RESUMO
Collagen derived from chum salmon (Oncorhynchus keta) was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) during collagen fibrillogenesis and applied to an in vitro cell culture to evaluate its potential use as a scaffold for vascular tissue engineering. Human umbilical vein endothelial cells (HUVEC) were cultured on the crosslinked salmon collagen fibrillar gel (EDC-SC gel), and their growth rates and production levels of cytokines, including platelet-derived growth factor-BB and von Willebrand factor, were measured. Comparison was also made with bovine collagen gel crosslinked with EDC (EDC-BC gel). The growth and cytokine production of the HUVEC cultured on the EDC-SC gel were higher than those on the EDC-BC gel. In addition, HUVEC were found to attach to the EDC-BC gel through alpha2beta1 integrin for native collagen, whereas they attached to the EDC-SC gel through alphavbeta3 integrin for denatured collagen as well as the alpha2beta1 integrin, indicating that HUVEC recognized denatured domains in the EDC-SC gel. In conclusion, the EDC-SC gel can be used as a scaffold to support HUVEC growth, although the integrin-mediated attachment manner differs between the two gels.
Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Colágenos Fibrilares/química , Salmão/metabolismo , Engenharia Tecidual/métodos , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Reagentes de Ligações Cruzadas/química , Géis/química , Humanos , Teste de MateriaisRESUMO
Marine-derived collagen is expected to be a much safer alternative to calf collagen, which in medical applications carries the risk of bovine spongiform encephalopathy. In this study, acid-soluble collagen was extracted from salmon skin and crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide during fibril formation to produce a crosslinked salmon collagen (SC) gel. The growth rates and the differentiated functions of human periodontal ligament fibroblasts (HPdLFs) cultured on the SC gel were investigated. Growth was faster on the SC gel than on porcine collagen (PC) gel. In addition, the HPdLFs cultured on the SC gel exhibited higher alkaline phosphatase (ALP) activity than those cultured on the PC gel. Quantitative RT-PCR revealed higher mRNA expression of type I collagen, ALP, and osteocalcin in the HPdLFs cultured on the SC gel. HPdLFs had a flat shape on the SC gel and a spindle shape on the PC gel, as revealed by observation with scanning electron microscopy and immunostaining with cytoskeletal protein and vinculin. The results showed that HPdLFs could grow and show highly differentiated activity on the SC gel as well as on the PC gel.
Assuntos
Materiais Biocompatíveis , Colágeno , Ligamento Periodontal/citologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Materiais Biocompatíveis/isolamento & purificação , Diferenciação Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Colágeno/isolamento & purificação , Colágeno Tipo I/genética , Primers do DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Géis , Humanos , Técnicas In Vitro , Teste de Materiais , Microscopia Eletrônica de Varredura , Osteocalcina/genética , Ligamento Periodontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Salmão , SuínosRESUMO
Elastic collagen gel (e-gel) was prepared from salmon atelocollagen fibrillar gel reinforced by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) mediated cross-linking (f-gel). The preparation consisted of a simple heat treatment of the f-gel at 80 degrees C, in which the f-gel drastically shrank and the collagen fibril structure was deformed. The e-gel obtained showed rubber-like elasticity; its stress-strain behavior little changed through repeated stretching. The elongation at the breaking point was approximately 230%. Furthermore, normal human osteoblasts showed good attachment and proliferation on the e-gel. These results suggest its potential to be utilized for the development of tissue engineering.