Macroporous PHBV Matrices For Cartilage Tissue Engineering

Objective To investigate the possibility of using PHBV as scaffolds for cartilage tissue engineering and compare the engineered cartilage generated in vitro with those in nude mice modles. Methods PHBV porous scaffolds were fabricated using a compression moulding, thermal processing and salt particulate leaching method. Surface and cross-section morphologies were observed by scanning electron microscope (SEM) and the porosity of PHBV was measured by measuring the dimensions and mass of the scaffolds. The cytocompatibility of scaffolds was evaluated by the proliferation of L929 mouse flbroblasts cultured with maceration extract of PHBV and flbroblasts morphology in the scaffolds observed with SEM. After chondrocytes isolated from cartilage slices by enzymatic digestion and expansed in monolayer culture, the growth status, morphologic features and differentiation properties of the cells were observed, then passage III chondrocytes were seeded in porous PHBV in an appropriate density(50 106cells/ml). Chondrocytes cultured in vitro for 3,7 and 14 days on the scaffolds were examined by SEM. After incubation for 2 weeks in vitro, chondrocytes-PHBV constructs were implanted subcutaneously in the dorsum of athymic nude mice. Controlgroups were established by subcutaneous implantation of PHBV scaffolds alone. The implants harvested after in vivo incubation of 4 and Sweeks and cell-scaffolds cultured in vitro for 2,6 and lOweeks were respectively examined macroscopically, histologically and immunohistochemically. Results The PHBV scaffolds exhibited highly porous and open-cellular pore structure. The pore size in the scaffolds ranged from 200um~300um and the porosity was about 80%. The maceration extract of PHBV did not influence L929 cell growth and SEM observation revealed L929 cells seeded onto PHBV scaffolds could maintain normal morphology. The toxicity gradation of PHBV was 0. At the appropriate plating density(2~3 X 104/cm2), primary cells and passage I ~III articular cartilage cells could retain rounded morphology, while after division , dedifferentiation of chondrocytes into a fibroblastic-like phenotype was detected. The growth curves showed the cells proliferated vigorously from primary to the fourth passage. SEM showed chondrocytes could aggregate and synthesize extracellular matrix on PHBV scaffolds. At the end of the culture periods, both specimens harvested from nude mice and cultured in vitro demonstrated the presence of cartilaginous layer in the superficial area of the scaffolds and the scaffolds maintained their original shapes. Histological examination confirmed morphology of the chondrocytes and the expression of the cartilaginous matrices. The amount of glycosaminoglycans(GAG) and collagen increasedwith an increase in the culture periods, while histological staining for cartilaginous extracellular matrices of GAG and collagen revealed that more GAG and collagen were detected in vivo than in vitro and characteristics of the engineered cartilage generated in vivo were more typical. Immunohistochemical staining of collagen type II indicated low levels of the type II collagen expressed in both groups. No cartilage was histologically detected in the implants without chondrocytes. Conclusion PHBV scaffold is suitable to be applied in cartilage tissue engineering as it has optimal porous structure and biocompatibility. Nude mice modle seems to facilitate chondrogenesis compared with in vitro system.