Development Of Methaccrylic Alginate Gel Microspheres Using Mixture Metal-Cation Crosslinker And Effect Of The Microspheres On The Proliferation Of Chondrocyte

This study aims at fabricating a carrier suitable for three-dimensional(3D) culture of chondrocyte, which can be applied in cartilage tissue engineering. Methods:1) The methacrylic alginate (MA) beads was prepared through photocross-linking reaction between methacrylic alginate and the mixture cationic crosslinker of barium ions and calcium ions at the ratio of5:5. The compressive modulus and permeability of the methacrylic alginate gel beads were investigated. Alginate gel beads serves as a control group.2) Chondrocytes were isolated from rabbit knee joint, and digested with0.25%trypsin when cells reached70%-80%, conflence After digestion, the cells were designed as first generation (P1).3)The PI chondrocytes were then encapsulated into MA gel beads by photocrosslinking-reaction. After culturing a designated time, cell morphology in the two groups was observed under an inverted microscope and scanning electron microscope,Hematoxylin and eosin (HE) staining, alamar blue, and immunohistochemistry assay were performed to determine the cartilage cells growth, proliferation and secretion of extracellular matrix of type Ⅱ collagen. Results:1) The methacrylic alginate beads were successfully developed through photocross-linking reaction. Compared to the alginate gel beads, the methacrylic alginate gel beads revealed a significant increase of stiffness without sacrifice in permeability.2) Chondrocytes were successfully isolated from the rabbit’s knee joint and passaged to P1.3) With time, chondrocytes cultured in MA beads showed significant cell proliferation and proliferated into cell clusters. More cell clusters were observed in MA beads at21day,compared to that in the alginate group. Under the observation of the scanning electron microscopy. Chondrocytes in both of groups adhered and grew well on the surface of gel beads. Histological examination revealed that chondrocytes were uniformly distributed in the beads and gradually proliferated into cell clusters and that cell clusters were much larger in MA beads, compared to that in the control group. The result of immunohistochemistry staining of collagen type Ⅱ was positive at21day in the MA group Alamar blue assay showed constant cells proliferation in both group, and that MA beads showed significantly high cell number than that in alginate beads. The assay of extracellular matrix secretion showed that GAG production increased with time and that MA beads significantly promoted more GAG secretion compared with alginate beads. Conclusion:The carrier developed through photo- cross-linking reaction between methacrylic alginate and the mixture cationic crosslinker was more suitable for3D culture chondrocytes...