Kato Hiroko, Marcelo Cynthia L, Washington James B, Bingham Eve L, Feinberg Stephen E
1 Department of Oral and Maxillofacial Surgery, University of Michigan Health System, Ann Arbor, Michigan.
2 Course for Oral Life Science, Department of Biomimetics, Postgraduate School of Medical and Dental Sciences, Niigata University , Niigata, Japan .
Tissue Eng Part C Methods. 2015 Sep;21(9):872-80. doi: 10.1089/ten.tec.2014.0600. Epub 2015 Apr 15.
The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm(®), was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3×10(5) cells/cm(2)) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0 μm in thickness. During the air-liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550-1020 μm) to the thinnest (228.6-330.2 μm) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME graft for reconstructing large mucosa defects.
对大面积撕脱性和/或手术造成的组织缺损进行软组织重建，需要具备制造大量软组织构建物以修复由此产生的伤口的能力。在本研究中，我们详细阐述了将之前用于较小构建物的方法扩大规模，在体外制造更大的组织工程装置（体外产生的口腔黏膜等效物 [EVPOME]）时需要解决的问题。制造更大尺寸的EVPOME是为了重建大型临床缺损，它由自体人口腔角质形成细胞和真皮替代物AlloDerm®组成。作为一种自体体细胞治疗产品，其制造过程遵循美国食品药品监督管理局（FDA）生物制品评估和研究中心（CBER）要求的现行良好生产规范和现行良好组织规范。成功制造大型EVPOME采用了更高的细胞接种密度（5.3×10⁵个细胞/cm²）以及相对较薄的AlloDerm，厚度在356.6至508.0μm之间。在气液界面培养过程中，支架的厚度影响培养基扩散速率，进而导致上皮分层的变化。组织学上，角质形成细胞祖细胞（p63）、增殖（Ki-67）和晚期分化标志物（丝聚蛋白）的表达显示出差异，这些差异与从最厚（550 - 1,020μm）到最薄（228.6 - 330.2μm）的AlloDerm支架的EVPOME中葡萄糖转运蛋白-1（GLUT1）的表达相关。葡萄糖消耗和2-脱氧葡萄糖（2DG）摄取与支架厚度直接相关。由于扩散速率影响葡萄糖的可及性，在EVPOME制造过程中，支架尺寸和厚度对细胞表型和上皮成熟有影响。这些结果为制造用于重建大型黏膜缺损的大型、健康的EVPOME移植物提供了基本策略。
