Experimental Study On Repairing Of Osteochondral Defect Of Knee Joint In Rabbits With Tissue Engineered Osteochondral Composite And Related Research

Articular cartilage defect caused by trauma, inflammation, degeneration or tumor resection is very common in clinical practice. Articular cartilage is a unique tissue with a limited healing capacity and cannot be adequately replaced with de novo synthesized normal articular cartilage after injury. If not treated properly, not only can the damaged cartilage not be repaired with hyaline cartilage, but cartilage injury also gradually deteriorates because of abrasion degeneration of the neighboring tissues. Finally, cartilage injury may lead to severe osteoarthritis of the joint which can cause pain, dysfunction even loss of function of the joints. Traditional treatment options, including chondral shaving, subchondral drilling, subchondral microfracture, implantation of periosteum with or without injection of isolated autologous chondrocytes, has poor outcome, and none of these is a satisfied therapy for cartilage repair. There are more difficulties in treating cartilage defect with loss of subchondral bone. Mosaicplasty may be a promising approach forcorrection of such defects. But the extra injury of resection and the limited availability of donor tissue make it difficult to apply clinically, moreover, this technique cannot be applied when the damaged area is more than 2 cm. The most commonly practiced approach for large osteochondral defect is joint replacement, which has some complications such as infection, loosening, sinking, et al. At present, the research of treatment for cartilage defect focuses on regeneration and reconstruction. Tissue engineered cartilage is constructed and cultivated in vitro and implanted into the articular cartilage defect to restore the function of the joint. The interface between tissue engineered cartilage and the defect is a cartilage-to-cartilage one treating a cartilage defect, and with another cartilage-to-bone interface in treating an osteochondral defect. The integration of these two interfaces is slow. The potentially mechanical instability of the graft body in defect may lead to the failure of repair. It is well known that a bone-to-bone interface integrates faster and better than the above two. Consequently , a mechanical stability is provided, and the graft can structurally and functionally integrate with the host tissue if there is a faster integration and an early neoformed subchondral bane.Bone marrow stromal cells has strong proliferation and differentiation capacity. The harvest of a limited bone marrow sample is an easy and relatively safe procedure. Large numbers of BMSCs can be obtained in culture, making it possible to use BMSCs as seed cells of bone and cartilage tissue engineering. PLGA and PLGA/TCP, which are porous biomaterials produced by rapid prototyping technique, have the characteristic of biodegradability, biocompatibility and good 3-D space structure. RP PLGA and PLGA/TCP are ideal scaffolds for bone and cartilage tissue engineering.In this paper, we studied the biocompatibility of RP PLGA andPLGA/TCP, explored the possibility of inducing adult rabbit BMSCs to express and maintain chondrocyte phenotype by TGF~￡,, investigated the possibility of construction of tissue engineered cartilage using BMSCs as seed cell and biocompatibility rapid prototyping porous poly PLGA and PLGA/TCP as scaffolds, explored the feasibility of construction of an osteochndral composite using tissue engineering technique, and we also tried to repair a surgically created femoropatellar groove defect in rabbit with tissue engineered osteochondral composite. The ultimate objective of this paper is to construct a graft that can repair cartilage and subchondral bone defect simultaneously, provide mechanical stability for graft in defect and physiologic loading conditions for new cartilage forming and remodeling in early times after operation. The methods and results of this.study are as follows:1. Biocompatibility study of rapid prototyping porous poly ( PLGA/TCP and PLGA) scaffolds Cultured BMSCs were seeded onto polymer scaffolds (PLGA/TCP and PLGA), then cultured in DMEM with 10% FBS .The implanted cells and scaffold materials were observed at differion times with phase contrast microscope, scanning electron microscope and cell growth curves to study cell growth, morphology and adherent to the scaffolds. The sdudy indicated that BMSCs grew well around the scaffolds and then adhered to them, gradually linked with each other and formed a film on the surface of scaffolds. Growth curves of experimental group (PLGA/TCP, PLGA) and control group (without scaffold) were alike and had no statistical difference.2. Study on expressing and maintaining of chondrocyte phenotype of cells differentiated from adult rabbit BMSCs BMSCs which derived from adult rabbit bone marrow were induced in chondrogenic medium with rhTGF-Pi, dexamethasone and ascorbic acid. The cell morphology was observed with phase contrast microscope. Toluidine blue staining andimmunohistochemistry of collagen II were applied to detect the expression of chondrocyte phenotype at 7 and 14d after inducing. The induced cells with chondrocyte phenotype were induced and cultured continually in medium with rhTGF-fi, or alternately in medium with a low concentration of rhTGF-￡ ,, or medium without rhTGF-P i. The change of cell phenotype was observed after a long period of culturing in vitro. After 7 days of inducing, BMSCs changed from a spindle—like fibroblast appearance to polygonal shape. The differentiation of BMSCs was verified by the positive result of collage typeII through immunohistochemistry and the metachromatic matrix, which toluidine staining were positive. The induced cells could maintain the chondrocyte phenotype after a long period of culturing in vitro in medium with low concentration of rhTGF-Pi.3. Construction of tissue engineered cartilage with BMSCs seeding onto RP PLGA. Adult rabbit BMSCs were cultured in chondrogenic medium with rhTGF- P to induce them to express chondrocyte phenotype. The chondrogenic induced cells were seeded onto PLGA, then cultured for 2 weeks in vitro. The cell-loaded scaffolds were implanted into muscle pouches. The cell-free scaffolds served as controls. Specimens were harvested at 8 weeks after implantation, examined histologically for morphologic features, stained with toluidine blue and immunohistochemically for collagen type II. The examination revealed that : BMSCs of adult rabbit cultured in chondrogenic medium could express chondrocyte phenotype. Observing the cell-loaded PLGA cultured in vitro f or 2weeks with canning electron microscope, we found that BMSCs could expanded on PLGA. Cartilaginous tissue was observed with examination of histology and toluidine blue stain in the cell-loaded scaffolds at 8 weeks after implantation. Type II collagen was identified in the cartilaginous tissue.4. Experimental study on construction of tissue engineeredosteochondral composite Cartilage constructs were created by seeding chondrocyte induced from BMSCs onto PLGA; While bone-like constructs were created by seeding oeosteoblast, also induced from BMSCs, onto PLGA/TCP. Pairs of constructs were sutured together to form a tissue engineered osteochondral composite after 2 weeks of isolated culture in vitro. Cartilage constructs, bone-like constructs and the resulting composites were implanted into muscle pouches separately. The cell-free scaffolds served as controls. Specimens were harvested at 8 weeks after implantation and subjected to histological examination. Cartilaginous tissue was observed in the cartilage constructs, and bone was detected in the bone-like constructs. New cartilaginous tissue integrated well with new bone-like tissue in tissue engineered osteochondral composites at 8 weeks after implantation.5. Repairing of osteochondral defect of knee joint in rabbits with tissue engineered osteochondral composite Tissue engineered osteochondral composites were constructed as described in experiment 4. Femoropatellar groove defect in rabbit, which were 4 mm in diameter and deep into medullary canal, were created surgically and repaired with tissue engineered osteochondral composites. The cell-loaded scaffolds were incubated in medium with BrdU for 24h to label the cells. Defects that were left empty or treated with cell-free scaffold served as controls. Histological examination, including haematoxylin and eosin staining, toluidine blue staining and safranin O-fast green staining were applied to measure the repair of defects at 12, 24, 48 weeks after operation. The examination revealed that repairs of defects in experimental groups were superior to those in the two control groups with respect to histologic score, fraction of hyaline cartilage cartilage thickness, thickness uniformity , structural integrity, bony filling, and tidemark. The tidemark and subchondral...