Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

软骨细胞接种小孔径聚乳酸-羟基乙酸共聚物支架的方法选择

目的 探讨软骨细胞接种小孔径聚乳酸-羟基乙酸共聚物(PLGA)支架的最佳接种方法. 方法 实验分3组(n=9):注射组、负压组、振荡组,取纤维蛋白原溶液混悬第2代软骨细胞,采用上述3种方法对孔隙率为92％、孔径为50 ～ 100 μm的PLGA支架进行细胞接种.48 h后,Hoechst33258法检测支架内DNA含量;接种并观察支架内含异硫氰酸荧光素的无细胞纤维蛋白原凝胶的分布;硬组织切片、4’,6-二脒基-2-苯基吲哚(DAPI)染色观察支架内细胞分布.7d后,扫描电镜观察支架表面及内部的细胞形态.各组部分支架(n=3)植入裸鼠皮下,以无细胞PLGA支架为空白对照组,术后8周取出支架行甲苯胺蓝、Ⅱ型胶原免疫组织化学染色,并计算累积吸光度(IOD)值. 结果 注射组、负压组、振荡组平均DNA含量分别为(755.79±80.50)、(657.32±89.68)、(650.18±106.33)ng/mg,各组比较差异均无统计学意义(F=1.214,P=0.361).无细胞纤维蛋白凝胶在各组中都均匀分布,DAPI染色显示注射组细胞分布较其他两组均匀.扫描电镜显示负压组和振荡组外周细胞较注射组多,而支架孔隙内仅注射组可见细胞黏附.注射组甲苯胺蓝、Ⅱ型胶原免疫组织化学染色的IOD值均优于其他3组,差异有统计学意义(P＜0.05). 结论 对于50 ～ 100 μm的小孑L径PLGA支架,注射法是一种快捷、高效的细胞接种方法.     

Yes相关蛋白通过Wnt/β-catenin信号通路调控软骨细胞Sox9表达的研究

目的研究Yes相关蛋白（Yes?associated protein, YAP）通过Wnt/β?catenin信号通路调控软骨细胞Sox9表达的机制。方法用siRNA分别沉默YAP基因和Lats1基因来调控YAP的活性,通过Real?time PCR检测软骨特异性基因和Wnt/β?catenin信号通路下游基因的表达;通过Western Blot检测GSK3β的磷酸化水平、活化β?catenin的蛋白水平以及Sox9的蛋白水平;并通过阿利新蓝染色检测软骨细胞外基质的分泌情况。用siRNA沉默YAP基因后,再用氯化锂干预细胞,通过Western Blot检测Sox9的表达和GSK3β的磷酸化水平。结果沉默YAP基因后,磷酸化GSK3β和活化β?catenin的蛋白水平减少,c?Myc和Nanog基因表达减少,Sox9、Col2和Aggrecan基因表达升高,并且软骨细胞分泌基质增多;沉默Lats1基因后,磷酸化GSK3β和活化β?catenin的蛋白水平增加,c?Myc和Nanog基因表达上调,Sox9、Col2和Aggre?can基因表达减少,并且软骨细胞分泌基质减少。此外,氯化锂可以阻断沉默YAP引起的Sox9表达上调。结论 YAP通过Wnt/β?catenin信号通路调控软骨细胞Sox9的表达。     

自体软骨细胞联合Ⅰ型胶原治疗剥脱性膝骨软骨炎

目的探讨自体软骨细胞联合Ⅰ型胶原蛋白三维支架治疗膝关节剥脱性骨软骨炎的疗效。 方法选取近5年来在青岛市黄岛区中心医院采用自体软骨细胞联合Ⅰ型胶原蛋白三维支架治疗膝关节剥脱性骨软骨炎的患者12例,用单因素方差分析评估术前与术后6个月、12个月国际膝关节文献委员会(IKDC)膝关节评估表、Lysholm膝关节功能评分。术后12个月磁共振成像(MRI)评估软骨修复情况。 结果12例患者术后6个月、12个月的IKDC评分分别为(83.7±5.6)、(91.7±3.7),Lysholm评分分别为(87.5±5.2)、(93.6±2.1),均较术前IKDC评分(53.9±6.7)(F＝158.877)、Lysholm评分(59.1±7.2)(F＝104.258)明显改善(均为P<0.05);每2个时间点之间的IKDC评分、Lysholm评分,差异均具有统计学意义(P<0.05)。术后12个月MRI检查显示,所有患者的移植软骨恢复良好,均未出现移植物脱落或局部水肿。术后随访期内,所有患者均未出现膝关节感染。 结论自体软骨细胞联合Ⅰ型胶原蛋白三维支架能有效治疗膝关节剥脱性骨软骨炎。     

大鼠软骨细胞与兔软骨细胞的培养与比较

目的 体外分离培养大鼠及兔膝关节软骨细胞,观察并比较两种软骨细胞的培养特点。方法 采用机械-酶消化法处理SD大鼠幼鼠和新西兰兔的软骨组织,传代培养,倒置显微镜观察细胞形态,细胞计数法测定生长曲线,实时定量PCR测定不同代数的软骨细胞Ⅱ型胶原与含血小板结合蛋白基序的解聚蛋白样金属蛋白酶5（a disintegrin and metalloproteinase with thrombospondin motifs 5, ADAMTS5）表达水平,甲苯胺蓝染色及Ⅱ型胶原酶免疫荧光染色对细胞进行鉴定。结果 原代培养大鼠软骨细胞12 h,内贴壁成多角形或不规则型,排列成“铺路石”状,培养3~5 d后进入快速增殖期,1周后进行细胞传代培养。兔软骨细胞相对贴壁缓慢,生长滞后。两类软骨细胞随着培养代数的增加,Ⅱ型胶原蛋白的mRNA表达量逐渐减少,ADAMTS5的表达逐代升高,表明随着培养代数的增加,软骨细胞活力逐渐下降。相同代数的大鼠软骨细胞活性较兔软骨细胞更高。甲苯胺蓝染色可见大鼠软骨细胞内成蓝色异染的糖胺多糖成分。不同波段的荧光激发下,Ⅱ型胶原酶免疫荧光染色可见大鼠原代培养的软骨细胞胞浆和胞膜呈清晰的绿色荧光,兔软骨细胞呈红色荧光,细胞核为明亮的蓝色荧光。结论 本实验通过比较大鼠及新西兰兔软骨细胞的培养与特点,证实随培养代数增加,软骨细胞活性逐渐降低,且相同代数大鼠软骨细胞较兔软骨细胞具备更高活性。     

自噬在张力诱导终板软骨细胞退变过程中的变化

 目的 探讨自噬在张力诱导终板软骨细胞退变过程中的变化及作用。方法 取10只清洁级SD大鼠腰椎终板软骨进行细胞培养。对P1代终板软骨细胞分别加载间歇循环张力（10%伸长率,0.5 Hz）0 h、3 h、12 h、24 h、48 h。以倒置相差显微镜观察细胞形态学变化,实时PCR与蛋白印迹法检测软骨标志基因Ⅱ型胶原、转录因子SOX-9及蛋白多糖转录因子、Beclin-1和LC3基因表达的变化,以单丹磺酰戊二胺染色观察自噬小体。MTT（3-2,5-二苯基四氮唑溴盐染色）法检测3-甲基腺嘌呤（自噬抑制剂）刺激前后的细胞存活率。结果 间歇循环张力诱导后0 h组与3 h组为正常终板软骨细胞形态,呈多角形;12 h组略呈不规则形;24 h组和48 h组呈梭形改变。实时PCR显示24 h组和48 h组中Ⅱ型胶原、转录因子SOX-9及蛋白多糖的表达量降低;自噬相关基因LC3和Beclin-1表达量呈时间依赖性增加。单丹磺酰戊二胺染色显示24 h组和48 h组自噬发生率呈时间依赖性增加。MTT结果显示细胞存活率呈降低趋势;添加3-甲基腺嘌呤刺激后细胞活性减弱、存活率降低。结论 间歇循环张力刺激下终板软骨细胞表型逐渐丧失;自噬相关基因LC3与Beclin-1表达明显上调,但细胞活性降低;抑制自噬水平可降低细胞存活率,提示自噬参与了间歇循环张力诱导的终板软骨细胞退变过程。     

羧甲基壳聚糖抑制白细胞介素-1β诱导的软骨细胞凋亡及作用机制的研究

目的评价羧甲基壳聚糖对重组人白细胞介素(rhIL)-1β刺激下软骨细胞凋亡的影响,并探讨其作用机制。方法体外培养兔软骨细胞,加入不同浓度羧甲基壳聚糖预处理1h后,加入rhIL-1β共培养24h,通过Annexin V-FITC和PI双标及Hoechst 33342荧光染色检测软骨细胞凋亡；以罗丹明-123荧光染色检测线粒体膜电位和荧光素酶反应检测线粒体合成ATP能力来评价线粒体功能。结果羧甲基壳聚糖能明显抑制IL-1β诱导的软骨细胞凋亡,呈剂量依赖性；羧甲基壳聚糖可恢复IL-1β对软骨细胞线粒体功能的损害。结论羧甲基壳聚糖通过保护线粒体功能抑制IL-1β诱导的软骨细胞凋亡。     

Senkyunolide A inhibits the progression of osteoarthritis by inhibiting the NLRP3 signalling pathway

ContextOsteoarthritis (OA) is a degenerative disease. Senkyunolide A (SenA) is an important phthalide from Ligusticum chuanxiong Hort (Umbelliferae) with anti-spasmodic and neuroprotective effects.ObjectiveWe explored the effect of SenA on IL-1β-stimulated chondrocytes and OA miceMaterials and methodsChondrocytes were stimulated by IL-1β (10 ng/mL) to establish an OA model in vitro. Cells were treated with SenA (20, 40, 80 and 160 μg/mL) for 48 h. The in vivo OA model was established by cutting off the medial meniscus tibial ligament (MMTL) at right knee incision of male C57BL/6 mice. One week after surgery, mice were injected with SenA (intraperitoneally one week) and divided into four groups (n = 6 per group): Sham, OA, OA + SenA 20 mg/kg and OA + SenA 40 mg/kg. The OA progression was examined by haematoxylin and eosin (H&E) staining.ResultsSenA treatment increased cell viability (33%), proliferation (71%), inhibited apoptosis (21%), decreased levels of catabolic marker proteins (MMP13, 23%; ADAMTS4, 31%; ADAMTS5, 19%), increased levels of anabolic marker proteins (IGF-1, 57%; aggrecan, 75%; Col2a1, 48%), reduced levels of inflammation cytokines (TNF-α, 31%; IL-6, 19%; IL-18, 20%) and decreased levels of NLRP3 (21%), ASC (20%) and caspase-1 (29%) of chondrocytes. However, NLRP3 agonist nigericin increased levels of MMP13 (55%), ADAMTS4 (70%), ADAMTS5 (53%), decreased levels of IGF-1 (36%), aggrecan (26%), Col2a1 (25%), inhibited proliferation (61%) and promoted apoptosis (76%).Discussion and conclusionsSenA alleviates OA progression by inhibiting NLRP3 signalling pathways. These findings provide an experimental basis for the clinical application of drugs in the treatment of OA.