MSCs From Bone Marrow Seeded In ADM Scaffold Combining With PRP For Cartilage Regeneration

Articular cartilage provides near frictionless motion between articulating surfaces and protects the bones of synovial joints from being damaged when subjected to impact and load bearing. Although articular cartilage is highly susceptible to damage, it has limited intrinsic regeneration and self-repair capacity due to its innate avascular nature and low cell-to-matrix ratio. Several methods to improve the cartilage repair, including subchondral abrasion,microfracture, transplantion of osteochondral plugs, and autologous chondrocyte implantation, have had limited success. Tissue engineering has proved to be themost promising alternative therapy that combines cells, scaffolds and environmental factors for repair of articular cartilage defects.Part I Preparation of a three-dimentional Acellular dermal matrix(ADM) scaffolds and its characteristics.Objective To prepare a cartilage acellular matrix scaffold and to evaluate its characteristics. Methods Firstly,Calf full-thickness back skin was treated by a modified Courtman, s four-step method which was improved to produce a acellular dermal matrix. After the freeze-drying method,the3-D acellular dermal matrix (ADM) was prepared.The scaffolds were then cross-linked by a neotype crosslinking agent genepin for48h, and then placed into glycine solution server times for removing redundant genepin.The freeze-drying method was used to prepare ADM scaffold before which were investigated by gross observation,scanning electronmicroscope(SEM) observation and pore size, porosity measurement,water absorptionrate and degradation rate analysis.Secondly, Acellular dermal matrix (ADM) scaffolds were seeded with BMSCs(6X105/ml), which were obtained from the2-month-old New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope. Results The ADM scaffold had (99.2±9.3)%pore size,90%porosity, and(13.83±7.12)%or (25.66±8.19)%degradation rate at2or4weeks.MTT test showed that BMSCs grew well in the3-D ADM scaffolds of logarithmic trend, supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that the scaffolds were porous and the cells covered the scaffolds firmly with cell processes.Conclusion The Courtman’s four-step method which is improved makes acellular effects more thoroughly and a retention of the acellular dermal matrix components.The neotype crosslinking agent Genepin makes scaffolds’mechanical strength and resistanceto degradation be enhanced.Part II MSCs from bone marrow seeded in ADM scaffold combining with PRP for cartilage regenerationObjective To investigate the feasibility of MSCs from bone marrow seeded in ADM scaffold for cartilage regeneration in vivo.Methods Acellular dermal matrix (ADM) scaffolds were seeded with BMSCs(5X106/ml), which were obtained from the2-month-old New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope.Twenty-seven healthy3-month-old rabbits (female, weighing2.4kg±0.2kg) were randomly divided into3groups, They were left unfilled (Untreated group), or were filled with ADM(ADM group), ADM with BMSCs (ADM-BMSCs group) in a randomized manner.At4weeks after transplantation,9rabbits were euthanized with an overdose of anesthesia.The clinical parameters, and histological and immunohistochemical examinations were evaluated at weeks4,8,12post-treatment, respectively. Samples were harvested for evaluation, according to the recommended guidelines by the International Cartilage Repair Society (ICRS).Results A significant increase in proliferation of BMSCs in PRP group was measured by MTT assay, which proliferation was higher than the control group from day3to day11(p<0.05),SEM revealed that the ADM scaffold appeared to provide an effective substrate for MSCs to adhere and proliferate. ICRS scoring (Macroscopic and Histological scores.)for evaluation of cartilage repair in PRP-ADM-BMSC groups resulted in higher grades at each time point than the ADM-BMSC groups(p<0.05), which achieved better results than the ADM groups (p<0.05).After12weeks post-implantation,Defects in the PRP+ADM+BMSCs group were fully filled with cartilage-like tissue, well-integrated with the normal cartilage,although the content of cartilaginous matrix was not as abundant as that of normal.The value of Young’s modulus of the PRP-ADM-BMSC group was higher than that of the ADM-BMSC group At12weeks after implantation(p<0.05). Conclusion Our findings suggest that PRP can promote BMSCs proliferation and differentiation.MSCs from bone marrow seeded in ADM scaffold combining with PRP can enhance the biomechanical property of tissue engineered cartilage and thus it represents a promising approach to cartilage tissue engineering.