Preparation And Properties Evaluation Of Methacrylic Alginate Gel Beads And The Application In Culturing Chondrocyte As A Scafford

The aim of this study is to obtain the alginate gel which have proper compressive strength and excellent permeability for cell proliferation, and can be more promising potentials in the application of tissue engineering.Methods:1) The modification research of the alginate gel: Through the reaction of the carboxyl of the alginate and the amino of methacrylic acid, methylacrylic is generated into alginate long chain which can be then enhanced through the polymerization of double bond under thermal reaction condition.Finally, alginate gel beads were prepared using the mixture of calcium chloride and barium chloride solution as cationic crosslinker, the compressive modulus and permeability of the prepared alginate gel beads were investigated.2) Isolation and culture of the rabbit knee joint chondrocytes:Chondrocytes were aseptically harvested from the1-month old rabbit’s knee joint.The cartilage was minced with ophthalmic scissor about I mm’size of small pieces.Then moved to a conical flask,according to the amount of the pieces,add10-15ml1g/L collagenase Ⅱ to be digested for6hours at86r/min under37℃incubator.When we harvest the chondrocytes,they should be cultured in incubator with5%CO2and under37℃.chondrocytes were digested with0.25%trypsin to P2.3) The toluidine blue staining and immunohistochemistry for type II collagen were used to identify the chondrocytes.4)Preparation of the alginate gel beads and the culture of chondrocytes in alginate gel beads.Results:1)The results of the modification research of the alginate gel:When the ratio of barium ions to calcium ions was5:5, the compression modulus was189.7Kpa, and it showed the best permeability for trypsin with molecular weight of24KDa and entrapment effect for bovine serum albumin with molecular weight of67kda. Compared to compositions of another ratios, the alginate gel beads made in5:5mixture indicate excellent compressive modulus and permeability.2)The result of cell culturing and identification:It was succeeded to isolate chondrocytes from the rabbit’s knee joint. Toluidine blue staining and immunohistochemistry for type II collagen indicated that the cells have the features of chondrocytes.3) Preparation of the alginate gel beads encapsulating chondrocytes:the alginate gell beads with the diameter of1mm were prepared successfully. But the alginate gell beads with the diameter of400um cann’t get a satisfactory results. Conclusion:1)The alginate hydrogel beads with the ration of barium ions to calcium ions being5:5have the potential application in tissue engineering as support materials and encapsulating materials in cell culture.2)using digestion method, chondrocytes can be isolated successfully from rabbits’knee joint,futher efforts need to be done in encapsulating chondrocytes into alginate beads.