RESUMO
We aimed to quantify bone colonization toward an untreated titanium implant with primary stability following filling of the defect with micromacroporous biphasic calcium phosphate (MBCP) granules (TricOs) or MBCP granules mixed with fibrin sealant (Tisseel). Medial arthrotomy was performed on the knees of 20 sheep to create a bone defect (16 mm deep; 10 mm diameter), followed by anchorage of a titanium screw. Defects were filled with TricOs or TricOs-Tisseel granules, a perforated MBCP washer, a titanium washer and titanium screw. Sheep were euthanized at 3, 6, 12 and 26 weeks. From Week 12 onwards, the percentage of bone in contact with the 8 mm anchorage part of the screw increased in both groups, confirming its primary stability. At 26 weeks, whereas bone colonization was similar in both groups, biodegradation of ceramic was more rapid in the TricOs-Tisseel group (P = 0.0422). The centripetal nature of bone colonization was evident. Bone contact with the titanium implant surface was negligible. In conclusion, the use of a model that reproduces a large metaphyseal bone defect around a titanium implant with primary stability, filled with a mixture of either TricOs ceramic granules or TricOs granules mixed with Tisseel fibrin sealant, suggests that the addition of fibrin to TricOs enhances bone filling surgical technology.
Assuntos
Osso e Ossos/cirurgia , Fosfatos de Cálcio/química , Cerâmica/química , Adesivo Tecidual de Fibrina/química , Fibrina , Animais , Feminino , Próteses e Implantes , OvinosRESUMO
Calcium phosphate bioceramic granules associated with hydrosoluble polymers were developed as bone substitutes for various maxillofacial and orthopaedic applications. These injectable bone substitutes, support and regenerate bone tissue and resorb after implantation. The efficiency of these multiphasic materials is due to the osteogenic and osteoconductive properties of the microporous biphasic calcium phosphate. The associated hydrosoluble polymers are considered as carriers in order to achieve the rheological properties of injectable bone substitutes (IBS). In this study, we used 2 semi synthetic hydrosoluble polymers of polysaccharidic origin. The hydroxy propyl methyl cellulose (HPMC), with and without silane, was combined with microporous BCP granules. The presence of silane induced considerable gelation of the suspension. The 2 IBS used (without gelation, IBS1, with gelation, IBS2) were implanted in critical size femoral epiphysis defects in rabbits. No foreign body reactions were observed in either sample. However, because of the higher density from gelation, cell colonisation followed by bone tissue ingrowth was delayed over time with IBS2 compared to the IBS1 without gelation. The results showed resorption of the BCP granule and bone ingrowth at the expense of both IBS with different kinetics. This study demonstrates that the hydrogel cannot be considered merely as a carrier. The gelation process delayed cell and tissue colonisation by slow degradation of the HPMC Si, compared to the faster release of HPMC with IBS1, in turn inducing faster permeability and spaces for tissue ingrowth between the BCP granules.
Assuntos
Materiais Biocompatíveis/química , Fosfatos de Cálcio/química , Hidrogéis/química , Animais , Substitutos Ósseos/química , Osso e Ossos/patologia , Feminino , Derivados da Hipromelose , Teste de Materiais , Metilcelulose/análogos & derivados , Metilcelulose/química , Músculos/patologia , Ortopedia , Osseointegração/efeitos dos fármacos , Polímeros/química , Coelhos , Silanos/químicaRESUMO
INTRODUCTION: Mandibular reconstruction in head and neck oncology uses a number of techniques whose results are inconsistent and whose indications do not benefit from consensus. MATERIALS AND METHODS: A review of the literature allowed us to assemble the available knowledge on current mandibular reconstruction techniques, their functional results, and the research perspectives. RESULTS AND DISCUSSION: Marginal resections lead to dental rehabilitation problems, which can be palliated by alveolar enhancement techniques but whose results have not been validated in cases subjected to irradiation. Reconstruction of segmental substance loss is warranted by the repercussions on the vital prognosis when it is anterior and on the quality of life when it is posterior. The ideal means of reconstruction is the free fibular flap, which is limited by cost, morbidity of the donor site, and selection of the patient's surgical team. For these reasons, it may be necessary to turn to pedicled osteo-myocutaneous flaps, abandoned because of their reputedly very high failure rate, but few have been reported in the literature. CONCLUSION: Poor functional results of mandibular reconstruction plates make this a last-resort solution. Tissue engineering is currently the most promising line of research. It runs counter to the principles of oncology itself because postoperative radiotherapy reduces the osteoinduction potential of the biomaterials proposed.
Assuntos
Transplante Ósseo/métodos , Carcinoma/cirurgia , Mandíbula/cirurgia , Neoplasias Bucais/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Retalhos Cirúrgicos , Humanos , Procedimentos Cirúrgicos Bucais/métodos , Satisfação do Paciente , Resultado do TratamentoRESUMO
Interconnection, one of the main structural features of macroporous calcium-phosphate ceramics, contributes to the biological and physicochemical properties of bone substitutes. As no satisfactory method exists for evaluating this feature, analysis was performed to determine the permeability, tortuosity, and equivalent diameter of interconnecting channels, that is the parameters that appear to be representative of the way pores are linked. The testing of two ceramics with similar porosity levels revealed important differences in all three interconnection parameters. One ceramic showed poor permeability, corresponding to a small equivalent diameter for interconnecting channels in conjunction with a high tortuosity factor, while the other displayed high permeability, a large diameter for interconnecting channels, and a low tortuosity factor. The methodology used, which can be applied to the quantification of interconnection in all calcium-phosphate ceramics, constitutes the first step in a complete study of the role of this feature in cellular colonization of the ceramic, matrix dissolution, and drug release from the calcium-phosphate matrix.
Assuntos
Materiais Biocompatíveis/química , Fosfatos de Cálcio/química , Cerâmica/química , Algoritmos , Materiais Biocompatíveis/síntese química , Fosfatos de Cálcio/síntese química , Cerâmica/síntese química , Difusão , Iodetos/química , Mercúrio/química , Modelos Químicos , Permeabilidade , Porosidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.
Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Hidrogéis , Metilcelulose/análogos & derivados , Engenharia Tecidual , Agrecanas/biossíntese , Cartilagem/citologia , Cartilagem/lesões , Técnicas de Cultura de Células , Células Cultivadas , Condrócitos/citologia , Colágeno Tipo II/biossíntese , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrogéis/química , Derivados da Hipromelose , Metilcelulose/química , Mucosa Nasal/metabolismo , Nariz/citologiaRESUMO
The use of injectable biomaterials is of interest in osteoporotic patients to locally restore bone mass in sites at risk of fracture. An injectable bone substitute (IBS1 made of betaTCP/hydroxyapatite as a calcium phosphate substitute and hydroxy-propyl-methyl-cellulose as a polymer carrier) was used in a severely osteopenic rat model obtained by combining orchidectomy (ORX) and disuse (paralysis induced by botulinum toxin - BTX). Fifty-six aged male rats were randomized into three groups: 18 were SHAM operated; 38 were ORX and BTX injected in the right hindlimb; they constituted the OP (osteoporotic) group. One month after ORX-BTX surgery, 20 of these OP rats received a IBS1 injection in the right femur (OP-IBS1 rats). Animals were studied at the time of IBS1 injection 1 month post ORX-BTX (M1), 1 month (M2) and 2 months (M3) after IBS1 injection. Bone mass (BV/TV) and microarchitectural parameters were measured by microCT. BV/TV was decreased after ORX-BTX; ORX and BTX had cumulative effects on bone loss (differences maximized on the right femur). BV/TV (combining the volume of both bone and material in OP-IBS1 rats) was elevated at M1 but decreased at M2. Marked bone formation was found onto the biomaterial granules but bone had a woven texture. A marked increase in the number of nonosteoclastic TRAcP+ cells was found in the implanted area. IBS1 induced new bone formation shortly after implantation but both IBS1 and woven bone were resorbed without inducing lamellar bone. Biomaterial trials must be conducted with long-term implantation periods, in aged osteoporotic animals.
Assuntos
Envelhecimento , Substitutos Ósseos , Durapatita , Metilcelulose/análogos & derivados , Osteoporose/terapia , Animais , Substitutos Ósseos/administração & dosagem , Modelos Animais de Doenças , Derivados da Hipromelose , Injeções Intralesionais , Masculino , Osteoporose/fisiopatologia , Ratos , Ratos Wistar , Índice de Gravidade de DoençaRESUMO
Tissue engineering is an emerging field of regenerative medicine which holds promise for the restoration of tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity and trauma. During the past decade, tissue engineering has evolved from the use of naked biomaterials, which may just replace small area of damaged tissue, to the use of controlled three-dimensional scaffolds in which cells can be seeded before implantation. These cellularized constructs aims at being functionally equal to the unaffected tissue and could make possible the regeneration of large tissue defects. Among the recently developed scaffolds for tissue engineering, polymeric hydrogels have proven satisfactory in cartilage and bone repair. Major technological progress and advances in basic knowledge (physiology and developmental biology) are today necessary to bring this proof of concept to clinical reality. The present review focuses on the recent advances in hydrogel-based tissue engineered constructs potentially utilizable in bone and cartilage regenerative medicine.
Assuntos
Materiais Biocompatíveis/química , Cartilagem/química , Hidrogéis/química , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Engenharia Biomédica/métodos , Regeneração Óssea , Substitutos Ósseos , Osso e Ossos/patologia , Cartilagem/metabolismo , Tecido Conjuntivo/metabolismo , HumanosRESUMO
PURPOSE OF THE STUDY: Certain confirmation of bone fusion remains difficult to obtain after arthrodesis despite progress in imaging techniques. Microscanning enables both qualitative and quantitative analysis of the bone microarchitecture. The purpose of this study was to evaluate this technique using a cervical arthrodesis with an intersomatic cage on an animal model and to validate results with histological analysis and electron scan microscopy (SEM). MATERIAL AND METHODS: C3-C4 discectomy was performed in 8 goats divided into two groups. In group 1 (3 animals), PEEK cages were inserted without bone graft. In group 2 (5 goats) the same cage was inserted and filled with an autologous iliac graft. The animals were sacrificed at six months. The instrumented levels were analyzed with a microscan. Histological slides were obtained and SEM performed. RESULTS: Nonunion was observed in the three animals with an empty cage (group 1) while only one animal in group 2 presented nonunion. Histology and SEM confirmed the diagnosis established with the microscan which also enabled a 3D analysis of the sample and study of the trabecular architecture of the intersomatic graft. DISCUSSION: The microscan enabled a micrometric analysis of the sample. This is the only technique enabling 3D analysis (slices can be obtained in the three planes for 3D reconstruction) for both qualitative and quantitative assessment. Analysis of the trabecular microstructure constitutes a major progress in evaluating the mechanical value of the fusion. The sample is not destroyed and can be studied further with other biomechanical techniques. CONCLUSION: Microscanning is an important technical advancement for the analysis of bone fusion. Future applications will undoubtedly be numerous (follow-up after arthrodesis, analysis of the mechanical quality of a graft). In vivo applications will probably be adapted soon.
Assuntos
Imageamento Tridimensional , Fusão Vertebral , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/cirurgia , Tomografia Computadorizada por Raios X/métodos , Animais , Cabras , Microscopia Eletrônica de Varredura , Coluna Vertebral/ultraestruturaRESUMO
Mineralization and crystal deposition are natural phenomena widely distributed in biological systems from protozoa to mammals. In mammals, normal and pathological calcifications are observed in bones, teeth, and soft tissues or cartilage. We review studies on the adaptive apatite crystal formation in enamel compared with those in other calcified tissues (e.g., dentin, bone, and fish enameloids) and in pathological calcifications, demonstrating the adaptation of these crystals (in terms of crystallinity and orientation) to specific tissues that vary in functions or vary in normal or diseased conditions. The roles of minor elements, such as carbonate, magnesium, fluoride, hydrogen phosphate, pyrophosphate, and strontium ions, on the formation and transformation of biologically relevant calcium phosphates are summarized. Another adaptative process of crystals in biology concerns the recent development of calcium phosphate ceramics and other related biomaterials for bone graft. Bone graft materials are available as alternatives to autogeneous bone for repair, substitution, or augmentation. This paper discusses the adaptive crystal formation in mineralized tissues induced by calcium phosphate and related bone graft biomaterials during bone repair.
Assuntos
Materiais Biocompatíveis/química , Calcificação Fisiológica , Calcinose , Fosfatos de Cálcio/química , Animais , Cristalização , Cristalografia por Raios X , HumanosRESUMO
For the repair of bone defects, a tissue engineering approach would be to combine cells capable of osteogenic (i.e. bone-forming) activity with an appropriate scaffolding material to stimulate bone regeneration and repair. Human mesenchymal stem cells (hMSCs), when combined with hydroxyapatite/beta-tricalcium phosphate (HA/TCP) ceramic scaffolds of the composition 60% HA/40% TCP (in weight %), have been shown to induce bone formation in large, long bone defects. However, full repair or function of the long bone could be limited due to the poor remodeling of the HA/TCP material. We conducted a study designed to determine the optimum ratio of HA to TCP that promoted hMSC induced bone formation yet be fully degradable. In a mouse ectopic model, by altering the composition of HA/TCP to 20% HA/80% TCP, hMSC bone induction occurred at the fastest rate in vivo over the other formulations of the more stable 100% HA, HA/TCP (76/24, 63/37, 56/44), and the fully degradable, 100% TCP. In vitro studies also demonstrated that 20/80 HA/TCP stimulated the osteogenic differentiation of hMSCs as determined by the expression of osteocalcin.
Assuntos
Células da Medula Óssea/citologia , Substitutos Ósseos/química , Osso e Ossos/citologia , Fosfatos de Cálcio/química , Transplante de Células-Tronco Mesenquimais/métodos , Osteogênese/fisiologia , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Diferenciação Celular , Células Cultivadas , Cerâmica/química , Humanos , Teste de Materiais , Camundongos , Camundongos SCIDRESUMO
Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Metilcelulose/análogos & derivados , Silanos/química , Animais , Cartilagem/metabolismo , Cartilagem Articular/citologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Dimerização , Glicosaminoglicanos/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Derivados da Hipromelose , Metilcelulose/química , Microscopia de Fluorescência , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Engenharia TecidualRESUMO
The aim of this study was to compare the bone colonization of a macroporous biphasic calcium phosphate (MBCP) ceramic in different sites (femur, tibia, and calvaria) in two animal species (rats and rabbits). A critical size defect model was used in all cases with implantation for 21 days. Bone colonization in the empty and MBCP-filled defects was measured with the use of backscattered electron microscopy (BSEM). In the empty cavities, bone healing remained on the edges, and did not bridge the critical size defects. Bone growth was observed in all the implantation sites in rats (approximately 13.6-36.6% of the total defect area, with ceramic ranging from 46.1 to 51.9%). The bone colonization appeared statistically higher in the femur of rabbits (48.5%) than in the tibia (12.6%) and calvaria (22.9%) sites. This slightly higher degree of bone healing was related to differences in the bone architecture of the implantation sites. Concerning the comparison between animal species, bone colonization appeared greater in rabbits than in rats for the femoral site (48.5% vs. 29.6%). For the other two sites (the tibia and calvaria), there was no statistically significant difference. The increased bone ingrowth observed in rabbit femurs might be due to the large bone surface area in contact with the MBCP ceramics. The femoral epiphysis of rabbits is therefore a favorable model for testing the bone-bonding capacity of materials, but a comparison with other implantation sites is subject to bias. This study shows that well-conducted and fully validated models with the use of small animals are essential in the development of new bone substitutes.
Assuntos
Substitutos Ósseos/metabolismo , Cerâmica/metabolismo , Implantes Experimentais , Modelos Animais , Animais , Substitutos Ósseos/química , Osso e Ossos/citologia , Cerâmica/química , Feminino , Microscopia Eletrônica de Varredura , Porosidade , Coelhos , RatosRESUMO
Fourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel. Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA). Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin. The v1v3 PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO4(2-) groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues.
Assuntos
Colágeno/metabolismo , Dentina/metabolismo , Durapatita/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Calcificação de Dente/fisiologia , Adolescente , Adulto , Artefatos , Colágeno/efeitos dos fármacos , Dentina/ultraestrutura , Dessecação , Humanos , Metacrilatos , Dente Serotino/crescimento & desenvolvimento , Dente Serotino/metabolismo , Desnaturação Proteica , Manejo de Espécimes , Inclusão do Tecido/métodos , Dente não Erupcionado/metabolismo , Dente não Erupcionado/ultraestruturaRESUMO
The study of bone mineralization processes is of considerable interest in understanding bone diseases and developing new therapies for skeletal disorders, particularly since bone homeostasis requires numerous cell types and a large cytokine network. Cell culture models of mineralization have often been used to study the cellular mechanisms of mineralization, but few data have been reported concerning the influence of extracellular matrix components and cytokines on the physicochemical properties of mineral. The purpose of this study was to analyze the effects of two cytokines, leukemia inhibitory factor (LIF) and oncostatin M (OSM), involved in bone metabolism on the physicochemical properties of bone mineral formed in a murine in vivo mineralization model. Murine bone marrow cells implanted under the kidney capsule in the presence or absence of cytokines led to heterotopic ossicle formation. A scanning electron microscopic microprobe revealed that heterotopic calcification had a lower (approximately 20%) Ca/P ratio after cytokine treatment as compared with the control without cytokine. Transmission electron microscopic analysis of cytokine-treated ossicles showed numerous areas with low mineral density, whereas electron diffraction pattern revealed an apatitic phase. These areas were not observed in the absence of cytokine. Moreover, Fourier transform-infrared microspectroscopy showed at the molecular level that the presence of either cytokine induced many microscopic areas in which short-range order organization, such as incorporation of carbonate and crystallinity/maturity of ossicle mineral, were modified. LIF and OSM influenced mineral phase formation in the present model and may thus be key protagonists in bone mineral development and skeletal diseases.
Assuntos
Calcinose , Inibidores do Crescimento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Minerais/metabolismo , Peptídeos/metabolismo , Animais , Células da Medula Óssea/química , Células da Medula Óssea/ultraestrutura , Transplante de Medula Óssea , Células Cultivadas , Feminino , Rim/química , Rim/diagnóstico por imagem , Rim/ultraestrutura , Fator Inibidor de Leucemia , Camundongos , Microquímica , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Nefrocalcinose/patologia , Oncostatina M , Radiografia , Espectroscopia de Infravermelho com Transformada de Fourier , Transplante HeterotópicoRESUMO
Calcium-phosphate bone replacement biomaterial has been used as a drug carrier for therapeutic agents. This study investigated the efficacy of local administration of human growth hormone (hGH) by macroporous biphasic calcium phosphate (MBCP) implants in improving the bone substitution qualities of ceramics. hGH release from MBCP implants loaded with 1 microg of hGH was rapid during the first 48 h and then sustained for a total of 9 days. Immunolocalization of hGH in vitro and in vivo by transmission electron microscopy showed its presence inside the material, indicating that it was able to penetrate within the porosity of the ceramic during the adsorption process. MBCP cylinders (6 x 6 mm) were loaded with 0.1, 1, and 10 microg of hGH and implanted into rabbit femurs (n = 40). The effects of locally released hGH on bone ingrowth and ceramic resorption were evaluated by scanning electron microscopy and image analysis. The results indicated that hGH increased bone ingrowth (+65%) and ceramic resorption (+140%) significantly in comparison with control implants and that the increase was dose dependent. Biochemical parameters monitored in rabbit plasma and urine, as well as the absence of any significant difference between contralateral implants and the control, indicated that hGH did not produce detectable systemic effects. Thus, the use of MBCP appears to be effective for local delivery of hGH, resulting in improved bone substitution.
Assuntos
Sistemas de Liberação de Medicamentos , Hormônio do Crescimento Humano/administração & dosagem , Animais , Materiais Biocompatíveis/administração & dosagem , Reabsorção Óssea , Fosfatos de Cálcio/administração & dosagem , Cerâmica , Implantes de Medicamento , Feminino , Fêmur/efeitos dos fármacos , Fêmur/ultraestrutura , Prótese de Quadril , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Porosidade , Próteses e Implantes , Coelhos , RadioimunoensaioRESUMO
UNLABELLED: Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. INTRODUCTION: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. MATERIALS AND METHODS: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-(EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). RESULTS: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces pi-induced mineralization. CONCLUSIONS: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.
Assuntos
Apoptose/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Fosfatos/farmacologia , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Condrócitos/enzimologia , Condrócitos/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Although the effects of growth hormone on bone metabolism are well-documented, their role in the regulation of immune responses such as the inflammatory process has not been thoroughly explored. This study investigated the formation of multinucleated cells (MNC) in long-term human bone marrow cultures. Experiments using 1 and 100 ng/ml of human recombinant growth hormone (hGH) and 10(-7) M of 1,25 dihydroxyvitamin D3 (VD3) showed that hGH increased the total number and nucleation of MNC. The effects of hGH were generally greater than those observed with VD3. Cytological and immunological characterization of MNC revealed several macrophage polykaryon features. MNC did not respond to calcitonin in a cyclic adenosine monophosphate assay and failed to resorb dentin slices. These results demonstrate that MNC formed in the presence of hGH and VD3 present an essentially macrophage polykaryon phenotype. In this context, growth hormone may be involved in the inflammatory process through upmodulation of macrophage polykaryon formation.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Hormônio do Crescimento Humano/farmacologia , Fosfatase Ácida/metabolismo , Antígenos de Superfície/biossíntese , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Calcitonina/farmacologia , Calcitriol/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Células Gigantes/enzimologia , Histocitoquímica , Humanos , Isoenzimas/metabolismo , Fosfatase Ácida Resistente a TartaratoRESUMO
Fourier transform infrared microspectroscopy (FTIRM) was used to study carbonated apatite/collagen interactions and maturation in horse secondary dentin. Unlike human dentin, this model contains no peritubular material around the odontoblastic processes and is thus quite similar to bone in composition, but not subject to tissue turnover. Crystals close to the mineralization front were very immature, showing high HPO(4) and very low CO(3) levels. Carbonate ions were located essentially in very labile, reactive environments, probably on the crystal surface. Removal of some of the HPO(4) ions from crystals during maturation was linked to an increase in total carbonate content. The CO(3) ions in labile environments decreased, probably after incorporation into more organized regions of the lattice. However, this increase of total carbonate content was associated with greater mineral crystallinity, confirming findings in other studies of synthetic apatite maturation in vitro. The good correlation between these results and those of in vitro experiments suggests that crystal maturation is essentially due to physicochemical processes and that the organic matrix controls only crystal size, multiplication, and/or organization.
Assuntos
Apatitas/química , Dentina/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Cavalos , Microscopia Eletrônica de VarreduraRESUMO
We studied a new injectable biomaterial for bone and dental surgery consisting of a hydrophilic polymer as matrix and bioactive calcium phosphate (CaP) ceramics as fillers. This material is composed of complex fluids whose flow is determined by the laws of rheology. We investigated the macromolecular effects on this composite in a tube. The stability of the polymer and the mixture is essential to the production of a ready-to-use injectable biomaterial. These flow properties are necessary to obtain CaP bioactivity in a dental canal or bone defect during percutaneous surgery. Macromolecules provide spaces between CaP ceramic granules and facilitate the role of the biological agents of bone substitution.
Assuntos
Substitutos Ósseos/química , Fosfatos de Cálcio/química , Lactose/análogos & derivados , Metilcelulose/análogos & derivados , Animais , Materiais Biocompatíveis , Substitutos Ósseos/farmacologia , Fosfatos de Cálcio/farmacologia , Fêmur/efeitos dos fármacos , Fêmur/ultraestrutura , Implantes Experimentais , Injeções , Lactose/química , Lactose/farmacologia , Teste de Materiais , Metilcelulose/química , Metilcelulose/farmacologia , Oxazinas , Coelhos , Reologia , ViscosidadeRESUMO
Bisphosphonates, potent inhibitors of bone resorption, have been used clinically to correct the continued loss of bone mass in osteoporosis and in other conditions. However, there has been some concern that long-term treatment with these compounds, as well as more recently developed drugs, may also decrease the rate of bone formation. Bisphosphonates, which are strongly bound to hydroxyapatite crystals, may alter the structure and reactivity of the crystals, interfere with new crystal nucleation and growth, as well as alter the short-range order of newly formed crystals. We have investigated the chemistry and structure of the solid calcium-phosphate mineral phase of lumbar vertebrae of ovariectomized, 6.5-month-old rats treated with bisphosphonates for 1 year after onset of osteopenia. Appropriate control groups were used for comparison. The techniques used to assess the mineral phase were chemical analyses, Fourier transform-infrared (FT-IR) and FT-Raman spectroscopy, FT-IR microspectroscopy, and phosphorus-31 magic-angle-sample spinning nuclear magnetic resonance spectroscopy ((31)P MAS NMR). The (31)P MAS NMR spectra of trabecular bone of lumbar vertebrae of control, ovariectomized, and treated animals were similar. However, there were several significant differences in the results obtained by FT-IR spectroscopy of the whole tissue samples, FT-IR microspectroscopy of sections of bone, and chemical analyses. For example, whereas chemical analyses demonstrated that the CO(3) content of the mineral phase of the ovariectomized animals was decreased compared with controls, FT-IR microspectroscopy of bone sections showed no changes in the relative CO(3) content, but some changes in the environment of the CO(3) groups. However, chemical analyses of the crystals, combined with data from all three spectroscopic methods and with data from serum analysis, did indicate small changes in the mineral phase after ovariectomy, corrected after treatment with bisphosphonates. In any event, the chemical and structural data in the present studies demonstrate that the bisphosphonate, tiludronate, does not significantly alter the mineral components of bone after 1 year of treatment during the course of which bone loss was reversed.