Repair of liver injury in rats by adipose-derived mesenchymal stem cells combined with porous chitosan microspheres

doi:10.3969/j.issn.2095-4344.1562

Abstract

Abstract:

BACKGROUND: With the development of stem cell therapy and tissue engineering, adipose-derived mesenchymal stem cells combined with biocompatible scaffolds have been introduced to constructing liver tissue engineering micro-units as a new strategy for the treatment of liver diseases.
OBJECTIVE: To investigate the repairing ability of adipose-derived mesenchymal stem cells combined with porous chitosan microspheres on acute liver injury.
METHODS: Adipose-derived mesenchymal stem cells from neonatal Sprague-Dawley rats and porous chitosan microspheres were co-cultured in vitro to observe the proliferation of adipose-derived mesenchymal stem cells on the microspheres on the 1^st, 3^rd and 5^th days. Thirty-six Sprague-Dawley rats (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. in China) were randomly divided into three groups (n=12 per group): control group was intraperitoneally injected with normal saline; acute liver injury group was intraperitoneally administered with 10% carbon tetrachloride-olive oil; and cell-microsphere group was intraperitoneally given 10% carbon tetrachloride-olive oil and then adipose-derived mesenchymal stem cells-porous chitosan microspheres were implanted into the liver surface on the 1^st day after injection. Gross, serological and histological evaluations were performed on the 1^st, 2^nd, 3^rd and 7^th days.
RESULTS AND CONCLUSION: Adipose-derived mesenchymal stem cells-porous chitosan microspheres were stained with live/dead staining on the 1^st, 3^rd and 5^th days. The cells grew normally on the microspheres as the number of dead cells was much less than that of living cells, and the cells proliferated continuously over time. The gross score, liver function score and histological score of the cell-microsphere group were higher than those of the acute liver injury group on the 1^st, 2^nd, 3^rd and 7^th days. There was no significant difference between the cell-microsphere group and control group since the 3^rdday. All these experimental findings indicate that adipose-derived mesenchymal stem cells-porous chitosan microspheres implanted onto the liver surface can accelerate the repair of liver injury.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Liver Diseases, Carbon Tetrachloride, Adipose Tissue, Mesenchymal Stem Cells, Chitosan, Tissue Engineering

CLC Number:

Huang Kun, Liu Chengli, Han Gonghai, Liu Rui, Chen Weiwei, Ding Xiao, Tang He, Peng Jiang. Repair of liver injury in rats by adipose-derived mesenchymal stem cells combined with porous chitosan microspheres[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 716-722.

Figures/Tables 2

2.1 ADSCs的形态接种2 h后细胞开始大量贴壁，12 h内完成贴壁，然后开始大量增殖，24 h细胞增殖可达80%。PBS洗掉未贴壁细胞，在显微镜下观察到ADSCs贴壁生长，呈梭形，并逐渐融合，见图1A。在进行稳定传代后，可观察到细胞形态和生长速度没有明显改变，见图1B。 2.2 ADSCs成脂、成骨、成软骨三系诱导分化成脂诱导3 d后细胞回缩成多角形形态，细胞内可见少量脂滴，随着诱导时间的延长脂滴逐渐增多并相互融合，诱导完成后油红O染色可见细胞内脂滴成红色，见图2A。成骨诱导3 d后细胞逐渐成长梭形，10 d左右细胞变为多边形并可见少量钙结节沉积。诱导21 d后茜素红染色可见橘红色钙结节，见图2B。成软骨诱导3 d后出现肉眼可见的团块，14 d后细胞团渐成透明状，诱导28 d后经阿利新蓝染色可见软骨组织内酸性黏多糖，见图2C。 2.3 ADSCs在多孔壳聚糖微球上的活/死染色结果多孔壳聚糖微球无明显的细胞毒作用，ADSCs可在微球上贴附生长及增殖，用共聚焦显微镜进行扫描时，可在不同深度的层面观察到微球内部仍有活细胞，但细胞数量较少，见图3。复合第1天干细胞形态由圆形转为长梭形贴附在微球表面，第3天ADSCs贴附在微球上具有明显的增殖趋势，第5天ADSCs在微球上细胞数量明显增加。 2.4 ADSCs-多孔壳聚糖微球对肝损伤大鼠血清肝功能指标的影响谷丙转氨酶、谷草转氨酶作为肝细胞内酶，在氨基酸的合成与分解代谢中起着非常重要的作用，是诊断肝细胞受损程度最敏感的指标。正常情况下，血清中两种酶的活性很低。在肝细胞损伤时，细胞膜通透性增加，谷丙转氨酶、谷草转氨酶释放入血，血清酶活性显著升高，并且反映肝细胞受损程度。ADSCs-多孔壳聚糖微球对肝损伤大鼠血清谷丙转氨酶、谷草转氨酶活力的影响，见图4。结果显示，大鼠在治疗第3天时肝功能恢复至接近正常水平(P > 0.05)，急性肝损伤组肝功能与空白对照组差异有显著性意义(P < 0.05)。 2.5 ADSCs-多孔壳聚糖微球移植入肝脏表面后组织形态学变化正常对照组大鼠腹腔内无腹水，肝脏表面鲜红，肝包膜完整，大网膜和肝脏表面无明显粘连，见图5A。注射四氯化碳后SD大鼠腹腔内积水超过5 mL，肝脏表面由红变灰白色，肝包膜破损明显，大网膜与肝脏表面有一定的粘连，见图5B。治疗3 d后取出大鼠肝脏，观察到ADSCs-多孔壳聚糖微球仍贴附在肝脏表面，见图5C。正常对照组肝脏经苏木精-伊红染色后可观察到肝细胞排列整齐，结构完整，胞质丰富，肝细胞核清晰，大而圆，无明显肿胀及脂肪变性，肝小叶内及汇管区无炎症细胞浸润，未见肝细胞坏死变性及异常，见图6A，B。急性肝损伤组注射10%四氯化碳-橄榄油24 h，可以观察到肝细胞排列紊乱，肝细胞有明显的坏死，肝细胞弥漫性水肿，肝小叶内有明显的坏死灶，炎细胞浸润严重，细胞核大小不一，有浓缩及破碎的核，组织形态证明造模成功，见图6C，D；在第2天时，ADSCs-多孔壳聚糖微球组肝细胞坏死数较多，存在着大小不一的脂肪空泡样变化，但较急性肝损伤组有一定的改善，见图6E，F，急性肝损伤组肝细胞仍有坏死，炎细胞浸润较严重，见图6G，H。在第3天时，ADSCs-多孔壳聚糖微球组肝窦内仍有出血，但无明显的炎细胞浸润，较急性肝损伤组有明显改善，见图6I，J，急性肝损伤组仍有少量的脂肪变性，见图6K，L。在第7天时，ADSCs-多孔壳聚糖微球组肝细胞排列相对整齐，胞质均匀清晰，只有部分细胞坏死，少量的空泡现象，见图6M，N，比急性肝损伤组肝脏修复效果好，见图6O，P。肝功能及病理学切片结果表明ADSCs-多孔壳聚糖微球对大鼠急性肝损伤有明显的修复作用。"

References

[1] Bernal W, Wendon J. Acute liver failure. N Engl J Med. 2013;369(26): 2525-2534.

[2] Kadota Y, Yagi H, Inomata K, et al. Mesenchymal stem cells support hepatocyte function in engineered liver grafts. Organogenesis. 2014;10(2):268-277.

[3] An SY, Han J, Lim HJ, et al. Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells. Tissue Cell. 2014;46(2):127-135.

[4] Ortiz LA, Dutreil M, Fattman C, et al. Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci U S A. 2007;104(26): 11002-11007.

[5] Gilsanz C, Aller MA, Fuentes-Julian S, et al. Adipose-derived mesenchymal stem cells slow disease progression of acute-on-chronic liver failure. Biomed Pharmacother. 2017;91:776-787.

[6] Nicolas CT, Wang Y, Nyberg SL. Cell therapy in chronic liver disease. Curr Opin Gastroenterol. 2016;32(3):189-194.

[7] Xu L, Wang S, Sui X, et al. Mesenchymal Stem Cell-Seeded Regenerated Silk Fibroin Complex Matrices for Liver Regeneration in an Animal Model of Acute Liver Failure. ACS Appl Mater Interfaces. 2017;9(17):14716-14723.

[8] Wiebe J, Nef HM, Hamm CW. Current status of bioresorbable scaffolds in the treatment of coronary artery disease. J Am Coll Cardiol. 2014;64(23):2541-2551.

[9] Malcor JD, Juskaite V, Gavriilidou D, et al. Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF. Biomaterials. 2018;182:21-34.

[10] Subbarayan R, Girija DM, Rao SR. Human umbilical cord tissue stem cells and neuronal lineages in an injectable caffeic acid-bioconjugated gelatin hydrogel for transplantation. J Cell Physiol. 2018 Aug 24. doi: 10.1002/jcp.26834. [Epub ahead of print]

[11] Liu L, You Z, Yu H, et al. Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis. Nat Mater. 2017;16(12):1252-1261.

[12] Etter JN, Karasinski M, Ware J, et al. Dual-crosslinked homogeneous alginate microspheres for mesenchymal stem cell encapsulation. J Mater Sci Mater Med. 2018;29(9):143.

[13] Chander V, Singh AK, Gangenahalli G. Cell encapsulation potential of chitosan-alginate electrostatic complex in preventing natural killer and CD8+ cell-mediated cytotoxicity: an in vitro experimental study. J Microencapsul. 2018:1-27.

[14] Wang Y, Yuan X, Yu K, et al. Fabrication of nanofibrous microcarriers mimicking extracellular matrix for functional microtissue formation and cartilage regeneration. Biomaterials. 2018;171:118-132.

[15] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970;3(4):393-403.

[16] Fan CG, Tang FW, Zhang QJ, et al. Characterization and neural differentiation of fetal lung mesenchymal stem cells. Cell Transplant. 2005;14(5):311-321.

[17] Caplan AI, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011;9(1):11-15.

[18] Manuguerra-Gagné R, Boulos PR, Ammar A, et al. Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment. Stem Cells. 2013;31(6):1136-1148.

[19] Guo DB, Zhu XQ, Li QQ, et al. Efficacy and mechanisms underlying the effects of allogeneic umbilical cord mesenchymal stem cell transplantation on acute radiation injury in tree shrews. Cytotechnology. 2018 Jul 31. doi: 10.1007/s10616-018-0239-z. [Epub ahead of print]

[20] Chang SH, Huang HH, Kang PL, et al. In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering. Acta Biomater. 2017;63:261-273.

[21] Hoyer DP, Munteanu M, Canbay A, et al. Liver transplantation for acute liver failure: are there thresholds not to be crossed. Transpl Int. 2014; 27(6):625-633.

[22] Pan XN, Zheng LQ, Lai XH. Bone marrow-derived mesenchymal stem cell therapy for decompensated liver cirrhosis: a meta-analysis. World J Gastroenterol. 2014;20(38):14051-14057.

[23] Kumari S, Vermeulen S, van der Veer B, et al. Shaping Cell Fate: Influence of Topographical Substratum Properties on Embryonic Stem Cells. Tissue Eng Part B Rev. 2018;24(4):255-266.

[24] Li Y, Hermanson DL, Moriarity BS, et al. Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell. 2018;23(2):181-192.e5.

[25] Fujita Y, Kawamoto A. Stem cell-based peripheral vascular regeneration. Adv Drug Deliv Rev. 2017;120:25-40.

[26] Perico L, Morigi M, Rota C, et al. Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function. Nat Commun. 2017;8(1):983.

[27] Soliman MG, Mansour HA, Hassan WA, et al. Mesenchymal stem cells therapeutic potential alleviate lipopolysaccharide-induced acute lung injury in rat model. J Biochem Mol Toxicol. 2018:e22217. doi: 10.1002/jbt.22217. [Epub ahead of print]

[28] Jiang W, Tan Y, Cai M, et al. Human Umbilical Cord MSC-Derived Exosomes Suppress the Development of CCl4-Induced Liver Injury through Antioxidant Effect. Stem Cells Int. 2018;2018:6079642.

[29] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2): 211-228.

[30] Padalhin AR, Lee BT. Hemostasis and Bone Regeneration Using Chitosan/Gelatin-BCP Bi-layer Composite Material. ASAIO J. 2018 Aug 25. doi: 10.1097/MAT.0000000000000850. [Epub ahead of print]

[31] Shi D, Zhang J, Zhou Q, et al. Quantitative evaluation of human bone mesenchymal stem cells rescuing fulminant hepatic failure in pigs. Gut. 2017;66(5):955-964.