Preparation And Biocompatibility Assessment Of Articular Cartilage Extracellular Matrix-derived Microcarriers

Background：The damages of articular cartilage caused by trauma, inflammation anddegeneration, are the most common joint diseases in the world. Because articularcartilage is an avascular tissue, it has very little tendency to self-repair and this leads tofurther degeneration without treatment. Currently, a number of surgical options includemicrofracture, osteochondral grafting, and cell-based techniques. However, articularcartilage injuries treated with microfracture or osteochondral grafting deteriorate withtime as a result of the high proportion of fibrocartilage or a lack of lateral integrationbetween host and donor cartilage. As an alternative treatment, tissue-engineering hasbeen demonstrated to be a promising approach to restoring articular cartilage defects.The most common and most-studied problems in tissue engineering include how toobtain a sufficient number of cells with the original phenotype, how to prepare the bestscaffold and how to load the factors into the scaffold to achieve long-term controlledrelease of factors–the seeds, soil and fertilizer. The autologous chondrocytes are themost suitable seed cells of tissue engineering and used in the clinic. Now, how to obtainbetter sufficient numbers of seed-cells in a short-time and preserve their characteristicmorphology during in vitro expansion culture of cells is a top issue in tissue engineering.Micro-carriers can help face the challenge in amplifying cell numbers and maintainingthe appropriate phenotype for tissue repair and restoration of function. Micro-carrierscan serve as substrates for the propagation of seed cells, especially extracellularmatrix-derived.At present, in the field of regenerations of skin and adipose tissues, tissueextracellular matrix-derived micro-carriers have successfully obtained, but in the field of cartilage tissue not been reported. This study was divided into three parts. The firstpart includes preparation and observation of cartilage extracellular matrix-derivedmicro-carriers (CEMMs). The second part includes observation of biocompatibility ofarticular extracellular matrix-derived micro-carriers. The third part is to study the effectsof articular extracellular matrix-derived micro-carriers on the phenotypes of rabbitchondrocytes.Objective: This study was divided into three parts. The first part includes preparationand observation of cartilage extracellular matrix-derived micro-carriers (CEMMs). Thesecond part includes observation of biocompatibility of articular extracellularmatrix-derived micro-carriers. The third part is to study the effects of articularextracellular matrix-derived micro-carriers on the phenotypes of rabbit chondrocytes.Methods: The flesh cartilage tablets were smashed into cartilage slurry by dishesgrinder. The cartilage slurry was diluted with distilled water and was filtered throughmeshes. And then we harvested the CEMMs. The CEMMs was evaluated by histologyquantitive biochemical measure of collagen, glycosaminoglycan, and DNA content. Theconfluence of proliferation from CEMMs on chondrocytes was assessed by MTT andDead/Live viability staining in vitro. Under the dynamicthree-dimensional microgravity conditions, the phenotypes and cell cycles ofchondrocytes, cultured on the CEMMs, were observed by Rt-PCR and flow cytometry,Ordinary static culture and Cytodex-3micro-carrier as control.Result: Scanning electron microscopy (SEM) showed that the surface of articularcartilage extracellular matrix-derived micro-carriers were covered with microfilaments.The Hoechst33258fluorescent staining was negative after decellularization. The alcianblue staining, safaranin O staining and collagen Ⅱ immunohistochemistry stainingwere positive. Dead/Live viability staining showed that the chondrocytes loaded onCEMMs were live and proliferated quickly. In MTT test, the OD value of CEMMsgroup were higher than complete medium group. Rt-PCR results showed that the CEMMs promote the expression of collagen-Ⅱ, SOX-9and aggrecan genes andinhibited the expression of collagen-I. The test of cell cycle showed the chondrocytesloaded on CEMMs were mostly in S or G2-M phases.Conclusions: The CEMMs,220-300μm diameter, were harvested successfully by theimproved preparation and were oval in shape. The CEMMs were covered withmicrofilaments. The CEMMs had ECM components after decellularization by SafraninO, tuoluidine blue staining and collagen-II immunohistochemistry staining. TheCEMMs, which had no cytotoxicity, promoted cell proliferation and possessed goodbiocompatibility, could serve as excellent amplification carrier of seed cells for cartilagetissue engineering. The CEMMs can effectively maintain the chondrocytes phenotypes,which was better than Cytodex-3.