Comparison of chondrocyte differentiation ability using three-dimensional culture in an atelocollagen sponge on dedifferentiated fat cells and adipose-derived stem cells from the human buccal fat pad

Abstract

Statement of the Problem: Dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs) are reported to show the same high proliferation ability andmultilineage potential as bone marrow mesenchymal stem cells. On the other hand, there are few reports of studies that used human buccal fat pad (hBFP), and no reported studies compared the differentiation ability to cartilage by three-dimensional culture. Therefore, the purpose of this studywas to evaluate and compare the chondrocyte differentiation ability of DFAT cells and ASCs from the hBFP in a 3D construct. Materials and Methods: We isolated human DFAT cells and ASCs from the hBFP of a patient who underwent oral and maxillofacial surgery. hBFP was digested with collagenase solution and subjected to centrifugation. The floating top layer containing mature adipocytes and precipitated stromal-vascular fraction (SVF) was collected. We isolated DFAT cells from mature adipocytes by ceiling culture and ASCs from SVF by conventional culture. Furthermore, DFAT cells and ASCs were seeded on a collagen sponge and cultured threedimensionally in chondrogenic induction medium for 7, 14 and 21 days. Methods of Data Analysis:We performed hematoxylin and eosin (HE) staining, Alcian blue staining and realtime PCR. Results: The cells attached to the upper surface of the flasks by ceiling culture. The cells exhibited the characteristic features of cytoplasmic extension, reduction and loss of lipid droplets and fibroblast-like morphology. The cells in the 3D construct were evenly seeded in the collagen sponge and round chondrocyte-like cells were exhibited by HE staining at 7, 14 and 21 days. The cells were also positive for Alcian blue staining. Real-time PCR showed increased expression of aggrecan, collagen type 2 and SOX9 for 7, 14 and 21 days. In addition, DFAT cells showed significantly higher gene expression of aggrecan, collagen type 2 and sox9 than ASCs at 14 and 21 days in real-time PCR. Conclusions: We were able to isolate DFAT cells and ASCs from the human buccal fat pad and produced chondrocyte differentiation in a 3D construct. In addition, DFAT cells showed higher chondrocyte differentiation ability than ASCs.