背景:不断增长的人群面临着皮肤伤口愈合受损的挑战,经常导致身体残疾。脂肪来源的干细胞(ASCs),特别是在单元格表格格式中,已成为伤口愈合受损的有希望的补救措施。人血小板裂解物(HPL)提供了用于培养临床级ASC的胎牛血清(FBS)的有吸引力的替代方案。然而,HPL片促进伤口愈合的潜力尚未得到充分研究。本研究旨在探讨HPL培养的ASC片的抗纤维化和促血管生成能力,并探讨其分子机制。
方法:采用大鼠烧伤模型评价HPL培养的ASC片促进创面愈合的效果。ASC板材用HPL制造,和FBS患者进行比较。进行各种分析以评估HPL片对伤口愈合的影响。伤口组织的组织学检查提供了对伤口闭合等方面的见解,胶原蛋白沉积,和整体组织再生。采用免疫荧光来评估治疗后移植的ASC的存在和分布。进行了进一步的体外研究以破译HPL片中有助于血管生成的特定因子。
结果:HPL培养的ASC片显着加速伤口闭合,在新真皮中培养充足和有组织的胶原蛋白沉积。与FBS对应物相比,在用HPL片处理的伤口组织中观察到显著更多保留的ASC。此外,HPL片减轻了巨噬细胞募集并减少了随后的体内伤口组织纤维化。免疫组织化学还表明HPL片组的血管生成增强。体外分析显示HPL片中C-C基序趋化因子配体5(CCL5)和血管生成素上调,包括基因表达和蛋白质分泌。与补充有CCL5或血管生成素的培养基相比,在条件培养基中培养内皮细胞表明CCL5与HPL片的促血管生成作用之间存在相关性。此外,通过中和抗体实验,我们进一步验证了CCL5在体外HPL片介导的血管生成中的关键作用。
结论:本研究强调CCL5是HPL培养的ASC片层在伤口愈合过程中的促血管生成作用的重要因素。这些发现凸显了HPL培养的ASC片作为临床环境中愈合受损皮肤伤口的有希望的治疗选择的潜力。此外,机制探索为优化ASC产品的再生策略提供了有价值的信息。
■这项研究得到了国家科学技术委员会的支持,台湾(NSTC112-2321-B-002-018),台湾大学医院(111C-007),和E大医院-国立台湾大学医院联合研究计划(111-EDN0001,112-EDN0002)。
BACKGROUND: A rising population faces challenges with healing-impaired cutaneous wounds, often leading to physical disabilities. Adipose-derived stem cells (ASCs), specifically in the cell sheet format, have emerged as a promising remedy for impaired wound healing. Human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for culturing clinical-grade ASCs. However, the potential of HPL sheets in promoting wound healing has not been fully investigated. This study aimed to explore the anti-fibrotic and pro-angiogenic capabilities of HPL-cultured ASC sheets and delve into the molecular mechanism.
METHODS: A rat burn model was utilized to evaluate the efficacy of HPL-cultured ASC sheets in promoting wound healing. ASC sheets were fabricated with HPL, and those with FBS were included for comparison. Various analyses were conducted to assess the impact of HPL sheets on wound healing. Histological examination of wound tissues provided insights into aspects such as wound closure, collagen deposition, and overall tissue regeneration. Immunofluorescence was employed to assess the presence and distribution of transplanted ASCs after treatment. Further in vitro studies were conducted to decipher the specific factors in HPL sheets contributing to angiogenesis.
RESULTS: HPL-cultured ASC sheets significantly accelerated wound closure, fostering ample and organized collagen deposition in the neo-dermis. Significantly more retained ASCs were observed in wound tissues treated with HPL sheets compared to the FBS counterparts. Moreover, HPL sheets mitigated macrophage recruitment and decreased subsequent wound tissue fibrosis in vivo. Immunohistochemistry also indicated enhanced angiogenesis in the HPL sheet group. The in vitro analyses showed upregulation of C-C motif chemokine ligand 5 (CCL5) and angiogenin in HPL sheets, including both gene expression and protein secretion. Culturing endothelial cells in the conditioned media compared to media supplemented with CCL5 or angiogenin suggested a correlation between CCL5 and the pro-angiogenic effect of HPL sheets. Additionally, through neutralizing antibody experiments, we further validated the crucial role of CCL5 in HPL sheet-mediated angiogenesis in vitro.
CONCLUSIONS: The present study underscores CCL5 as an essential factor in the pro-angiogenic effect of HPL-cultured ASC sheets during the wound healing process. These findings highlight the potential of HPL-cultured ASC sheets as a promising therapeutic option for healing-impaired cutaneous wounds in clinical settings. Furthermore, the mechanism exploration yields valuable information for optimizing regenerative strategies with ASC products.
UNASSIGNED: This research was supported by the National Science and Technology Council, Taiwan (NSTC112-2321-B-002-018), National Taiwan University Hospital (111C-007), and E-Da Hospital-National Taiwan University Hospital Joint Research Program (111-EDN0001, 112-EDN0002).