RESUMO
BACKGROUND: We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17ß (E2) modulate bone morphology in rat female model. AIM: We now analyze the effects of phytoestrogens, E2, selective E2 re ceptor modulators, and the less-calcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). MATERIALS AND METHODS: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. RESULTS: In OVX young adults there is a decreased bone formation and a 10-fold increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or DT56a resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. CONCLUSIONS: Our results demonstrate that adipogenesis induced by OVX is a reversible process which can be corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone loss as well as a novel approach to diagnosis and treatment of postmenopausal osteoporosis.
Assuntos
Adipócitos/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Calcitriol/análogos & derivados , Estrogênios/farmacologia , Adipócitos/citologia , Animais , Calcitriol/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Feminino , Genisteína/farmacologia , Isoflavonas/farmacologia , Ovariectomia , Fitoestrógenos/farmacologia , Cloridrato de Raloxifeno/farmacologia , Ratos , Ratos WistarRESUMO
Recent studies successfully demonstrated induction of new bone formation in the maxillary sinus by mucosal membrane lifting without the use of any graft material. The aim of this work was to test the osteogenic potential of human maxillary sinus Schneiderian membrane (hMSSM) using both in vitro and in vivo assays. Samples of hMSSM were used for establishment of cell cultures and for histological studies. Flow cytometry analysis was performed on P(0), P(1), and P(2) cultures using established mesenchymal progenitor cell markers (CD 105, CD 146, CD 71, CD 73, CD 166), and the ability of hMSSM cells to undergo osteogenic differentiation in culture was analyzed using relevant in vitro assays. Results showed that hMSSM cells could be induced to express alkaline phosphatase, bone morphogenic protein-2, osteopontin, osteonectin, and osteocalcin and to mineralize their extracellular matrix. Inherent osteogenic potential of hMSSM-derived cells was further proven by in vivo experiments, which demonstrated the formation of histology-proven bone at ectopic sites following transplantation of hMSSM-derived cells in conjunction with an osteoconductive scaffold. This study provides the biological background for understanding the observed clinical phenomena in sinus lifting. Our results show that a genuine osteogenic potential is associated with the hMSSM and can contribute to development of successful sinus augmentation techniques.
Assuntos
Mucosa Nasal/citologia , Osteoblastos/citologia , Células-Tronco/citologia , Animais , Calcificação Fisiológica , Diferenciação Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Nus , Mucosa Nasal/química , Mucosa Nasal/metabolismo , Osteoblastos/metabolismo , Osteoblastos/transplante , Osteogênese , Células-Tronco/metabolismoRESUMO
Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol-17beta (E(2)) restored bone structure of ovariectomized (Ovx) female rats to values obtained in intact sham-operated female rats. E(2) also selectively stimulated creatine kinase (CK) specific activity a hormonal-genomic activity marker. In the present study, we compared the effects of E(2) and the phytoestrogens: daidzein (D), biochainin A (BA), genistein (G), carboxy-derivative of BA (cBA), and the SERM raloxifene (Ral) in Ovx, on both histological changes of bones and CK, when administered in multiple daily injections for 2.5 months. Bone from Ovx rats, showed significant disrupted architecture of the growth plate, with fewer proliferative cells and less chondroblasts. The metaphysis underneath the growth plate, contained less trabeculae but a significant increased number of adipocytes in the bone marrow. D like E(2) and Ral but not G, BA, or cBA, restored the morphology of the tibiae, similar to that of control sham-operated animals; the bony trabeculeae observed in the primary spongiosa was thicker, with almost no adipocytes in bone marrow. Ovariectomy resulted also in reduced CK, which in both epiphysis and diaphysis was stimulated by all estrogenic compounds tested. In summary, only D stimulated skeletal tissues growth and differentiation as effectively as E(2) or Ral, suggesting that under our experimental conditions, D is more effective in reversing menopausal changes than any of the other isolated phytoestrogens which cannot be considered as one entity.
Assuntos
Osso e Ossos/efeitos dos fármacos , Creatina Quinase/metabolismo , Isoflavonas/farmacologia , Fitoestrógenos/farmacologia , Adipócitos/patologia , Animais , Medula Óssea/patologia , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Estradiol/administração & dosagem , Estradiol/farmacologia , Feminino , Genisteína/administração & dosagem , Genisteína/farmacologia , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Isoflavonas/administração & dosagem , Ovariectomia , Fitoestrógenos/administração & dosagem , Cloridrato de Raloxifeno/administração & dosagem , Cloridrato de Raloxifeno/farmacologia , Ratos , Ratos Wistar , Tíbia/efeitos dos fármacos , Tíbia/enzimologia , Tíbia/patologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/enzimologia , Malha Trabecular/patologiaRESUMO
The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.
Assuntos
Embrião de Mamíferos/citologia , Miocárdio/citologia , Células-Tronco/citologia , Actinina/genética , Actinina/metabolismo , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Sequência de Bases , Cálcio/metabolismo , Agregação Celular , Diferenciação Celular , Células Cultivadas , Primers do DNA/genética , Desmina/genética , Desmina/metabolismo , Expressão Gênica , Humanos , Microscopia Eletrônica , Contração Miocárdica , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Células-Tronco/metabolismo , Troponina I/genética , Troponina I/metabolismoRESUMO
This pilot study aimed at determining serum VEGF levels (S-VEGF) at diagnosis and at restaging in children with Hodgkin lymphoma, and investigating whether this parameter provides prognostic information for remission after 2 courses of chemotherapy. PET-CT fusion was performed to assess response to treatment. Changes in S-VEGF levels were found to correlate with response to treatment for most of the children. This provides a rationale for exploring clinical interest in S-VEGF measurements in a larger group of children with Hodgkin lymphoma, and using the test for clinical trials of anti-angiogenic therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/sangue , Doença de Hodgkin/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Adolescente , Criança , Pré-Escolar , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Feminino , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/tratamento farmacológico , Humanos , Masculino , Estadiamento de Neoplasias/métodos , Projetos Piloto , Tomografia por Emissão de Pósitrons , Indução de Remissão , Tomografia Computadorizada por Raios XRESUMO
Mandibular condyles of late embryonic NMRI mice were used to study the effect of the FBR murine osteosarcoma virus in an in vitro tissue culture system. Chondroprogenitor cells and chondroblastic cells present in the condylar tissue normally undergo rapid differentiation in vitro which results in an advanced stage of bone formation. The infection of condyles with FBR murine osteosarcoma virus induced the transformation of bone progenitor cells and the formation of an atypical proliferative osseous lesion. In markedly disorganized tissue many spindle-like cells, giant cells, and pleomorphic cells were seen together with the formation of large bone spicules and the heavy mineralization of osteoid-like material and of the remaining cartilage. Fibroblast-like cells were found to penetrate from the perichondrial zone into the condylar mass and also into the underlying collagen sponge. The in vivo growth characteristics of FBR murine osteosarcoma virus-infected condyles after 3 days in culture were studied via s.c. transplantation into syngeneic mice. Control condyles developed normal trabecular bone, whereas the infected condyles induced a strong cellular response with the presence of atypical cells and newly formed connective tissue and bone in situ. These observations raise the possibility of a novel approach for further investigations related to numerous aspects of virus-induced osteosarcomagenesis.
Assuntos
Neoplasias Ósseas/patologia , Osteossarcoma/patologia , Animais , Anticorpos Antivirais/análise , Neoplasias Ósseas/etiologia , Neoplasias Ósseas/ultraestrutura , Modelos Animais de Doenças , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Técnicas de Cultura de Órgãos , Osteossarcoma/etiologia , Osteossarcoma/ultraestrutura , Vírus do Sarcoma Murino/imunologiaRESUMO
The loss of bone mass observed in aging enhances the risk of fractures. The process of bone repair in aging is slow and limited due to reduced activity of the osteoblasts. Bone marrow stem cells (MSCs) residing in the bone marrow are the progenitors for osteoblasts. The ability to enhance healing of bone defect in aging by MSCs can contribute in the prevention of the complications resulting from long-term immobilization that are especially fatal in old age. Our aim was to test the ability of MSCs inserted into a biological scaffold to enhance bone defect repair. Osteoprogenitor cells were selected from rat bone marrow stem cells cultured in DMEM medium supplemented with FCS, antibiotics, ascorbic acid, beta-glycerophosphate, and dexamethasone. The selected osteogenic subpopulation was identified by osteocalcin immunohistochemistry as well as Alizarin red S and von Kossa staining which are specific for bone matrix and mineral deposition. Committed osteoprogenitor cells cultured on the hydrogel scaffold were transplanted into the area of a rat tibia segmental bone defect and examined after 6 weeks. Radiology images revealed that 6 weeks post-implantaion, calcified material was present in the site of the defect, indicating new bone formation. It is concluded that committed osteogenic MSCs contained in a biocompatible scaffold can provide a promising surgical tool for enhancement of bone defect healing that will minimize the complications of bone repair in aging and disease.
Assuntos
Envelhecimento , Células da Medula Óssea/citologia , Osso e Ossos/patologia , Consolidação da Fratura , Células-Tronco/citologia , Animais , Antibacterianos/farmacologia , Ácido Ascórbico/farmacologia , Proteínas Sanguíneas/farmacologia , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/ultraestrutura , Células Cultivadas , Dexametasona/farmacologia , Glicerofosfatos/farmacologia , Substâncias de Crescimento , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Imuno-Histoquímica , Células-Tronco Mesenquimais/metabolismo , Mesoderma/citologia , Microscopia Eletrônica de Varredura , Modelos Biológicos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , CicatrizaçãoRESUMO
Bone marrow contains progenitor cells that are able to differentiate into several mesenchymal lineages, including bone. These cells may also provide a potential therapy for bone repair. The purpose of this study was to select the osteoprogenitor cell subpopulation from bone marrow-derived mesenchymal stem cells (MSCs) and to test the ability of a hydrogel scaffold to support growth and osteogenic differentiation. MSCs isolated from rat femur bone marrow were cultured in DMEM medium supplemented with antibiotics, FCS, and L-glutamine. Osteogenic supplements (dexamethasone, sodium beta-glycerophosphate, and ascorbic acid) were added for one, two or three weeks. A selective subpopulation of osteoprogenitor cells was identified by immunohistochemistry, general morphology, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Committed osteogenic cells were transferred to a 3-D hydrogel scaffold and cultured for an additional week. In standard culture, the osteoprogenitor cells formed cell clusters identified by Alizarin red S staining and by positive osteocalcin immunostaining. The number of osteoprogenitor cells, matrix synthesis, and mineralization increased gradually up to three weeks in culture. Mineral deposition in the matrix analyzed by EDS revealed the presence of calcium and phosphate ions at a Ca/P molar ratio of 1.73 in both the osteogenic cultures and the scaffold osteoprogenitor culture. Histological preparations revealed cell clusters within the hydrogel scaffold and SEM analysis revealed cell clusters attached to the scaffold surface. It is concluded that the hydrogel scaffold can support growth and differentiation of osteogenic cultures including mineralization and can potentially serve as a bone graft substitute containing committed osteoprogenitor cells.
Assuntos
Células da Medula Óssea/citologia , Calcificação Fisiológica , Hidrogel de Polietilenoglicol-Dimetacrilato , Células-Tronco Mesenquimais/citologia , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Imuno-Histoquímica , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Osteocalcina/metabolismo , Ratos , Ratos Sprague-Dawley , Análise Espectral , Engenharia TecidualRESUMO
Mouse mandibular condyles develop spontaneous degenerative changes by 6 months of age, hence providing a good in vivo model for studies related to processes associated with the onset and progression of age-related osteoarthritis. Further, this joint provides an appropriate system to investigate the potential of articular cartilage to respond to hormones and local growth factors in old age. The present study examined (1) the age-related changes in [3H]thymidine incorporation by articular chondrocytes in the mouse mandibular condyle, and (2) the effect of systemic and local factors upon the tissue's ability to resume DNA synthesis. Condyles of female CW-1 mice ranging from 3 to 18 months of age were cultured in the presence of PTH-(1-84) (2 micrograms/ml), PGE1 (20 micrograms/ml), dexamethasone (10(-7) M), and MSA (5 micrograms/ml) and were concomitantly labeled with [3H]thymidine. Autoradiographs were analyzed quantitatively and revealed (1) a significant (p less than 0.01) age-related decrease (-80%) in the labeling index of the articular cartilage, and (2) the ability of old tissues to resume DNA synthesis following in vitro treatment with PTH-(1-84), PGE1, and dexamethasone. Concomitant quantitative incorporation studies further substantiated the autoradiographic findings. Hence, these factors possess a direct stimulatory effect upon senescent chondrocytes involved in an advanced stage of spontaneous osteoarthritis.
Assuntos
Envelhecimento/patologia , Alprostadil/farmacologia , Cartilagem Articular/patologia , Dexametasona/farmacologia , Hormônio Paratireóideo/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Histocitoquímica , Côndilo Mandibular/citologia , Côndilo Mandibular/metabolismo , Camundongos , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoblastos/metabolismo , Somatomedinas/farmacologia , Timidina/metabolismoRESUMO
The zone of progenitor cells of mandibular condyles of neonatal mice was kept in an organ culture system for up to 8 days. Qualitative and quantitative determinations indicated a pronounced proliferative activity during the initial phases of the culture followed by a differentiation phase and the acquisition of typical hyaline cartilage. The mature hypertrophic chondrocytes were found to be surrounded by cartilage-specific macromolecules such as type II collagen, cartilage proteoglycans, and cartilage anchorin. The extracellular mineralization proceeded along matrix vesicles as is usually noted in vivo. A unique finding in this study was the observation that explants comprising cartilage progenitor cells and their adjacent extracellular matrix succeeded in repairing the damaged condylar in vitro.
Assuntos
Cartilagem Articular/citologia , Animais , Animais Recém-Nascidos , Cartilagem Articular/ultraestrutura , Divisão Celular , Matriz Extracelular/ultraestrutura , Cinética , Côndilo Mandibular , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Técnicas de Cultura de ÓrgãosRESUMO
The ability of parathyroid hormone (PTH 1-84), dexamethasone, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2) and human transforming growth factor beta (hTGF-beta) to stimulate the synthesis of matrical components in articular cartilage of aging mice, was studied in an organ culture system. A marked age-dependent decrease was observed in the synthesis of sulfated glycosaminoglycans (GAGs), protein, collagen digestible protein (CDP) and non-collagen protein (NCP) between 1 and 18 months of age. The addition of hTGF-beta (1 ng/ml) into the culture medium resulted in a significant (P < 0.01) increase of both protein and sulfated GAGs in condylar cartilage from animals aged 1, 3, 6 and 12 months. PGE2 (10 micrograms/ml) induced [3H]leucine and [35S]SO4 incorporation into condylar cartilage from 1, 3 and 6 months old animals. A stimulatory effect of PGE1 (10 micrograms/ml) on [3H]leucine incorporation was noted at 1 and 3 months of age. The effect of PTH appeared to be stimulatory only for protein synthesis in young (1 and 3 months old) animals, whereas it had no effect at 6, 12 and 18 months of age. In contrast, dexamethasone exerted a stimulatory effect on young adults (6 months old) and in matured (12 months old) animals, respectively and a slight inhibitory effect on young (1 and 3 months old) animals. [3H]Proline incorporation was enhanced by all the factors tested in 1-month-old animals. In cultures from 6- and 18-month-old animals only PGE1 and PGE2 appeared to be stimulatory. It is concluded that synthesis of protein, sulfated GAGs and collagen by chondrocytes from maturing and osteoarthritic senescent animals can be stimulated by hormones and growth factors. The efficiency of this response, however, varied according to the animal's age and the factor studied.
Assuntos
Envelhecimento/metabolismo , Cartilagem Articular/metabolismo , Substâncias de Crescimento/farmacologia , Hormônios/farmacologia , Animais , Autorradiografia , Cartilagem Articular/citologia , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Glicosaminoglicanos/metabolismo , Leucina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Prolina/metabolismo , Proteínas/metabolismo , Sulfatos/metabolismo , Radioisótopos de Enxofre , Fatores de Tempo , TrítioRESUMO
Osteoarthritic lesions were observed in the mandibular condyle cartilage of mice aged 7 months and older. These lesions appeared as fibrillations along the articular surface and were accompanied by reduced extracellular matrix synthesis and chondrocyte proliferation. Explants of mandibular condyle cartilage were cultured in serum-free BGJb medium supplemented with ascorbic acid (300 micrograms/ml), penicillin (100 U/ml) and streptomycin (100 micrograms/ml) for up to 72 h. Cultures were further supplemented with either hTGF-beta 1 (0.1-5.0 ng/ml) or human IL-1 alpha (40 U/ml). [3H]thymidine (2 microCi/ml) and [35S]SO4 (10 microCi/ml) were added to the culture medium for the last 24 h of culture and incorporation into DNA and sulfated proteoglycans, respectively, studied. The results indicated that protein and DNA contents, [3H]thymidine and [35S]SO4 incorporation, as well as the specific activity of alkaline phosphatase, were increased by TGF-beta 1. Addition of 1.0 or 5.0 ng/ml hTGF-beta 1 to the cultures for 48 h, had the most stimulatory effect on matrix synthesis and cell proliferation, whereas 0.1 ng/ml hTGF-beta 1 appeared to be inhibitory when compared to controls. Increased [35S]SO4 labeling of chondrocyte clusters was observed by autoradiography in tissue sections from cultures treated with TGF-beta 1 (1.0 and 5.0 ng/ml). In contrast, IL-1 alpha exerted inhibitory effects on cell proliferation and matrix synthesis. However, it induced the activity of acid phosphatase in these cultures. The results of the present study show that IL-1 alpha had catabolic effect on his tissue, while TGF-beta 1 enhanced proliferation and induced synthetic activity of the cartilage cells. Such action by TGF-beta suggests the existance of a possible repair process in osteoarthritic cartilage of the temporo-mandibular joint of aged mice.
Assuntos
Envelhecimento/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Interleucina-1/farmacologia , Osteoartrite/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Envelhecimento/patologia , Animais , Cartilagem Articular/patologia , DNA/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Côndilo Mandibular/efeitos dos fármacos , Côndilo Mandibular/metabolismo , Côndilo Mandibular/patologia , Camundongos , Camundongos Endogâmicos ICR , Osteoartrite/patologia , Proteínas/metabolismo , Articulação Temporomandibular/efeitos dos fármacos , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/patologia , Transtornos da Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/patologiaRESUMO
It is well documented that various organs and tissues in the body show a differential, non-homogeneous pattern of development and aging. The present study evaluates some of the morphological and metabolic changes characterizing the aging of different types of cartilages in normal male mice. Representatives of hyaline, fibrous, articular and elastic types of cartilage were obtained from animals ranging from 1 week to 1 year of age. Our determinations included the following: total body and organ weights, proteins and DNA concentrations, [3H]leucine and [35S]sulfate uptake. The biochemical assays were accompanied by morphological examinations of corresponding tissue specimens. This investigation clearly indicates that the growth and maturational activities of the various cartilages examined attained their completion at a very early stage of life (1-3 months postnatally). Thereafter, a phase of steady state prevails, followed by a gradual but continuous decline in the various metabolic activities. The morphological findings illustrate the nature of the age-related structural changes. The present findings indicate that a skeletal tissue such as cartilages of various types, tends to age early during the lifespan of the animal.
Assuntos
Envelhecimento , Cartilagem/crescimento & desenvolvimento , Animais , Peso Corporal , Cartilagem/anatomia & histologia , Cartilagem/metabolismo , DNA/metabolismo , DNA/fisiologia , Disco Intervertebral/anatomia & histologia , Disco Intervertebral/crescimento & desenvolvimento , Disco Intervertebral/metabolismo , Masculino , Côndilo Mandibular/anatomia & histologia , Côndilo Mandibular/crescimento & desenvolvimento , Côndilo Mandibular/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Tamanho do Órgão , Sulfatos/metabolismo , Processo Xifoide/anatomia & histologia , Processo Xifoide/crescimento & desenvolvimento , Processo Xifoide/metabolismoRESUMO
Mandibular condyles of fetal mice 19-20 days in utero were kept in a serum-free organ culture system for up to 14 days and were investigated for their capacity to develop osteoid and to mineralize in vitro. After 3 days in culture, the cartilage of the mandibular condyle appeared to have maintained all its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic. After 7-9 days in culture, no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. In addition, various areas throughout the explant revealed the appearance of osteoidlike material. The process of matrix mineralization progressed with time, and by the 14th day new bonelike material was found to occupy a larger portion of the explant. The newly formed matrix reacted positively with antibodies against type I and type III collagens, as well as against bone alkaline phosphatase. Electron microscopic examination showed that the mineralization appeared to be associated with collagen fibers as well as the matrix vesicles. In composition, the in vitro-formed mineral deposits resembled hydroxyapatite crystals. Biochemical assays indicated that the newly formed tissue reacted strongly for alkaline phosphatase and incorporated 45Ca. The findings of the present study imply that fetal condylar cartilage possesses the potential to develop in vitro osseouslike tissue even in a system that is serum-free. Due to the fact that the newly formed extracellular matrix mineralized and reacted positively to bone markers as well as to cartilage macromolecules, it would seem justifiable to define the new tissue as chondroid bone.
Assuntos
Cartilagem/embriologia , Côndilo Mandibular/embriologia , Fosfatase Alcalina/metabolismo , Animais , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Colágeno/metabolismo , Meios de Cultura , Técnicas In Vitro , Côndilo Mandibular/metabolismo , Côndilo Mandibular/ultraestrutura , Camundongos , Microscopia Eletrônica , Minerais/metabolismoRESUMO
The internalization of cationized ferritin (CF) was studied in isolated pancreatic acinar cells in vitro. Horseradish peroxidase (HRP) was used in conjunction with CF to compare internalization of soluble-phase and membrane-bound tracers. The mode of internalization of CF was dependent upon tracer concentration and origin of the plasma membrane (apical vs. lateral-basal). At the lower tracer concentrations (0.19 and 0.38 mg/ml), internalization from the apical cell surface occurred via small vesicles. The tracer then appeared in multivesicular bodies, in tubules, and in irregular membrane-bound structures. After 15 min, CF particles were seen in many small vesicles near the Golgi apparatus, but not in the Golgi saccules. In contrast, at the lateral-basal cell surface the CF particles tended to form clusters. These clusters were more pronounced at higher CF concentrations (0.76 and 1.5 mg/ml) and were associated with elongated cellular processes, which seemed to engulf CF accumulations in a phagocytic manner. Once internalized, CF was found primarily in large irregular structures which appeared to migrate slowly toward the nucleus, reaching a juxtanuclear position after approximately 30 min. CF was observed in lysosomes after 30-45 min and by 90 min most of the CF was confined to large vacuoles and to trimetaphosphatase-positive lysosomes. Similar routes were observed when cells were double-labeled with CF and HRP, where endocytic structures showed co-localization of both tracers. The results of this study indicate the importance of the Golgi region in the intracellular sorting of internalized apical membrane. Furthermore, this work confirms the presence of distinct endocytic pathways at the apical and lateral-basal cell surfaces.
Assuntos
Ferritinas/metabolismo , Pâncreas/metabolismo , Animais , Cátions , Endocitose , Peroxidase do Rábano Silvestre , Pâncreas/ultraestrutura , FagocitoseRESUMO
Alkaline phosphatase (AP), a membrane-associated glycoprotein which enhances the hydrolysis of monophosphate esters at alkaline pH, is widely distributed in animal tissues. AP activity is increased in a variety of muscle disorders, i.e., myopathies and denervation. Established histochemical methods at the light microscopy level failed to demonstrate AP in skeletal muscles. In the present study we applied the Gomori lead nitrate method for ultrastructural examination of AP in rat gastrocnemius muscles and showed that the enzyme was linked to the sarcolemma of the striated muscle and to the membranes of endothelial cells in adjacent capillaries. In comparison with ATPase activity, AP activity was inhibited by both levamisole and a pH of 7.2, but not by ouabain. Hence, it appears that in skeletal muscles AP is active at a high pH and is bound to cell membranes.
Assuntos
Fosfatase Alcalina/metabolismo , Músculos/enzimologia , Sarcolema/enzimologia , Fosfatase Alcalina/antagonistas & inibidores , Animais , Membrana Celular/enzimologia , Endotélio/enzimologia , Feminino , Histocitoquímica , Microscopia Eletrônica , Músculos/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
Bone defects are often created in order to repair bone pathologies. In the aging population, the healing of such defects is very limited. Bone healing in aging depends on the availability of various hormone and growth factors. The ability of growth factors to enhance bone formation in femoral defects in old rats was tested. Bone defects were induced in femurs of old rats. A single dose of transforming growth factor-beta (TGF-beta), IGF-1, TGF-beta+IGF-1 or saline was inserted in the defect and bones were tested after 2 and 4 weeks. Radiology revealed that mineralization appeared in the 2 weeks group in defects treated with TGF-beta and in defects treated with TGF-beta, TGF-beta+IGF-1 in the 4 weeks groups. Computerized tomography (CT) coronal and axial images revealed that 4 weeks after treatment with TGF-beta+IGF-1, a complete bone bridge was observed. Morphology revealed that these defects were filled with trabecular bone. A less pronounced bone healing was observed after TGF-beta or IGF-1, while control specimens revealed partial healing of the bone defect. Biomechanical tests indicated that treatment with TGF-beta, IGF-1 or TGF-beta+IGF-1 resulted in a significant increase of bone bending rigidity compared to control in the 4 weeks group and that TGF-beta+IGF-1 was the most inductive in this respect. The ability to induce bone healing in aging by TGF-beta+IGF-1 is of a great clinical importance for restoration of bone strength and biomechanical properties of bone defects in aging.
Assuntos
Envelhecimento/fisiologia , Remodelação Óssea/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Fenômenos Biomecânicos , Feminino , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
The appearance of age-related ulcerative changes in the mouse mandibular condyle were evaluated by light and electron microscopy examinations. Fibrillations appeared along the articular surface and in deeper tissue regions, as early as at six months of age. Such changes were characterized by a marked loss of the tissue's cellularity and by a marked reduction in matrix metachromasia and safranin-0 staining. These microscopical changes were accompanied by a reduced reactivity for both ruthenium red and colloidal iron binding, as noted ultrastructurally. At the same time, increasing numbers of erythrocytes appeared to be adhered to the surface irregularities and were also found in deeper regions within the articular lesions. Using morphological criteria, it became apparent that the degenerative changes of aging articular cartilage started at the more superficial regions of the tissue and only thereafter proceeded toward the chondro-osseous junction. Also, with the advancement of age, the degenerative changes became more severe.
Assuntos
Envelhecimento , Artrite/veterinária , Cartilagem Articular/patologia , Côndilo Mandibular/patologia , Doenças dos Roedores/patologia , Animais , Cartilagem Articular/ultraestrutura , Glicosaminoglicanos/análise , Côndilo Mandibular/ultraestrutura , Camundongos , Camundongos Endogâmicos ICR , Fenazinas , Rutênio Vermelho , Cloreto de TolônioRESUMO
The temporo-mandibular joint of aged mice develops osteoarthritic (OA) degenerative lesions. Adult chondrocytes have a low rate of cell replication, and cartilage repair potential is very limited. One of the major problems in OA is the low rate of matrix synthesis and the inability of the chondrocytes to exceed the rate of matrix degradation. These combined factors lead to the overall destruction of the cartilage as seen in OA. Cartilage degradation is mediated by elevated proteolytic activity of enzymes. Among the enzymes degrading cartilage are the metalloproteinases, stromelysin and collagenase. Other proteinases that may potentially participate in matrix degradation are the lysosomal enzymes cathepsin B, D, and L, and acid phosphatase. On the other hand, alkaline phosphatase (ALP) is an enzyme that has been shown to be a marker for anabolic activity in skeletal tissues such as bone and cartilage. The cartilage of the mandibular condyle in the T-M-J from aged mice reveals OA lesions. An overall reduction of cell proliferation and sulfated proteoglycan synthesis has been also shown in this joint. In the present study the effects of hTGF-beta on the stimulation of DNA and sulfated GAG synthesis and ALP activity were studied. Mandibular condyle cartilage obtained from 12-month-old ICR male mice were cultured in BGJb serum-free medium for 24-72 hours, supplemented with 0.1-10 ng/ml hTGF-beta 1. 3H-thymidine and 35S-sulfate were added for the last 24 hours of the culture and their incorporation into DNA and sulfated GAGs respectively, as well as the activity of ALP, were determined. Results indicated that hTGF-beta 1 enhanced the incorporation of both 3H-thymidine and of 35S-sulfate into cartilage cultures of aged mice, and also induced ALP activity. It thus appeared that in OA degenerating articular cartilage, the chondrocytes could be stimulated in vitro to proliferate and to synthesize new matrix, thus indicating induced anabolic activity in the tissue.
Assuntos
Fosfatase Alcalina/metabolismo , Cartilagem Articular/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Osteoartrite/metabolismo , Articulação Temporomandibular/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Cartilagem Articular/citologia , Cartilagem Articular/ultraestrutura , Divisão Celular/efeitos dos fármacos , Técnicas de Cultura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Histocitoquímica , Masculino , Camundongos , Microscopia Eletrônica , Osteoartrite/patologia , Proteoglicanas/efeitos dos fármacos , Proteoglicanas/metabolismo , Articulação Temporomandibular/patologiaRESUMO
Immobilization of limbs of aged animals is associated with swift muscular damage and atrophy. We investigated the effect of rat growth hormone (rGH) on immobilized hindlimb muscles of 26-mo-old rats. Administration of rGH significantly reduced muscle weight loss and muscle protein oxidation caused by immobilization. Capillary blood volume, measured by photoplethysmography of the hindlimb, showed a 34% reduction in immobilized animals, which was eliminated by rGH. The activity of creatine phosphokinase in immobilized gastrocnemius muscle was significantly reduced by immobilization. This damage was diminished by rGH administration. Similarly, the increase in acid phosphatase activity in immobilized muscle was reduced after rGH treatment. Morphologically, marked muscle atrophy and fiber disorientation were observed in immobilized limbs. Therapy with rGH prevented some of these changes. These results indicate that administration of rGH may provide a useful means to attenuate the degenerative effects of limb immobilization of aged rats, as evident from physiological, biochemical, and morphological parameters.