摘要
目的 研究力学刺激对猪骨髓基质干细胞 (BMSC)体外构建组织工程化软骨的影响。方法 取第二代猪BMSC以 5× 10 7/cm3 的密度接种于聚羟基乙酸 /聚羟基乳酸 (PGA/PLA)圆盘形支架上 ,一周后均改用软骨诱导液 [高糖DMEM培养基含 10 %胎牛血清 (FBS)、转化生长因子 (TGF) β110ng/ml、胰岛素样生长因子 (IGF) I 5 0ng/ml、地塞米松 4 0ng/ml]体外培养。根据不同的施加力分为 3组 :离心组设定离心力 10 0g ,2次 /d ,30min/次 ,间隔 12h ;摇床组设定为 80r/min ,每天施力 8h ;静止培养作为对照组。分别于第 4周、第 8周进行大体观察、组织学、组织化学、免疫组织化学 ,蛋白聚糖(GAG)定量等检测 ,比较不同力学刺激对诱导BMSC体外软骨形成的影响。结果第 4周时两实验组 ,即摇床组及离心组形成的细胞材料复合物基本保持原来的体积与外形 ,软骨陷窝形成 ,呈结节状或巢状分布并伴有GAG沉积及胶原合成 ,Ⅱ型胶原免疫组化染色阳性。静止组体积缩小 ,有少量软骨陷窝结构形成 ;8周时两实验组形成的细胞材料复合物形状保持良好 ,浅灰白色 ,光泽好 ;HE染色结果显示大量成熟软骨陷窝形成 ,核仁清楚并有多处分裂相 ,细胞外基质沉积均匀 ;Safranin O染色显示有大量的GAG形成 ,Masson染色显示有较多新生胶原组织形成 ,免疫?
Objective To evaluate the influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells (BMSC). Methods Porcine BMSC of passage 2 were seeded onto a cylinder-shaped PGA/PLA scaffold, 8mm in diameter and 3mm in thickness, at a density of 5×10 7/ cm 3. After the cell-scaffold constructs were cultured for one week, the primary medium, high-glucose DMEM medium with 10% fetal bovine serum (FBS), was replaced by chondrogenically inductive medium containing TGFβ 1 (10 ng/ml), IGF-I (50 ng/ml), and dexamethasone (40 ng/ml) in addition to DMEM+10% FBS. The constructs were randomly divided into three groups according to the imposed stress: experimental group A in which a centrifugal stress was imposed at 100 g, 30 min, 2/d; experimental group B in which a rotative stress was imposed at 80rpm,8h/d by a shaker; and control group in which the constructs were statically cultured. The gross view, histology, histochemistry, immunohistochemistry and glycosamino-glycan(GAG)content were evaluated after 4 and 8 weeks respectively. Results Four weeks later, the constructs in both experimental groups maintained their original sizes and shapes. Histology showed nodular lacuna-like structures, in company with GAG deposition and collagen synthesis. In addition, collagen type II was detected by immunohistochemistry. In the control group, however, the constructs shrunk to a little smaller size than those in the experimental groups, and histological staining showed a little amount of lacuna. Eight weeks later, the constructs in both experimental groups still maintained the original sizes and shapes with good elasticity. HE staining showed massive lacuna-like structures in most areas of the construct and extracellular matrix deposited evenly. Fibrous tissues were only observed in some areas. Safranin-O staining showed massive GAG formation and Masson staining showed much more collagen formation than those in the control group. Immunohistochemical staining of collagen type II showed strong positive expression. In the control group the constructs showed massive fibrous tissues, with a small amount of lacuna-like structures in the peripheral areas. GAG contents in the 2 experimental groups were 5.98 mg/g and 5.62 mg/g respectively,both significantly higher than that in the control group (4.73 mg/g) without a difference between the 2 experimental groups.Conclusion Mechanical stress promotes chondrogenesis and cartilage maturation of BMSC in vitro.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2004年第23期1997-2001,共5页
National Medical Journal of China
基金
国家高技术研究发展计划 (863计划)基金资助项目 (2 0 0 2AA2 0 5 0 2 1)
国家重点基础研究发展规划(973计划)基金资助项目(G19990 5 43 0 8 2 )
上海市"重中之重"重点学科基金资助项目
