MSCs-loaded Collagen Scaffolds In The Regeneration Of Full-thickness Articular Cartilge Defect

Introduction:Articular Cartilage (AC) has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic and labrotary. When the size of the cartilage defect diameter exceeds 2–4 mm, damaged cartilage rarely heals in the absence of treatment. Many methods have been involved in an attempt to repair articular cartilage defects, including microfracture, osteochondral allografting, and the use of periosteal or perichondrial tissue. However, all of these techniques possess disadvantages and the long-term outcome may include cartilage degeneration, including fibrosis and calcification.A biologic repair process that regenerates (not simply repairs) normal AC has not been achieved at this time. However, an acceptable outcome may be a functional cartilage surface that provides pain relief. An ideal AC substitute would have a nμmber of desirable characteristics, including (1) availability in an off-the-shelf variety of sizes to fill different size lesions, (2) adheres or fixes easily to surfaces to be repaired, (3) handles immediate weight bearing, (4) immediately stabilizes the bone and AC from further degradation, (5) promotes biologic repair that is durable, and, finally and most importantly, (6) provides pain relief for the patient.Tissue engineering is a rapidly developing field offering new perspectives in the treatment of damaged or diseased tissues. The basic premise of tissue engineering relies on the use of scaffolds to encourage cells to proliferate and organize their extracellular matrix (ECM) in order to form ex vivo a clinically functional tissue, exhibiting histochemical, biochemical and biomechanical properties identical to native, healthy tissue. Three key constituents form the basis of a tissue engineering approach, namely, cells, a scaffold, and signaling molecules. In recent years, The use of mesenchymal stem cells (MSCs) as a cell source is attractive because they can be harvested with less morbidity from a patient than most differentiated cells.The objectives of this study were to determin the repair abilities of two kinds of nature three-dimensional collagen scaffolds, one is the bilayer designed typeⅠ/Ⅲcollagen scaffold, and the other is typeⅡporous scaffold. Meanwhile, add the process of using bone marrow-derived MSCs grown in two scaffolds as a new technique to hypothesis better results. The typeⅠ/Ⅲscaffold has a rough side on which the chondrocytes are seeded and a smooth, more dense side that is placed facing the articular cavity, inhibiting the migration of the seeded cells into the joint cavity. The rough side has a larger pore size compared with that of the smooth side, as it is characterized by a looser collagen fiber arrangement that facilitates chondrocyte attachment.Materials and Methods:1. Mesenchymal Stem Cells (MSCs) derived from a goat multipotent bone marrow was used for this study. Complete culture mediμm consisted of DMEM, supplemented with 5%, 10%, 15% goat autologous serμm or 10% fetal bovine serμm respectively, 100 U/ml penicillin, and 100μg/ml streptomycin, and to investigate the opitimized concentration for cells culture. Cells were identified by cells growth cycle and osteoblasts inducement.2. 15 skeletally mature goats, 30 knees were used. Prepare a 6mm diameter cartilage defect on the TR, then treated with relatively detect. Scaffold was structured by steriling, and then seeded with sheep bone marrow derived mesenchymal stem cells, had histology, histocompatibility measurements after cultured 10 days in vitro. Defects were examined arthroscopically at 6w, 24w and with gross, histological analysis.3. TypeⅡcollagen was isolated and purified from the knee joints of the pigs. Scaffolds was made by vacuμm freeze-drying, and valued the scaffold by SEM, histochemical staining, cell biocompatibility and animal experiments, then data were analysed by SPSS17.0.Results:1. The proliferation of 5% autologous serμm group was slower compared with that of 10% fetal bovine serμm group.(P <0.05).Both the 10% and 15% autolous serμm groups had the similar proliferation of BMSCs as 10% fetal bovine serμm group, and had no significant deviation between groups. The standard method of MSCs harvesting and preparation for transplantation is well established.2. After 6 months, there were few filled with repaired tissue in control group, and the histological analysis showed itwas fibrocartilage-like tissue. In Scaffold only group, scaffold was almost fully filled, and it was combined with little hyaline cartilage-like but mainly fibrocartilage-like tissue. In MSCs-loaded group, scaffold was fully filled, and the histological analysis showed it was hyaline cartilage-like and fibrocartilage-like tissue. According to the score, MSCs-loaded scaffold group is superior to other 2 groups.3. Type II collagen scaffold was porous, porosity and pore ratio higher scaffold material, and has good biological compatibility. Animal experiments show that, type II collagen scaffold with MSCs transplantation is an effective treatment modality, and the repaired tissue thickness was increase. In the early stage of degradation rate was too fast and host integration interface still need further improvement.Conclusion:1. Autologous serum (AS) preparation technique for the MSCs culture was well established. 80ml goat blood can got 38.2±0.6ml serum, which accounting for about 50% of whole blood.2. Successfully isolated and purified BMSCs from bone-marrow. 8ml bone marrow can be isolated 2×106 nucleated cells. Cell number was increased 6 times by using 10% autologous serum cultured for 7 days, and there was no significant difference between the 10% AS and 10% FBS group. BMSCs have strong ability of proliferation and multi-potential differentiation.3. We improved the protocol anesthesia and resuscitation techniques for the goats. Goat knee model of full-thickness articular cartilag defects was well made for the cartilage tissue engineering; it will provide a good evaluation platform for the further study.4. CollagenⅠ/Ⅲbilayer scaffold with MSCs transplantation is an easy and efficient technique for treating cartilage defect, and the effect of MSCs transplantation is not enhanced in the early repair process.5. Preparation of type II collagen scaffold is a feasible method for cartilage tissue engineering; it provides a new train of thought to solve the articular cartilage defect.6. Results of Wakitani score showed that the CollagenⅠ/Ⅲbilayer scaffold group was 1.86±0.28, the Collagen 2 scaffold was 2.55±1.02 and control group was 11.42±1.26. MSCs-loaded CollagenⅠ/Ⅲbilayer scaffold was inferior to the Collagen 2 scaffold group in the vivo experiments.