| Chick bone is the main by-products of chick manufacturing industry, most of chick bone have not been exploited and are directly discharged into estuaries resulting in environmental pollution. Type II collagen is the main structural component of chick sternal cartilage and is used in various food applications, pharmaceutical applications and chemical industrial with specific molecular structure. If the type II collagen can be extracted from the chick sternal cartilage, not only the polluting problem can be resolved, but also can obtain a kind of biological activity product with high appending value.Pepsin was used to extract type II collagen from chick sternal cartilage and the biological activities and the structure of type II collagen were studied in this paper. Major content of this paper as following:Reversed-phase high performance liquid chromatography (RP-HPLC) method was demonstrated to determine the content of type II collagen. Chromatography was performed with an injection volume of 10μl. The mobile phase consisted of two solvent: (A) 5% acetonitrile and 0.05% trifluoroacetic (TFA) and (B) 80% acetonitrile (v/v). The separation was performed using the linear gradient of A-B (v/v). Flow rate was maintained 1 ml/min. Absorbance was monitored at 220 nm. The RP-HPLC method had a good linearity correlation with Woessner I method (R2>0.99). As compared with the conventional method, the proposed method has proved to be convenient, sensitive, accurate and reproducible on base of avoiding the use of a time-consuming pretreatment procedure.Technical route of extracting type II collagen from chick sternal cartilage were confirmed. The chick sternal cartilage was defatted with chloroform-methanol (2:1, v/v) and the residual fat was 0.072%. Then, the sternal cartilage was extracted using 1.0 mol/L NaCl in 0.05 mol/L Tris-HCl (pH 7.5) at 4°C for 24 h to remove contaminating protein. After the extracts were aggregated by centrifugation, the digestion was tested by mixing precipitation with pepsin to assess the temperature, times and the ratio of enzyme to precipitation effect on the extracting ratio and secondary structure of type II collagen. The optimum technical parameters for pepsin digestion were studied: extraction times for 32h, pepsin concentration for 0.5% and temperature for 20°C. It seemed, therefore, the pepsin-solubilized type II collagen of higher stability and extracting ratio can be obtained (43.49%).NaCl was applied to precipitate type II collagen. The optimum technical parameters for NaCl salting out were studied: extraction times for 32h, pepsin concentration for 0.5%, temperature for 20°C and the recovery ratio was 85.93%.DEAE-sepharose CL 6B chromatography provided a rapid and efficient method for separating collagen from proteoglycans in cartilage extracts. The results of SDS-PAGE and RP-HPLC suggested the prepared type II collagen essentially was free from contaminating proteins by the method used in this study for purification. All these collagen samples purified had similar migration bands and consisted ofαchain and their dimers (βchains) with a subunit Mr of 110 kDaThe structure and composition of type II collagen were studied. Type II collagen had a high content of glycine, hydroxyproline and proline residues, with 310, 117 and 115 residues per 1000 amino acids residues respectively, and small amounts of tyrosine, cysteine, histidine and methionine residues with 5, 18 and 10 residues per 1000 amino acids residues respectively. The Td was determined to be about 42°C for purified type II collagen and about 44°C for intact cartilage. The minimal difference between denaturation temperature of purified type II collagen and intact cartilage indicate that purification process has little influence for stability of collagen.Type II collagen molecules consisted of three polypeptide chains, which self-assemble into D-periodic cross-striated fibrils with 65 nm. Collagen molecules, forming the fibril, consisted of an uninterrupted right handed triple helix called tropocollagen, approximately 300 nm in length and 1.5 nm in diameter. The stagger of molecules gives rise to a characteristic band pattern of light and dark regions and viewed using an electron microscope.The porous type II collagen-chondroitin sulfate (CS) scaffold were prepared using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results suggested that EDC - crosslinking increased the denaturation temperature (Td) and resistance to enzymatic degradation by bacterial collagenase. A significant decrease in the swelling ratio and free amino groups per 1000 amino acid residues was also detected. The mechanical stability of collagen-CS scaffolds were improved with EDC concentration for 7 mg/ml. Then, isolated chondrocytes were cultured in porous type II collagen scaffolds either in the presence and/or absence of covalently attached CS up to 14 days. Cell proliferation and the total amount of proteoglycans and type II collagen retained in the scaffolds were higher in type II collagen-CS scaffolds. Histological analysis showed the formation of a denser cartilaginous layer at the scaffold periphery. Scanning electron microscopy revealed chondrocytes distributed the porous surface of both scaffolds maintained their spherical morphology. The results of the present study also indicate that type II collagen-CS scaffolds have potential for use in tissue engineering.The effect of type II collagen (CII) prepared from the chick sternal cartilage and Glucosamine Sulfate (GLS) on collagen-induced rat rheumatoid arthritis (RA) model (CIA) was studied. CIA was established in male Sprague-Dawley (SD) rats with intradermal injection of chicken type II collagen at the left hind paw of the animals, and then CIA rats were treated daily using oral administration of different doses of CII with or without GLS beginning on the day of the induction of arthritis (day 0, the prophylactic treatment) until day 45. Histopathologic sections of diarthrodial joints showed that injection of type II collagen with CFA produced a chronic proliferative synovitis which secondarily destroyed articular cartilage and bone. Synovium of the onset of arthritis showed marked edema and infiltration by dense aggregates of mononuclear cells and occasional neutrophils. Prophylactic treatment with type II collagen significantly suppressed the onset of arthritis and markedly reduced paw swelling even in the established CIA. The therapy may be achieved by restraining cytokines excreted by synoviocyte and balance the Th1 and Th2 cell. Hence, our studies demonstrate the quality, safety, and effectiveness of type II collagen as an anti-arthritic agent, which makes type II collagen a strong candidate for further clinical trials on rheumatoid arthritis (RA) patients.
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