MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China.
Biomaterials. 2011 Feb;32(4):1019-31. doi: 10.1016/j.biomaterials.2010.08.087. Epub 2010 Nov 10.
Repair of deep burn by use of the dermal equivalent relies strongly on the angiogenesis and thereby the regeneration of dermis. To enhance the dermal regeneration, in this study plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165)/N,N,N-trimethyl chitosan chloride (TMC) complexes were loaded into a bilayer porous collagen-chitosan/silicone membrane dermal equivalents (BDEs), which were applied for treatment of full-thickness burn wounds. The DNA released from the collagen-chitosan scaffold could remain its supercoiled structure but its degree was decayed along with the prolongation of incubation time. The released DNA could transfect HEK293 cells in vitro with decayed efficiency too. Human umbilical vein endothelial cells (HUVECs) in vitro cultured in the scaffold loaded with TMC/pDNA-VEGF complexes expressed a significantly higher level of VEGF and showed higher viability than those cultured in the controls, i.e. blank scaffold, and scaffolds loaded with naked pDNA-VEGF and TMC/pDNA-eGFP, respectively. The four different BDEs were then transplanted in porcine full-thickness burn wounds. Results showed that the TMC/pDNA-VEGF group had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of the dermis. RT-qPCR and western blotting found that the experimental group also had the highest expression of VEGF, CD31 and α-SMA in both mRNA and protein levels. Furthermore, ultra-thin skin grafting was performed on the regenerated dermis 14 days later, leading to complete repair of the burn wounds with normal histology. Moreover, the tensile strength of the repaired tissue increased along with the time prolongation of post grafting, resulting in a value of approximately 70% of the normal skin at 105 days.
真皮等效物修复深度烧伤强烈依赖于血管生成，从而实现真皮再生。为了增强真皮再生，本研究将编码血管内皮生长因子-165（VEGF-165）/N，N，N-三甲基壳聚糖氯化物（TMC）复合物的质粒 DNA 载入双层多孔胶原-壳聚糖/硅橡胶膜真皮等效物（BDE）中，用于治疗全厚烧伤创面。从胶原-壳聚糖支架中释放的 DNA 可以保持其超螺旋结构，但随着孵育时间的延长，其程度会衰减。释放的 DNA 也可以以衰减的效率转染体外培养的 HEK293 细胞。体外培养在负载 TMC/pDNA-VEGF 复合物的支架中的人脐静脉内皮细胞（HUVEC）表达了显著更高水平的 VEGF，并且比在对照物（即空白支架）以及分别负载裸 pDNA-VEGF 和 TMC/pDNA-eGFP 的支架中培养的细胞具有更高的活力。然后将四种不同的 BDE 移植到猪全厚烧伤创面中。结果表明，TMC/pDNA-VEGF 组具有更高数量的新形成和成熟的血管，并且真皮的再生速度最快。RT-qPCR 和 Western blot 发现实验组在 mRNA 和蛋白水平上 VEGF、CD31 和 α-SMA 的表达也最高。此外，在再生的真皮上 14 天后进行超薄皮肤移植，导致烧伤创面完全修复，具有正常的组织学。此外，随着移植后时间的延长，修复组织的拉伸强度增加，在 105 天时达到正常皮肤的约 70%。
