中国修复重建外科杂志

中国修复重建外科杂志

脂肪间充质干细胞-壳聚糖凝胶复合物治疗大鼠深Ⅱ度烫伤创面实验研究

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目的制备脂肪间充质干细胞(adipose-derived stem cells,ADSCs)与温敏氯化壳聚糖(chitosan chloride,CSCl)凝胶复合物,研究其生物相容性及对大鼠深Ⅱ度烫伤创面修复的可行性。 方法取 SPF 级 6 周龄雄性 SD 大鼠皮下脂肪组织,采用酶原消化并差速贴壁法制备 ADSCs。将 CSCl、β-甘油磷酸钠和羟乙基纤维素按 8∶2∶2.5 比例混合,制作温敏 CSCl 凝胶;采用 Live/Dead 染色和细胞计数试剂盒 8(cell counting kit 8,CCK-8)法观察 ADSCs 在其中的存活和增殖情况。取 SPF 级 8 周龄雄性 SD 大鼠 72 只,采用开水接触烫伤法制备深Ⅱ度烫伤创面模型,随机分为 3 组,每组 24 只,A 组为空白对照组,B 组用温敏 CSCl 凝胶涂抹创面,C 组用 ADSCs/温敏 CSCl 凝胶复合物涂抹创面。术后 3、7、14、21 d 观察创面闭合情况并计算创面闭合率;术后 7 d 行 HE 染色观察炎性细胞数,21 d 行 HE 染色观察新生表皮厚度;同时于术后 7 d 行 CD31 免疫组织化学染色观察新生微血管情况。 结果CSCl 有温度敏感性,4℃ 为液态,37℃ 时成凝胶。Live/Dead 染色和 CCK-8 法结果显示 ADSCs 在温敏 CSCl 凝胶中存活并持续增殖。大体观察示,术后各时间点 C 组创面闭合率均高于 A、B 组(P<0.05)。HE 染色示,术后 7 d,3 组大鼠创面修复进入纤维增生期,各组可见胶原沉积,真皮层可见炎性细胞浸润,C 组炎性细胞数显著小于 A、B 组,B 组小于 A 组(P<0.01)。术后 21 d,B、C 组新生上皮及真皮层纤维结缔组织排列整齐,可见成纤维细胞及新生毛细血管;A 组亦可见新生表皮覆盖,但真皮层纤维结缔组织排列较紊乱,见零星毛细血管。C 组新生表皮厚度显著大于 A、B 组,B 组大于 A 组(P<0.01)。术后 7 d,CD31 免疫组织化学染色示各组均可见新生微血管,C 组大鼠创面新生微血管数显著高于 A、B 组,B 组高于 A 组(P<0.05)。 结论ADSCs/温敏 CSCl 凝胶复合物具有良好的组织相容性,并且能加快大鼠深Ⅱ度烫伤创面修复。

ObjectiveTo prepare adipose-derived stem cells (ADSCs) and chitosan chloride (CSCl) gel complex to study the biocompatibility and the feasibility of repairing the wounds of deep partial thickness scald in rats. MethodsADSCs were prepared by enzymogen digestion and differential adherence method from the subcutaneous adipose tissue of SPF grade 6-week-old male Sprague Dawley (SD) rats. Temperature sensitive CSCl gel was prepared by mixing CSCl, β glycerol phosphate, and hydroxyethyl cellulose in 8∶2∶2.5 ratio. The proliferation of ADSCs was measured by cell counting kit 8 (CCK-8) assay and the survival of ADSCs was detected by the Live/Dead flurescent stainingin vitro. A deep partial thickness burn animal model was made on the back of 72 SPF grade 6-week-old male SD rats by boiled water contact method and randomly divided into 3 groups (n=24). Group A was blank control group, group B was CSCl hydrogel group, group C was ADSCs/CSCl gel group. The wound closure rate at 3, 7, 14, 21 days was observed after operation. The number of inflammatory cells at 7 days and epidermal thickness at 21 days were observed by HE staining after operation. The angiogenesis at 7 days was evaluated by immunohistochemistry staining with CD31 expression. ResultsCSCl had a temperature sensitivity, at 4℃, the temperature-responsive hydrogel was liquid and became solid at 37℃. The CCK-8 assay and Live/Dead flurescent staining confirmed that ADSCs could grow and proliferate in the ADSCs/CSCl hydrogel complex. General observation showed the wound closure ratio in group C was superior to groups A and B after operation (P<0.05). HE staining showed that at 7 days after operation, the wound healing of the three groups entered fibrous proliferation stage. Collagen deposition and inflammatory cell infiltration were observed in the dermis of each group. The proportion of inflammatory cells in group C was significantly lower than that in groups A and B, and in group B than in group A (P<0.01). At 21 days after operation, the fibrous connective tissues of neoepithelium and dermis in groups B and C were arranged neatly, and fibroblasts and neocapillaries could be seen. In group A, neoepidermis could also be seen, but the fibrous connective tissues in dermis were arranged disorderly and sporadic capillaries could be seen. The thickness of neonatal epidermis in group C was significantly larger than that in groups A and B, and in group B than in group A (P<0.01). CD31 immunohistochemistry staining showed that the neovascularization could be seen in all groups. The number of neovascularization in group C was significantly higher than that in groups A and B, and in group B than in group A (P<0.05). ConclusionThe ADSCs/CSCl hydrogel complex has a good biocompatibility and possessed positive effects on promoting the deep partial thickness scald wound repairing in rats.

关键词: 氯化壳聚糖; 凝胶; 脂肪间充质干细胞; 深Ⅱ度烫伤; 创面修复; 大鼠

Key words: Chitosan chloride; hydrogel; adipose-derived stem cells; deep partial thickness burn; wound repair; rat

引用本文: 廖筱梅, 罗兴前, 代蕾, 黄海峻, 郭杏. 脂肪间充质干细胞-壳聚糖凝胶复合物治疗大鼠深Ⅱ度烫伤创面实验研究. 中国修复重建外科杂志, 2019, 33(1): 101-109. doi: 10.7507/1002-1892.201804109 复制

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1. Hüging M, Biedermann T, Sobrio M, et al. The effect of wound dressings on a bio-engineered human dermo-epidermal skin substitute in a rat model. J Burn Care Res, 2017, 38(6): 354-364.
2. Steffens D, Mathor MB, Soster PRDL, et al. Treatment of a burn animal model with functionalized tridimensional electrospun biomaterials. J Biomater Appl, 2017, 32(5): 663-676.
3. Caliari-Oliveira C, Yaochite JN, Ramalho LN, et al. Xenogeneic mesenchymal stromal cells improve wound healing and modulate the immune response in an extensive burn model. Cell Transplant, 2016, 25(2): 201-215.
4. Motamed S, Taghiabadi E, Molaei H, et al. Cell-based skin substitutes accelerate regeneration of extensive burn wounds in rats. Am J Surg, 2017, 214(4): 762-769.
5. Dashtimoghadam E, Bahlakeh G, Salimi-Kenari H, et al. Rheological study and molecular dynamics simulation of biopolymer blend thermogels of tunable strength. Biomacromolecules, 2016, 17(11): 3474-3484.
6. Naderi-Meshkin H, Andreas K, Matin MM, et al. Chitosan-based injectable hydrogel as a promising in situ forming scaffold for cartilage tissue engineering. Cell Biol Int, 2014, 38(1): 72-84.
7. 程凤, 贺金梅, 李纪伟, 等. 壳聚糖基抗菌型创伤敷料的研究进展. 高分子通报, 2016, 27(7): 46-52.
8. Bajek A, Gurtowska N, Olkowska J, et al. Adipose-derived stem cells as a tool in cell-based therapies. Arch Immunol Ther Exp (Warsz), 2016, 64(6): 443-454.
9. Duscher D, Barrera J, Wong VW, et al. Stem cells in wound healing: the future of regenerative medicine? A mini-review Gerontology, 2016, 62(2): 216-225.
10. Teng M, Huang Y, Zhang H. Application of stems cells in wound healing—an update. Wound Repair Regen, 2014, 22(2): 151-160.
11. Jeong JH. Adipose stem cells and skin repair. Curr Stem Cell Res Ther, 2010, 5(2): 137-140.
12. Kaisang L, Siyu W, Lijun F, et al. Adipose-derived stem cells seeded in Pluronic F-127 hydrogel promotes diabetic wound healing. J Surg Res, 2017, 217: 63-74.
13. Vidor SB, Terraciano PB, Valente FS, et al. Adipose-derived stem cells improve full-thickness skin grafts in a rat model. Res Vet Sci, 2018, 118: 336-344.
14. Liu S, Jiang L, Li H, et al. Mesenchymal stem cells prevent hypertrophic scar formation via inflammatory regulation when undergoing apoptosis. J Invest Dermatol, 2014, 134(10): 2648-2657.
15. 周虹, 郭杏, 李丹, 等. 大鼠脂肪间充质干细胞体外分离培养及 CM_DiI 标记后的传代示踪. 安徽医科大学学报, 2016, 51(11): 1590-1595.
16. Hasdemir M, Agir H, Eren GG, et al. Adipose-derived stem cells improve survival of random pattern cutaneous flaps in radiation damaged skin. J Craniofac Surg, 2015, 26(5): 1450-1455.
17. Pu CM, Liu CW, Liang CJ, et al. Adipose-derived stem cells protect skin flaps against ischemia/reperfusion injury via IL-6 expression. J Invest Dermatol, 2017, 137(6): 1353-1362.
18. Xu P, Yu Q, Huang H, et al. Nanofat increases dermis thickness and neovascularization in photoaged nude mouse skin. Aesthetic Plast Surg, 2018, 42(2): 343-351.
19. Wang H, Shi J, Wang Y, et al. Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction. Biomaterials, 2014, 35(13): 3986-3998.
20. Gao J, Liu R, Wu J, et al. The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury. Biomaterials, 2012, 33(14): 3673-3681.
21. Song K, Li L, Yan X, et al. Characterization of human adipose tissue-derived stem cells in vitro culture and in vivo differentiation in a temperature-sensitive chitosan/β-glycerophosphate/collagen hybrid hydrogel. Mater Sci Eng C Mater Biol Appl, 2017, 70(Pt 1): 231-240.
22. Hoemann CD, Chenite A, Sun J, et al. Cytocompatible gel formation of chitosan-glycerol phosphate solutions supplemented with hydroxyl ethyl cellulose is due to the presence of glyoxal. J Biomed Mater Res A, 2007, 83(2): 521-529.