Microenvironment Induce Neovascularization Process In Human Umbilical Vein Endothelial Cells

A cell-scaffold construct larger than a few cubic millimeters demands the ingrowth of blood vessels. Without proper blood supply, seeded cells in the middle of porous scaffolds would be subject to necrosis due to ineffective transportation of oxygen, nutrients and metabolites, thus resulting in the ultimate failure of the engineered tissue. Therefore, a vascular network that can provide nutrient and oxygen to maintain cell viability might be a prerequisite for the success of engineering large tissues. One potential solution is the application of endothelial cells and tissue specific cells onto the scaffold to construct a vascular network within the three-dimensional tissue construct before implantation. Although this approach has shown promising, human endothelial cells in the scaffold are not survival very well and more prone to apoptosis, thus it is difficult to construct a vascular network that can provide nutrient and oxygen to maintain cell viability within the three-dimensional tissue construct. So, acellular derm matrix and the extremely frequency electromagnetic treatment were employed to facilitate endothelial cells survival in the scaffold and blood vessels formation in vitro. Our results provide an interesting progress in the engineering thick, complex tissues and present a new strategy for the ultimate clinical applications.The study was composed of three sections as follows:PartⅠ. Preparation and related characteristics evluation of xenogeneic acellular dermal matrix and its micronized microcarriersIn order to testify the security of xenogeneic acellular dermal matrix (ADM) fabricated by a decellularization protocol incorporating trypsin, NaOH, and freeze thawing, we investigated their alpha-Gal epitope, DNA remnants, endogenous growth factors, cell toxicity, and in vivo biocompatibility. Following that, ADM was homogenized and the resulting particles were used as a cell culture substrate to evaluate the viability of expanded human fibroblasts. We further investigated the effectiveness of such cell-microcarriers constructers and feasibility for surgical applications by using an athymic murine subcutaneous injection and full-thickness cutaneous wound model. Conclusion:①Xenogeneic ADM fabricated by trypsin, NaOH, and freeze thawing protocol did not express of alpha-Gal epitope, retained lower DNA remnants,endogenous growth factors such as bFGF as well as TGF-β, and had good biocompatibility. These indicated that this ADM maybe serve as a scaffold for tissue engineering.②Micronized ADM microcarriers supported human fibroblast expansion, and they simultaneously formed engineered particulate dermal substitutes. In addition, such dermal substitutes had the potential to promote soft tissue augmentation and accelerate healing of the full-thickness wound.PartⅡ. Effects of acellular dermal matrix on the proliferation and vascular formation of human umbilical vein endothelial cells.To study the effects of the microenvironment created by ADM on the proliferation and vascular formation of human umbilical vein endothelial cells (HUVECs), HUVECs were seeded directly onto the ADM scaffold. Our observations showed that the ADM microenvironment can effectively stimulate the proliferation activity and blood vessel formation of HUVECs. These indicate ADM seeded with HUVECs may be an ideal vascularization of the scaffold for successful engineering of large, thick and complex tissue such as liver, kidney, and cardiac muscle.PartⅢ. Effects of ELF-EMF microenvironment on the vascular formationConsidering that electromagnetic fields are endogenous feature of all organisms, we proposed a novel engineering vascularized tissue protocol by using the exogenous electromagnetic treatment and explored a feasible strategy to enhance vessel formation in single as well as coculture cell, and three-dimensional complicated tissue by using the extremely frequency electromagnetic fields (ELF-EMF) treatment. In order to testify HUVECs treated by ELF-EMF are more easily to form blood vessel, the temporal effects of ELF-EMF on HUVECs were evaluated by the status of proliferation, apoptosis, migration, tube formation within the Matrigel, and gene expression of extracellular matrix, firstly. Following treated by ELF-EMF, some of the coculture of HUVECs and fibroblasts were investigated by the status of blood vessel formations. Others were implanted to subcutaneous pocket of nude mice and also evaluated by the status of blood vessel formations after 1 week operation. Finally, engineering dermal substitutes were constructed with HUVECs and fibroblasts coseeded on collagen and ADM scaffold fabricated in Part One and treated by ELF-EMF. The effect of ELF-EMF on engineering dermal substitutes was investigated by the status of blood vessel formations. In addition, engineering dermal substitutes using collagen scaffold were implanted to full-thickness cutaneous wound model and also evaluated by the status of blood vessel formations after 2 and 4 weeks operation. Conclusion:①ELF-EMF treatment significantly accelerated HUVECs proliferation, migration, tube formation and stability, and expression of extracellular matrix such as collagen, VCAM, as well as bFGF, but depressed HUVECs apoptosis.②ELF-EMF treatment not only increased the blood vessel formation in the coculture of HUVECs and fibroblasts, but also enhanced the blood vessel of coculture grafts inosculating with recipient host circulation.③ELF-EMF treatment increased the blood vessel formation in the engineering dermal substitutes using collagen and ADM scaffold.④The blood vessels treated by ELF-EMF were more easily to inosculate with recipient host circulation after engineering dermal substitutes implanted to mice.In summary, ADM prepared by improved solution method had very lower antigenicity as well as good biocompatibility, retained endogenous growth factors, and also effectively enhanced the proliferation activity and blood vessel formation of HUVECs. ELF-EMF treatment not only increased the blood vessel formation of HUVECs, but also enhanced a functioning vascular network inosculating with recipient host circulation. The according results we present in the text give an indication of possible application as a promising candidate for successful engineering of large, thick and complex tissue.