基于可主动调控创面微环境的天然活性微载体构建可注射性原位成型真皮替代物的实验研究

Traditional living dermal substitutes constructed by two steps have many disadvantages, such as long construction time, poor cell activity, low survival rate after transplantation and inconvenient clinical use. Our pre-research which has been published on Biomaterials（IF:8.38）demonstrated that living micronized amnion(LMAM) have the characteristics of microcarrier and actively modulating local wound microenvironment. We hypothesize to use LMAM as a natural three-dimensional microcarrier to amplify large amounts of human dermal fibroblasts (HDF) by using the rotating cell culture system for quickening the construction of HDF-LMAM composite skin by one step, thus shortening cell culture time in vitro and avoiding damage to cells because of repeated trypsinization. A natural temperature-sensitive human amniotic extracellular matrix-derived hydrogel (haECM) is prepared and then mixed evenly with HDF-LMAM to construct HDF-LMAM-haECM composite skin. HDF-LMAM-haECM could increase cell survival rate after transplantation by actively modulating bad microenvironment of chronic wound and evenly distributing HDF-LMAM in the wounds. In addition, HDF-LMAM-haECM could be convenient in clinical use by treating arbitrarily shaped wounds through injection and then in-situ forming dermal substitute. The present study would abandon construction model of traditional living dermal substitutes and provide a new way for treating chronic wounds.

基于两步法构建的传统活性真皮替代物存在构建时间长、细胞活性差、移植存活率低、临床使用不便等缺点。前期我们发表于Biomaterials（IF:8.38）的研究表明300–600μm大小的活性微粒羊膜兼具微载体特征及主动调控创面微环境的特性。本项目设想：以活性微粒羊膜为三维微载体，结合旋转细胞培养系统一步法实现成纤维的大量扩增与成纤维细胞-活性微粒羊膜微型复合皮的快速构建，从而缩短体外培养时间、避免反复消化培养对细胞活性的损伤。制备天然温敏型羊膜基质水凝胶，与成纤维细胞-活性微粒羊膜复合皮均匀混合后通过注射可填充任意形状创面缺损，在创面原位成型为能主动调控慢性创面恶性微环境的成纤维细胞-活性微粒羊膜-羊膜水凝胶三位一体复合皮，提高移植后细胞存活率，实现成纤维细胞-活性微粒羊膜微型复合皮在创面的均匀分布及临床应用的简便性。本研究将摒弃传统活性真皮替代物的构建模式，为慢性创面的治疗提供新的思路。

基于两步法构建的传统活性真皮替代物存在构建时间长、细胞活性差、移植存活率低、临床使用不便等缺点。本研究以300–600μm大小的活性微粒羊膜（LMAM）作为三维微载体，结合旋转细胞培养系统（RCCS）一步法实现成纤维的大量扩增与成纤维细胞-活性微粒羊膜微型复合皮（HFB-LMAM）的快速构建，从而缩短体外培养时间、避免反复消化培养对细胞活性的损伤。种植1天后成纤维细胞大量粘附于活性微粒羊膜表面，培养4天时可见细胞密布，且细胞维持良好的增殖活性。免疫荧光及ELISA检测发现HFB-LMAM能旁分泌bFGF、IL-6、IL-8、Laminin、TGF-β1等多种因子，复合皮中羊膜上皮细胞干性维持良好。进一步我们制备天然温敏型羊膜基质水凝胶，发现10mg/ml浓度羊膜水凝胶（AM gel）凝胶时间为5分钟，并具有良好的存储模量、溶胀率及降解率，且细胞相容性良好。HFB-LMAM与AM gel在液态条件下均匀混合，37℃条件下固态成型为HFB-LMAM-AM gel三位一体复合皮，检测发现复合皮中HFB-LMAM的旁分泌特性及羊膜上皮细胞干性维持良好。HFB-LMAM-AM gel裸鼠皮下埋植，发现第7天复合皮中AM gel大部分发生降解，但细胞存活良好；第14天复合皮中细胞大部分发生凋亡，而活性微粒羊膜未发生降解；第60天活性微粒羊膜大部分发生降解。以HFB-LMAM-AM gel修复糖尿病小鼠全层皮肤缺损创面，发现第7天复合皮中细胞在创面存活良好， 但羊膜水凝胶基本已降解完毕；第21天复合皮中大部分细胞凋亡，复合皮开始出现降解，周围肉芽组织生长良好。计算创面愈合率发现HFB-LMAM-AM gel能明显促进创面愈合，形态学检测发现其可能机制与增加创面真皮厚度及促进新生血管化有关。本研究摒弃了传统活性真皮替代物的构建模式，为慢性创面的治疗提供新的思路。