Conclusion
The ADSCs/CSCl hydrogel complex has a good biocompatibility and possessed positive effects on promoting the deep partial thickness scald wound repairing in rats. 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。.
Methods
ADSCs 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 staining in 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.
Objective
To 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.
Results
CSCl 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).