RESUMO
Nanometer scale surface features on implants and prostheses can potentially be used to enhance osseointegration and may also add further functionalities, such as infection resistance, to the implant. In this study, a nanostructured noble metal coating consisting of palladium, gold and silver, never previously used in bone applications, was applied to machined titanium screws to evaluate osseointegration after 6 and 12 weeks in rabbit tibiae and femurs. Infection resistance was confirmed by in vitro adhesion test. A qualitatively and quantitatively similar in vivo bone response was observed for the coated and uncoated control screws, using histology, histomorphometry and electron microscopy. The bone-implant interface analysis revealed an extensive bone formation and direct bone-implant contact. These results demonstrate that the nanostructured noble metal coating with antimicrobial properties promotes osseointegration and may therefore be used to add extra implant functionality in the form of increased resistance to infection without the use of antibiotics. FROM THE CLINICAL EDITOR: The authors of this paper demonstrate that nanostructured noble metal coating of implants and prostheses used in orthopedic procedures promotes osseointegration and may be used to add extra implant functionality in the form of increased resistance to infection without the use of antibiotics.
Assuntos
Anti-Infecciosos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Metais/farmacologia , Nanoestruturas/química , Osseointegração/efeitos dos fármacos , Titânio/farmacologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Contagem de Colônia Microbiana , Fêmur/efeitos dos fármacos , Fêmur/fisiologia , Fêmur/ultraestrutura , Implantes Experimentais , Interferometria , Nanoestruturas/ultraestrutura , Osteogênese/efeitos dos fármacos , Espectroscopia Fotoeletrônica , Coelhos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Propriedades de Superfície , Tíbia/efeitos dos fármacos , Tíbia/fisiologia , Tíbia/ultraestruturaRESUMO
Calcium phosphates (CaP) represent an important class of osteoconductive and osteoinductive biomaterials. As proof-of-concept, we show how a multi-component CaP formulation (monetite, beta-tricalcium phosphate, and calcium pyrophosphate) guides osteogenesis beyond the physiological envelope. In a sheep model, hollow dome-shaped constructs were placed directly over the occipital bone. At 12 months, large amounts of bone (â¼75%) occupy the hollow space with strong evidence of ongoing remodelling. Features of both compact bone (osteonal/osteon-like arrangements) and spongy bone (trabeculae separated by marrow cavities) reveal insights into function/need-driven microstructural adaptation. Pores within the CaP also contain both woven bone and vascularised lamellar bone. Osteoclasts actively contribute to CaP degradation/removal. Of the constituent phases, only calcium pyrophosphate persists within osseous (cutting cones) and non-osseous (macrophages) sites. From a translational perspective, this multi-component CaP opens up exciting new avenues for osteotomy-free and minimally-invasive repair of large bone defects and augmentation of the dental alveolar ridge.
RESUMO
The early cell and tissue interactions with nanopatterned titanium implants are insufficiently described in vivo. A limitation has been to transfer a pre-determined, well-controlled nanotopography to 3D titanium implants, without affecting other surface parameters, including surface microtopography and chemistry. This in vivo study aimed to investigate the early cellular and molecular events at the bone interface with screw-shaped titanium implants superimposed with controlled nanotopography. Polished and machined titanium implants were firstly patterned with 75-nm semispherical protrusions. Polished and machined implants without nano-patterns were designated as controls. Thereafter, all nanopatterned and control implants were sputter-coated with a 30nm titanium layer to unify the surface chemistry. The implants were inserted in rat tibiae and samples were harvested after 12h, 1d and 3d. In one group, the implants were unscrewed and the implant-adherent cells were analyzed using quantitative polymerase chain reaction. In another group, implants with surrounding bone were harvested en bloc for histology and immunohistochemistry. The results showed that nanotopography downregulated the expression of monocyte chemoattractant protein-1 (MCP-1), at 1d, and triggered the expression of osteocalcin (OC) at 3d. This was in parallel with a relatively lower number of recruited CD68-positive macrophages in the tissue surrounding the nanopatterned implants. Moreover, a higher proportion of newly formed osteoid and woven bone was found at the nanopatterned implants at 3d. It is concluded that nanotopography, per se, attenuates the inflammatory process and enhances the osteogenic response during the early phase of osseointegration. This nanotopography-induced effect appeared to be independent of the underlying microscale topography. STATEMENT OF SIGNIFICANCE: This study provides a first line of evidence that pre-determined nanopatterns on clinically relevant, screw-shaped, titanium implants can be recognized by cells in the complex in vivo environment. Until now, most of the knowledge relating to cell interactions with nanopatterned surfaces has been acquired from in vitro studies involving mostly two-dimensional nanopatterned surfaces of varying chemical composition. We have managed to superimpose pre-determined nanoscale topography on polished and micro-rough, screw-shaped, implants, without changes in the microscale topography or chemistry. This was achieved by colloidal lithography in combination with a thin titanium film coating on top of both nanopatterned and control implants. The early events of osseointegration were evaluated at the bone interface to these implants. The results revealed that nanotopography, as such, elicits downregulatory effects on the early recruitment and activity of inflammatory cells while enhancing osteogenic activity and woven bone formation.
Assuntos
Substitutos Ósseos/química , Nanopartículas/química , Osseointegração/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Tíbia/fisiologia , Titânio/química , Animais , Adesão Celular/fisiologia , Células Cultivadas , Masculino , Nanopartículas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Tíbia/citologiaRESUMO
Bone insufficiency remains a major challenge for bone-anchored implants. The combination of guided bone regeneration (GBR) and bone augmentation is an established procedure to restore the bone. However, a proper understanding of the interactions between the bone substitute and GBR membrane materials and the bone-healing environment is lacking. This study aimed to investigate the early events of bone healing and the cellular activities in response to a combination of GBR membrane and different calcium phosphate (CaP) materials. Defects were created in the trabecular region of rat femurs, and filled with deproteinized bovine bone (DBB), hydroxyapatite (HA) or strontium-doped HA (SrHA) or left empty (sham). All the defects were covered with an extracellular matrix membrane. Defects were harvested after 12h, 3d and 6d for histology/histomorphometry, immunohistochemistry and gene expression analyses. Histology revealed new bone, at 6d, in all the defects. Larger amount of bone was observed in the SrHA-filled defect. This was in parallel with the reduced expression of osteoclastic genes (CR and CatK) and the osteoblast-osteoclast coupling gene (RANKL) in the SrHA defects. Immunohistochemistry indicated fewer osteoclasts in the SrHA defects. The observations of CD68 and periostin-expressing cells in the membrane per se indicated that the membrane may contribute to the healing process in the defect. It is concluded that the bone-promoting effects of Sr in vivo are mediated by a reduction in catabolic and osteoblast-osteoclast coupling processes. The combination of a bioactive membrane and CaP bone substitute material doped with Sr may produce early synergistic effects during GBR. STATEMENT OF SIGNIFICANCE: The study provides novel molecular, cellular and structural evidence on the promotion of early bone regeneration in response to synthetic strontium-containing hydroxyapatite (SrHA) substitute, in combination with a resorbable, guided bone regeneration (GBR) membrane. The prevailing view, based mainly upon in vitro data, is that the beneficial effects of Sr are exerted by the stimulation of bone-forming cells (osteoblasts) and the inhibition of bone-resorbing cells (osteoclasts). In contrast, the present study demonstrates that the local effect of Sr in vivo is predominantly via the inhibition of osteoclast number and activity and the reduction of osteoblast-osteoclast coupling. This experimental data will form the basis for clinical studies, using this material as an interesting bone substitute for guided bone regeneration.
Assuntos
Implantes Absorvíveis , Antígenos de Diferenciação/metabolismo , Regeneração Óssea , Substitutos Ósseos , Regeneração Tecidual Guiada/métodos , Membranas Artificiais , Osteoclastos/metabolismo , Animais , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Bovinos , Masculino , Osteoclastos/patologia , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: In orthopaedic surgery, cobalt chromium (CoCr) based alloys are used extensively for their high strength and wear properties, but with concerns over stress shielding and bone resorption due to the high stiffness of CoCr. The structural stiffness, principally related to the bulk and the elastic modulus of the material, may be lowered by appropriate design modifications, to reduce the stiffness mismatch between metal/alloy implants and the adjacent bone. Here, 3D printed CoCr and Ti6Al4V implants of similar macro-geometry and interconnected open-pore architecture prepared by electron beam melting (EBM) were evaluated following 26week implantation in adult sheep femora. Despite higher total bone-implant contact for Ti6Al4V (39±4%) than CoCr (27±4%), bone formation patterns were similar, e.g., densification around the implant, and gradual ingrowth into the porous network, with more bone in the outer half (periphery) than the inner half (centre). Raman spectroscopy revealed no major differences in mineral crystallinity, the apatite-to-collagen ratio, or the carbonate-to-phosphate ratio. Energy dispersive X-ray spectroscopy showed similar Ca/P ratio of the interfacial tissue adjacent to both materials. Osteocytes made direct contact with CoCr and Ti6Al4V. While osteocyte density and distribution in the new-formed bone were largely similar for the two alloys, higher osteocyte density was observed at the periphery of the porous network for CoCr, attributable to slower remodelling and a different biomechanical environment. The results demonstrate the possibility to achieve bone ingrowth into open-pore CoCr constructs, and attest to the potential for fabricating customised osseointegrated CoCr implants for load-bearing applications. STATEMENT OF SIGNIFICANCE: Although cobalt chromium (CoCr) based alloys are used extensively in orthopaedic surgery, stress shielding due to the high stiffness of CoCr is of concern. To reduce the stiffness mismatch between CoCr and bone, CoCr and Ti6Al4V implants having an interconnected open-pore architecture were prepared by electron beam melting (EBM). After six months of submerged healing in sheep, both alloys showed similar patterns of bone formation, with densification around the implant and gradual ingrowth into the porous network. The molecular and elemental composition of the interfacial tissue was similar for both alloys. Osteocytes made direct contact with both alloys, with similar overall osteocyte density and distribution. The work attests to the potential for achieving osseointegration of EBM manufactured porous CoCr implants.
Assuntos
Substitutos Ósseos/química , Interface Osso-Implante , Ligas de Cromo/química , Fêmur/metabolismo , Osteócitos/metabolismo , Ligas , Animais , Porosidade , Ovinos , Titânio/químicaRESUMO
Infection constitutes a major risk for implant failure, but the reasons why biomaterial sites are more vulnerable than normal tissue are not fully elucidated. In this study, a soft tissue infection model was developed, allowing the analysis of cellular and molecular responses in each of the sub-compartments of the implant-tissue interface (on the implant surface, in the surrounding exudate and in the tissue). Smooth and nanostructured titanium disks with or without noble metal chemistry (silver, gold, palladium), and sham sites, were inoculated with Staphylococcus epidermidis and analysed with respect to number of viable bacteria, number, viability and gene expression of host cells, and using different morphological techniques after 4 h, 24 h and 72 h. Non-infected rats were controls. Results showed a transient inflammatory response at control sites, whereas bacterial administration resulted in higher recruitment of inflammatory cells (mainly polymorphonuclear), higher, continuous cell death and higher gene expression of tumour necrosis factor-alpha, interleukin-6, interleukin-8, Toll-like receptor 2 and elastase. At all time points, S. epidermidis was predominantly located in the interface zone, extra- and intracellularly, and lower levels were detected on the implants compared with surrounding exudate. This model allows detailed analysis of early events in inflammation and infection associated to biomaterials in vivo leading to insights into host defence mechanisms in biomaterial-associated infections.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Inflamação/patologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus epidermidis/efeitos dos fármacos , Animais , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Materiais , Microscopia de Força Atômica , Nanoestruturas/ultraestrutura , Espectroscopia Fotoeletrônica , Projetos Piloto , Próteses e Implantes , Ratos Sprague-Dawley , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/ultraestrutura , Propriedades de SuperfícieRESUMO
Commercially pure titanium (cp-Ti) is regarded as the state-of-the-art material for bone-anchored dental devices, whereas the mechanically stronger alloy (Ti-6Al-4V), made of titanium, aluminum (Al) and vanadium (V), is regarded as the material of choice for high-load applications. There is a call for the development of new alloys, not only to eliminate the potential toxic effect of Al and V but also to meet the challenges imposed on dental and maxillofacial reconstructive devices, for example. The present work evaluates a novel, dual-stage, acid-etched, Ti-Ta-Nb-Zr alloy implant, consisting of elements that create low toxicity, with the potential to promote osseointegration in vivo. The alloy implants (denoted Ti-Ta-Nb-Zr) were evaluated after 7 days and 28 days in a rat tibia model, with reference to commercially pure titanium grade 4 (denoted Ti). Analyses were performed with respect to removal torque, histomorphometry and gene expression. The Ti-Ta-Nb-Zr showed a significant increase in implant stability over time in contrast to the Ti. Further, the histological and gene expression analyses suggested faster healing around the Ti-Ta-Nb-Zr, as judged by the enhanced remodeling, and mineralization, of the early-formed woven bone and the multiple positive correlations between genes denoting inflammation, bone formation and remodeling. Based on the present experiments, it is concluded that the Ti-Ta-Nb-Zr alloy becomes osseointegrated to at least a similar degree to that of pure titanium implants. This alloy is therefore emerging as a novel implant material for clinical evaluation.
Assuntos
Ligas/farmacologia , Osso e Ossos/efeitos dos fármacos , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Contagem de Células , Morte Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Implantes Experimentais , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Espectroscopia Fotoeletrônica , Ratos Sprague-Dawley , Propriedades de SuperfícieRESUMO
Bisphosphonates reverse the negative effects of ovariectomy on bone, but they have also been associated with adverse processes in human jawbone. The molecular events determining bone regeneration and implant integration in osteoporotic conditions, with and without bisphosphonate treatment, are unclear. In this study, ovariectomised rats, to which a single dose of saline (NaCl) or zoledronic acid (Zol) was administered, received titanium alloy implants in their tibiae and mandibles. An enzyme-linked immunosorbent assay, gene expression analysis and histomorphometry were performed. The results show that ovariectomy, per se, upregulated the expression of genes denoting bone formation in the tibia, bone remodelling in the mandible and apoptosis in the tibia and mandible. Zoledronic acid administration resulted in lower levels of a remodelling marker in serum and downregulated gene expression for inflammation, bone formation, angiogenesis and apoptosis, mainly in the mandible, after 28 d of healing. Histomorphometry revealed improved bone-to-implant contact in the tibia, while the opposite was observed in the mandible. The present data show that a systemic single dose of zoledronic acid, in ovariectomised animals, results in site-specific differences in the regulation of genes involved in bone healing and regeneration in association with implant installation. These events occur in parallel with site-specific differences in the rate of osseointegration, indicating diverse tissue responses in the tibia and mandible after zoledronic acid treatment. The zoledronic acid effect on gene expression, during the late phase of healing in the mandible, suggests negative effects by the anti-resorptive agent on osseointegration at that particular site.
Assuntos
Difosfonatos/administração & dosagem , Fraturas Ósseas/terapia , Imidazóis/administração & dosagem , Osteíte/prevenção & controle , Osteogênese/efeitos dos fármacos , Ovariectomia , Próteses e Implantes , Animais , Conservadores da Densidade Óssea/administração & dosagem , Terapia Combinada , Feminino , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Osteíte/metabolismo , Osteíte/patologia , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ácido ZoledrônicoRESUMO
Osseointegration, the direct contact between an implant surface and bone tissue, plays a critical role in interfacial stability and implant success. Analysis of interfacial zones at the micro- and nano-levels is essential to determine the extent of osseointegration. In this paper, a series of state-of-the-art microscopy techniques are used on laser-modified implants retrieved from humans. Partially laser-modified implants were retrieved after two and a half months' healing and processed for light and electron microscopy. Light microscopy showed osseointegration, with bone tissue growing both towards and away from the implant surface. Transmission electron microscopy revealed an intimate contact between mineralized bone and the laser-modified surface, including bone growth into the nano-structured oxide. This novel observation was verified by three-dimensional Z-contrast electron tomography, enabling visualization of an apatite layer, with different crystal direction compared with the apatite in the bone tissue, encompassing the nano-structured oxide. In conclusion, the present study demonstrates the nano-scale osseointegration and bonding between apatite and surface-textured titanium oxide. These observations provide novel data in human specimens on the ultrastructure of the titanium-bone interface.