Fabrication Of Fibrin Gel Filled PLGA Sponge For Cartilage Regeneration

A composite scaffold was fabricated by fibrin gel filled poly(lactide-co-glycolide) (PLGA) sponge for cartilage tissue engineering. The PLGA sponge with an average pore size of 350μm and a porosity of 87% was fabricated by a gelatin porogen leaching method. Via a process of sol-gel of fibrin gel, it was filled into the PLGA sponge. The fibrin gel evenly distributed in the composite scaffold with visible fibrinogen fibers with a diameter about 200nm after drying. In vitro co-culture with chondrocytes found that in the PLGA/fibrin gel the chondrocytes distributed more evenly and kept a round morphology as that in the normal cartilage. Although the chondrocytes seeded in the PLGA sponges showed similar proliferation behavior with that in the PLGA/fibrin gel, they were remarkably elongated, forming a fibroblast-like morphology. Moreover, a larger amount of glycosaminoglycans (GAGs) was secreted in the PLGA/fibrin gel than that in the PLGA sponges after 4wk. The results suggest that the fibrin/PLGA may be more favorably applied for cartilage tissue engineering than the PLGA sponge.Degradation of the PLGA sponges was investigated in PBS (pH=7.4) at 37℃and in cartilage defects, respectively. In vitro, the number-average molecular weight (Mn) of the scaffold decreased almost exponentially along with the incubation time. After 24wk, the Mn decreased from 76kDa to 5.6kDa. Meanwhile, Mn of the sponges decreased to 3.3kDa at 12wk post-implantion in cartilage defect, showing a faster degradation rate.BMSCs were employed as seed cell for the animal experiment. The PLGA/fibrin gel/BMSCs was implanted into the full-thickness cartilage defects made in New Zealand white rabbit joints (3mm in diameter and 4mm in thickness), while the PLGA/BMSCs served as the control. At 12wk post-implantation, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the experimental group, whereas only a little bit of cartilage-like tissue and fibrous tissue was observed in the group absent from fibrin gel. These results imply that the PLGA/fibrin gel may be a better choice for cartilage restoration than the PLGA sponge too, when the BMSCs are used as the seed cells.The effectiveness of any cellular repair approach depends on the retention of cell viability after implantation. To evaluate the cell viability, allogenic BMSCs were labeled with CM-Dil fluorochrome, seeded in PLGA/fibrin gel scaffolds and implanted into the full-thickness cartilage defects (4mm in diameter and 4mm in thickness). The red fluorescence in the defects zone and in BMSCs was observed by small animal in vivo fluorescence imaging system and laser scanning confocal microscope after frozen section, respectively. The results showed that even after 12wk post-implantation, the transplanted BMSCs still localized and kept alive in the defects.Then the composite scaffold was upgraded by incorporating with transforming growth factor-β1 (TGF-β1). The PLGA/fibrin gel/BMSCs/TGF-β1 composite constructs were implanted into the full-thickness cartilage defects (4mm in diameter and 4mm in thickness), while the constructs absent from TGF-β1 served as the control. At 12wk post-implantation, the generated neo-cartilage integrated with its surrounding normal cartilage and subchondral bone in the experimental group, whereas only a little bit of cartilage-like tissue was observed in the group absent from TGF-β1. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data also showed that the cartilage special genes expressed in the neo-tissue were higher than those of the control group. The composite scaffold incoporated with TGF-β1 improved cartilage restoration substantially.Growth factors are expensive and generally have a short-half life in the order of minutes because of rapid clearance via the lymphatic system. Another way to solve the problem is the use of gene therapy, which was incorporated into this composite system in the next study. A cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) was employed as a carrier to condense DNA forming nano-complexes. In vitro, BMSCs were transfected by the TMC/DNA complexes with an efficiency of 9% and showed heterogeneous TGF-β1 expression in a 10 day culture period after transefected by TMC/pDNA encoding TGF-β1 (pDNA-TGF-β1). The PLGA/fibrin gel/BMSCs/(TMC/pDNA-TGF-β1) constructs were implanted into the full-thickness cartilage defects (4mm in diameter and 4mm in thickness), while the scaffolds absent from pDNA-TGF-β1 or BMSCs served as the control. In vivo heterogeneous TGF-(31 was expressed in the experimental group at least lasting for 4wk detected by western-blotting and qRT-PCR. At 12wk post-implantation, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in experimental group, whereas only a little bit of cartilage-like tissue and fibrous tissue was observed in the group absent from pDNA-TGF-β1 and BMSCs, respectively. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The qRT-PCR data also showed that cartilage special genes expressed in the neo-tissue were comparable to those of the normal cartilage and were much higher than those of the control groups. The successful repair thus evinces the potentiality of using this composite construct for cartilage regeneration.