Phillips Jennifer E, Guldberg Robert E, García Andrés J
Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
Tissue Eng. 2007 Aug;13(8):2029-40. doi: 10.1089/ten.2006.0041.
Cell-based bone tissue engineering strategies have been effectively applied toward the development of grafting templates for skeletal repair and regeneration, but remain limited by inadequate availability of a robust mineralizing cell source. Dermal fibroblasts have emerged as a particularly promising cell alternative because they are harvested from autologous donors through minimally invasive skin biopsy and display a high capacity for in vitro expansion. In the present study, we investigated retroviral gene delivery of the osteogenic transcription factor Runx2 as a mineralization induction strategy in primary dermal fibroblasts. We demonstrate that constitutive overexpression of Runx2 induced osteogenic gene expression and mineralized nodule deposition in fibroblasts cultured on 3-dimensional fibrous collagen disks in vitro. Fourier transform infrared analysis revealed that Runx2 expressing fibroblasts deposit a carbonate-containing, poorly crystalline hydroxyapatite, whereas control constructs did not contain biologically-equivalent mineral. Importantly, Runx2-transduced fibroblasts formed mineralized templates in vivo after implantation in a subcutaneous, heterotopic site, whereas minimal mineralization was evident in control constructs. Furthermore, immunohistochemical analysis indicated that Runx2-engineered cells co-localized with mineral deposits in vivo, suggesting that nodule formation primarily originated from transplanted donor cells. These results establish Runx2-genetic engineering as a strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with compromised ability to recruit endogenous osteoprogenitors to the site of injury as a result of extreme trauma, age, radiation treatment, or osteolytic disease.
基于细胞的骨组织工程策略已有效地应用于骨骼修复和再生移植模板的开发，但仍受限于强大的矿化细胞来源供应不足。真皮成纤维细胞已成为一种特别有前景的细胞替代物，因为它们通过微创皮肤活检从自体供体获取，并且在体外显示出高扩增能力。在本研究中，我们研究了作为原代真皮成纤维细胞矿化诱导策略的成骨转录因子Runx2的逆转录病毒基因递送。我们证明，Runx2的组成型过表达在体外培养于三维纤维胶原盘上的成纤维细胞中诱导了成骨基因表达和矿化结节沉积。傅里叶变换红外分析表明，表达Runx2的成纤维细胞沉积了含碳酸盐的、结晶度差的羟基磷灰石，而对照构建体不含生物学等效的矿物质。重要的是，Runx2转导的成纤维细胞在皮下异位部位植入后在体内形成了矿化模板，而对照构建体中仅有极少的矿化明显可见。此外，免疫组织化学分析表明，Runx2工程化细胞在体内与矿物质沉积共定位，表明结节形成主要源于移植的供体细胞。这些结果确立了Runx2基因工程作为一种将非成骨细胞表型转化为用于骨修复应用的矿化细胞来源的策略。基于原代真皮成纤维细胞的细胞疗法对于因极端创伤、年龄、放射治疗或溶骨性疾病而使内源性骨祖细胞募集到损伤部位的能力受损的患者将特别有益。
