Heidari Moghadam Abbas, Bayati Vahid, Orazizadeh Mahmoud, Rashno Mohammad
Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Cell J. 2022 Jul 27;24(7):380-390. doi: 10.22074/cellj.2022.8098.
The main objective of this study is to determine the myogenic effects of skeletal muscle extracellular matrix, vascular endothelial growth factor and human umbilical vein endothelial cells on adipose-derived stem cells to achieve a 3-dimensional engineered vascular-muscle structure.
The present experimental research was designed based on two main groups, i.e. monoculture of adipose tissue-derived stem cells (ADSCs) and co-culture of ADSCs and human umbilical vein endothelial cells (HUVECs) in a ratio of 1:1. Skeletal muscle tissue was isolated, decellularized and its surface was electrospun using polycaprolactone/gelatin parallel nanofibers and then matrix topography was evaluated through H and E, trichrome staining and SEM. The expression of MyHC2 gene and tropomyosin protein were examined through real-time reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence, respectively. Finally, the morphology of mesenchymal and endothelial cells and their relationship with each other and with the engineered scaffold were examined by scanning electron microscopy (SEM).
According to H and E and Masson's Trichrome staining, muscle tissue was completely decellularized. SEM showed parallel Polycaprolactone (PCL)/gelatin nanofibers with an average diameter of about 300 nm. The immunofluorescence proved that tropomyosin was positive in the ADSCs monoculture and the ADSCs/HUVECs coculture in horse serum (HS) and HS/VEGF groups. There was a significant difference in the expression of the MyHC2 gene between the ADSCs and ADSCs/HUVECs culture groups (P<0.05) and between the 2D and 3D models in HS/ VEGF differentiation groups (P<001). Moreover, a significant increase existed between the HS/VEGF group and other groups in terms of endothelial cells growth and proliferation as well as their relationship with differentiated myoblasts (P<0.05).
Co-culture of ADSCs/HUVECs on the engineered cell-free muscle scaffold and the dual effects of VEGF can lead to formation of a favorable engineered vascular-muscular tissue. These engineered structures can be used as an acceptable tool for tissue implantation in muscle injuries and regeneration, especially in challenging injuries such as volumetric muscle loss, which also require vascular repair.
本研究的主要目的是确定骨骼肌细胞外基质、血管内皮生长因子和人脐静脉内皮细胞对脂肪来源干细胞的成肌作用，以构建三维工程化血管肌肉结构。
本实验研究基于两个主要组进行设计，即脂肪组织来源干细胞（ADSCs）的单培养以及ADSCs与人脐静脉内皮细胞（HUVECs）按1:1比例的共培养。分离骨骼肌组织，进行脱细胞处理，然后使用聚己内酯/明胶平行纳米纤维对其表面进行静电纺丝，随后通过苏木精和伊红染色、三色染色及扫描电子显微镜对基质形貌进行评估。分别通过实时逆转录聚合酶链反应（RT-PCR）和免疫荧光检测肌球蛋白重链2（MyHC2）基因的表达及原肌球蛋白蛋白。最后，通过扫描电子显微镜（SEM）检查间充质细胞和内皮细胞的形态及其相互之间以及与工程支架的关系。
根据苏木精和伊红染色及Masson三色染色，肌肉组织完全脱细胞。扫描电子显微镜显示平均直径约为300 nm的平行聚己内酯（PCL）/明胶纳米纤维。免疫荧光证明在马血清（HS）和HS/VEGF组中，原肌球蛋白在ADSCs单培养以及ADSCs/HUVECs共培养中呈阳性。ADSCs与ADSCs/HUVECs培养组之间MyHC2基因的表达存在显著差异（P<0.05），在HS/VEGF分化组的二维和三维模型之间也存在显著差异（P<0.01）。此外，HS/VEGF组与其他组相比，在内皮细胞生长和增殖及其与分化成肌细胞的关系方面有显著增加（P<0.05）。
在工程化无细胞肌肉支架上进行ADSCs/HUVECs共培养以及VEGF的双重作用可导致形成良好的工程化血管肌肉组织。这些工程化结构可作为肌肉损伤和再生组织植入的可接受工具，特别是在诸如大面积肌肉缺损等具有挑战性的损伤中，这类损伤也需要血管修复。
