Cultivation and Transplantation of Three-Dimensional Skins with Laser-Processed Biodegradable Membranes
Cultivation and Transplantation of Three-Dimensional Skins with Laser-Processed Biodegradable Membranes
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激光加工可生物降解膜三维皮肤的培养和移植
DOI：
10.1089/ten.tea.2022.0208
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发表时间：
2023
影响因子：
4.1
通讯作者：
Sato Shunichi
中科院分区：
文献类型：
--
作者：
Tsunoi Yasuyuki;Takayama Izumi;Kondo Naonari;Nagano Yo;Miyazaki Hiromi;Kawauchi Satoko;Akashi Mitsuru;Saitoh Daizoh;Terakawa Mitsuhiro;Sato Shunichi
对于不可逆的大面积皮肤损伤的治疗，在没有同种异体皮肤的情况下，人工皮肤或培养皮肤是有用的。然而，它们大多缺乏血管系统，导致灌注延迟，从而导致组织植入延迟或失败。我们之前开发了一种基于逐层细胞包膜技术（LbL-3D皮肤）的预血管化三维（3D）培养皮肤，其中细胞被播种并层压在多孔聚合物膜上，为厚培养组织提供培养基。最近的动物研究表明，LbL-3D皮肤在移植后可以实现快速灌注和较高的移植物存活率。然而，在移植前将LbL-3D皮肤从膜上分离出来，以及分离后的LbL-3D皮肤移植后的处理存在实际问题。为了解决这些问题，在本研究中，我们研究了使用可生物降解的多孔聚合物膜，使LbL-3D皮肤和膜一起移植，并且移植后可以分解。用飞秒激光脉冲照射聚乳酸-羟基乙酸（PLGA）薄膜，形成微通孔，制备多孔膜。我们设计并制作了PLGA膜的培养插入物，并在20层细胞中培养了2 × 106个新生儿正常人真皮成纤维细胞和1 × 104个人脐静脉内皮细胞，在表皮中培养了1 × 105个新生儿表皮角质形成细胞的lb - 3d皮肤。组织学分析表明，在PLGA膜上培养的皮肤厚度约为400 μm，与在常规（不可生物降解）商业膜上培养的皮肤相比，在PLGA膜上培养的皮肤质量没有缺陷。将培养的LbL-3D皮肤与PLGA膜一起移植到小鼠全层切除创面上。在小鼠身上移植7天后，膜上和膜下的组织通过孔连接，胶原阳性纤维似乎从宿主和供体两侧迁移，并且在整个移植皮肤区域观察到良好的再上皮形成。然而，在某些情况下，观察到植入不足。因此，需要进一步优化膜条件以改善移植效果。
For the treatment of irreversible, extensive skin damage, artificial skins or cultured skins are useful when allogeneic skins are unavailable. However, most of them lack vasculature, causing delayed perfusion and hence delay or failure in engraftment of the tissues. We previously developed a prevascularized three-dimensional (3D) cultured skin based on the layer-by-layer cell coating technique (LbL-3D skin), in which cells are seeded and laminated on a porous polymer membrane for medium supply to the thick cultured tissue. Recent animal studies have demonstrated that LbL-3D skin can achieve rapid perfusion and high graft survival after transplantation. However, there were practical issues with separating LbL-3D skins from the membranes before transplantation and the handling separated LbL-3D skins for transplantation. To address these problems, in this study, we examined the use of biodegradable porous polymer membranes that enabled the transplantation of LbL-3D skins together with the membranes, which could be decomposed after transplantation. Thin films made from poly (lactic-co-glycolic acid) (PLGA) were irradiated with femtosecond laser pulses to create micro through-holes, producing porous membranes. We designed and fabricated culture inserts with the PLGA membranes and cultivated LbL-3D skins with 2 × 106neonatal normal human dermal fibroblasts and 1 × 104human umbilical vein endothelial cells in the dermis of 20 cell layers and 1 × 105neonatal human epidermal keratinocytes in the epidermis. Histological analyses revealed that the skins cultured on the PLGA membranes had thickness of about 400 μm and that there were no defects in the quality of the skins cultured on the PLGA membranes when compared with those cultured on the conventional (nonbiodegradable) commercial membranes. The cultured LbL-3D skins were then transplanted together with the PLGA membranes onto full-thickness excisional wounds in mice. At 7 days posttransplantation onto a mouse, the tissues above and below the membrane were connected through the holes with collagen-positive fibers that appeared to migrate from both the host and donor sides, and favorable reepithelization was observed throughout the transplanted skin region. However, insufficient engraftment was observed in some cases. Thus, further optimization of the membrane conditions would be needed to improve the transplantation outcome.