Osteoblast response and osseointegration of a Ti–6Al–4V alloy implant incorporating strontium

This study investigated the surface characteristics, in vitro and in vivo biocompatibility of Ti–6Al–4V alloy implants incorporating strontium ions (Sr), produced by hydrothermal treatment using a Sr-containing solution, for future biomedical applications. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, contact angle and surface energy measurement and inductively coupled plasma-mass spectroscopy (ICP-MS). Human osteoblast-like cell (MG63) attachment, proliferation, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on Sr-containing Ti–6Al–4V surfaces were compared with untreated Ti–6Al–4V surfaces. Fifty-six screw implants (28 control and 28 experimental) were placed in the tibiae and femoral condyles of seven New Zealand White rabbits. The osteoconductivity of Sr-containing Ti–6Al–4V implants was evaluated by removal torque testing and histomorphometric analysis after 4 weeks implantation. Hydrothermal treatment produced a crystalline SrTiO₃ layer. ICP-MS analysis showed that Sr ions were released from treated surfaces into the solution. Significant increases in ALP activity (P = 0.000), mRNA expressions of key osteoblast genes (osterix, bone sialoprotein, and osteocalcin), removal torque values (P < 0.05) and bone–implant contact percentages (P < 0.05) in both cortical and cancellous bone were observed for Sr-containing Ti–6Al–4V surfaces. The results indicate that the Sr-containing oxide layer produced by hydrothermal treatment may be effective in improving the osseointegration of Ti–6Al–4V alloy implants by enhancing differentiation of osteoblastic cells, removal torque forces and bone apposition in both cortical and cancellous bone.     

Osteoconductivity of hydrophilic microstructured titanium implants with phosphate ion chemistry

This study investigated the surface characteristics and bone response of titanium implants produced by hydrothermal treatment using H₃PO₄, and compared them with those of implants produced by commercial surface treatment methods – machining, acid etching, grit blasting, grit blasting/acid etching or spark anodization. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, contact angle measurement and stylus profilometry. The osteoconductivity of experimental implants was evaluated by removal torque testing and histomorphometric analysis after 6 weeks of implantation in rabbit tibiae. Hydrothermal treatment with H₃PO₄ and subsequent heat treatment produced a crystalline phosphate ion-incorporated oxide (titanium oxide phosphate hydrate, Ti₂O(PO₄)₂(H₂O)₂; TiP) surface approximately 5 μm in thickness, which had needle-like surface microstructures and superior wettability compared with the control surfaces. Significant increases in removal torque forces and bone-to-implant contact values were observed for TiP implants compared with those of the control implants (p < 0.001). After thorough cleaning of the implants removed during the removal torque testing, a considerable quantity of attached bone was observed on the surfaces of the TiP implants.     

UV photoactivation of 7-dehydrocholesterol on titanium implants enhances osteoblast differentiation and decreases Rankl gene expression

Vitamin D plays a central role in bone regeneration, and its insufficiency has been reported to have profound negative effects on implant osseointegration. The present study aimed to test the in vitro biological effect of titanium (Ti) implants coated with UV-activated 7-dehydrocholesterol (7-DHC), the precursor of vitamin D, on cytotoxicity and osteoblast differentiation. Fourier transform infrared spectroscopy confirmed the changes in chemical structure of 7-DHC after UV exposure. High-pressure liquid chromatography analysis determined a 16.5 ± 0.9% conversion of 7-DHC to previtamin D₃ after 15 min of UV exposure, and a 34.2 ± 4.8% of the preD₃ produced was finally converted to 25-hydroxyvitamin D₃ (25-D₃) by the osteoblastic cells. No cytotoxic effect was found for Ti implants treated with 7-DHC and UV-irradiated. Moreover, Ti implants treated with 7-DHC and UV-irradiated for 15 min showed increased 25-D₃ production, together with increased ALP activity and calcium content. Interestingly, Rankl gene expression was significantly reduced in osteoblasts cultured on 7-DHC-coated Ti surfaces when UV-irradiated for 15 and 30 min to 33.56 ± 15.28% and 28.21 ± 4.40%, respectively, compared with the control. In conclusion, these findings demonstrate that UV-activated 7-DHC is a biocompatible coating of Ti implants, which allows the osteoblastic cells to produce themselves active vitamin D, with demonstrated positive effects on osteoblast differentiation in vitro.     

Enhanced osteoblast response to an equal channel angular pressing-processed pure titanium substrate with microrough surface topography

This study investigated the surface characteristics and in vitro biocompatibility of ultrafine-grain pure titanium substrates produced by equal channel angular pressing (ECAP) using MC3T3-E1 pre-osteoblast cells, compared with those of conventional coarse-grain pure titanium (CP) and Ti–6Al–4V (Ti64) substrates. All Ti surfaces were grit-blasted with hydroxyapatite particles to produce microrough surfaces. The surface characteristics were evaluated by electron back-scattered diffractometry, scanning electron microscopy, contact angle and surface energy measurements, and optical profilometry. The morphology of spread cells, cell attachment, viability, alkaline phosphatase (ALP) activity, quantitative analysis of osteoblastic gene expression and mineralization nodule formation on different surfaces were evaluated. ECAP-processed substrates showed a significantly lower water contact angle and higher surface energy compared with coarse-grain CP and Ti64 substrates (p < 0.05). They also showed enhanced cell spreading, attachment, viability and ALP activity compared with the CP and Ti64 surfaces (p < 0.05). Real-time polymerase chain reaction analysis showed notably higher ALP, osteopontin and osteocalcin mRNA levels in cells grown on the ECAP surfaces than on the CP and Ti64 surfaces, and the ECAP surfaces showed significantly greater mineralization nodule formation compared with the CP and Ti64 substrates (p < 0.05). These results demonstrate the superior osteoblast cell compatibility of microroughened Ti surface made of ECAP-processed ultrafine-grain pure Ti substrates over coarse-grain pure Ti and Ti64 substrates.     

Phase composition,mechanical performance and in vitro biocompatibility of hydraulic setting calcium magnesium phosphate cement

Brushite (CaHPO₄·2H₂O)-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. However, their short setting times and low mechanical strengths limit broad clinical application. In this study, we showed that a significant improvement of these properties of brushite cement could be achieved by the use of magnesium-substituted β-tricalcium phosphate with the general formula Mg_xCa_(3–x)(PO₄)₂ with 0 < x < 3 as cement reactants. The incorporation of magnesium ions increased the setting times of cements from 2 min for a magnesium-free matrix to 8–11 min for Mg_2.25Ca_0.75(PO₄)₂ as reactant. At the same time, the compressive strength of set cements was doubled from 19 MPa to more than 40 MPa after 24 h wet storage. Magnesium ions were not only retarding the setting reaction to brushite but were also forming newberyite (MgHPO₄·3H₂O) as a second setting product. The biocompatibility of the material was investigated in vitro using the osteoblast-like cell line MC3T3-E1. A considerable increase of cell proliferation and expression of alkaline phosphatase, indicating an osteoblastic differentiation, could be noticed. Scanning electron microscopy analysis revealed an obvious cell growth on the surface of the scaffolds. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium, magnesium and phosphate ions were altered markedly due to the chemical solubility of the scaffolds. We conclude that the calcium magnesium phosphate (newberyite) cements have a promising potential for their use as bone replacement material since they provide a suitable biocompatibility, an extended workability and improved mechanical performance compared with brushite cements.     

Synthesis and properties of hydroxyapatite-containing porous titania coating on ultrafine-grained titanium by micro-arc oxidation

Equal channel angular pressing results in ultrafine-grained (～200–500 nm) Ti with superior mechanical properties without harmful alloying elements, which benefits medical implants. To further improve the bioactivity of Ti surfaces, Ca/P-containing porous titania coatings were prepared on ultrafine-grained and coarse-grained Ti by micro-arc oxidation (MAO). The phase identification, composition, morphology and microstructure of the coatings and the thermal stability of ultrafine-grained Ti during MAO were investigated subsequently. The amounts of Ca, P and the Ca/P ratio of the coatings formed on ultrafine-grained Ti were greater than those on coarse-grained Ti. Nanocrystalline hydroxyapatite and α-Ca₃(PO₄)₂ phases appeared in the MAO coating formed on ultrafine-grained Ti for 20 min (E20). Incubated in a simulated body fluid, bone-like apatite was completely formed on the surface of E20 after 2 days, thus evidencing preferable bioactivity. Compared with initial ultrafine-grained Ti, the microhardness of the E20 substrate was reduced by 8% to 2.9 GPa, which is considerably more than that of coarse-grained Ti (～1.5 GPa).     

Influence of a zirconia sandblasting treated surface on peri-implant bone healing: An experimental study in sheep

A sandblasting process with round zirconia (ZrO₂) particles might be an alternative surface treatment to enhance the osseointegration of titanium dental implants. Our previous study on sheep compared smooth surface titanium implants (control) with implant surfaces sandblasted with two different granulations of ZrO₂. As the sandblasted surfaces proved superior, the present study further compared the ZrO₂ surface implant with other surface treatments currently employed: machined titanium (control), titanium oxide plasma sprayed (TPS) and alumina sandblasted (Al-SL) at different times after insertion (2, 4 and 12 weeks). Twelve sheep were divided into three groups of four animals each and underwent implant insertion in tibia cortical bone under general anaesthesia. The implants with surrounding tissues were subjected to histology, histomorphometry, scanning electron microscopy and microhardness tests. The experimentation indicated that at 2 weeks Zr-SL implants had the highest significant bone ingrowth (p < 0.05) compared to the other implant surfaces, and a microhardness of newly formed bone inside the threads significantly higher than that of Ti. The present work shows that the ZrO₂ treatment produces better results in peri-implant newly formed bone than Ti and TPS processing, whereas its performance is similar to the Al-SL surface treatment.     

An electrochemical study on self-ordered nanoporous and nanotubular oxide on Ti–35Nb–5Ta–7Zr alloy for biomedical applications

Highly ordered nanoporous and nanotubular oxide layers were developed on low-rigidity β Ti–35Nb–5Ta–7Zr alloy by controlled DC anodization in electrolyte containing 1 M H₃PO₄ and 0.5 wt.% NaF at room temperature. The as-formed and crystallized nanotubes were characterized by electron microscopy, energy-dispersive X-ray spectrometry and X-ray diffraction. The electrochemical passivation behavior of the nanoporous and nanotubular oxide surfaces were investigated in Ringer’s solution at 37 ± 1 °C employing a potentiodynamic polarization technique and impedance spectroscopy. The diameters of the as-formed nanotubes were in the range of 30–80 nm. The nanotubular surface exhibited passivation behavior similar to that of the nanoporous surface. However, the corrosion current density was considerably higher for the nanotubular alloy. The surface after nanotube formation seemed to favor an immediate and effective passivation. Electrochemical impedance spectra were simulated by equivalent circuits and the results were discussed with regard to biomedical applications.     

Anti-infective and osteointegration properties of silicon nitride,poly(ether ether ketone), and titanium implants

Silicon nitride (Si₃N₄) is an industrial ceramic used in spinal fusion and maxillofacial reconstruction. Maximizing bone formation and minimizing bacterial infection are desirable attributes in orthopedic implants designed to adhere to living bone. This study has compared these attributes of Si₃N₄ implants with implants made from two other orthopedic biomaterials, i.e. poly(ether ether ketone) (PEEK) and titanium (Ti). Dense implants made of Si₃N₄, PEEK, or Ti were surgically implanted into matching rat calvarial defects. Bacterial infection was induced with an injection of 1 × 10⁴ Staphylococcus epidermidis. Control animals received saline only. On 3, 7, and 14 days, and 3 months post-surgery four rats per time period and material were killed, and calvariae were examined to quantify new bone formation and the presence or absence of bacteria. Quantitative evaluation of osteointegration to adjacent bone was done by measuring the resistance to implant push-out (n = 8 rats each for Ti and PEEK, and n = 16 rats for Si₃N₄). Three months after surgery in the absence of bacterial injection new bone formation around Si₃N₄ was ～69%, compared with 24% and 36% for PEEK and Ti, respectively. In the presence of bacteria new bone formation for Si₃N₄, Ti, and PEEK was 41%, 26%, and 21%, respectively. Live bacteria were identified around PEEK (88%) and Ti (21%) implants, whereas none were present adjacent to Si₃N₄. Push-out strength testing demonstrated statistically superior bone growth onto Si₃N₄ compared with Ti and PEEK. Si₃N₄ bioceramic implants demonstrated superior new bone formation and resistance to bacterial infection compared with Ti and PEEK.     

Titanium phosphate glass microspheres for bone tissue engineering

We have demonstrated the successful production of titanium phosphate glass microspheres in the size range of ～10–200 μm using an inexpensive, efficient, easily scalable process and assessed their use in bone tissue engineering applications. Glasses of the following compositions were prepared by melt-quench techniques: 0.5P₂O₅–0.4CaO–(0.1 ? x)Na₂O–xTiO₂, where x = 0.03, 0.05 and 0.07 mol fraction (denoted as Ti3, Ti5 and Ti7 respectively). Several characterization studies such as differential thermal analysis, degradation (performed using a novel time lapse imaging technique) and pH and ion release measurements revealed significant densification of the glass structure with increased incorporation of TiO₂ in the glass from 3 to 5 mol.%, although further TiO₂ incorporation up to 7 mol.% did not affect the glass structure to the same extent. Cell culture studies performed using MG63 cells over a 7-day period clearly showed the ability of the microspheres to provide a stable surface for cell attachment, growth and proliferation. Taken together, the results confirm that 5 mol.% TiO₂ glass microspheres, on account of their relative ease of preparation and favourable biocompatibility, are worthy candidates for use as substrate materials in bone tissue engineering applications.     

The influence of poly(ethylene oxide) grafting via siloxane tethers on protein adsorption

Amphiphilic PEO–silanes (a–c) having siloxane tethers of varying lengths with the general formula α-(EtO)₃Si–(CH₂)₂–oligodimethylsiloxane_n-block-poly(ethylene oxide)₈–OCH₃ [n = 0 (a), n = 4 (b), and n = 13 (c)] were grafted onto silicon wafers and resistance to adsorption of plasma proteins was measured. Distancing the PEO segment from the hydrolyzable triethoxysilane [(EtO)₃Si] grafting group by a oligodimethylsiloxane tether represents a new method of grafting PEO chains to surfaces. Properties of surfaces grafted with a–c were compared to surfaces grafted with a traditional PEO–silane containing a propyl spacer [(EtO)₃Si–(CH₂)₃–poly(ethylene oxide)₈–OCH₃, PEO control]. As the siloxane tether length increased, chain density of PEO–silanes grafted onto oxidized silicon wafers decreased and hydrophobicity of the PEO–silane increased which led to a decrease in surface hydrophilicity. Despite decreased surface hydrophilicity, resistance to the adsorption of bovine serum albumin (BSA) increased in the order: PEO control < a < b ≈ c and to human fibrinogen (HF) increased in the order: PEO control < a < b < c.     

Bioactive polymer grafting onto titanium alloy surfaces

Bioactive polymers bearing sulfonate (styrene sodium sulfonate, NaSS) and carboxylate (methylacrylic acid, MA) groups were grafted onto Ti6Al4V alloy surfaces by a two-step procedure. The Ti alloy surfaces were first chemically oxidized in a piranha solution and then directly subjected to radical polymerization at 70 °C in the absence of oxygen. The grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the toluidine blue colorimetric method. Toluidine blue results showed 1–5 μg cm^?2 of polymer was grafted onto the oxidized Ti surfaces. Grafting resulted in a decrease in the XPS Ti and O signals from the underlying Ti substrate and a corresponding increase in the XPS C and S signals from the polymer layer. The ToF-SIMS intensities of the S^? and SO^? ions correlated linearly with the XPS atomic percent S concentrations and the ToF-SIMS intensity of the TiO₃H₂^? ion correlated linearly with the XPS atomic per cent Ti concentration. Thus, the ToF-SIMS S^?, SO^? and TiO₃H₂^? intensities can be used to quantify the composition and amount of grafted polymer. ToF-SIMS also detected ions that were more characteristic of the polymer molecular structure (C₆H₄SO₃^? and C₈H₇SO₃^? from NaSS, C₄H₅O₂^? from MA), but the intensity of these peaks depended on the polymer thickness and composition. An in vitro cell culture test was carried out with human osteoblast-like cells to assess the influence of the grafted polymers on cell response. Cell adhesion after 30 min of incubation showed significant differences between the grafted and ungrafted surfaces. The NaSS grafted surfaces showed the highest degree of cell adhesion while the MA-NaSS grafted surfaces showed the lowest degree of cell adhesion. After 4 weeks in vivo in rabbit femoral bones, bone was observed to be in direct contact with all implants. The percentage of mineralized tissue around the implants was similar for NaSS grafted and non-grafted implants (59% and 57%). The MA-NaSS grafted implant exhibited a lower amount of mineralized tissue (47%).     

Fluoridated hydroxyapatite coatings on titanium obtained by electrochemical deposition

Hydroxyapatite (HA) and fluoridated hydroxyapatite (FHA) coatings were deposited on titanium substrates using an electrochemical technique. Different concentrations of F^? ions were incorporated into the apatite structure by adding NaF into the electrolyte. Typical apatite structures were obtained for all the coatings after electrodeposition and subsequent post-treatment, including alkaline immersion and vacuum calcination. The coatings were uniform and dense, with a thickness of ～5 μm. When the F-concentration was higher than 0.012 M in the electrolyte, a saturation of F in the coating occurred and the F/Ca ratio in the coatings became almost constant (F/Ca ratio = 0.125). The FHA coatings showed higher bonding strength and lower dissolution rate than HA coating, particularly for those with a fluoridation level of 0.5–0.625. Compared with pure Ti, FHA and HA coatings exhibited higher biological affinity like cell proliferation and alkaline phosphatase activity. Regarding clinical application, it is suggested that a moderate content of F, such as Ca₅(PO₄)₃(OH)_0.375?0.5F_0.5?0.625, be most suitable as a compromise among cell attachment, cell proliferation, apatite deposition and dissolution resistance.     

In vitro cytotoxicity evaluation of porous TiO2–Ag antibacterial coatings for human fetal osteoblasts

Implant-associated infections (IAIs) may be prevented by providing antibacterial properties to the implant surface prior to implantation. Using a plasma electrolytic oxidation (PEO) technique, we produced porous TiO₂ coatings bearing various concentrations of Ag nanoparticles (Ag NPs) (designated as 0 Ag, 0.3 Ag and 3.0 Ag) on a Ti–6Al–7Nb biomedical alloy. This study investigates the cytotoxicity of these coatings using a human osteoblastic cell line (SV-HFO) and evaluates their bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA). The release of Ag and the total amount of Ag in the coatings were determined using a graphite furnace atomic absorption spectrometry technique (GF-AAS) and flame-AAS, respectively. Cytotoxicity was evaluated using the AlamarBlue assay coupled with the scanning electron microscopy (SEM) observation of seeded cells and by fluorescence microscopy examination of the actin cytoskeleton and nuclei after 48 h of incubation. Antibacterial activity was assessed quantitatively using a direct contact assay. AlamarBlue viability assay, SEM and fluorescence microscopy observation of the SV-HFO cells showed no toxicity for 0 Ag and 0.3 Ag specimens, after 2, 5 and 7 days of culture, while 3.0 Ag surfaces appeared to be extremely cytotoxic. All Ag-bearing surfaces had good antibacterial activity, whereas Ag-free coatings showed an increase in bacterial numbers. Our results show that the 0.3 Ag coatings offer conditions for optimum cell growth next to antibacterial properties, which makes them extremely useful for the development of new antibacterial dental and orthopedic implants.     

Zinc-containing apatite layers on external fixation rods promoting cell activity

Zinc-containing apatite layers were successfully formed on commercially available anodically oxidized Ti external fixation rods using ZnCl₂-containing supersaturated calcium phosphate solutions. With an increase in concentration of ZnCl₂ in the supersaturated calcium phosphate solutions, the amounts of zinc that precipitated on the Ti external fixation rods increased (from 0 to 0.195 ± 0.020 μg cm^?2); meanwhile, the amounts of calcium and phosphorus that precipitated on the Ti external fixation rods decreased (from 11.2 ± 1.5 and 4.8 ± 0.5 μg cm^?2 to 2.9 ± 1.6 and 1.3 ± 0.9 μg cm^?2, respectively). The zinc-containing apatite layers precipitated on the Ti external fixation rods caused a significant increase in fibroblastic proliferation, osteoblastic proliferation and differentiation in vitro. The Ti external fixation rods coated with zinc-containing apatite layers are expected to be more effective in accelerating the tissue regeneration around the surgical site than those coated with an apatite layer.     

Preliminary investigation of rate of torque development deficits following total knee arthroplasty

BackgroundTo assess changes in maximal strength and rate of torque development (RTD) following TKA, and examine the relationships between these measures and physical function.MethodsThirty-five TKA patients and 23 controls completed isometric knee extensor torque testing preoperatively, 1, and 6 months after surgery. Maximal strength was calculated as the peak torque during a maximal voluntary isometric contraction (MVIC) of the knee extensor muscles, peak RTD (RTD_peak) was calculated as the maximum value from the 1st derivative of the isometric knee extension torque data, RTD_25% and RTD _50% were calculated as the change in force over the change in time from force onset to 25% and 50% MVIC. Physical function was measured using a timed-up-and-go (TUG) and stair climbing test (SCT).ResultsRTD was significantly lower in the TKA group, at all-time points, compared to the Controls. MVIC and RTD significantly decreased 1-month following surgery (p = 0.000 for all measures). RTD_peak measures added to linear regressions with strength improved the prediction of TUG scores (p = 0.006) and the SCT scores (p = 0.015) 1-month post-surgery. Adding RTD_50% to the regression model, following MVIC, improved predicting both TUG (p = 0.033) and SCT (p = 0.024). At 6-months, the addition of RTD_25% to the regression model, following MVIC, improved the prediction of TUG (p = 0.037) and SCT (p = 0.036).ConclusionFollowing TKA, physical function is influenced by both the maximal strength and the rate of torque development of the knee extensors, and the prediction of function is improved with the addition of RTD compared to that of maximal strength alone.     

Electrically polarized HAp-coated Ti: In vitro bone cell–material interactions

Electrically polarized bulk sintered hydroxyapatite (HAp) compacts have been shown to accelerate mineralization and bone tissue ingrowth in vivo. In this work, a comprehensive study has been carried out to investigate the influence of surface charge and polarity on in vitro bone cell adhesion, proliferation and differentiation on electrically polarized HAp-coated Ti. Uniform and crack free sol–gel derived HAp coatings of 20 ± 1.38 μm thickness were polarized by application of an external d.c. field of 2.0 kV cm^?1 at 400 °C for 1h. In vitro bioactivity of polarized HAp coatings was evaluated by soaking in simulated body fluid, and bone cell–material interactions were studied by culturing with human fetal osteoblast cells (hFOB) for a maximum period of 11 days. Scanning electron microscopic observation showed that accelerated mineralization on negatively charged surfaces favored rapid cell attachment and faster tissue ingrowth over non-polarized HAp coating surfaces, while positive charge on HAp coating surfaces restricted apatite nucleation with limited cellular response. Immunochemistry and confocal microscopy confirmed that the cell adhesion and early stage differentiation were more pronounced on negatively charged coating surfaces as hFOB cells expressed higher vinculin and alkaline phosphatase proteins on negatively charged surface compared to cells grown on all other surfaces. Our results in this study are process independent and potentially applicable to any other commercially available coating techniques.     

Peri-implant reactivity and osteoinductive potential of immobilized rhBMP-2 on titanium carriers

Recombinant human BMP-2 (rhBMP-2) was immobilized non-covalently and covalently as a monolayer on plasma vapour deposited (PVD) porous commercially pure titanium surfaces in amounts of 5–8 μg cm^?2, providing a ca. 10-fold increase vs. previously reported values [37]. Dissociation of the immobilized [¹²⁵I]rhBMP-2 from the surface occurred in a two-phase exponential decay: a first rapid phase (ca. 15% of immobilized BMP-2) with a half-life of 1–2 days and a second slow sustained release phase (ca. 85% of immobilized BMP-2) with a half-life of 40–60 days. Dissociation rate constants of sustained release of k_?1 = 1.3–1.9 × 10^?7 s^?1 were determined, allowing an estimation of the binding constants (K_A) for the adsorbed rhBMP-2 monolayer, to be around 10¹² M^?1. The rhBMP-2-coated surfaces showed a high level of biological activity, as demonstrated by in vitro epifluorescence tests for alkaline phosphatase with MC3T3-E1 cells and in vivo experiments. In vivo osteoinductivity of rhBMP-2-coated implants was investigated in a gap-healing model in the trabecular bone of the distal femur condylus of sheep. Healing occurred without inflammation or capsule formation. The calculated concentration of released rhBMP-2 in the 1 mm gap ranged from 20 to 98 nM – well above the half-maximal response concentration (K_0.5) for inducing alkaline phosphatase in MC3T3-E1 cells. After 4, 9 and 12 weeks the bone density (BD) and bone-to-implant contact (BIC) of the explanted implants were assessed histomorphometrically. Implants with immobilized rhBMP-2 displayed a significant (2- to 4-fold) increase in BD and BIC values vs. negative controls after 4–9 weeks. Integration of implants by trabecular bone was achieved after 4 weeks, indicating a mean “gap-filling rate” of ～250 μm week^?1. Integration of implants by cortical bone was observed after 9 weeks. Control implants without rhBMP-2 were not osseointegrated. This study demonstrates the feasibility of enhancing peri-implant osseointegration and gap bridging by immobilized rhBMP-2 on implant surfaces which may serve as a model for future clinical applications.     

Effect of silicon content on the sintering and biological behaviour of Ca10(PO4)6-x(SiO4)x(OH)2-x ceramics

Silicated hydroxyapatite powders (Ca₁₀(PO₄)_6-x(SiO₄)_x(OH)_2-x; Si_xHA) were synthesized using a wet precipitation method. The sintering of Si_xHA ceramics with 0 ? x ? 1 was investigated. For 0 ? x ? 0.5, the sintering rate and grain growth decreased slightly with the amount of silicate. For larger amounts, the sintering behaviour differed with the formation of secondary phases before total densification. Sintering parameters (temperature and time) were adjusted to each composition to produce dense materials having similar microstructure without formation of these secondary phases. Dense ceramics made of pure hydroxyapatite and Si_xHA containing various amounts of silicate (up to x = 0.6) were biologically tested in vitro with human osteoblast-like cells. The proliferation of cells on the surface of the ceramics increased up to 5 days of culture, indicating that the materials were biocompatible. However, the silicon content did not influence the cell proliferation.