Department of Otolaryngology Head and Neck Surgery, The Sheba Medical Center, Tel-Hashomer.
Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv.
Otol Neurotol. 2019 Dec;40(10):e1030-e1036. doi: 10.1097/MAO.0000000000002367.
The scarred rim of chronic tympanic membrane (TM) perforation contains keratinocytes with potential for regeneration while maintaining their morphological and genetic characteristics.
The squamous epithelium of the TM has a good regeneration capacity. Successful isolation and expansion of human TM keratinocytes (hTMKR) was reported from a full, en-bloc, healthy TM.
Trimmed margins of the TM perforation (harvested during tympanoplasty) underwent enzymatic digestion (collagenase or trypsin) and were seeded either with serum-containing medium (SCM) or keratinocyte serum-free medium (KSFM) and progenitor cell growth medium (PR) (KSFM:PR, 1:1). Gene expression analysis by real-time qRT-PCR was used to compare between human TM cells derived from scarred perforation margins (hTMKR), normal human skin keratinocytes (NhSKR), and human fibroblasts.
Twelve patients were included in the study. In 9 of 12 cases (75%) single-cell isolation with fibroblastic or epithelial cell morphology (or both) was achieved. Cells seeded with KSFM:PR yielded epithelial morphology (hTMKR) while SCM culturing resulted in a fibroblastic morphology (hTMFib). Gene expression analysis revealed significant higher expression of VCAN (p = 0.002) and FOXC2 (p = 0.015) at the mRNA levels (normal hTMKR markers) in hTMKR compared to NhSKR. In addition, a comparison of gene expression between hTMKR and hTMFib revealed significantly higher levels of both VCAN (p = 0.045) and SLC6A14 (p = 0.036) among hTMKR.
For the first time, we developed a protocol to isolate hTMKR from scarred TM perforation margins. Furthermore, we succeeded in achieving tissue expansion that preserved the characteristic of healthy TM cells. This study bridges "regenerative medicine" approach with clinical and surgical objectives.
慢性鼓膜（TM）穿孔的瘢痕边缘含有具有再生潜力的角蛋白细胞，同时保持其形态和遗传特征。
TM 的鳞状上皮具有良好的再生能力。据报道，从完整的、整体的、健康的 TM 中成功分离和扩增了人 TM 角蛋白细胞（hTMKR）。
TM 穿孔的修剪边缘（在鼓室成形术中采集）进行酶消化（胶原酶或胰蛋白酶），并在含有血清的培养基（SCM）或无血清角质形成细胞培养基（KSFM）和祖细胞生长培养基（PR）（KSFM：PR，1:1）中接种。实时 qRT-PCR 基因表达分析用于比较源自瘢痕穿孔边缘的人 TM 细胞（hTMKR）、正常人皮肤角质形成细胞（NhSKR）和人成纤维细胞。
本研究纳入了 12 名患者。在 12 例中有 9 例（75%）实现了单细胞分离，具有成纤维细胞或上皮细胞形态（或两者兼有）。用 KSFM：PR 接种的细胞产生上皮形态（hTMKR），而 SCM 培养导致成纤维细胞形态（hTMFib）。基因表达分析显示，与 NhSKR 相比，hTMKR 在 mRNA 水平（正常 hTMKR 标志物）上显著更高表达 VCAN（p=0.002）和 FOXC2（p=0.015）。此外，hTMKR 和 hTMFib 之间的基因表达比较显示，hTMKR 中 VCAN（p=0.045）和 SLC6A14（p=0.036）的水平均显著更高。
我们首次开发了一种从瘢痕 TM 穿孔边缘分离 hTMKR 的方案。此外，我们成功实现了组织扩张，保留了健康 TM 细胞的特征。这项研究将“再生医学”方法与临床和手术目标联系起来。
