Experimental Studies On Cytocompatibility Of Polyhydroxybutyrate (PHB) In Vascular Tissue Engineering

One of the important parts studied tissue engineering (TE) is the three-dimensional biodegradable material that can be used to provide scaffold for cells. Although many efforts have been done, the ideal material hasn't found yet. In this experiment, polyhydroxybutyrate (PHB) ,a new degradable polymer, was studied as a scaffold in vascular tissue engineering (VTE). Part oneObjective: To provide cells resource for the study in VTE. Methods: Vascular endothelial cells (VEC) and smooth muscle cells (VSMC) are respectively isolated from rabbit thoracic aorta with enzyme digestion and tissue plant methods, subcultured, purified and identified. Phase contrast microscope was used to observe the cell's morphological characters. Results: The morphological characteristics of two kinds of cells are accord to those of VEC and VSMC. Both of the reactions were positive on cells with immunocytochemistry technique. Conclusion: The cultured cells can meet the experimental expectations in VTE. Part twoObjective: To study the cytocompatibility of PHB combined with VSMC and VEC in VTE. Methods: VSMC and VEC were seeded on PHB polymers respectively, then compared with PGLA, the morphological characters were observed with phase contrast microscope and scanning electron microscope (SEM), cell adhere rate and cell proliferation were detected with MTT assay, cell cycle and DNA index (DI) were detected with flow cytometer (FCM). Result: The adhere rate of VSMC which were seeded on PHB polymers was77%, was less than PGLA, but the cells seeded on PHB grew well and were confirmed as diploid with FCM. Conclusion: The results show that PHB has a good cytocompatibility with VSMC, but the adhere degree between cell and material should be further improved. Part threeObjective: To look for a ideal functionalization technique to improve the chemical and physical characters and degradation rate of PHB. Methods: PHB were coated with collagen, FN and PLYS respectively in adequate concentration, and then VSMC were seeded on them. The morphological characters were observed with phase contrast microscope, the cell adhere rate and cell proliferation were detected with MTT assay, cell cycle and DI were detected with FCM. Result: The cell adhere rates of VSMC were 94.5%, 98.2% and 95.1%, were much more than the non-functionlized PHB. The cells seeded on the materials grew well, and they were confirmed as diploid with FCM. The cells in S stage in the group with FN were more than the others. Conclusion: Collagen, FN and PLYS could improve the cell adhere degree and accelerate cell proliferation. FN is the best in the three. Part fourObjective: To assess the interaction between PHB and VSMC in vivo. Methods: VSMC were seeded on non-coated PHB scaffold and PHB scaffold coated with FN, then cultured in vitro. After one week, the composite were implanted in the back of nude mice. Six weeks later, the materials were removed from those mice. Histological observation was proceeded by HE staining and immunocytochemistry. Result: The wound healed well. The materials were enveloped in the thin fibrous capsule in the original shape. Smooth muscles had been constructed, and those with FN were better. Conclusion: PHB has a good Cytocompatibility in vivo. It can be used to build the tissue-engineered blood smooth muscle.There are a few innavations in the experiment. The first is that PHB were studied in VTE at first time. The second is surface functionalization of PHB with collagen, FN and PLYS, and the composing smooth muscle with PHB and VMSC in vivo is the third.In conclusion, PHB, a new biodegradable material, can meet the need as a scaffold in VTE. Collagen, FN and PLYS could improve the cell adhere degree, accelerate cell proliferation and tissue construct. FN is the best one in the three .