大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.     

全髋关节置换术治疗强直性脊柱炎合并髋关节骨性融合的中期随访研究CSCD

目的:评价全髋关节置换术(THA)治疗合并髋关节骨性融合的强直性脊柱炎(AS)患者的中期随访结果。方法:回顾性分析2003年1月至2020年9月因AS合并单侧或双侧髋关节受累而行THA的431例(627髋)患者的临床资料。根据术前骨盆正位X线片判定是否存在髋关节骨性融合,并将患者分成骨性融合组137例(218髋)和非骨性融合组294例(409髋)。收集并比较两组患者的基线资料,包括人口学资料、疾病相关数据、实验室检查结果及手术相关数据。结果:全部患者获得103(61,146)个月随访。围手术期并发症方面,骨性融合组包括术中假体周围骨折10例、坐骨神经损伤2例、脱位2例及术后关节弹响2例,非骨性融合组包括术中假体周围骨折12例、坐骨神经损伤2例、假体周围感染2例、术后关节弹响3例,两组总的并发症发生率差异无统计学意义(P=0.239)。临床随访结果显示,骨性融合组患者Harris髋关节评分(HHS)从术前33.0(17.5,44.0)分提高至末次随访时的86.0(78.0,94.0)分(P<0.001),但显著低于非骨性融合组(P<0.001)。骨性融合组的巴氏AS疾病活动度指数(BASDAI)和巴氏AS功能指数(BASFI)均显著高于非骨性融合组(P均<0.001);术后穿鞋袜困难程度显著高于非骨性融合组(P=0.035)。结论:AS合并髋关节骨性融合患者进行THA重建股骨近端存在多种并发症风险,总体临床疗效逊于非骨性融合患者,但整体疗效值得肯定。     

大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.     

不同种植密度对大鼠脂肪间充质干细胞三维培养下成软骨能力的影响

背景：软骨组织工程要求植入的软骨细胞在三维支架材料中能够合成与软骨相同的软骨基质，而植入密度是成功的关键点之一。 目的：探讨壳聚糖-胶原-硫酸软骨素支架上不同种植密度对大鼠脂肪间充质干细胞成软骨能力的影响。 设计、时间及地点：细胞-支架学体外观察，于2007-11/2008-07在中国医科大学细胞生物实验室完成。 材料：清洁级雄性SD大鼠6只，由中国医科大学实验动物中心提供。 方法：室温下以乙酸为溶剂的5 g/LⅠ型胶原溶液与20 g/L壳聚糖按7∶3体积比置于预冷的模具中混合，冷冻干燥后将支架切成5 mm×5 mm×2 mm，浸入含20 g/L硫酸软骨素的乙醇中室温交联，双蒸水冲洗至中性，再次冷冻干燥即为壳聚糖-胶原-硫酸软骨素支架。切取大鼠腹股沟脂肪组织，通过胰蛋白酶和Ⅰ型胶原酶消化后得到脂肪间充质干细胞。将制备的壳聚糖-胶原-硫酸软骨素支架分为3组，分别调整第3代细胞密度为2×109 L-1，2×1010 L-1，2×1011 L-1，吸取细胞悬液50 μL均匀的种植于每个支架上，加入成软骨诱导培养基培养3周。 主要观察指标：取样制成切片进行苏木精-伊红染色、Ⅱ型胶原免疫组化染色，RT-PCR检测软骨特异性基因的表达。 结果：诱导培养3周后，各组细胞在支架中生长黏附良好，且高种植密度2×1011 L-1组细胞排列紧密，有较多的基质形成，并有软骨陷窝样结构；各组Ⅱ型胶原均呈阳性表达，并随细胞种植密度的升高呈递增趋势。RT-PCR结果显示，随着细胞种植密度的升高，蛋白聚糖、Ⅱ型胶原mRNA的表达逐渐增强，而Ⅹ型胶原mRNA的表达逐渐下降。 结论：壳聚糖-胶原-硫酸软骨素复合支架材料可为脂肪间充质干细胞生长分化及组织形成提供一个良好的环境，2×1011 L-1高密度种植有利于脂肪间充质干细胞的成软骨分化。     

大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.