Hu Rong, Ling Wei, Xu Wen, Han Demin
Department of Otorhinolaryngology-Head Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, The People's Republic of China.
Department of Anatomy, Capital Medical University, Beijing, The People's Republic of China.
PLoS One. 2014 Mar 24;9(3):e92676. doi: 10.1371/journal.pone.0092676. eCollection 2014.
Tissue engineering has revealed the potential to regenerate injured vocal folds, and identification of the most suitable seed cells has remained a hot topic of research. The aim of this study was to implant fibroblast-like cells differentiated from adipose-derived mesenchymal stem cells (ADSCs) in a canine acute vocal fold wound model. We then sought to characterize changes in the extracellular matrix (ECM) proteins of vocal fold lamina propria. For this purpose, ADSCs were induced to differentiate into fibroblasts under the regulation of connective tissue growth factor in vitro. Cell surface proteins were identified by immunofluorescence staining. Thirty vocal folds of 17 canines were injured by localized resection and injected with fibroblast-like cells (differentiated ADSCs, dADSCs), ADSCs or vocal fold fibroblasts (VFFs). The morphology of vocal folds was observed, and the characteristics of ECM protein components (collagen, elastin, hyaluronic acid, decorin and fibronectin) were evaluated by immunofluorescence staining from 15 days to 6 months following implantation. The results showed that in vitro, the dADSCs showed morphology and cell surface protein expression similar to those of VFFs. After implantation in vivo, the surfaces of the recipient vocal folds became almost smooth in the dADSCs and ADSCs groups at 6 months but remained slightly concave and stiff in the VFFs group. The elastin fluorescence intensity increased significantly and was maintained at a high level in the dADSCs group. The collagen fluorescence intensity increased slightly in the dADSCs and ADSCs groups, whereas it demonstrated a more irregular arrangement in the VFFs group. The fluorescence intensity of hyaluronic acid, decorin and fibronectin was similar between the three implanted groups. These results indicated that dADSCs may confer an advantage for vocal fold wound healing. Furthermore, dADSCs have the ability to secrete ECM components in vivo, particularly elastin, which may be beneficial for vocal fold vibration recovery.
组织工程学已显示出再生受损声带的潜力，而确定最合适的种子细胞一直是研究的热点。本研究的目的是在犬急性声带伤口模型中植入由脂肪来源的间充质干细胞（ADSCs）分化而来的成纤维样细胞。然后，我们试图描述声带固有层细胞外基质（ECM）蛋白的变化特征。为此，在体外通过结缔组织生长因子的调控将ADSCs诱导分化为成纤维细胞。通过免疫荧光染色鉴定细胞表面蛋白。对17只犬的30条声带进行局部切除损伤，并注射成纤维样细胞（分化的ADSCs，dADSCs）、ADSCs或声带成纤维细胞（VFFs）。观察声带的形态，并在植入后15天至6个月通过免疫荧光染色评估ECM蛋白成分（胶原蛋白、弹性蛋白、透明质酸、核心蛋白聚糖和纤连蛋白）的特征。结果显示，在体外，dADSCs的形态和细胞表面蛋白表达与VFFs相似。在体内植入后，6个月时dADSCs组和ADSCs组受体声带的表面几乎变得光滑，而VFFs组则仍略呈凹陷且僵硬。dADSCs组弹性蛋白荧光强度显著增加并维持在高水平。dADSCs组和ADSCs组胶原蛋白荧光强度略有增加，而VFFs组胶原蛋白排列更不规则。三个植入组之间透明质酸、核心蛋白聚糖和纤连蛋白的荧光强度相似。这些结果表明，dADSCs可能在声带伤口愈合方面具有优势。此外，dADSCs在体内具有分泌ECM成分的能力，尤其是弹性蛋白，这可能有利于声带振动恢复。
