RESUMO
The high morbidity of tendon injuries and the poor outcomes observed following repair or replacement have stimulated interest in regenerative approaches to treatment and, in particular, the use of cell-based analogues as alternatives to autologous and allogeneic graft repair. Given the known regenerative properties of fetal tissues, the objective of this study was to assess the biological and mechanical properties of tissue-engineered three-dimensional (3D) composites seeded with fetal skin cells. Dermal fibroblasts were isolated from pregnant rats and their fetuses and characterized in monolayer culture and on 3D resorbable polyester scaffolds. To determine the differences between fetal and adult fibroblasts, DNA, total protein and types I and III collagen production were measured. In addition, morphology and mechanical properties of the 3D constructs were examined. In monolayer culture, fetal fibroblasts produced significantly more types I and III collagen and displayed serum-independent growth, while adult fibroblasts elaborated less collagen and exhibited reduced cell spreading and attachment under low-serum conditions. In 3D culture, fetal constructs appeared more developed based on gross examination, with significantly more total DNA, total protein and normalized type I collagen production compared to adult specimens. Finally, after 35 days, fetal fibroblast-seeded constructs possessed superior mechanical properties compared to adult samples. Taken together, these findings indicate that fetal dermal fibroblasts may be an effective source of cells for fabricating tissue equivalents to regenerate injured tendons.
Assuntos
Células Cultivadas/citologia , Colágeno/metabolismo , Fibroblastos/metabolismo , Pele/embriologia , Animais , Técnicas de Cultura de Células/métodos , Colágeno Tipo I/química , Colágeno Tipo III/química , DNA/química , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica/métodos , Poliésteres/química , Ratos , CicatrizaçãoRESUMO
Cell migration and extracellular matrix synthesis play an important role in the wound-healing response to injury. Several studies have described differences in migratory behavior and collagen biosynthetic activity in adult vs. fetal skin fibroblasts. The objective of this study was to examine the serum- and age-dependent effects on cell migration and collagen expression in tendon fibroblasts. Medial tendon fibroblasts were isolated from pregnant ewes and their fetuses, and cultured with and without serum for up to 7 days. Cell migration was determined by quantitative image analysis, and collagen expression was assessed by reverse transcription-polymerase chain reaction and immunohistochemical staining. In serum-containing medium, tendon fibroblasts migrated significantly faster than cells in serum-free medium. Additionally, fetal tendon fibroblasts migrated significantly faster than adult tendon fibroblasts under both culture conditions. The expression of types I and III collagen mRNA was significantly up-regulated in tendon cell populations in serum-free medium compared with those in serum-containing medium. Quantitative assessment of collagen staining indicated that fetal tenocytes produced more type I collagen than adult tenocytes under both culture conditions. These findings suggest that there is an inherent difference between adult and fetal tendon fibroblasts, which may have implications in the wound-healing response in tendons.
Assuntos
Movimento Celular/fisiologia , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/fisiologia , Soro/fisiologia , Tendões/citologia , Cicatrização/fisiologia , Animais , Células Cultivadas , Feminino , Feto/citologia , Fibroblastos/metabolismo , Imuno-Histoquímica , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , OvinosRESUMO
The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater fraction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.