Promotion Of Angiogenesis By Controlled Release Of RhGM-CSF From Porous Collagen-Chitosan Scaffolds

Cytokine therapy and tissue-engineered scaffolds were combined to fabricate bioactive scaffolds.As a pleiotropic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the proliferation, differentiation, and survival of hematopoetic cells by mobilizing and recruiting hematopoietic and endothelial stem cells from the bone marrow. And it specifically stimulates bone marrow progenitor cells destined to become granulocytes and macrophages. Shortly after the skin is injured, GM-CSF, secreted by keratinocytes, mediates epidermal cell proliferation in an autocrine manner and acted the immune system. Applied GM-CSF is beneficial for wounds healing, especially for deep partial-thickness burn wounds and chronic leg ulcers.A novel strategy of combining recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in a porous collagen-chitosan scaffold with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and heparin was developed, considering the slow angiogenesis during repair of full-thickness skin defects. We examined the basic scaffold properties in vitro and biological responses in vivo. In vitro, the controlled release pattern of rhGM-CSF from scaffolds was measured by ELISA, while an implantation model was used in vivo. In vivo, four groups of scaffolds were fabricated:heparinized or unheparinized scaffolds loaded with or without rhGM-CSF. Tissue specimens were harvested at different time points after implantation for histopathological, immunohistochemical observation and Western blotting analysis. The results showed that the heparinized scaffolds (H1E1) had slower biodegradation and a more stable release of rhGM-CSF than the unheparinized scaffolds (H0E1). The in vivo investigation revealed that the heparinized scaffolds loaded with rhGM-CSF (H1E1/rhGM-CSF) had the most cellular adhesion and migration, new vessel formation, and expression of VEGF and TGF-β1, which were beneficial for vascularization. The results indicate that H1E1/rhGM-CSF induced inflammatory cell infiltration towards the wound site and enhanced angiogenesis. Thus, this composite dermal substitute could be a potential therapeutic agent for full-thickness skin defects due to its sustained delivery of rhGM-CSF from porous collagen-chitosan scaffolds, which promoted angiogenesis.