The Preparation Of Collagen Type ? From Bovine Articular Cartilage And Its Effects On Bone Formation

Collagen is the main component of all mammal tissues.Collagen type ? are mainly distributed in the cartilage,and have significant curative effect on rheumatoid arthritis.Traditional methods for identifying collagen type ? could not quantify and recognize the collagen type at the same time,and had a low accuracy.In this article,collagen type ? were extracted from bovine articular cartilage,and a method for quantitation of collagen type ? was established by detecting marker peptide using high performance liquid chromatography–mass spectrometry(HPLC–MS).Afterwards,the collagen cladding materials was used in mice pre-osteoblast culture.The influence of different types of collagen to different cells functional protein expression was examined on the basis of differences in protein recognition based on mass spectrometry.On the basis of the study the influence of the collagen type ? in bone formation was discovered,which has broad application prospects in the future of functional food,medicine,biological materials and other fields.The main conclusions are summarized as follows:1.Collagen type ? was purified from bovine articular cartilage and characterized using mass spectrometry and other analysis methods.The bovine articular cartilage was pretreated using chloroform-methanol and guanidine hydrochloride to remove fat and the proteoglycan respectively.Then the miscellaneous proteins were digested by pepsin,and the type ? collagen was purified by salting out,dialysis and freeze-dried.The purified collagen showed a single band(?-chain)on SDS-PAGE with a subunit Mr of 130 kDa.The amino acid composition showed that the content of type ? collagen was 93.0%.Marker peptides typical for collagen type ? derived from bovine cartilage were identified with HPLC-MS,and the quantitative analysis results show that the content of type ? collagen was 92.2%,with the yield of 9.2%.TEM analysis indicated that the prepared type ? collagen had a supramolecular structure.Differential scanning calorimetry(DSC)analysis revealed that the denaturation temperatures of type ? collagen were 42.82 ?,43.68 ? and 43.78 ? in buffer solution with pH 4.5,7.0 and 8.0,respectively.Properties of collagen purified from bovine cartilage corresponded to bovine type ? collagen.2.The silicon wafer was coated by collagen,and when the N content was 8.41% on silicon wafer surface(?10cm),the collagen type I cladding quantity was 0.81 mg,and the collagen type ? cladding quantity was 0.67 mg;3.The surface biocompatibility of silicon wafers was evaluated by mice pre-osteoblasts culture.Compared with the bare silicon wafer,collagen coated titanium alloy material performed a better state of cell adhesion,a faster proliferation rate,and a higher activity.4.The cellular proteins extracted from the cells cultured from the titanium alloy material surface was analyzed by HPLC-MS,and the results showed that the cells cultured from collagen type I cladding materials expressed proteins associated with matrix synthesis,and the cells cultured from collagen type ? cladding materials expressed proteins associated with mineralization.