Biedermann Thomas, Klar Agnieszka S, Böttcher-Haberzeth Sophie, Schiestl Clemens, Reichmann Ernst, Meuli Martin
Tissue Biology Research Unit, University Children's Hospital Zurich, Zurich, Switzerland.
Pediatr Surg Int. 2014 Feb;30(2):165-72. doi: 10.1007/s00383-013-3446-x.
Human autologous tissue-engineered skin grafts are a promising way to cover skin defects. Clearly, it is mandatory to study essential biological dynamics after transplantation, including reinnervation. Previously, we have already shown that human tissue-engineered skin analogs are reinnervated by host nerve fibers as early as 8 weeks after transplantation. In this study, we tested the hypothesis that there is a de novo formation of a "classical" neurovascular link in tissue-engineered and then transplanted skin substitutes.
Keratinocytes, melanocytes, and fibroblasts were isolated from human skin biopsies. After expansion in culture, keratinocytes and melanocytes were seeded on dermal fibroblast-containing collagen type I hydrogels. These human tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds on the back of immuno-incompetent rats. Grafts were analyzed after 3 and 10 weeks. Histological sections were examined with regard to the ingrowth pattern of myelinated and unmyelinated nerve fibers into the skin analogs using markers such as PGP9.5, NF-200, and NF-160. Blood vessels were identified with CD31, lymphatic vessels with Lyve1. In particular, we focused on alignment patterns between nerve fibers and either blood and/or lymphatic vessels with regard to neurovascular link formation.
3 weeks after transplantation, blood vessels, but no nerve fibers or lymphatic vessels could be observed. 10 weeks after transplantation, we could detect an ingrowth of myelinated and unmyelinated nerve fibers into the skin analogs. Nerve fibers were found in close proximity to CD31-positive blood vessels, but not alongside Lyve1-positive lymphatic vessels.
These data suggest that host-derived innervation of tissue-engineered dermo-epidermal skin analogs is initiated by and guided alongside blood vessels present early post-transplantation. This observation is consistent with the concept of a cross talk between neurovascular structures, known as the neurovascular link.
人自体组织工程皮肤移植物是覆盖皮肤缺损的一种有前景的方法。显然，研究移植后的基本生物学动态，包括再神经支配，是必不可少的。此前，我们已经表明，人组织工程皮肤类似物在移植后8周就已被宿主神经纤维再神经支配。在本研究中，我们测试了这样一个假设，即在组织工程化然后移植的皮肤替代物中存在“经典”神经血管连接的从头形成。
从人皮肤活检组织中分离角质形成细胞、黑素细胞和成纤维细胞。在培养扩增后，将角质形成细胞和黑素细胞接种在含真皮成纤维细胞的I型胶原水凝胶上。这些人组织工程化的真皮 - 表皮皮肤类似物被移植到免疫缺陷大鼠背部的全层皮肤伤口上。在3周和10周后对移植物进行分析。使用PGP9.5、NF - 200和NF - 160等标记物，对组织学切片进行检查，以观察有髓和无髓神经纤维向皮肤类似物内生长的模式。用CD31鉴定血管，用Lyve1鉴定淋巴管。特别是，我们关注神经纤维与血管和/或淋巴管之间关于神经血管连接形成的排列模式。
移植后3周，可观察到血管，但未观察到神经纤维或淋巴管。移植后10周，我们可以检测到有髓和无髓神经纤维向皮肤类似物内生长。神经纤维靠近CD31阳性血管，但不与Lyve1阳性淋巴管并行。
这些数据表明，组织工程化真皮 - 表皮皮肤类似物的宿主源性神经支配是由移植后早期存在的血管启动并沿着血管引导的。这一观察结果与神经血管结构之间的相互作用概念一致，即所谓的神经血管连接。
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