Improvement Of PHBV Scaffolds With Bioglass For Cartilage Tissue Engineering

PartⅠPreparation of poly(hydroxybutyrate-co-hydroxyvalerate)/bioglass composite scaffoldsObjective: To study the modification effects of bioglass on enhancing thehydrophilic ability of poly(hydroxybutyrate-co-hydroxyvalerate)/bioglass compositescaffolds and to analyze the biocompatibility and physical characteristic of the compositescaffolds. Methods: Composite scaffolds of poly(hydroxybutyrate-co-hydroxyvalerate)(PHBV) with sol-gel-derived bioglass (BG) were fabricated by compression molding,thermal processing, and salt particulate leaching method. The hydrophilic ability of simplePHBV and PHBV/BG composite scaffold were detected by static water contact angle andwater absorption method. The biocompatibility of the composite scaffold was evaluated bycell adhesion rate test and pore size and interpores of the scaffolds were observed byscanning electron microscope. Results: There were no significant differences inmacroporous structure, the pore size and the porosity between the simple PHBV andPHBV/BG composite scaffolds. The compressive yield strength of the scaffolds increasedfrom0.16to0.41MPa with increased BG contents from0to20wt%(p <0.05), whereasthe static water contact angle decreased from65°to32°(p <0.05). Compared with purePHBV, the rate of cell adhesion to the composite material was significantly increased withthe increase of the content of BG in the composites. Scanning electron microscopy showedPHBV/BG composite scaffolds could promote the adhension of chondrocytes. Conclusion:The incorporation of BG into PHBV could improve the hydrophilicity and mechanicalproperties of the composites and PHBV/BG composites, as porous scaffolds, were highly biocompatible. Part II Tissue engineered cartilage constructed byPHBV/BG composite scaffolds with chondrocytesObjective: To evaluate the feasibility of PHBV/BG composite material asa biocompatible scaffold for construction of tissue-engineered cartilage．Methods: Thefluorescent labeled chondrocytes were seeded onto the PHBV and PHBV/BG compositescaffolds respectively and cell distribution was observed under fluorescence microscopeafter4h or12h culture. Chondrocytes cultured with PHBV and PHBV/BG scaffold extractrespectively were used to assess cytotoxicity of the scaffolds and the proliferation of thechondrocytes on the scaffolds were examined using CCK-8method. The chondrocyteswere seeded onto the PHBV (the control group) and PHBV/BG composite scaffolds (theexperimental group) respectively and after3weeks’ culture in vitro, they were implantedsubcutaneously into nude mice. These specimens were collected after in vivo implantationfor12weeks and were analyzed by observation, histology and quantitative detection of theextracellular matrix components. Results: The fluorescence microscope revealed thatcompared with those on the PHBV, the chondrocytes seeded onto PHBV/BG compositescaffolds migrated faster into the scaffolds and distributed in the inner frame moreuniformly. CCK test showed the chondrocytes cultured with PHBV/BG scaffold extractproliferated faster. During in vitro culture, the DNA content of the chondrocytes from theexperimental group was significantly more than those from the control group. Withprolonged incubation time in vivo, the chondrocyte-scaffold constructs in the PHBV/BGgroup formed thicker cartilage-like tissue with better biomechanical properties and ahigher cartilage matrix content than the constructs in the PHBV group. Conclusion:PHBV/BG scaffolds, which could be used to prepare better tissue-engineered cartilage, arerelatively more promising than pure PHBV scaffolds. Part III Induced differentiation of bone marrow mesenchymal stemcells into chondrocytesObjective: To observe the chondrogenic differentiation of bone marrowmesenchymal stem cells (BMSCs) by cytokines or by co-culturing with chondrocytes.Methods: BMSCs were isolated and purified by density-gradient centrifugation andadherent culture in vitro. After complete confluency, BMSCs at passage2were identifiedby flow cytometry and were induced to differentiate into chondrocytes-like using DMEMcontaining TGF-1，IGF, dexamethasone and fetal bovine serum. The glycosaminoglycanand collagen typeⅡsynthesized by the induced BMSCs were detected with toluidine bluestaining and immunohistochemistry staining. The mRNA expressions of collagen typeⅡwere detected with real-time fluorescent quantitative PCR. Rabbit BMSCs (5.0×107/ml)were seeded onto a polyglycolic acid/polyactic acid (PGA/PLA) scaffold. The cell-scaffoldconstructs were put into a transwell, under transwell were adherent chondrocytes, asexperimental group. The control group had no adherent chondrocytes under transwell（containing10%fetal calf serum）. All specimens were harvested after in vitro culture for8weeks and implanted subcutaneously in the dorsum of athymic nude mice for6weeks.Gross observation, RT-PCR, histology and immunohistolochemistry were used to evaluatethe results. Results: Flow cytometry analysis showed that in vitro expanded BMSCsexpressed mesenchymal cell marker, including CD29, CD105, CD166. For chondrogenesiscells，the results of the toluidin blue staining and collegen Ⅱimmunocytochemical stainwere positive. The results of real-time fluorescent quantitative PCR indicated theexpression of collagen typeⅡby the induced BMSCs. In experimental and control groups，the cells adhered to the scaffold well and produced abundant extracellular matrices afterone week of in vitro culture. RT-PCR showed TypeⅡcollagen and aggrecan demonstratedstronger expression in experimental group, while that expressed little in control group after8weeks of in vitro culture, which suggested BMSCs in experimental group underwentchondrogenic differentiated stage. After6weeks of subcutaneous implantation into nude mice, specimen in experimental group could form cartilage-like tissue with typicalcartilage lacuna, and maintain the original size and shape. The cell-scaffold constructs incontrol group shrunk gradually and could not form cartilage. The results were furthersupported by histological feature and immunohistochemistry. Conclusion: BMSCs candifferentiate into chondrocytes-like by co-culturing with chondrocytes or by cytokines. PartⅣRepair of articular cartilage defect using BMSCsloaded on PHBV/BG composite scaffoldsObjective: To investigate the effect of bone marrow mesenchymal stemcells seeded on PHBV/BG composite scaffolds on repair of articular cartilage defect.Methods: The BMSCs derived from20rabbits were cultured in vitro and seeded onPHBV/BG composite scaffolds to fabricate BMSCs-PHBV/BG constructs TheBMSCs-PHBV/BG constructs after being in vitro cultured for72h were transplanted intofull thickness articular defects in non-weight bearing area in rabbit model.20rabbits with20knee joint were made models of cartilage defects in the intercondylar fossa. These kneejoints were divided into3groups according to the repair materials with10in each group:group A: BMSCs-PHBV/BG complex; group B: only PHBV/BG; and group C: nothing.The articular defects of group B and Group C were made in the same joints. At12weeksafter operation, the gross and histological observations were made. The mRNAexpressionsof collagen typeⅡwere detected with real-time fluorescent quantitative PCR. Results: At12weeks after transplantation, the defects in group A were repaired by the hyline-liketissue and defects in groups B and C were repaired by the fibrous tissues. The HE stainsshowed normal cartilage histologic appearance in group A, however in group B and Cthere was much fibrous tissue. RT-PCR showed TypeⅡcollagen demonstrated strongerexpression in group A, while that expressed little in group B and C, which suggestedBMSCs in group A underwent chondrogenic differentiated stage. Conclusion: BMSCs combined PHBV/BG composite scaffolds could repair cartilage defect.