壳聚糖凝胶活性载体与骨髓间充质干细胞的相容性

背景：壳聚糖-β-甘油磷酸钠凝胶（Chitosan—disodiumB—glycerolphosphate，C／GP）与软骨细胞显示了良好的相容性，因此假设作为组织工程的种子细胞——骨髓间充质干细胞也与C／GP凝胶有较好的细胞相容性。目的：观察骨髓间充质干细胞在C／GP凝胶中生长、增殖和成软骨分化，探讨C／GP凝胶与骨髓间充质干细胞的细胞相容性，为软骨组织工程材料寻找新的适宜细胞载体。设计、时间及地点：完全随机对照，细胞组织工程实验，于2005—10／2006—05在解放军第三军医大学西南医院中心实验室完成。材料：成年雌性小型猪6只用于收集骨髓，培养骨髓间充质干细胞。方法：取培养后传至第3代的骨髓间充质干细胞用于实验。在成骨培养条件下检测骨髓间充质干细胞碱性磷酸酶活性和钙沉积，在成软骨培养条件F行甲苯胺蓝染色和Ⅱ型胶原免疫组织化学检测。壳聚糖盐酸溶液与β-甘油磷酸钠溶液混匀后，置37℃孵箱内5～10min即形成壳聚糖β甘油磷酸钠凝胶。成软骨培养3周，倒置显微镜观察骨髓间充质干细胞在C／GP凝胶内黏附及成活情况，组织免疫组织化学法分析骨髓间充质干细胞在凝胶内成软骨分化情况，MTT法检测骨髓间充质干细胞种植2，5和8d后增殖情况。主要观察指标：①猪骨髓间充质干细胞的特征。②骨髓间充质干细胞在C／GP内的黏附和成活。⑧骨髓间充质干细胞在C／GP内的成软骨分化。④骨髓间充质干细胞在C／GP内的增殖。结果：①猪骨髓间充质干细胞的特征：体外培养的骨髓间充质干细胞呈成纤维细胞样形态，在成骨和成软骨诱导条件下可分别向成骨样细胞和软骨细胞分化。②骨髓间充质干细胞在C／GP内的黏附和成活：MTT染色发现，骨髓间充质干细胞植入C／GP凝胶21d内，细胞黏附于凝胶内并保持90％以上成活。③骨髓间充质十细胞在C／GP内的成软骨分化：体外培养21d后成软骨诱导的骨髓间充质干细胞在C／GP凝胶内产生大量软骨基质。④骨髓间充质干细胞在C／GP内的增殖：成软骨诱导骨髓间充质干细胞在C／GP凝胶内持续增殖，细胞种植后2，5和8d，细胞增殖程度差异明显（P〈0．05）。结论：C／GP凝胶与骨髓间充质干细胞显示了良好的细胞相容性，有利于骨髓间充质干细胞在其内生长、增殖和成软骨分化，有孳作为软骨组织工种的细胞载体。

Chitosan-based gels as bioactive carriers for bone marrow mesenchymal stem cells:A cytocompatibility study

BACKGROUND: Chitosan-disodium β-glycerol phosphate (C/GP) gel has been shown to be compatible with the entrapment of viable chondrocytes, and bone marrow mesenchymal stem cells (BMSCs) are considered to be the potential cells used in tissue engineering. This experiment is aimed to observe the cytocompatibility of BMSCs with C/GP gel.OBJECTIVE: To study the effect of C/GP gel on the growth, proliferation and chondrogenic differentiation in vitro cultured BMSCs and explore a new carrier for the application of cartilage tissue engineering.DESIGN: Completely randomized controlled experiment.SETTING: Department of Bone and Joint Surgery, Affiliated Hospital of Luzhou Medical College; Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA.MATERIALS: The experiment was performed in Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA between October 2005 and April 2006, Six adult female mini-pigss were employed. C/GP gel is prepared by mixture the HCI solution of chitosan with salt solution of β-glycerol phosphate, allowed gel at 37 ℃ in incubator for about 5 to 10 minutes.METHODS: ① BMSCs culture: 4-6 mL of bone marrow harvested from the posterior superior lilac crest were plated at 20 ×10<'6>/100 mm dish and then grown for 14 days in complete media, consisting of DMEM/F-12 supplemented with 10% fetal ovine serum. Cells were harvested and re-seeded for subculture. ② BMSCs differentiation assays: Osteogenic differentiation was assessed by histologic detection of alkaline phosphatase activity and calcium in cultures under osteogenic conditions. Chondrogenic differentiation was evaluated by histology for toluidine blue and immunohistochemistry for type Ⅱ collagen in cultures under chondrogenic conditions. ③ In vitro assays, expanded BMSCs were suspended in C/GP solution and allowed gel at 37 ℃ in incubator for about 5 to 10 minutes, then cultured under chondrogenic conditions for 3 weeks. Cells attached to and viability in C/GP gel was monitored with the aid of an inverted light microscope. Chondrogenic differentiation of cell in C/GP gel were assessed by histological and immunohistocbemistry. The cell proliferated was monitored by MTT after 2, 5, 8 days seeding.MAIN OUTCOME MEASURES: ① Characterization of mini-pigs' BMSCs; ② BMSCs attached to and viability in C/GP gel; ③ Chondrogenic differentiation of BMSCs in C/GP gel; ④ BMSCs proliferated in C/GP gel.RESULTS: ① Characterization of mini-pigs' BMSCs: Cultured BMSCs showed fibroblastic morphology and were able to differentiate to chondrocytes or osteogenic cells under chondrgenic or osteogenic cultured condition respectively. ②BMSCs attached to and viability in C/GP gel: BMSCs attached to and remained > 90% viable in C/GP gels immediately post-casting, and throughout the 21 days, using MTT staining. ③ Chondrogenic differentiation of BMSCs in C/GP gel: During 21 days culture period in vitro, chondrogenic induced BMSCs produced amounts of de novo cartilage matrix in the chitosan, as assessed by histological and biochemical criteria. ④ BMSCs proliferated in C/GP gel: Chondrogenic induced BMSCs cultured in C/GP gels continued to proliferate. There was a significant difference among the values of optical density in the cells-gel constructs compared to the controls without cells after 2, 5, and 8 days of culture (P<0.05).CONCLUSION: It is confirmed that C/GP gel shows good cytocompatibility with BMSCs and contributes to the growth, proliferation and chondrogenic differentiation for BMSCs in vitro culture. C/GP gel can be a potential cell-carrier for tissue engineering of articular cartilage.