Xie Jinbo, Wan Jian, Tang Xuemin, Li Wei, Peng Bo
Department of Urology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
Center for Difficult and Complicated Abdominal Surgery, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
Transl Androl Urol. 2021 Sep;10(9):3656-3668. doi: 10.21037/tau-21-703.
Constructing tissue-engineered kidneys using decellularized kidney scaffolds (DKS) has attracted widespread attention as it is expected to be the key to solving the shortage of donor kidneys. However, thrombosis and the host inflammatory response are unfavorable factors that hider the re-endothelialization and vascularization of the decellularized scaffolds.
Heparin was immobilized into the DKS using the method of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) activation. Fourier-transform infrared (FTIR) spectra were used to verify the heparinization of DKS. Human umbilical vein endothelial cells (HUVECs) were seeded and cultured in the DKS, then the sliced scaffolds were transplanted subcutaneously into nude mouse. Scanning electron microscopy and a series of histochemical stains including hematoxylin and eosin (H&E), elastic Verhöeff-Van Gieson (EVG), Sirius red, Masson's trichrome, and toluidine blue (TB) staining were used for morphological characterization. The qRT-PCR analysis, immunohistochemistry (IHC), and immunofluorescence (IF) staining were used to determine the expression of related molecular markers.
The rat DKS completely retained the extracellular matrix and heparinized modification. The H&E staining results showed there were more HUVECs covering the internal surfaces of tubular structures in the HEP-DKS group compared with the DKS group. The IF analysis results revealed that CD31, Ki67, and CD206 had higher positive rates in HUVECs in the HEP-DKS group compared to the DKS group. Both groups of scaffolds showed blood vessel formation via H&E staining, and there were more blood vessels in the HEP-DKS group compared with the native DKS group (P<0.05). The qRT-PCR results showed that the levels of IL-1β, IL-6, and TNF-α in the HEP-DKS group were significantly lower than those of the native DKS group, while the expression level of IL-10 was significantly higher than that in the native DKS group (P<0.05).
Heparin modification improves the re-endothelialization and vascular regeneration of the DKS through anticoagulation and . The anti-inflammatory effect of heparin on the transplanted host was initially confirmed, and it is considered that this effect may play a non-negligible role in promoting DKS re-endothelialization and angiogenesis. Heparinized DKS is therefore a promising candidate for kidney tissue engineering.
利用去细胞肾支架（DKS）构建组织工程肾已引起广泛关注，因为它有望成为解决供肾短缺问题的关键。然而，血栓形成和宿主炎症反应是阻碍去细胞支架再内皮化和血管化的不利因素。
采用1-乙基-3-（3-二甲基氨基丙基）-碳二亚胺/N-羟基琥珀酰亚胺（EDC/NHS）活化法将肝素固定到DKS中。利用傅里叶变换红外（FTIR）光谱验证DKS的肝素化。将人脐静脉内皮细胞（HUVECs）接种到DKS中进行培养，然后将切片后的支架皮下移植到裸鼠体内。采用扫描电子显微镜以及包括苏木精和伊红（H&E）、弹性韦尔霍夫-范吉森（EVG）、天狼星红、马森三色染色和甲苯胺蓝（TB）染色在内的一系列组织化学染色进行形态学表征。采用qRT-PCR分析、免疫组织化学（IHC）和免疫荧光（IF）染色来确定相关分子标志物的表达。
大鼠DKS完全保留了细胞外基质并实现了肝素化修饰。H&E染色结果显示，与DKS组相比，HEP-DKS组中覆盖肾小管结构内表面的HUVECs更多。IF分析结果显示，与DKS组相比，HEP-DKS组中HUVECs的CD31、Ki67和CD206阳性率更高。两组支架经H&E染色均显示有血管形成，且HEP-DKS组的血管数量比天然DKS组更多（P<0.05）。qRT-PCR结果显示，HEP-DKS组中IL-1β、IL-6和TNF-α的水平显著低于天然DKS组，而IL-10的表达水平显著高于天然DKS组（P<0.05）。
肝素修饰通过抗凝作用改善了DKS的再内皮化和血管再生。初步证实了肝素对移植宿主的抗炎作用，认为这种作用可能在促进DKS再内皮化和血管生成中发挥不可忽视的作用。因此，肝素化DKS是肾组织工程中有前景的候选材料。
