Influence of RGD-loaded titanium implants on bone formation in vivo

Little is known about the ability of peptide-coated surfaces to influence cell responses in vivo. Many studies have demonstrated that peptide-modified surfaces influence cell responses in vitro. Integrins, which bind specifically short peptide sequences, are responsible for these cell responses. In this way, information can be transmitted to the nucleus through several cytoplasmic signaling pathways. The peptide sequence Arg-Gly-Asp (RGD peptide) plays an important role in osteoblast adhesion. The present study was designed to investigate new bone formation in a porous titanium (Ti) fiber mesh implant, which was coated with cyclic RGD peptide. The RGD-Ti implants were inserted into the cranium of a rabbit and were compared with porous titanium fiber mesh disks without RGD sequence (Ti) and with an open control defect. Histologic and histomorphometric examinations were performed 2, 4, and 8 weeks postoperatively. A significant increase in bone formation, or bone ingrowth, was seen in the RGD-Ti group compared with the Ti group after 4 and 8 weeks. All control defects stayed open in all three periods. It was concluded that the use of cyclic RGD peptide in combination with titanium fiber mesh has a positive effect on bone formation in vivo in a rabbit animal model.     

Qualitative evaluation of titanium implant integration into bone by diffraction enhanced imaging

Diffraction enhanced imaging (DEI) uses refraction of x-rays at edges, which allows pronounced visualization of material borders and rejects scattering which often obscures edges and blurs images. Here, the first evidence is presented that, using DEI, a destruction-free evaluation of the quality of integration of metal implants into bone is possible. Experiments were performed in rabbits and sheep with model implants to investigate the option for DEI as a tool in implant research. The results obtained from DEI were compared to conventional histology obtained from the specimens. DE images allow the identification of the quality of ingrowth of bone into the hydroxyapatite layer of the implant. Incomplete integration of the implant with a remaining gap of less than 0.3 mm caused the presence of a highly refractive edge at the implant/bone border. In contrast, implants with bone fully grown onto the surface did not display a refractive signal. Therefore, the refractive signal could be utilized to diagnose implant healing and/or loosening.     

Hydrophilic interpenetrating polymer networks of poly(dimethyl siloxane) (PDMS) as biomaterial for cochlear implants

Poly(dimethyl siloxane) (PDMS) was bulk-modified to develop a new intra-cochlear electrode that can closely hug the inner wall of scala tympani (ST). The hydrophilicity of bulk and surface of PDMS was changed using a sequential method for preparation of interpenetrating polymer networks (IPNs). A series of IPNs, based on PDMS and poly(acrylic acid) (PAAc), was synthesized and characterized by means of attenuated total reflectance Fourier transform infrared spectroscopy, water contact-angle measurement, dynamic mechanical thermal analysis and peel strength tests. The performances of actual-sized fabricated electrodes were assessed inside a transparent model of ST, which was filled with saline. The cell behavior of L929 fibroblasts on materials was studied in vitro.     

Hydrophilic interpenetrating polymer networks of poly(dimethyl siloxane) (PDMS) as biomaterial for cochlear implants

Poly(dimethyl siloxane) (PDMS) was bulk-modified to develop a new intra-cochlear electrode that can closely hug the inner wall of scala tympani (ST). The hydrophilicity of bulk and surface of PDMS was changed using a sequential method for preparation of interpenetrating polymer networks (IPNs). A series of IPNs, based on PDMS and poly(acrylic acid) (PAAc), was synthesized and characterized by means of attenuated total reflectance Fourier transform infrared spectroscopy, water contact-angle measurement, dynamic mechanical thermal analysis and peel strength tests. The performances of actual-sized fabricated electrodes were assessed inside a transparent model of ST, which was filled with saline. The cell behavior of L929 fibroblasts on materials was studied in vitro.     

Hydroxyapatite grafting promotes new bone formation and osseointegration of smooth titanium implants

Titanium is the ideal metal for intra-osseous dental implants. It permits the natural formation of an oxide layer on its surface and thereby it prevents the release of potentially toxic molecules. New formation of bone around implants, partially placed into the bone marrow cavity, is a gradual process that runs from the endosteum to the surface of the implant. Deposition of hydroxyapatite crystals on collagen type I fibrils is initiated by acidic proteins and leads to bone mineralization. This study analyzed the effects of hydroxyapatite upon peri-implant bone formation after insertion of smooth titanium implants. Screw-shaped smooth titanium implants of 3.75 mm thickness and 8.5 mm length were inserted into the metaphysis of rabbit tibia, either together with bovine hydroxyapatite into the right tibia or in controls without hydroxyapatite into the left tibia. Polyfluorochrome tracers (alizarin complex, calcein, tetracycline) were injected subcutaneously at different time intervals after implantation to evaluate the time frame of bone new formation over a period of 8 weeks. All samples were processed for histology and analyzed by fluorescence and polarizing microscopy. Our results showed a higher quantity of mature type I collagen fibers around implants and an acceleration of bone formation in the presence of hydroxyapatite. Mainly immature organic matrix was formed at the surface of implants in controls. The presence of hydroxyapatite seems to promote the maturation of collagen fibers surrounding the titanium implants and to support osteoconduction. Moreover, new formation of bone was faster in all samples where implants were inserted together with hydroxyapatite.     

Controlled electro-implementation of fluoride in titanium implant surfaces enhances cortical bone formation and mineralization

Previous studies have shown that bone-to-implant attachment of titanium implants to cortical bone is improved when the surface is modified with hydrofluoric acid. The aim of this study was to investigate if biological factors are involved in the improved retention of these implants. Fluoride was implemented in implant surfaces by cathodic reduction with increasing concentrations of HF in the electrolyte. The modified implants were placed in the cortical bone in the tibias of New Zealand white rabbits. After 4 weeks of healing, wound fluid collected from the implant site showed lower lactate dehydrogenase activity and less bleeding in fluoride-modified implants compared to control. A significant increase in gene expression levels of osteocalcin and tartrate-resistant acid phosphatase (TRAP) was found in the cortical bone attached to Ti implants modified with 0.001 and 0.01 vol.% HF, while Ti implants modified with 0.1% HF showed only induced TRAP mRNA levels. These results were supported by the performed micro-CT analyses. The volumetric bone mineral density of the cortical bone hosting Ti implants modified with 0.001% and 0.01% HF was higher both in the newly woven bone (<100 μm from the interface) and in the older Haversian bone (>100 μm). In conclusion, the modulation of these biological factors by surface modification of titanium implants with low concentrations of HF using cathodic reduction may explain their improved osseointegration properties.     

Interface analysis of titanium and zirconium bone implants

A thin layer of titanium or zirconium was evaporated onto the surface of a plastic implant which was then inserted in the rabbit tibial metaphysis for six months. The implants and surrounding bone were cut out en bloc and sectioned for phase contrast microscopy, scanning- and transmission-electron microscopy. The intact bone-to-metal interface in the case of titanium revealed a fibrous tissue-free boundary zone with a 200-400 A thick proteoglycan coat immediately adjacent to the titanium oxide. Thereafter collagen filaments were seen and, at approximately 1000 A from the interface, collagen bundles. The tissues surrounding the zirconium-coated implant consisted of a 300-500 A thick proteoglycan layer, followed by a zone with collagen filaments and collagen bundles, not closer to the zirconium oxide than a few thousand A.     

Migration, matrix production and lamellar bone formation of human osteoblast-like cells in porous titanium implants

The goal of this study was to characterize growth, mineralization and bone formation of osteoblast-like cells in titanium pore channels of defined diameter. Titanium implants with continuous drill channels of diameters of 300, 400, 500, 600 and 1,000 microm were inserted into human osteoblast-like cell cultures. The ingrowth of the cells into the drill channels was investigated by transmitted-light microscopy and scanning electron microscopy. Immunofluorescence and histological analysis of 15-channel sections of each diameter were used to investigate the growth behavior and the matrix protein patterns. Mineralization was evidenced by Alizarin red staining and high-resolution microradiography. The ingrowth of human osteoblast-like cells in the drill channels occurred in a sequence of four characteristic stages. In stage 1, osteoblast precursor cells adhered to the wall of the channel and migrated three-dimensionally into the channel by forming foot-like protoplasmic processes. For all 15 sample drill channels that were investigated, the cell ingrowth over 20 days amounted on average to 793 microm (+/- 179) into 600-microm-diameter channels, where they migrated significantly faster than in all the other channels. In stage 2, approximately on day 5-7, the osteoblast-like cells began to anchor on the substrate wall by matrix proteins and to build up a dense network of matrix proteins in the drill channel. The mineralization of the extracellular matrix, while depending on cell stimulation, was initiated in stage 3, on average after 4 weeks. In drill channels of a diameter of 1,000 microm the cell growth was incomplete and no mineralization was found by radiological assessment. Starting in week 6, in the drill channels of diameters ranging from 300 to 600 microm, the network of extracellular matrix proteins and osteoblast-like cells began to form an osteon-like structure. Neither the highly developed migration behavior of osteoblastic cells nor the reorganization from a fiber-like matrix to a lamellar structure have so far been described for cell cultures.     

Trabecular bone response to surface roughened and calcium phosphate (Ca-P) coated titanium implants.

The influence of calcium phosphate (Ca-P) coating and surface roughness on the trabecular bone response of titanium implants was investigated. Four types of titanium implants, i.e. blasted with titanium powder, sintered with titanium beads, titanium powder blasted and provided with an additional Ca-P coating, and titanium beads with Ca-P coating, were prepared. The Ca-P coating was deposited by ion beam dynamic mixing method. The Ca-P coating was rapid heat-treated with infrared radiation at 700 degrees C. The implants were inserted into the trabecular bone of the left and right femoral condyles of 16 rabbits. After implantation periods of 2, 3, 4 and 12 weeks, the bone-implant interface was evaluated histologically and histomorphometrically. Histological evaluation revealed new bone formation around different implant materials after already 3 weeks of implantation. After 12 weeks, mature trabecular bone surrounded all implants. At 3 and 4 weeks of implantation, no difference existed in bone contact to the various implant materials. On the other hand, after 12 weeks of implantation the highest percentage of bone contact was found around the Ca-P coated beads implants. Supported by the results, we concluded that the combination of surface geometry and Ca-P coating benefits the implant-bone response during the healing phase.     

Interface between bone tissue and implants of solid hydroxyapatite or hydroxyapatite-coated titanium implants

Loaded prestressed implants of dense hydroxyapatite and non-loaded hydroxyapatite-coated titanium implants were placed in edentulous regions of the lower jaw of dogs. After 6 month the jaw specimens were fixed and embedded in methyl-methacrylate. Thin non-decalcified ground sections were made for histology. Although the hydroxyapatite showed histological differences between the coated implants and the prestressed solid ones, both had an extensive apposition of normal lamellar bone on the whole surface of the bone-buried part of the implant. The bone contact was very intimate and without any visible intermediate tissue layer. The tissue response observed forms a good biological base for the clinical application of hydroxyapatite-coated titanium implants.     

Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs

The purpose of the present study was to evaluate the influence of different surface characteristics on bone integration of titanium implants. Hollow-cylinder implants with six different surfaces were placed in the metaphyses of the tibia and femur in six miniature pigs. After 3 and 6 weeks, the implants with surrounding bone were removed and analyzed in undecalcified transverse sections. The histologic examination revealed direct bone-implant contact for all implants. However, the morphometric analyses demonstrated significant differences in the percentage of bone-implant contact, when measured in cancellous bone. Electropolished as well as the sandblasted and acid pickled (medium grit; HF/HNO3) implant surfaces had the lowest percentage of bone contact with mean values ranging between 20 and 25%. Sandblasted implants with a large grit and titanium plasma-sprayed implants demonstrated 30-40% mean bone contact. The highest extent of bone-implant interface was observed in sandblasted and acid attacked surfaces (large grit; HCl/H2SO4) with mean values of 50-60%, and hydroxylapatite (HA)-coated implants with 60-70%. However, the HA coating consistently revealed signs of resorption. It can be concluded that the extent of bone-implant interface is positively correlated with an increasing roughness of the implant surface.     

Ultrastructural characterization of the implant/bone interface of immediately loaded dental implants

Primary stability and an optimized load transfer are assumed to account for an undisturbed osseointegration process of implants. Immediate loaded newly designed titanium dental implants inserted in the mandible of minipigs were used for the characterization of the interfacial area between the implant surface and the surrounding bone tissue during the early healing phase. Histological and electron microscopical studies were performed from implant containing bone specimens. Two different load regimens were applied to investigate the load related tissue reaction. Histological and electron microscopical analysis revealed a direct bone apposition on the implant surfaces, as well as the attachment of cells and matrix proteins in the early loading phase. A striking finding of the ultrastructural immunocytochemical investigations was the synthesis and deposition of bone related proteins (osteonectin, fibronectin, fibronectin receptor) by osteoblasts from day one of bone/biomaterial interaction. Calcium-phosphate needle-like crystallites were newly synthesized in a time-related manner directly at the titanium surface. No difference in the ultrastructural appearance of the interface was found between the two loading groups. Our experimental data suggest that loading of specially designed implants can be performed immediately after insertion without disturbing the biological osseointegration process.     

The roles of surface chemistry and topography in the strength and rate of osseointegration of titanium implants in bone

The present study investigated the effects of surface chemistry and topography on the strength and rate of osseointegration of titanium implants in bone. Three groups of implants were compared: (1) machine-turned implants (turned implants), (2) machine-turned and aluminum oxide-blasted implants (blasted implants), and (3) implants that were machine-turned, aluminum oxide-blasted, and processed with the micro-arc oxidation method (Mg implants). Three and six weeks after implant insertion in rabbit tibiae, the implant osseointegration strength and rate were evaluated. Surface chemistry revealed characteristic differences of nine at.% Mg for Mg implants and 11 at.% Al for blasted implants. In terms of surface roughness, there was no difference between Mg implants and blasted implants in developed surface ratio (Sdr; p = 0.69) or summit density (Sds; p = 0.96), but Mg implants had a significantly lower arithmetic average height deviation (Sa) value than blasted implants (p = 0.007). At both 3 and 6 weeks, Mg implants demonstrated significantly higher osseointegration strength compared with turned (p = 0.0001, p = 0.0001) and blasted (p = 0.0001, p = 0.035) implants, whereas blasted implants showed significantly higher osseointegration than turned implants at 6 weeks (p = 0.02) but not at 3 weeks (p = 0.199). The present results not only support the hypothesis that biochemical bonding facilitates rapid and strong integration of implants in bone, but also provide evidence for biochemical bonding theory previously proposed by Sul.     

The bone response of oxidized bioactive and non-bioactive titanium implants

A number of experimental and clinical data on so-called oxidized implants have reported promising outcomes. However, little is investigated on the role of the surface oxide properties and osseointegration mechanism of the oxidized implant. Sul [On the Bone Response to Oxidized Titanium Implants: The role of microporous structure and chemical composition of the surface oxide in enhanced osseointegration (thesis). G?teborg: Department of Biomaterials/Handicap Research, University of G?teborg, Sweden; 2002; Biomaterials 2003; 24: 3893-3907] recently proposed two action mechanisms of osseointegration of oxidized implants, i.e. mechanical interlocking through bone growth in pores/other surface irregularities (1) and biochemical bonding (2). The aim of the present study is two-fold: (i) investigating the role of the implant surface chemistry on bone responses; (ii) investigating the validity of the biochemical bonding theory of the oxidized, bioactive bone implants with specific implant surface chemistry. Two groups of oxidized implants were prepared using micro arc oxidation process and were then inserted in rabbit bone. One group consisted of magnesium ion incorporated implants (MgTiO implant), the other consisted of TiO2 stoichiometry implants (TiO implant). Surface oxide properties of the implants were characterized with various surface analytic techniques. After 6 weeks of follow up, the mean peak values of removal torque of Mg implants dominated significantly over TiO implants (p < or = 0.0001). Bonding failure generally occurred in the bone away from the bone to implant interface for the MgTiO implant and mainly occurred at the bone to implant interface for the TiO implant that consisted mainly of TiO2 chemistry and significantly rougher surface as compared to the MgTiO implant. Between bone and the Mg- incorporated implant surface, ionic movements and ion concentrations gradient were detected. The current in vivo experimental data may provide positive evidence for the surface chemistry-mediated biochemical bonding theory of oxidized bioactive implants. However, the present study does not rule out potential synergy effects of the oxide thickness, micro-porous structure, crystal structure and surface roughness on improvements of bone responses to oxidized bioactive implants.     

Saline irrigation does not affect bone formation or fixation strength of hydroxyapatite/tricalcium phosphate-coated implants in a rat model

Intramembranous bone regeneration is critical to implant fixation. In cementless joint replacement (as opposed to cemented joint replacement), saline irrigation is not typically performed during surgery so that the osteogenic stimulus provided by the marrow is preserved. Several groups are now using the rat marrow ablation model to study intramembranous bone regeneration and implant fixation. In this model, the marrow contents are mechanically disrupted, and debris is often cleared by saline irrigation, a step that appears inconsistent with the clinical situation. Furthermore, in contrast to conventional wisdom, it has been reported that saline irrigation enhanced bone-implant contact and peri-implant bone formation in the rat model (Ishizaka et al. Bone 1996;19:589-594), although mechanical fixation of the implant was not investigated. Accordingly, the present study was performed to determine if saline irrigation leads to enhanced mechanical fixation of implants in the rat model. Forty-eight 400 to 450 g male rats were divided equally into two groups. The treatment group, in contrast to the control group, received saline irrigation in the ablated medullary canal prior to placement of hydroxyapatite/tricalcium phosphate-coated implants. Eight animals in each group were killed at 2, 4, or 8 weeks after implantation, at which time the specimens were analyzed by micro computed tomography to measure bone formation around the implant, followed by a mechanical pull-out test to measure the strength of fixation of the implant. As expected, there was increased fixation strength over time, but there were no significant differences in peri-implant bone volume, bone-implant contact, or implant fixation strength between the two groups. Thus, we found no effect of saline irrigation on bone formation or implant fixation strength in this study in which the implant had an osteoconductive coating.     

Interfacial shear strength of titanium implants in bone is significantly improved by surface topographies with high pit density and microroughness

Surface structure of implants influences bone response and interfacial shear strength between implants and bone. The aim of this study was to find topographical parameters that correlate with the interfacial shear strength. Two groups of sand-blasted titanium screws were implanted in 17 sheep tibia, each for 2-52 weeks: (A) acid pickled with HF/HNO(3); (B) acid etched with HCl/H(2)SO(4). Screw removal torque was measured and surface topography of both implant groups was studied by scanning electron microscopy, optical profilometry, and scanning probe microscopy. The roughness as well as the surface area of type A surface was higher in the scan region of 100 microm, but the microroughness and surface area of type B surface was higher in the scan region of 10 microm. A significantly higher removal torque (interfacial shear strength) of the surface treatment type B (412 +/- 60 Ncm) compared to surface treatment type A (157 +/- 33 Ncm) was found after 52 weeks of implantation in sheep due to differences in microroughness of both types of screws. It was also shown that the specification of the parameters Delta(a), R(a) and R(q) was not sufficient to characterize the properties of the implant surfaces. The analysis of R(q) parameter over wavelengths was required to characterize the size, shape and distribution of the implant surface structures.     

The in vitro blood compatibility of poly(ethylene oxide)-grafted polyurethane/polystyrene interpenetrating polymer networks

Poly(ethylene oxide) (PEO)-grafted polyurethane (PU)/polystyrene (PS) interpenetrating polymer networks (IPNs) were synthesized. The effects of the mobile pendant PEO chains with their microphase separated structure on blood-compatibility were investigated. The morphology of both the fracture surface as well as the top surface indicate that the size of the dispersed domains of the PS-rich phase decreased as the grafting with the PEO was increased. The swelling ratio also decreased as the grafting with the PEO was increased. However, the dynamic contact angle and the interfacial energy between IPN surface and water decreased, due to the structural reorganization of the pendant PEO chains. PU/PS IPNs have an excellent mechanical property as compared with PU homopolymers. The adsorption of bovine plasma fibrinogen (BPF) onto the PU/PS IPNs and PU homopolymers was effectively suppressed by the PEO-grafting. In the platelet adhesion test, the amount of platelets adsorbed, activated, and/or coagulated upon the PEO-grafted PU/PS IPNs were reduced when compared to the ungrafted PU homopolymers.     

The effect of alendronate (Fosamax) and implant surface on bone integration and remodeling in a canine model.

Patients at high risk for osteoporosis and its associated morbidity, including postmenopausal women, are being pharmacologically managed to stabilize and improve bone mass. Alendronate sodium (Fosamax) is a commonly used antiresorptive agent effective in osteopenic women for reducing bone resorption, increasing bone density, and decreasing fracture incidence. With the increased incidence of alendronate-treated women who are undergoing hip replacement or fracture repair by prosthesis placement, data are needed to predict how alendronate affects host bone integration with uncemented surfaces. The aim of this study was to determine the effect of alendronate on new bone formation and attachment to implant surfaces in a normal and simulated estrogen-deficient, calcium-deficient canine model, using an implantable bone growth chamber. Alendronate did not affect host bone integration to surfaces commonly used in uncemented total joint arthroplasty, but there were significant differences dependent solely on the type of surface.