The cell adhesive properties of decellularized valve scaffolds were promoted by immobilization of valve scaffold with arginine-glycine-aspartic acid （RGD）-containing peptides. Porcine aortic valves were decellularized with trypsin/EDTA, and detergent Triton X-100. With the help of a coupling reagent Sulfo-LC-SPDP, the valve scaffolds were immobilized with glycine-arginine-glycine-aspartic acid-serine-proline-cysteine （GRGDSPC） peptide. X-ray photoelectron spectroscopy （XPS） was used for surface structure analysis. Myofibroblasts harvested from rats were seeded onto the valve scaffolds. Cell count by using microscopy and modified MTT assay were performed to assess cell adhesion. Based on the spectra of XPS, the conjugation of GRGDSPC peptide with decellularized valve scaffolds was confirmed. Both cell count and MTT assay showed that myofibroblasts were much easier to adhere to the modified valve scaffolds, which was also confirmed histologically. Our findings suggest that it is feasible to immobilize RGD-containing peptides onto decellularized valve scaffolds. And the technique can effectively promote cell adhesion, which is beneficial for in vitro tissue engineering of heart valves.
The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells （MSCs） onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly（3-hydroxybutyrate-co-4-hydroxybutyrate） using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically （hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy）, biochemically （DNA and 4-hydroxyproline） and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline （P〉0.05）. However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls （P〈0.05）. This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.
Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD （arginine-glycine-aspartic acid） containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.
