CHA implants was significantly greater. The results of this in vivo lapine study suggest that the presence of an hydroxyapatite coating enhances osseointegration despite similarities in average surface roughness.
Background and methods
Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells.
The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells.
This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.
micro/nanotexture; nanorod; titanium surface; bone marrow mesenchymal stem cells; adhesion; osteogenic differentiation
Microarc oxidation (MAO) is a surface treatment that provides nanoporous pits, and thick oxide layers, and incorporates calcium and phosphorus into the coating layer of titanium alloy. We presumed such modification on the surface of titanium alloy by MAO would improve the ability of cementless stems to osseointegrate. We therefore compared the in vitro ability of cells to adhere to MAOed titanium alloy to that of two different types of surface modifications: machined and grit-blasted. We performed energy-dispersive x-ray spectroscopy and scanned electron microscopy investigations to assess the structure and morphology of the surfaces. Biologic and morphologic responses to osteoblast cell lines (SaOS-2) were then examined by measuring cell proliferation, cell differentiation (alkaline phosphatase activity), and αvβ3 integrin. The cell proliferation rate, alkaline phosphatase activity, and cell adhesion in the MAO group increased in comparison to those in the machined and grit-blasted groups. The osteoblast cell lines of the MAO group were also homogeneously spread on the surface, strongly adhered, and well differentiated when compared to the other groups. This method could be a reasonable option for treating the surfaces of titanium alloy for better osseointegration.
Background: The goal of this study was to evaluate the behavior of neonatal rat calvarial osteoblast-like cells cultured on different implant surfaces and exposed once or three times to a 660-nm light-emitting diode (LED).
Methods: An LED with a 660-nm wavelength was applied once or three times to cultured cells on standard and modified sandblasted acid-etched surfaces (SLA and SLActive; Straumann, Basel, Switzerland). To analyze the effect of the LED on cell proliferation, numbers, and viability, cells were cultured on titanium discs, and measurements were taken after 72 h. Cell proliferation rates were assessed using a bromodeoxyuridine immunohistochemical technique. Cell morphologies were evaluated by scanning electron microscopy (SEM).
Results: Osteoblast-like cells proliferated on all tested surfaces, with differences among groups in cell counts and DNA synthesis values. The application of one LED treatment caused a significant increase in cell count in the SLActive group in comparison with the SLA group (p = 0.001), whereas the application of three LED treatments caused a significant decrease in cell count in the SLA group compared with the SLActive group (p < 0.001). After 72 h, the number of cells was highest in the SLActive group exposed once to the LED.
Conclusions: One LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak attachments between cells and to the surface. Thus, further studies using different LED application times are needed to clarify the reason for the increased number of cells that are apparently incapable of attaching to the titanium surfaces after 72 h.
Light-emitting diode; SLA and SLActive surface; primary osteoblast cell culture
Osseointegration is crucial for the long-term success of dental implants and depends on the tissue reaction at the tissue-implant interface. Mechanical properties and biocompatibility make zirconia a suitable material for dental implants, although surface processings are still problematic. The aim of the present study was to compare osteoblast behavior on structured zirconia and titanium surfaces under standardized conditions.
The surface characteristics were determined by scanning electron microscopy (SEM). In primary bovine osteoblasts attachment kinetics, proliferation rate and synthesis of bone-associated proteins were tested on different surfaces.
The results demonstrated that the proliferation rate of cells was significantly higher on zirconia surfaces than on titanium surfaces (p < 0.05; Student's t-test). In contrast, attachment and adhesion strength of the primary cells was significant higher on titanium surfaces (p < 0.05; U test). No significant differences were found in the synthesis of bone-specific proteins. Ultrastructural analysis revealed phenotypic features of osteoblast-like cells on both zirconia and titanium surfaces.
The study demonstrates distinct effects of the surface composition on osteoblasts in culture. Zirconia improves cell proliferation significantly during the first days of culture, but it does not improve attachment and adhesion strength. Both materials do not differ with respect to protein synthesis or ultrastructural appearance of osteoblasts. Zirconium oxide may therefore be a suitable material for dental implants.
Pure titanium is the material of choice for contemporary dental implants. However, superficial reaction of the moderately rough titanium surface with atmospheric components decreases its hydrophilicity. INICELL® represents a chemical alteration and hydrophilization of a moderately rough i. e. sand-blasted and acid-etched titanium surface. The hydrophilicity leads to a more homogenous adsorption of proteins on the implant surface in-vitro, supporting the activation of a higher number of platelets and the generation of a homogenous, complete fibrin matrix in the early phases of osseointegration. This in turn helps to reduce the healing time and enhances the predictability of osseointegration in compromised bony situations.
The objective of this case series trial was therefore to investigate if early loading (after 8 weeks) of hydrophilic INICELL implants is feasible in patients with reduced bone quality.
In 10 patients, 35 hydrophilic implants were placed in sites revealing bone quality class 3 and 4, and uncovered after 4 weeks. Eight weeks later implants were released for loading if the tactile resistance was ≥35 Ncm. Lower resistances resulted in 12 weeks initial healing period. Insertion torque, ISQ, tactile resistance and vertical bone level were evaluated at implant installation, after 4 weeks (uncovering), 8 or 12 weeks (loading), and 12 weeks and one year after loading.
Mean implant insertion torque was 21 Ncm. 31 (88.6%) showed a tactile resistance of >35 Ncm after eight weeks and were released for prosthetic loading. Eight weeks after insertion, one implant (2.9%) had to be removed following a soft tissue complication. One implant had to be removed after 4 weeks due to a technical complication (fractured Osstell-abutment), it was therefore excluded from the analysis.
33 of 34 implants (97%) were loaded to occlusion and were in situ/functional one year after implantation. ISQs increased from 43 at baseline to 63 at eight weeks, and 72 at three months after loading. Then, ISQ remained constant until one year after loading.
Within the limitations of this prospective case series, hydrophilic implants may allow for shortening of the initial healing period even in bone with compromised density.
Titanium implants; Hydrophilic surface; Healing time; Bone quality; Weak bone
Stainless steel is one of the most widely used biomaterials for internal fixation devices, but is not used in cementless arthroplasty implants because a stable oxide layer essential for biocompatibility cannot be formed on the surface. We applied a Ti electron beam coating, to form oxide layer on the stainless steel surface. To form a thicker oxide layer, we used a microarc oxidation process on the surface of Ti coated stainless steel. Modification of the surface using Ti electron beam coating and microarc oxidation could improve the ability of stainless steel implants to osseointegrate.
The ability of cells to adhere to grit-blasted, titanium-coated, microarc-oxidated stainless steel in vitro was compared with that of two different types of surface modifications, machined and titanium-coated, and microarc-oxidated.
We performed energy-dispersive x-ray spectroscopy and scanning electron microscopy investigations to assess the chemical composition and structure of the stainless steel surfaces and cell morphology. The biologic responses of an osteoblastlike cell line (SaOS-2) were examined by measuring proliferation (cell proliferation assay), differentiation (alkaline phosphatase activity), and attraction ability (cell migration assay).
Cell proliferation, alkaline phosphatase activity, migration, and adhesion were increased in the grit-blasted, titanium-coated, microarc-oxidated group compared to the two other groups. Osteoblastlike cells on the grit-blasted, titanium-coated, microarc-oxidated surface were strongly adhered, and proliferated well compared to those on the other surfaces.
The surface modifications we used (grit blasting, titanium coating, microarc oxidation) enhanced the biocompatibility (proliferation and migration of osteoblastlike cells) of stainless steel.
This process is not unique to stainless steel; it can be applied to many metals to improve their biocompatibility, thus allowing a broad range of materials to be used for cementless implants.
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of fibronectin-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically-translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed RGD peptides, serum proteins, and full-length FN. Differences in biological responses correlated with integrin binding specificity and signaling among surface coatings. This work validates a simple, clinically-translatable, surface biofunctionalization strategy to enhance biomedical device integration.
fibronectin; osseointegration; coating; integrins; biomimetic; implant
Competition occurs between the osteoblasts in regional microenvironments and pathogens introduced during surgery, on the surface of bone implants, such as joint prostheses. The aim of this study was to modulate bacterial and osteoblast adhesion on implant surfaces by using a nanotube array. Titanium oxide (TiO2) nanotube arrays, 30 nm or 80 nm in diameter, were prepared by a two-step anodization on titanium substrates. Mechanically polished and acid-etched titanium samples were also prepared to serve as control groups. The standard strains of Staphylococcus epidermidis (S. epidermidis, American Type Culture Collection [ATCC]35984) and mouse C3H10T1/2 cell lines with osteogenic potential were used to evaluate the different responses to the nanotube arrays, in bacteria and eukaryotic cells. We found that the initial adhesion and colonization of S. epidermidis on the surface of the TiO2 nanotube arrays were significantly reduced and that the adhesion of C3H10T1/2 cells on the surface of the TiO2 nanotube arrays was significantly enhanced when compared with the control samples. Based on a surface analysis of all four groups, we observed increased surface roughness, decreased water contact angles, and an enhanced concentration of oxygen and fluorine atoms on the TiO2 nanotube surface. We conclude that the TiO2 nanotube surface can reduce bacterial colonization and enhance C3H10T1/2 cell adhesion; multiple physical and chemical properties of the TiO2 nanotube surface may contribute to these dual effects.
bacterial adhesion; titanium implant; surface modification