RESUMO
BACKGROUND: We and others have reported that low-magnitude high-frequency dynamic loading has an osteogenic effect on alveolar bone. Since chondrocytes and osteoblasts originate from the same progenitor cells, we reasoned that dynamic loading may stimulate a similar response in chondrocytes. A stimulating effect could be beneficial for patients with damaged condylar cartilage or mandibular deficiency. METHODS: Studies were conducted on growing Sprague-Dawley rats divided into three groups: control, static load, and dynamic load. The dynamic load group received a dynamic load on the lower right molars 5 minutes per day with a 0.3 g acceleration and peak strain of 30 µÎµ registered by accelerometer and strain gauge. The static load group received an equivalent magnitude of static force (30 µÎµ). The control group did not receive any treatment. Samples were collected at days 0, 28, and 56 for reverse transcriptase polymerase chain reaction analysis, microcomputed tomography, and histology and fluorescent microscopy analysis. RESULTS: Our experiments showed that dynamic loading had a striking effect on condylar cartilage, increasing the proliferation and differentiation of mesenchymal cells into chondrocytes, and promoting chondrocyte maturation. This effect was accompanied by increased endochondral bone formation resulting in lengthening of the condylar process. CONCLUSIONS: Low-magnitude, high-frequency dynamic loading can have a positive effect on condylar cartilage and endochondral bone formation in vivo. This effect has the potential to be used as a treatment for regenerating condylar cartilage and to enhance the effect of orthopedic appliances on mandibular growth.
Assuntos
Condrócitos , Côndilo Mandibular , Animais , Cartilagem/patologia , Condrócitos/fisiologia , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
OBJECTIVES: Vibration, in the form of high frequency acceleration (HFA), stimulates alveolar bone formation under physiologic conditions and during healing after dental extractions. It is not known if HFA has an anabolic effect on osteoporotic alveolar bone. Our objective is to determine if HFA has a regenerative effect on osteoporotic alveolar bone. METHODS AND MATERIALS: Adult female Sprague-Dawley rats were divided into five groups: 1) Ovariectomized Group (OVX), 2) Sham-OVX Group that received surgery without ovariectomy, 3) OVX-HFA Group that was ovariectomized and treated daily with HFA, 4) OVX+Static Force Group that was ovariectomized and received the same force as HFA, but without vibration, and 5) Control Group that did not receive any treatment. All animals were fed a low mineral diet for 3 months. Osteoporosis was confirmed by micro-CT of the fifth lumbar vertebra and femoral head. HFA was applied to the maxillary first molar for 5 minutes/day for 28 and 56 days. Maxillae were collected for micro-CT, histology, fluorescent microscopy, protein and RNA analysis, and three-point bending mechanical testing. RESULTS: Micro-CT analysis revealed significant alveolar bone osteoporosis in the OVX group. Vibration restored the quality and quantity of alveolar bone to levels similar to the Sham-OVX group. Animals exposed to HFA demonstrated higher osteoblast activity and lower osteoclast activity. Osteogenic transcription factors (RUNX2, Foxo1, Osterix and Wnt signaling factors) were upregulated following vibration, while RANKL/RANK and Sclerostin were downregulated. HFA did not affect serum TRAcP-5b or CTx-1 levels. The osteogenic effect was highest at the point of HFA application and extended along the hemimaxillae this effect did not cross to the contra-lateral side. CONCLUSIONS: Local application of vibration generated gradients of increased anabolic metabolism and decreased catabolic metabolism in alveolar bone of osteoporotic rats. Our findings suggest that HFA could be a predictable treatment for diminished alveolar bone levels in osteoporosis patients.
Assuntos
Cabeça do Fêmur , Vértebras Lombares , Maxila , Osteogênese , Osteoporose , Vibração/uso terapêutico , Microtomografia por Raio-X , Animais , Feminino , Cabeça do Fêmur/diagnóstico por imagem , Cabeça do Fêmur/metabolismo , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/metabolismo , Maxila/diagnóstico por imagem , Maxila/metabolismo , Osteoporose/diagnóstico por imagem , Osteoporose/metabolismo , Osteoporose/terapia , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Orthodontic tooth movement results from increased inflammation and osteoclast activation. Since patients of all ages now routinely seek orthodontics treatment, we investigated whether age-dependent biologic responses to orthodontic force correlate with the rate of tooth movement. METHODS: We studied 18 healthy subjects, adolescents (11-14 years) and adults (21-45 years), with Class II Division 1 malocclusion requiring 4 first premolar extractions. Canines were retracted with a constant force of 50 cN. Gingival crevicular fluid was collected before orthodontic treatment and at days 1, 7, 14, and 28 after the canine retraction. Cytokine (IL-1ß, CCL2, TNF-α) and osteoclast markers (RANKL and MMP-9) were measured using antibody-based protein assays. Pain and discomfort were monitored with a numeric rating scale. The canine retraction rate was measured from study models taken at days 28 and 56. RESULTS: Although the cytokine and osteoclast markers increased significantly in both age groups at days 1, 7, and 14, the increases were greater in adults than in adolescents. Interestingly, the rate of tooth movement in adults was significantly slower than in adolescents over the 56-day study period. Adults also reported significantly more discomfort and pain. CONCLUSIONS: Age is a significant variable contributing to the biologic response to orthodontic tooth movement. Adults exhibited a significantly higher level of cytokine and osteoclasts activity but, counterintuitively, had a significantly slower rate of tooth movement.
Assuntos
Técnicas de Movimentação Dentária , Adolescente , Adulto , Fatores Etários , Biomarcadores/sangue , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Vibration in the form of High Frequency Acceleration (HFA) is anabolic on the craniofacial skeleton in the absence of inflammation. Orthodontic forces trigger an inflammation-dependent catabolic cascade that is crucial for tooth movement. It is unknown what effect HFA has on alveolar bone if applied during orthodontic treatment. The objectives of this study are to examine the effect of HFA on the rate of tooth movement and alveolar bone, and determine the mechanism by which HFA affects tooth movement. Adult Sprague Dawley rats were divided to control, orthodontic force alone (OTM), and different experimental groups that received the same orthodontic forces and different HFA regimens. Orthodontic tooth movement was assessed when HFA parameters, frequency, acceleration, duration of exposure, and direct or indirect application were varied. We found that HFA treatment significantly enhanced the inflammation-dependent catabolic cascade during orthodontic tooth movement. HFA treatment increased inflammatory mediators and osteoclastogenesis, and decreased alveolar bone density during orthodontic tooth movement. Each of the HFA variables produced significant changes in the rate of tooth movement and the effect was PDL-dependent. This is the first report that HFA enhances inflammation-dependent catabolic cascades in bone. The clinical implications of our study are highly significant, as HFA can be utilized to enhance the rate of orthodontic tooth movement during the catabolic phase of treatment and subsequently be utilized to enhance retention during the anabolic remodeling phase after orthodontic forces are removed.
Assuntos
Remodelação Óssea/fisiologia , Terapia por Radiofrequência , Técnicas de Movimentação Dentária/métodos , Processo Alveolar/fisiologia , Anabolizantes/metabolismo , Animais , Fenômenos Biomecânicos , Masculino , Ortodontia/métodos , Ligamento Periodontal/fisiologia , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Vibração/uso terapêuticoRESUMO
This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1ß, TGFß1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, 'multi-cell type' interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation.
Assuntos
Materiais Revestidos Biocompatíveis/química , Osteogênese , Titânio/química , Adesão Celular , Sobrevivência Celular , Cerâmica/química , Técnicas de Cocultura , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/fisiologia , Teste de Materiais , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Osteoblastos/fisiologia , Propriedades de SuperfícieRESUMO
Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed.
Assuntos
Diferenciação Celular/fisiologia , Proteínas da Matriz Extracelular/genética , Botões de Extremidades/citologia , Botões de Extremidades/metabolismo , Animais , Diferenciação Celular/genética , Embrião de Galinha , Galinhas , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited (â¼35%, p = 0.02), and antibodies to F-spondin increased (â¼30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-ß treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-ß from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-ß dependent pathways.
Assuntos
Diferenciação Celular/fisiologia , Condrócitos/citologia , Proteínas da Matriz Extracelular/fisiologia , Osteogênese/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Condrócitos/fisiologia , Feminino , Lâmina de Crescimento/citologia , Lâmina de Crescimento/fisiologia , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Modelos Animais , Gravidez , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologiaRESUMO
Skeletogenesis depends on the activity of bone-forming cells derived from mesenchymal cells. The pathways that control mesenchymal cell differentiation are not well understood. We propose that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblasts. In mouse embryos, Foxo1 expression is higher in skeletal tissues, while Foxo1 silencing has a drastic impact on skeletogenesis and craniofacial development, specially affecting pre-maxilla, nasal bone, mandible, tibia, and clavicle. Similarly, Foxo1 activity and expression increase in mouse mesenchymal cells under the influence of osteogenic stimulants. In addition, silencing Foxo1 blocks the expression of osteogenic markers such as Runx2, alkaline phosphatase, and osteocalcin and results in decreased culture calcification even in the presence of strong osteogenic stimulants. Conversely, the expression of these markers increases significantly in response to Foxo1 overexpression. One mechanism through which Foxo1 affects mesenchymal cell differentiation into osteoblasts is through regulation of a key osteogenic transcription factor, Runx2. Indeed, our results show that Foxo1 directly interacts with the promoter of Runx2 and regulates its expression. Using a tibia organ culture model, we confirmed that silencing Foxo1 decreases the expression of Runx2 and impairs bone formation. Furthermore, our data reveals that Runx2 and Foxo1 interact with each other and cooperate in the transcriptional regulation of osteoblast markers. In conclusion, our in vitro, ex vivo, and in vivo results strongly support the notion that Foxo1 is an early molecular regulator in the differentiation of mesenchymal cells into osteoblast.
Assuntos
Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Embrião de Mamíferos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Embrião de Mamíferos/citologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Inativação Gênica , Camundongos , Especificidade de Órgãos/fisiologia , Osteoblastos/citologia , Regiões Promotoras Genéticas/fisiologiaRESUMO
This study evaluated the effect of a bioactive ceramic coating, in the nanothickness range, onto a moderately rough surface on the osteogenic behavior of human bone cells. The cells were harvested from the mandibular mental region and were cultured over Ti-6Al-4V disks of different surfaces: as-machined (M), alumina-blasted/acid etched (AB/AE), and alumina-blasted/acid-etched + 300-500 nm thickness amorphous Ca- and P-based coating obtained by ion beam-assisted deposition (Nano). The culture was then evaluated regarding cell viability, adhesion, morphology, immunolocalization of osteopontin (OPN) and alkaline phosphatase (ALP). The results showed that the surface treatment did not interfere with cell viability. At 1 day, AB/AE and Nano showed higher adhesion than the M surface (p < 0.001). Higher adhesion was observed for the M than the Nano surface at 7 days (p < 0.005). The percentage of cells showing intracellular labeling for OPN at day 1 was significantly higher for the Nano compared to M surface (p < 0.03). The percentage of ALP intracellular labeling at 7 days was significantly higher for the AB/AE compared to the M surface (p < 0.0065); no differences were detected at 14 days. Our results suggest that the presence of a thin bioactive ceramic coating on a rough substrate did not favor the events related to in vitro osteogenesis. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.
Assuntos
Cerâmica/química , Materiais Revestidos Biocompatíveis/química , Osteoblastos/metabolismo , Osteogênese/fisiologia , Titânio/química , Adulto , Ligas , Animais , Células Cultivadas , Feminino , Humanos , Teste de Materiais , Osteoblastos/citologia , Osteopontina/genética , Osteopontina/metabolismo , Propriedades de SuperfícieRESUMO
Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation.
Assuntos
Regeneração Óssea/fisiologia , Osso e Ossos/fisiologia , Colágeno/metabolismo , Isoformas de Proteínas/metabolismo , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Osso e Ossos/citologia , Bovinos , Embrião de Galinha , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno/química , Força Compressiva , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Isoformas de Proteínas/química , Estresse MecânicoRESUMO
In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.
Assuntos
Cartilagem Articular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Osteoartrite/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/genética , Humanos , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
The use of biomaterials to replace lost bone has been a common practice for decades. More recently, the demands for bone repair and regeneration have pushed research into the use of cultured cells and growth factors in association with these materials. Here we report a novel approach to engineer new bone using a transient cartilage scaffold to induce endochondral ossification. Chondrocyte/chitosan scaffolds (both a transient cartilage scaffold-experimental-and a permanent cartilage scaffold-control) were prepared and implanted subcutaneously in nude mice. Bone formation was evaluated over a period of 5 months. Mineralization was assessed by Faxitron, micro computed tomography, backscatter electrons, and Fourier transform infrared spectroscopy analyses. Histological analysis provided further information on tissue changes in and around the implanted scaffolds. The deposition of ectopic bone was detected in the surface of the experimental implants as early as 1 month after implantation. After 3 months, bone trabeculae and bone marrow cavities were formed inside the scaffolds. The bone deposited was similar to the bone of the mice vertebra. Interestingly, no bone formation was observed in control implants. In conclusion, an engineered transient cartilage template carries all the signals necessary to induce endochondral bone formation in vivo.
Assuntos
Osso e Ossos/fisiologia , Cartilagem/fisiologia , Engenharia Tecidual/métodos , Animais , Osso e Ossos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Embrião de Galinha , Quitosana/farmacologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Minerais/metabolismo , Implantação de Prótese , Espectroscopia de Infravermelho com Transformada de Fourier , Alicerces TeciduaisRESUMO
Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.
Assuntos
Osso e Ossos/fisiologia , Cartilagem/fisiologia , Engenharia Tecidual/métodos , Fosfatase Alcalina/metabolismo , Animais , Fenômenos Biomecânicos , Osso e Ossos/efeitos dos fármacos , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Quitosana/farmacologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Colágeno Tipo X/metabolismo , DNA/metabolismo , Perfilação da Expressão Gênica , Poríferos , Porosidade/efeitos dos fármacos , Alicerces Teciduais , Tretinoína/farmacologiaRESUMO
Coordinated proliferation and differentiation of growth plate chondrocytes is required for endochondral bone growth, but the mechanisms and pathways that control these processes are not completely understood. Recent data demonstrate important roles for nitric oxide (NO) and C-type natriuretic peptide (CNP) in the regulation of cartilage development. Both NO and CNP stimulate the synthesis of cGMP and thus the activation of common downstream pathways. One of these downstream mediators, cGMP-dependent kinase II (cGKII), has itself been shown to be essential for normal endochondral bone formation. This review summarizes our knowledge of the roles and mechanisms of NO, CNP and cGKII signaling in cartilage and endochondral bone development.
Assuntos
Desenvolvimento Ósseo/fisiologia , GMP Cíclico/fisiologia , Peptídeo Natriurético Tipo C/fisiologia , Osteogênese/fisiologia , Animais , Condrócitos/fisiologia , Humanos , Modelos Animais , Óxido Nítrico/fisiologiaRESUMO
OBJECTIVE: To determine the role of mitochondria in chondrocyte apoptosis induced by inorganic phosphate (Pi). MATERIALS AND METHODS: Chondrocytes isolated from the growth plates of chick embryo tibia were treated with Pi in serum-free media; chondrocyte viability, mitochondrial membrane potential, cytochrome c release from mitochondria, caspase 3 activity, endonuclease activity, and DNA fragmentation were investigated. RESULTS: Exposure to Pi for 24 hours induced apoptosis in growth plate chondrocytes through a pathway that involved loss of mitochondrial function, release of cytochrome c into the cytoplasm, increases in caspase 3 and endonuclease activities, and fragmentation of DNA. CONCLUSIONS: This study suggests that mitochondria are important players in Pi-induced apoptosis.
Assuntos
Apoptose/fisiologia , Condrócitos/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Fosfatos/toxicidade , Animais , Western Blotting , Caspases/metabolismo , Embrião de Galinha , Condrócitos/enzimologia , Citocromos c/fisiologia , Endonucleases/metabolismo , Ativação Enzimática , Lâmina de Crescimento/citologia , Potenciais da Membrana/efeitos dos fármacos , TíbiaRESUMO
While skeletal development can occur by either intramembranous or endochondral bone formation, all current tissue engineering approaches for bone repair and regeneration try to mimic intramembranous ossification. In this study, we propose to create an in vitro cartilage template as the transient model for in vivo endochondral bone formation. The goals of this study are to (1) establish a method of growing chondrocytes in a well-characterized macroporous biphasic calcium phosphate (MBCP) scaffold and (2) induce maturation of chondrocytes grown in the MBCP scaffold. Chondrocytes isolated from chick embryonic tibia were grown on MBCP particles and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Chondrocytes were observed to attach and proliferate on the MBCP scaffold. The thickness of the chondrocyte and extracellular matrix layer increased in the presence of the retinoid. Alkaline phosphatase activity and expression, proteoglycans synthesis, cbfa1 and type I collagen mRNA levels also increased in the presence of retinoic acid. These results demonstrated for the first time the proliferation, maturation of chondrocytes, and matrix deposition on MBCP, suggesting the potential for such scaffold in tissue engineering via the endochondral bone formation mechanism.
Assuntos
Materiais Biocompatíveis , Fosfatos de Cálcio , Diferenciação Celular/fisiologia , Condrócitos/citologia , Lâmina de Crescimento/citologia , Animais , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Matriz Extracelular/metabolismoRESUMO
The goal of this investigation was to explore the mechanism by which NOS and NO serve to regulate events linked to chondrocyte terminal differentiation. NOS isoform expression and NO adducts in chick growth cartilage were detected by immunohistochemistry and Western blot analysis. All NOS isoforms were expressed in chick growth plate chondrocytes with the highest levels present in the hypertrophic region. The enzymes were active since nitrosocysteine and nitrotyrosine residues were detected in regions of the epiphysis with the highest levels of NOS expression. Maturing chick sternal chondrocytes evidenced an increase in NO release and a rise in NOS protein levels. When treated with NOS inhibitors, there was a decrease in the alkaline phosphatase activity of the hypertrophic cells. On the other hand, NO donors caused a small but significant elevation in alkaline phosphatase activity. Transient transfections of chondrocytes with an endothelial NOS isoform caused an increase in collagen type X promoter activity. Induction of both collagen type X expression and alkaline phosphatase activity was blocked by inhibitors of the cGMP pathway. These findings indicate that NO is generated by three NOS isoforms in terminally differentiated chondrocytes. The expression of NOS and the generation of NO enhanced maturation by upregulating alkaline phosphatase and collagen type X expression. Since expression of these two determinants was blocked by inhibitors of the cGMP pathway, it is concluded that NO metabolism is required for development of the mature chondrocyte phenotype.
Assuntos
Diferenciação Celular/fisiologia , Condrócitos/fisiologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Colágeno Tipo X/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Cisteína/análogos & derivados , Cisteína/metabolismo , Expressão Gênica/genética , Lâmina de Crescimento/enzimologia , Lâmina de Crescimento/metabolismo , Guanilato Ciclase/antagonistas & inibidores , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Luciferases/genética , Luciferases/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Nitritos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , S-Nitrosoglutationa/farmacologia , S-Nitrosotióis/metabolismo , Esterno/citologia , Transfecção , Tretinoína/farmacologia , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The major aim of the current investigation was to evaluate the role of thiols during chondrocyte maturation and apoptosis. Using a thiol-sensitive fluorescent probe, we found that in chick growth plate chondrocytes, hypertrophy is accompanied by a decrease in the glutathione content. In this study, we show that the maturation-dependent loss of thiol, although not causing death of maturing chondrocytes, drastically increases susceptibility to apoptosis by oxidative and nitrosoactive stress. To investigate how the loss of thiol content in cultured chondrocytes affects the expression of the hypertrophic phenotype, we chemically manipulated intracellular thiol levels and analyzed the expression of important maturation markers. We found that thiol depletion causes a decrease in the expression of osteopontin, type X and type II collagen and a significant loss of alkaline phosphatase activity, suggesting that the expression of the hypertrophic phenotype is tightly regulated by redox levels in chondrocytes. Furthermore, severe thiol depletion profoundly affected cell survival under oxidative and nitrosoactive stress. It was concluded that the loss of thiol reserve is not only linked to the expression of the hypertrophic phenotype but also influenced chondrocyte survival, linking chondrocyte maturation and the activation of the apoptotic pathway.
Assuntos
Condrócitos/citologia , Condrócitos/metabolismo , Compostos de Sulfidrila/metabolismo , Acetilcisteína/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Apoptose , Butionina Sulfoximina/farmacologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo IX/genética , Cisteína/metabolismo , Corantes Fluorescentes , Expressão Gênica , Glutationa/metabolismo , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Óxido Nítrico/metabolismo , Osteopontina , Oxirredução , Estresse Oxidativo , Sialoglicoproteínas/genética , Tretinoína/farmacologiaRESUMO
O referido estudo foi realizado em duas etapas, por alunos e professores da disciplina de parasitologia Curso Médico UNIMONTES, sendo a 1ª etapa (abril a agosto de 1996) e a 2ª etapa (outubro de 1997 a maio de 1998). Foram realizados exames parasitológicos pelo H.P.J. de 397 moradores da Vila Murici, sendo positivos 269 (67,76%); 125 adultos (46,47%) e 144 crianças até 12 anos (53,53%). As maiores prevalências foram de Entamoeba histolytica (50,56%), Entamoeba coli (45,35%) e Ancylostomatidae (16,36%). Foi feito o tratamento de todas as pessoas parasitadas, orientações de higiene e profilaxia através de audio-visual e cartilha educativa. Após 12 meses, repetimos os exames de 192 pessoas medicadas na 1ª etapa, das quais 77 positivas (40,1%), sendo 33 adultos (42,86%) e 44 crianças (57,14%$). Houve permanência de 40,8% do índice de parasitoses, o que se justifica, pela péssima condição sócio-econômica, carência de água potável, saneamento básico e o espaço decorrido entre a 1ª e 2ª etapa. Finalmente, repetimos o tratamento das pessoas e através da participação dos alunos do Curso Médico UNIMONTES e ROTARACT (Rotary Club de Jovens), doamos 1 filtro para cada moradia do bairro.