RESUMO
We determined conditions for effective transduction of multipotent mesenchymal stromal cells from human adipose tissue with adenoviral constructs carrying the gene of human bone morphogenetic protein BMP-2. The peak of transgene transcription and BMP-2 protein secretion in the transduced cultures was observed on day 6 after infection. The maximum transcription of BMP-2 gene and genes of osteogenic markers (bone sialoprotein, osteopontin, and osteocalcin) was observed in the medium containing sodium ß-glycerophosphate and ascorbic acid. Addition of D 3 vitamin did not enhance the expression of BMP-2 gene in transduced cells. The obtained cell cultures with high osteogenic potential can be used in bone tissue engineering.
Assuntos
Adenoviridae/genética , Proteína Morfogenética Óssea 2/genética , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo Branco/citologia , Antígenos CD/metabolismo , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Sialoproteína de Ligação à Integrina/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteocalcina/metabolismo , Osteopontina/metabolismo , Transcrição Gênica , Transdução GenéticaRESUMO
We studied of osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue. Experiments showed that 1α,25-dihydroxycalciferol is a more effective inductor of osteogenesis than dexamethasone. Comparative analysis revealed activation of gene expression for the major osteogenic markers on day 7 of culturing in a medium containing 1α,25-dihydroxycalciferol. It was found that transcription of genes encoding type 1 collagen proteins, osteopontin, osteocalcin, and bone sialoprotein peaked on day 14 in culture, while the expression of alkaline phosphatase and bone morphogenetic protein-2 genes increased over 21 days. Intensive mineralization of the extracellular matrix was observed starting from day 14 in culture. On the basis of the analysis of these data, optimal terms for osteogenic induction (day 14) and an optimal inductor (1α,25-dihydroxycalciferol) were chosen and the protocol of effective osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue was developed for creation of tissue-engineered bone equivalents.
Assuntos
Tecido Adiposo/citologia , Conservadores da Densidade Óssea/farmacologia , Calcitriol/farmacologia , Dexametasona/farmacologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese , Engenharia Tecidual , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Anti-Inflamatórios/farmacologia , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Matriz Extracelular/metabolismo , Humanos , Sialoproteína de Ligação à Integrina/biossíntese , Sialoproteína de Ligação à Integrina/genética , Células-Tronco Mesenquimais/citologia , Osteocalcina/biossíntese , Osteocalcina/genética , Osteopontina/biossíntese , Osteopontina/genéticaRESUMO
We developed a new method of creation of tissue engineering constructs for regeneration of the bone tissue based on the principle of free distribution of cells in a fibrin clot within a scaffold. The tissue engineering construct includes multipotent stromal adipose tissue cells committed in osteogenic lineage, platelet-rich plasma, and resorbed material on the basis of xenogeneic bone collagen. The culture of bone progenitor cells was characterized by the main markers of osteoblastic differon. The material meets all requirements for materials intended for tissue engineering. An innovative high-technological tissue engineering product for clinical application is prepared.
