| Objectives Fabrication and characterization of the PVA/ι-CA composite material. Investigation the effect of material on the attachment, proliferation, and differentiation of chondrocytes seeded in the scaffolds cultured in vitro. Assessment of the organize local reactions by implanting composite materials in vivo, including the early inflammatory response and vascularization and fiber hyperplasia in the late, in order to evaluate the histocompatibility of the material and the feasibility of constructing tissue engineering cartilage.Methods 1 PVA/ι-CA composite film and scaffold with different content of ι-CA were fabricated and the physical characteristics of the materials were detected. 2 The vascular endothelial cells were seeded on the composite materials and evaluated by the cell adhesion experiment, cell proliferation and SEM observation in order to select the optimal composite scaffold materials for further biological experiment. 3 Chondrocytes extracting from 3W SD rats were seeded into the composite scaffold and observed by SEM. The effect of the scaffold on the adhesion and proliferation was observed by of the conventional dyeing of the paraffin section. The function of the chondrocytes on the scaffolds were investigated by toluidine blue staining, collagen type Ⅱ immunohistochemical staining and collagen type Ⅱ immunofluorescence test. 4 Subcutaneous the optimization composite scaffold was implanted subcutaneously for 1, 2, 3 and 4 weeks, respectively. The histocompatibility of the composite scaffolds, vascularization and degradation in vivo were evaluated by the conventional dyeing of the paraffin section Masson staining, VG staining, immunohistochemistry and immunofluorescence staining.Results 1 The composite scaffolds were three-dimensional, with good porosity. 2 The vascular endothelial cells were cultured with composite membrane materials for 5 days and observed by SEM, which was shown that the number of cell on the composite membranes was increased with the increase of carrageenan content. Therefore, the scaffold with 30% content of carrageenan was chose for further experiment. 3 The HE staining result was shown that the chondrocytes grew well with polygon morphology. All samples stained positive for collagen type II by immunohistochemistry and immunofluorescence analysis, which verified the normal phenotype of chondrocytes on the scaffolds. 4 The results of subcutaneous implantation were shown as below: There was no tissue necrosis at 1 week and cells began to enter the hollows of the scaffold. Besides, a small amount of scattered neutrophils and lymphocytes could be seen, but there was no obvious evidence for the existence of phagocytes and multinucleated giant cells. The cells were ingrowth gradually into the scaffold and the inflammatory cells were decrease. Furthermore, new vasculature and fibroblasts was in growth in the scaffold at 2 weeks. For 3 weeks, part of the material was full of fibroblasts and capillary, with multinucleated giant cells and lymphocytes. Implanted for 4 weeks, the structure of the scaffold was intact, and collagen fibers were appeared in the gaps of the scaffold. However, the vessel was reduced.Conclusions 1 PVA/ι-CA composite materials were fabricated successfully by freezethaw method. 2 The ATDC-5 were proliferating not only on the outer surface, but also inside the micropores. As the cultivate time went by, the cells already began to grow around the corner of the pores, and multilayers of cells were visible, which indicated the materials were non-cytotoxicity and had good cell compatibility. 3 The results in vivo showed that inflammatory response caused by the materials was slight and the vascularization was good in the late. Besides, the materials possessed a slow degradation rate with good biocompatibility. |
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