The Construction Of Micro-vascular Engineered Tissue By HADSCs-PGA/PLA Complex

Background:Tissue transplantation is one of the commonly used treatments in plastic surgery. In recent years, the rapid developed tissue engineering technology turn to a promising new therapy to meet the challenge of tissue shortage. Just like autologous tissue transplantation, engineered tissue transplantation will also experience the process of revascularization. Nevertheless, the speed of vascularization for implanted engineered tissues is generally low. Vascularization remains one of the primary obstacles in tissue engineering. Studies have shown that 3D polymer mesh provides a better spatial guide to certain cell behaviors, especially like endothelial differentiation which is apt to organize into a tubular structure through cell migration. Human adipose-derived stem cells (hADSCs) can differentiated in the direction of cardiovascular and have the characteristics to promote vascular formation. Polyglycolic acid (PGA) is FDA approved and has long been used to construct tissues such as cartilage, bone, tendon in tissue engineering. To the best of our knowledge, there were no reports on the endothelial differentiation of hADSCs seeded on Polyglycolic acid/polylactic acid (PGA/PLA) mesh to construct micro-vascular engineered tissue. Our findings may bring new opportunity to improve the efficiency of vascularization for implanted engineered tissues and accelerate wound repair process.Objectives:1. To evaluate the potential of endothelial differentiation of hADSCs on PGA/PLA mesh, and attempt to construct induced hADSCs-PGA/PLA complex to micro-vascularized engineered tissues.2. To investigate the immunoregulation of hADSCs-PGA/PLA complex.3. To verify whether induced hADSCs-PGA/PLA complex is a viable therapeutic approach to improve wound angiogenesis and accelerate wound repair process in nude mice. Attempt to provide new experimental evidence for improving efficiency of engineered tissue vascularization.Methods and Results:1. Study on the ability of endothelial differentiation of human adipose-derived stem cells (hADSCs) seeded on PGA/PLA meshMethods:Human adipose tissue was obtained from liposuction procedures. Both density gradient centrifugation and adherent culture method were used to isolation, culture and purification hADSCs. P3 hADSCs were seeded on PGA/PLA mesh and cultured in endothelial differentiation medium supplemented with 20ng/mL VEGF. Cell adhesion, viability and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination. And the endothelial differentiation of hADSCs on 3D PGA/PLA mesh compared to 2D plastic under a supplement of VEGF was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings of vWF, VEGFR2, CD31, and gene expression analysis of endothelial related markers.Results:The hADSCs adhered well and distributed homogenously within the PGA/PLA fibers. With endothelial induction, hADSC cultured on the porous PGA/PLA mesh was able to gain an endothelial phenotype, demonstrated by the positive expressions of vWF, VEGFR2, CD31 and eNOS at either the cellular or the mRNA levels. Nonetheless,3D culture without induction and 2D culture still failed to develop any of the endothelial characteristics.2. The immunosuppression effects of induced hADSCs-PGA/PLA complex in vitroMethods:Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood, and induced hADSCs-PGA/PLA complex was co-culturing with PBMC in vitro. Proliferation of PBMC was detected by CFSE. The secretion of inflammatory cytokines IFN-y and IL-2 form PBMCs were detected by ELISA test. And the role of cell-cell interactions in immunosuppression was measured by transwell assay.Results:The induced hADSCs-PGA/PLA complex could neither stimulate the proliferation of PBMC nor increase the inflammatory factors secretion. The secretion of inflammatory cytokines IFN-y and IL-2 form PBMCs were decreased significantly after co-culturing with induced hADSCs-PGA/PLA (p< 0.05). Cell-cell interactions played an important role in immunosuppression of induced hADSCs-PGA/PLA.3. The effects of induced hADSCs-PGA/PLA complex in wound healing processMethods:Full-thickness skin defects murine model was created to analyze wound healing ability of induced hADSCs-PGA/PLA complex in combination with tissue-engineered skin substitute. The gross examination was supported by staining the harvested specimens with haematoxylin and eosin (HE), Masson trichrome and Picrosirius. And wound healing was evaluated by analyzing re-epithelialization speed, thickness of regenerated epidermis, and presence of collagen fibers. We also tracked GFP transgenic hADSCs to evaluate the function of hADSCs in neo-angiopoiesis during healing process of full thickness cutaneous injury.Results:The application of induced hADSCs-PGA/PLA complexes is a viable therapeutic approach to improve wound angiogenesis, regulate collagen deposition as well as accelerate wound repair. And some GFP transgenic hADSCs were detected position in newly formed blood vessels participated in forming capillaries in the wound site.Conclusion:1. PGA/PLA scaffolds possess appropriate degradation rate and favourable biological compatibility. And hADSCs adhered well and distributed homogenously within the PGA/PLA fibers. With endothelial induction, hADSC cultured on PGA/PLA mesh was able to gain an endothelial phenotype, and construct micro-vascularized engineered tissues.2. Growth factors and PGA/PLA mesh presented a synergistic effect on the acquisition of mature endothelial markers for hADSCs.3. Induced hADSCs-PGA/PLA complex have low immunogenicity, sound biocompatibility and great immunosuppressive effects on PBMC in vitro. Cell-cell interactions may be critical for induced hADSCs-PGA/PLA complex mediated immunosuppression.4. The induced hADSCs-PGA/PLA complex can improve wound angiogenesis and accelerate wound healing process remarkably.