RESUMO
The aim of this study is to determine if there is an antagonistic effect between tumour necrosis factor (TNF)-alpha and the immunoregulatory interleukin (IL)-10 on chondrocytes survival. Serum-starved primary human articular chondrocytes were stimulated with either 10 ng/ml recombinant TNF-alpha, IL-10 or a combination of both (at 10 ng/ml each). Chondrocyte apoptosis was determined by measuring caspase-3/7, -8 and -9 activities using caspase assays. Mitochondrial apoptotic inducer bax, and the suppressor bcl-2 were evaluated using western blotting at 48 h. Results indicated that TNF-alpha increased caspase activities and resulted in a significant (p = 0.001) increase in bax/bcl-2 ratio. Stimulation with IL-10 did not alter caspase activities, while co-treatment with IL-10 and TNF-alpha inhibited TNF-alpha induced caspase activities and significantly (p > 0.004) impaired bax/bcl-2 ratio. At 24 h, mRNA levels for collagen type II, TNF-alpha and IL-10 were determined using real-time RT-PCR. Stimulation with TNF-alpha or TNF-alpha and IL-10 significantly inhibited collagen type II and increased IL-10 and TNF-alpha mRNA expression. IL-10 modulated the pro-apoptotic capacity of TNF-alpha in chondrocytes as shown by the decrease in caspase activities and bax/bcl-2 ratio compared to TNF-alpha stimulated chondrocytes, suggesting a mostly antagonistic interplay of IL-10 and TNF-alpha on mitochondrial apoptotic pathways.
Assuntos
Apoptose/efeitos dos fármacos , Cartilagem Articular/imunologia , Condrócitos/imunologia , Interleucina-10/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Idoso , Apoptose/imunologia , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Caspases/imunologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/biossíntese , Colágeno Tipo II/imunologia , Antagonismo de Drogas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-10/antagonistas & inibidores , Interleucina-10/imunologia , Interleucina-10/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Proteína X Associada a bcl-2/imunologia , Proteína X Associada a bcl-2/metabolismoRESUMO
S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. Here we demonstrate S100A8 and S100A9 expression in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins. In keeping with their reported high-mRNA expression, S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption.
Assuntos
Osso e Ossos/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Cartilagem/metabolismo , Animais , Medula Óssea/metabolismo , Osso e Ossos/citologia , Calcificação Fisiológica/genética , Calcificação Fisiológica/fisiologia , Calgranulina A/análise , Calgranulina A/genética , Calgranulina B/análise , Calgranulina B/genética , Cartilagem/citologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Pré-Escolar , Condrócitos/citologia , Condrócitos/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 microm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + trade mark dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.
Assuntos
Biomarcadores/análise , Calcificação Fisiológica/fisiologia , Imuno-Histoquímica/métodos , Mandíbula/citologia , Osteogênese , Animais , Colágeno Tipo I/análise , Osteocalcina/análise , Osteopontina/análise , OvinosRESUMO
Establishing and maintaining mature bone at the bone-device interface is critical to the long-term success of prosthesis. Poor cell adhesion to orthopaedic and dental implants results in implant failure. Considerable effort has been devoted to alter the surface characteristics of these biomaterials in order to improve the initial interlocking of the device and skeleton. We investigated the effect of surface chemistry modification of titanium alloy (Ti-6Al-4V) with zinc, magnesium or alkoxide-derived hydroxy carbonate apatite (CHAP) on the regulation of key intracellular signalling proteins in human bone-derived cells (HBDC) cultured on these modified Ti-6Al-4V surfaces. Western blotting demonstrated that modifying Ti-6Al-4V with CHAP or Mg results in modulation of key intracellular signalling proteins. We showed an enhanced activation of Shc, a common point of integration between integrins and the Ras/Mapkinase pathway. Mapkinase pathway was also upregulated, suggesting its role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the Mg and CHAP modified Ti-6Al-4V. Thus surface modification with CHAP or Mg may contribute to successful osteoblast function and differentiation at the skeletal tissue-device interface.
Assuntos
Ligas/química , Osteoblastos/citologia , Transdução de Sinais , Titânio/química , Apatitas/química , Materiais Biocompatíveis/química , Western Blotting , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Carbonatos/química , Adesão Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Hidroxiapatitas/química , Integrinas/química , Integrinas/metabolismo , Íons , Sistema de Sinalização das MAP Quinases , Magnésio/química , Microscopia Eletrônica de Varredura , Osseointegração , Osteoblastos/metabolismo , Transição de Fase , Fosforilação , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Propriedades de Superfície , Fatores de Tempo , Regulação para Cima , Zinco/químicaRESUMO
Biomaterials research is investigating increasingly complex materials capable of mirroring the highly organized biochemical and architectural environments of the body. Accordingly, tissue scaffolds with nanoscale properties that mirror the fibrous proteins present in tissue are being developed. Such materials can benefit from the inherent dimensional similarities and nanocomposite nature of the cellular environment, altering nanoscale dimensional and biochemical properties to mimic the regulatory characteristics of natural cellular environments. One nanomaterial which demonstrates potential across a diverse range of biomaterial applications is carbon nanotubes (CNTs). Building on previous reports, a method to coat CNTs throughout 3D porous structures is developed. Through modifications to typical chemical vapour deposition (CVD), a high-quality uniform coating of carbon nanotubes (CNTs) is demonstrated over ß-tricalcium phosphate/hydroxyapatite (or TCP/HA), which is in clinical use; and the high-mechanical-strength multicomponent ceramic Ca2ZnSi2O7-ZnAl2O4, (or Sr-HT-Gah). The resulting materials address deficiencies of previously reported CNT biomaterials by simultaneously presenting properties of high porosity, biocompatibility and a mechanical stability. Together, this unique combination of properties makes these scaffolds versatile materials for tissue engineering in load bearing applications.
RESUMO
Implants, particularly joint replacement prostheses, are one of the great success stories of modern medicine. However, too many implants fail prematurely, mainly due to aseptic bone loss around the implant. This paper reviews our current understanding of the role of osteoclasts in this peri-implant bone lysis. Prosthetic particles, often produced by articulating prostheses, are one of the major causes of elevated osteoclast lysis of peri-implant bone. Over the past decade there have been major advances in our understanding of the factors that regulate osteoclast activity, many of which were found to be important in osteoclast formation and activity in the peri-implant tissues. These factors are targets of a number of recently developed drugs that have been used successfully to prevent and treat peri-implant bone lysis in experimental models. Treatments such as these are being used in a number of bone loss pathologies in humans and have the potential for successful treatment of peri-implant osteolysis. In addition, understanding how different biomaterials influence the expression of key osteoclastogenic factors may allow us to select biomaterials for implantation that will last the lifetime of the recipient.
Assuntos
Artroplastia de Quadril , Materiais Biocompatíveis , Osso e Ossos/química , Osteoclastos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Osseointegração , Osteólise , Osteoprotegerina , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose TumoralRESUMO
Calcium phosphate coated titanium and titanium alloy are widely used as dental and orthopaedic implants. This study examines the effect of novel calcium titanium and calcium titanium zirconium phosphates suitable for plasma-spraying onto titanium substrata on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that on native titanium and hydroxyapatite-coated titanium. Test materials were an acid etched and sand-blasted titanium surface (Ti-DPS), a plasma-sprayed hydroxyapatite coating (HA), and five materials which were created from CaTi(4)(PO(4))(6) (CTP) and CaZr(4)(PO(4))(6) (CZP): sintered CaTi(4)(PO(4))(6) (CTP-S1), sintered 46CaO.23TiO(2).31P(2)O(5) (CTP-S2), sintered CaTiZr(3)(PO(4))(6), (CTZP-S1), sintered 46CaO.23ZrO(2).31P(2)O(5) (CTZP-S2) and sintered 55CaO.20TiO(2).31P(2)O(5) (CTP-S3). HBDC were grown on the substrata for 3, 7, 14 and 21 d, counted and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrates significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. At 14 and 21 d, cells on CTP-S3 displayed significantly enhanced expression of all osteogenic mRNAs. Surfaces of CTP-S1 and CTP-S3 had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and HA, suggesting that these novel materials may possess a higher potency to enhance osteogenesis.
Assuntos
Substitutos Ósseos/química , Cálcio/química , Osteogênese , Titânio/química , Fosfatase Alcalina/química , Anticorpos/química , Fosfatos de Cálcio/química , Proliferação de Células , Células Cultivadas , Colágeno/química , Durapatita/química , Humanos , Imuno-Histoquímica , Hibridização In Situ , Sialoproteína de Ligação à Integrina , Osteoblastos/química , Osteocalcina/química , Osteonectina/química , Osteopontina , Próteses e Implantes , RNA Mensageiro/metabolismo , Sialoglicoproteínas/química , Fatores de TempoRESUMO
Loss of bone near joint prostheses is thought to be caused by activation of recruited osteoclasts by osteolytic mediators induced by wear particles. It is proposed that particles inhibit osteogenesis during bone remodelling causing a reduction in the levels of peri-implant bone. This study explores whether prosthetic particles modulate bone formation by affecting osteoblastic bone-related mRNAs (alkaline phosphatase, pro-collagen Ialpha1, osteopontin, osteonectin, osteocalcin, bone sialoprotein and thrombospondin) or their translated proteins using titanium alloy, commercially pure titanium, and cobalt-chrome particles. The direct effect of the particles revealed no change to the expression of the bone-related mRNAs in human bone-derived cells (HBDC) at the time points investigated; although non-collagenous translated proteins expressed by these HBDC were significantly effected (p<0.05). Different patterns of expression for bone-related proteins were induced by the different particles both directly and indirectly. Inflammatory mediators (interleukin-1beta, tumor necrosis factor alpha, interleukin-6, and prostaglandin E2) had similar effects on HBDC to the media obtained from monocytes incubated with particles. This study shows that prosthetic wear particles can significantly modify the expression of bone-related proteins by osteogenic cells in vitro. These alterations in osteogenic activity at the interface of the implant and bone may be an important factor in the failure of many orthopaedic implants.
Assuntos
Osso e Ossos/metabolismo , Prótese Articular , Osteoblastos/metabolismo , Proteínas/metabolismo , Osso e Ossos/citologia , Humanos , Hibridização In Situ , Técnicas In Vitro , Osteoblastos/citologia , Osteogênese , Proteínas/genética , RNA Mensageiro/genéticaRESUMO
The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.
Assuntos
Substitutos Ósseos , Fosfatos de Cálcio , Cerâmica , Osteoblastos/metabolismo , Técnicas de Cultura de Células , Humanos , RNA Mensageiro/metabolismoRESUMO
Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Osteoclastos/metabolismo , Osteólise/metabolismo , Falha de Prótese , Infecções Relacionadas à Prótese/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Reação a Corpo Estranho/etiologia , Reação a Corpo Estranho/metabolismo , Reação a Corpo Estranho/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Osteoclastos/patologia , Osteólise/etiologia , Osteólise/patologia , Osteonecrose/etiologia , Osteonecrose/metabolismo , Osteonecrose/patologia , Osteoprotegerina , Infecções Relacionadas à Prótese/etiologia , Infecções Relacionadas à Prótese/patologia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores do Fator de Necrose TumoralRESUMO
The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitative in situ hybridization (ISH) using either image analysis or fluorescence in situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by the p-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.
Assuntos
Hibridização In Situ/métodos , RNA Mensageiro/análise , Animais , Células COS/citologia , Células COS/metabolismo , Ensaio de Imunoadsorção Enzimática , Eritropoetina/genética , Vetores Genéticos/genética , Hibridização In Situ/instrumentação , Plasmídeos/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation because it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins by human bone-derived cells (HBDCs) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, two materials with a crystalline phase Ca(2)KNa(PO(4))(2) and with a small amorphous portion containing either magnesium potassium phosphate (material denominated GB14) or silica phosphate (material denominated GB9), and a calcium phosphate bone cement (material denominated Biocement D). HBDCs were grown on the substrata for 3, 7, 14, and 21 days, counted, and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All substrates supported continuous cellular growth for 21 days. In the presence of GB14 and Biocement D specimens cell proliferation was reduced and cell differentiation increased. At day 21, the greatest number of cells was found on GB9 expressing significantly higher levels of bone-related proteins than cells grown on all other surfaces. Because all novel materials facilitated the expression of the osteoblastic phenotype at least as much as TCP and the polystyrene control, these biomaterials can be regarded as excellent candidate bone substitute materials. GB9 induced the highest proliferation and cellular differentiation after 21 days of incubation, suggesting that this material may possess a higher potency for enhancing osteogenesis than TCP.
Assuntos
Cimentos Ósseos/farmacologia , Fosfatos de Cálcio/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Biossíntese de Proteínas , Adolescente , Feminino , Humanos , RNA Mensageiro , Coluna Vertebral/citologia , Coluna Vertebral/metabolismoRESUMO
Various surface modifications have been applied to titanium alloy (Ti-6Al-4V) implants, in an attempt to enhance osseointegration; crucial for ideal prosthetic fixation. Despite the numerous studies demonstrating that peptide-modified surfaces influence in vitro cellular behavior, there is relatively little data reporting their effects on bone remodeling. The objective of this article was to examine the effects of chemically modifying Ti-6Al-4V surfaces with a common RGD sequence, a 15-residue peptide containing GRGDSP (glycine-arginine-glycine-aspartate-serine-proline), on the modulation of bone remodeling. The expression of proteins known to be associated with osseous matrix and bone resorption were studied during the growth of human bone-derived cells (HBDC) on these peptide-modified surfaces. HBDC grown for 7 days on RGD surfaces displayed significantly increased levels of osteocalcin, and pro-collagen Ialpha1 mRNAs, compared with the production by HBDC grown on the native Ti-6Al-4V. A pattern that was also reflected at the protein levels for osteocalcin, type I collagen, and bone sialoprotein. Moreover, HBDC grown for 7 and 14 days on RGD-modified Ti-6Al-4V expressed significantly higher level of osteoclast differentiation factors and lower levels of osteoprotegerin and IL-6 proteins compared with other surfaces tested. These results suggest that different chemical treatments of implant material (Ti-6Al-4V) surface result in differential bone responses, not only their ability to form bone but also to stimulate osteoclastic formation.
Assuntos
Osso e Ossos/citologia , Diferenciação Celular , Peptídeos/química , Titânio/química , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Peptídeos/genética , Fenótipo , RNA Mensageiro/genéticaRESUMO
Aseptic loosening of prosthetic arthroplasty is the most common reason for implant failure in adult orthopaedic reconstruction. At the interface of aseptic loosened prostheses, there is an abundance of particle-activated macrophages and other inflammatory cells. The role of these particle-laden macrophages in the osteogenic arm of the remodeling skeleton in this pathological condition is poorly understood. Molecular signaling by mesenchymal cells and mononuclear inflammatory cells residing in the interfacial tissues between bone and cement or prosthetic material of aseptically loosened joint prostheses was studied using in situ hybridization and immunohistochemical techniques. We found that a range of collagenous and noncollagenous matrix proteins, including osteopontin, osteocalcin, bone sialoprotein, and type I collagen, were produced in the periprosthetic tissue by foamy macrophages, as well as nearby osteogenic cells. The former accumulated in profusion in the three zones of interfacial tissues: pseudomembranous, fibrous, and osseous. Spindle mesenchymal cells in the fibrous zone failed to express any of the osteogenic mRNAs or proteins sought. The expression of bone-related genes and proteins by foamy macrophages at the interface of an aseptic loosened prosthesis may contribute to the disturbance of bone remodeling at this site.
Assuntos
Osso e Ossos/citologia , Macrófagos/fisiologia , Osteogênese/genética , Próteses e Implantes , Adulto , Idoso , Idoso de 80 Anos ou mais , Compostos Azo , Materiais Biocompatíveis , Corantes , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Biossíntese de Proteínas , Fixação de TecidosRESUMO
The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non-articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell-cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid- (PGA) associated porcine heterotopic mono- and cocultures were assessed for cell vitality, types II, I, and total collagen-, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only ß1-integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species-dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan-rich hyaline-like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time-dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair.
Assuntos
Cartilagem/patologia , Condrócitos/citologia , Ácido Poliglicólico/química , Animais , Materiais Biocompatíveis/química , Técnicas de Cocultura , Colágeno Tipo II/metabolismo , Cartilagem da Orelha/patologia , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hidroxiprolina/metabolismo , Integrina beta1/metabolismo , Septo Nasal/patologia , Ácido Poliglicólico/metabolismo , Fatores de Transcrição SOX9/metabolismo , Suínos , Alicerces Teciduais/químicaRESUMO
During the past two decades, research on ceramic scaffolds for bone regeneration has progressed rapidly; however, currently available porous scaffolds remain unsuitable for load-bearing applications. The key to success is to apply microstructural design strategies to develop ceramic scaffolds with mechanical properties approaching those of bone. Here we report on the development of a unique microstructurally designed ceramic scaffold, strontium-hardystonite-gahnite (Sr-HT-gahnite), with 85% porosity, 500µm pore size, a competitive compressive strength of 4.1±0.3MPa and a compressive modulus of 170±20MPa. The in vitro biocompatibility of the scaffolds was studied using primary human bone-derived cells. The ability of Sr-HT-gahnite scaffolds to repair critical-sized bone defects was also investigated in a rabbit radius under normal load, with ß-tricalcium phosphate/hydroxyapatite scaffolds used in the control group. Studies with primary human osteoblast cultures confirmed the bioactivity of these scaffolds, and regeneration of rabbit radial critical defects demonstrated that this material induces new bone defect bridging, with clear evidence of regeneration of original radial architecture and bone marrow environment.
Assuntos
Regeneração Óssea/fisiologia , Substitutos Ósseos/síntese química , Cerâmica/química , Regeneração Tecidual Guiada/instrumentação , Fraturas do Rádio/fisiopatologia , Fraturas do Rádio/cirurgia , Alicerces Teciduais , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Consolidação da Fratura/fisiologia , Regeneração Tecidual Guiada/métodos , Masculino , Teste de Materiais , Coelhos , Fraturas do Rádio/diagnóstico , Resultado do TratamentoRESUMO
We here present the first successful report on combining nanostructured silk and poly(ε-caprolactone) (PCL) with a ceramic scaffold to produce a composite scaffold that is highly porous (porosity â¼85%, pore size â¼500 µm, â¼100% interconnectivity), strong and non-brittle with a surface that resembles extracellular matrix (ECM). The ECM-like surface was developed by self-assembly of nanofibrous structured silk (20-80 nm diameter, similar to native collagen found in ECM) over a thin PCL layer which is coated on biphasic calcium phosphate (BCP) scaffolds. The effects of different concentrations of silk solution on the mechanical and physical properties of the scaffolds were also comprehensively examined. Our results showed that using silk only (irrespective of concentration) for the modification of ceramic scaffolds could drastically reduce the compressive strength of the modified scaffolds in aqueous media, and the modification made a limited contribution to improving scaffold toughness. Using PCL/nanostructured silk the compressive strength and modulus of the modified scaffolds reached 0.42 MPa (compared with 0.07 MPa for BCP) and â¼25 MPa (compared with 5 MPa for BCP), respectively. The failure strain of the modified scaffold increased more than 6% compared with a BCP scaffold (failure strain of less than 1%), indicating a transformation from brittle to elastic behavior. The cytocompatibility of ECM-like composite scaffolds was investigated by studying the attachment, morphology, proliferation and bone-related gene expression of primary human bone-derived cells. Cells cultured on the developed scaffolds for 7 days had significant up-regulation of cell proliferation (â¼1.6-fold higher, P<0.001) and osteogenic gene expression levels (collagen type I, osteocalcin and bone sialoprotein) compared with the other groups tested.
Assuntos
Regeneração Óssea/fisiologia , Fosfatos de Cálcio/química , Nanofibras/química , Osteoblastos/fisiologia , Poliésteres/química , Seda/química , Alicerces Teciduais/química , Apatitas/metabolismo , Materiais Biocompatíveis/química , Líquidos Corporais/química , Osso e Ossos/química , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Células Cultivadas , Cerâmica/química , Força Compressiva , Elasticidade , Matriz Extracelular/química , Expressão Gênica , Humanos , Teste de Materiais , Osteoblastos/citologia , Porosidade , Estresse Mecânico , Engenharia Tecidual/métodosRESUMO
This is the first reported study to prepare highly porous baghdadite (Ca3ZrSi2O9) scaffolds with and without surface modification and investigate their ability to repair critical-sized bone defects in a rabbit radius under normal load. The modification was carried out to improve the mechanical properties of the baghdadite scaffolds (particularly to address their brittleness) by coating their surfaces with a thin layer (â¼400 nm) of polycaprolactone (PCL)/bioactive glass nanoparticles (nBGs). The ß-tricalcium phosphate/hydroxyapatite (TCP/HA) scaffolds with and without modification were used as the control groups. All of the tested scaffolds had an open and interconnected porous structure with a porosity of â¼85% and average pore size of 500 µm. The scaffolds (six per scaffold type and size of 4 mm × 4 mm × 15 mm) were implanted (press-fit) into the rabbit radial segmental defects for 12 weeks. Micro-computed tomography and histological evaluations were used to determine bone ingrowth, bone quality, and implant integration after 12 weeks of healing. Extensive new bone formation with complete bridging of the radial defect was evident with the baghdadite scaffolds (modified/unmodified) at the periphery and in close proximity to the ceramics within the pores, in contrast to TCP/HA scaffolds (modified/unmodified), where bone tended to grow between the ulna adjacent to the implant edge. Although the modification of the baghdadite scaffolds significantly improved their mechanical properties, it did not show any significant effect on in vivo bone formation. Our findings suggest that baghdadite scaffolds with and without modification can serve as a potential material to repair critical sized bone defects.