Department of Anaesthesiological, Surgical and Emergency Sciences, Second University of Naples, Naples, Italy.
J Mater Sci Mater Med. 2010 Jan;21(1):353-63. doi: 10.1007/s10856-009-3840-z. Epub 2009 Aug 5.
Human adipose tissues surgically resected from the subcutaneous abdominal region were enzymatically processed to obtain Human Adipose Stem cells (fibroblast-like adipose tissue-derived stromal cells-ADSC-FL) that were immunophenotypically characterized using a panel of mesenchymal markers by flow cytometry. The formation of new hydroxyapatite crystals in culture dishes, by differentiating cells, further demonstrate the osteogenic potential of purified cells. The aim of this study was to evaluate the osteogenic differentiation potential of ADSC-FL seeded onto a porous beta-tricalcium phosphate (beta-TCP) matrix. ADSC-FL was cultured on the beta-TCP matrix in medium with or without osteogenic differentiation additives. Time-dependent cell differentiation was monitored using osteogenic markers such as alkaline phosphatase (activity assay), osteocalcin and ostopontin (ELISA method) expression. Our results reveal that beta-TCP triggers the differentiation of ADSC-FL toward an osteoblastic phenotype irrespective of whether the cells are grown in a proliferative or a differentiative medium. Hence, a beta-TCP matrix is sufficient to promote osteoblastic differentiation of ADSC-FL. However, in proliferative medium, alkaline phosphatase activity was detected at lower level respect to differentiative medium and osteocalcin and osteopontin showed an expression delay in cells cultured in proliferative medium respect to differentiative one. Moreover, we observed an increase in FAK phosphorylation at level of tyrosine residue in position 397 (Western-blot) that indicates a good cell adhesion to beta-TCP scaffold. In conclusion, our paper demonstrates that a three-dimensional beta-TCP scaffold in vitro triggers on its own the differentiation of ADSC-FL toward an osteoblastic phenotype without the need to use differentiative media.
从皮下腹部区域手术切除的人体脂肪组织通过酶处理获得人脂肪干细胞（成纤维样脂肪组织来源的基质细胞-ADSC-FL），通过流式细胞术使用一组间充质标志物对其免疫表型进行特征描述。通过分化细胞在培养皿中形成新的羟基磷灰石晶体进一步证明了纯化细胞的成骨潜力。本研究的目的是评估接种在多孔β-磷酸三钙（β-TCP）基质上的 ADSC-FL 的成骨分化潜力。ADSC-FL 在含有或不含有成骨分化添加剂的培养基中在β-TCP 基质上培养。使用成骨标志物（如碱性磷酸酶（活性测定）、骨钙素和骨桥蛋白（ELISA 法）表达）监测时间依赖性细胞分化。我们的结果表明，β-TCP 触发 ADSC-FL 向成骨细胞表型分化，无论细胞在增殖培养基还是分化培养基中生长。因此，β-TCP 基质足以促进 ADSC-FL 的成骨细胞分化。然而，在增殖培养基中，碱性磷酸酶活性的检测水平低于分化培养基，并且在增殖培养基中培养的细胞中骨钙素和骨桥蛋白的表达延迟。此外，我们观察到在位置 397 的酪氨酸残基（Western blot）处 FAK 磷酸化水平增加，表明细胞对β-TCP 支架有良好的粘附。总之，我们的论文表明，体外三维β-TCP 支架本身可触发 ADSC-FL 向成骨细胞表型分化，而无需使用分化培养基。
