RESUMO
Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.
Assuntos
Envelhecimento/fisiologia , Transplante de Medula Óssea/métodos , Pesquisa Fetal , Osteogênese Imperfeita/prevenção & controle , Osteogênese Imperfeita/terapia , Útero/fisiologia , Animais , Células da Medula Óssea/citologia , Colágeno/metabolismo , Modelos Animais de Doenças , Espaço Extracelular/química , Feminino , Técnicas de Introdução de Genes , Genes Dominantes , Sobrevivência de Enxerto , Camundongos , Camundongos Transgênicos , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Fenótipo , Gravidez , Análise Espectral Raman , Taxa de Sobrevida , Doadores de TecidosRESUMO
Mutations in the diastrophic dysplasia sulphate transporter (dtdst) gene causes different forms of chondrodysplasia in the human. The generation of a knock-in mouse strain with a mutation in dtdst gene provides the basis to study developmental dynamics in the epiphyseal growth plate and long bone growth after impairment of the sulphate pathway. Our microscopical and histochemical data demonstrate that dtdst gene impairment deeply affects tissue organization, matrix structure, and cell differentiation in the epiphyseal growth plate. In mutant animals, the height of the growth plate was significantly reduced, according to a concomitant decrease in cell density and proliferation. Although the pathway of chondrocyte differentiation seemed complete, alteration in cell morphology compared to normal counterparts was detected. In the extracellular matrix, it we observed a dramatic decrease in sulphated proteoglycans, alterations in the organization of type II and type X collagen fibers, and premature onset of mineralization. These data confirm the crucial role of sulphate pathway in proteoglycan biochemistry and suggest that a disarrangement of the extracellular matrix may be responsible for the development of dtdts cartilage dysplasia. Moreover, we corroborated the concept that proteoglycans not only are structural components of the cartilage architecture, but also play a dynamic role in the regulation of chondrocyte growth and differentiation.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Lâmina de Crescimento/anormalidades , Osteocondrodisplasias/metabolismo , Sulfatos/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Transporte Biológico , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Lâmina de Crescimento/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Osteocondrodisplasias/genética , Proteoglicanas/metabolismo , Transportadores de SulfatoRESUMO
Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.
Assuntos
Colágeno/metabolismo , Células Epidérmicas , Adulto , Biomarcadores/metabolismo , Biópsia , Bisbenzimidazol/metabolismo , Proliferação de Células , Separação Celular , Células Cultivadas , Meios de Cultura , DNA/metabolismo , Procedimentos Cirúrgicos Dermatológicos , Epiderme/fisiologia , Epiderme/ultraestrutura , Proteínas Filagrinas , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/citologia , Queratinócitos/fisiologia , Queratinócitos/ultraestrutura , Microscopia Confocal , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Precursores de Proteínas/metabolismo , Tripsina/farmacologiaRESUMO
Cell kinetic studies provide important information on histogenesis in vivo and in vitro. In this regard, specific antibodies to cell cycle-related antigens have been raised and characterized, thus permitting the study of cell kinetics using immunochemical methods. Recent advances in culture technology permitted the generation of human skin equivalents in vitro. We here provide detailed practical procedures for the study of epidermal cell kinetics in a model of artificial skin using immunohistochemistry and flow cytometry. The combined application of both techniques allows a precise detection of tissue growth sites and a quantitative assessment of cell growth. Moreover, simultaneous analysis of differentiation markers and proliferation antigens may be useful to understand molecular mechanisms that regulate tissue growth and development.
Assuntos
Ciclo Celular/fisiologia , Epiderme/metabolismo , Queratinócitos/metabolismo , Antígeno Ki-67/imunologia , Pele Artificial , Células Epidérmicas , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Biological risk management is required in modern tissue engineering. Particular attention should be paid to the culture medium and the scaffold used. In this perspective, it is important to define minimal culture conditions which allow proper growth and differentiation of epithelial cells in vitro. We propose a simple experimental system which permits the generation of three-dimensional epidermal constructs using a collagen layer as a scaffold mimicking the entire dermal tissue and without the need of any feeder layer. Although showing significant differences compared to natural epidermis, these epidermal constructs appear useful to study keratinocyte differentiation and epidermis histogenesis.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/metabolismo , Citometria de Fluxo , Humanos , Engenharia TecidualRESUMO
The p63 gene encodes six splice variants expressed with transactivating or dominant-negative activities. Animal studies with p63 -/- mutants have suggested that p63 is important for proper development of several organs, including tooth and salivary gland. Moreover, mutations of p63 have been detected in patients affected by ectrodactyly, ectodermal dysplasia and facial clefts. To clarify the role of p63 in craniofacial development, we have studied the localization of p63 protein in human and rat oral tissues using immunohistochemistry. p63 immunostaining was identified in the enamel organ, oral epithelium and developing salivary glands. All compartments of the enamel organ were immunolabelled, whereas only basal and some suprabasal cells of the oral epithelium were stained. Ectomesenchyme-derived cells, including pulp cells, odontoblasts, bone cells and chondrocytes, were negative. The staining pattern was identical in human and rat tissues. These data lend support to the hypothesis that p63 is involved in growth and differentation of ectoderm-derived oral tissues and may be useful to clarify molecular and developmental aspects observed in animal knockout experiments and human syndromes related to p63 gene alteration.