Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan.
Department of Oral and Maxillofacial Reconstructive Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan.
Int J Mol Sci. 2021 Nov 17;22(22):12392. doi: 10.3390/ijms222212392.
We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs).
We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin.
The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference ( < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture.
DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.
我们研究并比较了去分化脂肪细胞（DFAT 细胞）和脂肪来源干细胞（ASCs）的成骨潜能和骨再生能力。
我们从 GFP 小鼠中分离 DFAT 细胞和 ASC。DFAT 细胞采用新的培养方法，使用网片培养代替天花板培养。将分离的 DFAT 细胞和 ASC 孵育在成骨培养基中，然后进行茜素红染色、碱性磷酸酶（ALP）测定和 RT-PCR（用于 RUNX2、骨桥蛋白、DLX5、osterix 和骨钙素），以评估两种细胞类型的体外成骨分化能力。在体内，将 DFAT 细胞和 ASC 孵育在成骨培养基中 4 周，接种在胶原复合支架上，然后皮下植入小鼠背部。然后进行苏木精和伊红染色以及 GFP 和骨钙素免疫染色。
DFAT 细胞的茜素红染色区域在两周时显示出较弱的钙化能力，但在三周时显示出较强的钙化能力，与 ASC 相似。ASC 的 ALP 水平在 6 天和 9 天更早增加，差异具有统计学意义（<0.05）。DFAT 的 ALP 水平在 12 天后高于 ASC。成骨分化培养后，DFAT 细胞和 ASC 的成骨标志物基因（osterix 和骨钙素）的表达水平升高。
DFAT 细胞易于从小鼠少量脂肪组织中分离，并且可以高纯度大量扩增，因此，DFAT 细胞适用于许多组织工程策略和基于细胞的治疗方法。
