Study On Articular Cartilage Derived Scaffold For Cartilage Tissue Engineering

Articular cartilage has a limited capacity for spontaneous self-repair and is consequently vulnerable to injuries and disease that may lead to irreversible damage. Numerous attempts have been made to promote reparation of the cartilage defect. Drilling, microfracture, autograft of periosteum, perichondrium and osteochondral autograft have been reported as potential procedure for the treatment of cartilage defects . All of these have been partially successful. With the development of tissue engineering , tissue engineering of cartilage seems to be a promising method for repairing cartilage defects. A scaffold plays a vital role in a tissue engineering approach to accommodate enough cells. The scaffold can be developed using either natural (i.e. chitosan, chitin, DBM, ascorbate, alginate and type I collagen ) and synthetic polymers (i.e. PLA, PGA, PLGA ). However, From biomimetic viewpoint, these polymers are devoid of chondrocyte-specific signals. A scaffold derived from extracellular matrices depleting the antigens may have good biocompatibility, and create an environment for cells which more closely resemble the extracellular matrix. The aim of this study was to develop a satisfactory scaffold from articular cartilage extracellular matrices.Firstly, we developed a cell extraction process to prepare human articular cartilage acellular matrix. A 3.5cm wide by 4.5cm long piece of fresh articular cartilage was cut after being washed in physiological saline, then , the samples were lyophilized in the FD-1 frozen-dried machine for 12 hours. The modified four -step detergent and enzymatic extraction process was as follows: The cartilage samples were immersed in a hypotonic Tris buffer(ph 7.4) containing a protease inhibitor (phenylmethyl-sulfonylfluoride, 0.35ml/L, ) for 24 hours at 4°C with constant stirring. The second stage used a 1% solution of octylphenoxypolyethoxyethanol (Triton X-100) in Tris-buffered salt solution with protease inhibition for 48 hours at 4℃ with constant stirring. Samples were thoroughly rinsed in distilled water before the third stage digestion with DNase and RNase overnight at 37℃. This was followed by a further 24 hours extraction with Triton X- 100 in Tris buffer. All samples were then washed for 48 hours in distilled water, then , the specimens were assessed by staining with haematoxylin-eosin, safranin-O (for GAGs), and by the immunolocation of aggrecan or type II collagen. Light microscopy confirmed that the celluar constituents of the specimens were removed , the specimens stained positively for GAGs and for aggrecan or type II collagen. The modified four-step detergent and enzymatic extraction process with lyophilization can remove the cellular constituents from human articular cartilage, in addition, structural extracellular matrix was retained, we have successfully prepared the articular cartilage acellular matrix. However, it has a low 45% porosity. We have to find a way to improve it.Then, we developed a method to prepare human articular cartilage derived microcarrier. The brief process was as follows: We crushed human articular cartilage into small pieces by muller after lyophilization, and sorted through two different meshes to collect only those specimens measuring 200-150 microns, then, in turn, the specimens were subjected to 0.25% trypsin at 37℃ for 24 hours and 1% Triton X-100 for 72 hours, respectively. The specimens were observed by inverted phase contrast microscopy, and assessed by staining with haematoxylin-eosin, safranin-O (for GAGs), as well as by the immunohistochemistry of aggrecan or collagen type II. We found that the cartilage pieces became flocculous or like a hairbrush by microscopy. HE stain of section confirmed that the celluar constituents of the specimens were removed, the specimens stained weakly positive forGAGs, negatively for aggrecan, and positively for collagen type II, respectively. So the detergent and trypsin can remove the cellular constituents and knock out the aggrecan from human articular cartilage, and made the cartilage pieces flocculous or hairbrush-like. Human articular cartilage derived microcarriers were prepared successfully. To evaluate the ability of human articular cartilage derived microcarriers to sustain repopulation by allogenic chondrocytes in vitro. We seeded the microcarriers with human sixth-passage (P6) chondrocytes in a flask after microcarriers being irradiated by Co⁶⁰, so they were observed immediately, after 2 hours , after 8 hours , and after 30 hours by inverted phase contrast light microscopy. We found that Some chondrocytes attached to the hman articular cartilage derived microcarriers immediately , and many spherical chondrocytes adhered to the microcarriers after 2 hours , The cells attached to the microcarriers remained chondrocyte morphology while those in monolayer became fibroblast-like cells after 8 hours . So it is with those cells after 30 hours. The chondrocytes repopulated well during observation. The chondrocytes can be well maintained in allogenic articular cartilage derived microcarriers in vitro.We developed a method to prepare human articular cartilage derived sponge based on the microcarrier. The brief process was as follows: We crushed human articular cartilage into small pieces by muller after lyophilization, and sorted through two different meshes to collect only those specimens measuring 38-25 microns, then, in turn , the specimens were subjected to 0.25% trypsin at 37°C for 24 hours and 1% Triton X-100 for 72 hours, respectively, and lyophilized for another 12 hours and irradiated by ultra-violet for 8 hours. The sponge' s anti-washout was good and nontoxic to cells ; The sponge consisted of a reasonably interconnected network of pores with 92% porosity , presenting a pore size between 100 and 200 um; Its degradation time in vivo was about 4 weeks; Immunological experiments...