应力环境下三维诱导组织工程种植细胞修复关节软骨缺损

目的:三维立体诱导非软骨来源种植细胞修复关节软骨损伤.方法:分离培养骨髓间充质干细胞,分组诱导,进行周期性应力刺激,设立对照.2周后,观测大体、组织学形态、生化指标,筛选最佳诱导方法修复关节软骨缺损模型,分别于12、24周取材,进行组织学观察及评分,验证长期修复效果.结果:与二维培养各组相反,立体培养各组出现明显软骨分化,并以凝胶微球悬浮细胞进行应力刺激培养组效果最佳.术后动物肢体功能优良,12周后软骨缺损被完整修复,表面光滑,质地坚硬,为透明软骨结构,与周围软骨结合紧密;24周后仍然保持透明软骨结构,组织学评分无变化.对照各组软骨缺损均未被修复.结论:立体软骨诱导优于平面诱导,应力环境提高诱导质量,获得的种植细胞可取得稳定的长期修复效果.     

脱细胞软骨支架材料修复兔关节软骨缺损

目的 观察异种异体脱细胞软骨支架材料(ACM)复合同种异体兔骨髓间充质干细胞(rBMSCs)修复兔股骨内髁关节软骨缺损的效果.方法 (1)密度梯度离心和差速贴壁法获得原代兔BMSCs,选择第3代BMSCs作为种子细胞;(2)利用冷冻干燥、胰酶消化和化学去垢剂等方法制备脱细胞软骨支架材料;(3)3个月龄新西兰兔股骨内髁制备直径4 mm,深3 mm砌关节软骨缺损模型,24只新西兰兔以2个时间段随机分为3组,Ⅰ ACM-BMSCs组:第3代BMSCs 1×106个/ml与ACM于37℃5%CO2饱和湿度复合48 h;Ⅱ ACM组;Ⅲ空白对照组.(4)移植6、12周后大体及组织学观察,免疫组织化学染色观察修复组织Ⅱ型胶原,Wakitani评分评估修复效果.结果 (1)大体观察及组织学观察:6和12周Ⅰ组再生组织与正常关节软骨面平齐,修复部位表面较平整,界限模糊,接近正常软骨.Ⅱ组修复组织表面不平整并有明显下陷,修复组织全层可见成纤维样细胞,深层可见极少数透明软骨样细胞.Ⅲ组未见明显修复,肉芽组织形成伴成纤维样细胞增生;(2)Wakitani组织学评分可见在不同的时间段I组和Ⅱ组均低于Ⅲ组,差异有统计学意义(P<0.05),Ⅰ组和Ⅱ组间组织学评分差异无统计学意义(P>0.05).(3)免疫组织化学:ACM-BMSCs组修复组织的细胞为软骨样细胞,可见柱状排列,周围软骨基质Ⅱ型胶原染色阳性.结论 以ACM为支架材料,同种异体BMSCs为种子细胞制备的组织工程化软骨对兔股骨内髁关节软骨缺损有修复作用,形成的新生软骨为透明软骨样组织.     

组织工程方法修复关节软骨缺损

骨性关节炎或外伤等造成软骨丢失而引起的关节疼痛是中老人残疾的重要原因.关节软骨自身修复能力十分有限,因此,关节软骨损伤通常会导致更严重的关节软骨退变[1,2].骨科医生们采用软骨下骨钻孔、微骨折、软骨移植、自体软骨细胞移植等方法来修复和重建关节软骨,许多技术已被广泛应用于临床,并且取得了良好的短期随访结果.组织工程学科的兴起为关节软骨修复提供了新的选择,其基本原理为体外培养扩增的细胞结合基质材料构建出新的软骨组组织以供移植.本文就关节软骨修复的现状,尤其是组织工程方法重建软骨的方法做一综述.     

"双相"组织工程软骨修复兔关节骨软骨缺损

目的探讨“双相”异体骨基质明胶（bonematrixgelatin,BMG）作为组织工程软骨载体,与同体骨髓间充质干细胞（marrowmesenchymalstemcells,MSCs）结合,构建组织工程软骨修复兔关节骨软骨缺损的效果。方法4月龄新西兰兔32只,雌雄不限,体重2～3kg。①体外实验：取5只新西兰兔,处死后取髂骨和四肢骨,制备一侧松质骨,一侧皮质骨的“双相”异体BMG载体,扫描电镜观察。另取新西兰兔18只,抽取骨髓,分离MSCs并诱导成软骨分化;将诱导而来的软骨前体细胞与“双相”BMG载体复合构建组织工程软骨,分别于1、3和5周取材行Masson、PAS染色和扫描电镜观察。②体内实验：将抽取骨髓的18只及余下的9只新西兰兔制成双侧股骨内髁骨软骨缺损模型,将前期制备的组织工程软骨同体植入18只兔的右股骨内髁骨软骨缺损（A组）,左侧缺损移植异体BMG（B组）,其余9只双侧软骨缺损未予处理作为空白对照（C组）,分别于术后1、3和6个月取材,行大体、组织学和Ⅱ型胶原mRNA原位杂交观察,改良Wakitani法评分,比较各组修复效果差异。结果①体外实验：“双相”BMG松质骨面孔隙大小100-800μm,细胞于其中增生,形成富含细胞的软骨层;皮质骨面孔隙大小10～40pm,细胞层状覆盖于其表面,可作为起支撑作用的软骨下骨。②体内实验：A组术后1个月即可重建关节骨软骨缺损;修复软骨在观察期内逐渐变薄,但在6个月内始终保持关节面及软骨下骨结构完整。B、C组未能修复缺损,缺损周边软骨磨损加剧。改良Wakitani评分显示A组在3个时间点的各项评分结果,除6个月软骨厚度外,其它指标均优于B、C组,且差异有统计学意义（P〈0．01）。Ⅱ型胶原mRNA原位杂交显示,A组缺损区修复组织中细胞阳性染色率明显高于B、C组,且差异有统计学意义（P〈0．01）。结论“双相”异体BMG可作为组织工程软骨载体材料,其结合自体MSCs诱导的软骨前体细胞制备的组织工程软骨,可修复兔关节软骨和软骨下骨。     

骨髓间充质干细胞修复关节软骨缺损研究进展

关节软骨损伤在临床上很常见．由于关节软骨的自身修复能力很差．关节软骨一一旦损伤即很难修复．继而会发生不可逆的病理变化，造成关节的退行性改变．严重影响患者的生活质量。目前．关节软骨损伤临床上尚无有效的治疗方法。近年发展起来的一门新兴学科一组织工程学．通过利用少量的种子细胞经过体外培养扩增，并附着在一定的吏架材料上，移植到体内而形成新的有生命力的组织。骨髓闸充质干细胞（Bone Marrow—derived Mesenchymal Stem Cells，BMSOs）用作种子细胞具有增殖能力强．多向分化潜能（可以分化为骨、软骨、肌肉、脂肪等多种组织），而且具有采集方便、对机体损伤小的特点．是软骨组织工程理想的种子细胞。     

关节软骨组织工程修复的种子细胞

随着材料学、细胞生物学、工程学以及相关物理、化学学科的发展,一种新的关节软骨缺损的修复方法-组织工程逐渐被提出并应用,本文就应用软骨组织工程技术修复关节缺损中种子细胞的研究进展进行综述。     

同种异体组织工程化软骨修复关节软骨缺损

目的：探讨应用同种异体组织工程化软骨修复软骨缺损的可行性。方法：取新西兰大白兔双膝关节软骨细胞，经体外培养扩增，与PlruonicF127混合，植入人为造成的异体兔膝关节软骨缺损。结果：空白对照组和材料对照组只见少许纤维组织修复，缺损凹陷。实验组8周后关节软骨缺损区由部分白色透明样软骨组织充填，Masson三色染色见胶原分布较均匀，软骨陷窝多见，未见明显炎症现象，16周后缺损完全修复，缺损表面较光滑，部分颜色呈淡兰色，软骨陷窝清晰，细胞与基质分布均匀，未见炎症和退变现象，结论：同种异体组织工程化软骨可用于修复关节软骨缺损。     

关节软骨干/祖细胞治疗关节软骨缺损研究进展

关节软骨由于无血管、神经和淋巴管,损伤后自我修复能力极差。目前软骨损伤修复主要采用其他组织来源的间充质干细胞体外大量扩增或自体移植,然而体外扩增容易去分化,自体移植需要多次手术。理想的关节软骨修复要求新生软骨具有与原关节软骨相同的分层结构、生物特性及基质组分,并实现功能学恢复。激活内源性关节软骨干/祖细胞促进缺损部位自我修复或组织工程化软骨为软骨再生提供可能性。前期研究表明,关节软骨干/祖细胞在多个组织存在。该文对关节软骨干/祖细胞治疗关节软骨缺损研究进展作一综述。     

不同应力环境对兔骨髓间充质干细胞修复关节软骨缺损的影响

目的探讨不同应力环境对骨髓间充质干细胞(MSCs)修复关节软骨缺损的影响. 方法将日本大耳白兔15只制成髌骨外侧脱位动物模型,平均分成3组,每组5只:即单纯载体脱位组(对照组)、移植物正常应力组及移植物脱位组.对兔MSCs进行分离、培养,以兔MSCs为种子细胞构建自体组织工程移植物修复关节软骨缺损.6周后处死动物,观察修复组织的成分和结构. 结果术后6周,移植物正常应力组修复组织浅层为软骨组织,甲苯胺蓝染色接近正常关节软骨;深层为软骨下骨,与正常关节软骨结构相似.移植物脱位组为骨组织所修复,缺损周围的正常关节软骨变薄,软骨下血管侵入正常关节软骨内,遗留在股骨髁滑车槽内的移植物在滑车槽正常关节软骨表面形成新生类透明软骨组织.单纯载体脱位组为纤维组织修复. 结论 MSCs修复关节软骨缺损,只有在正常应力状态下修复效果最佳;提示维持负重关节正常的应力刺激,对组织工程软骨修复组织的形成和维持必不可少.     

关节软骨组织工程研究进展

组织工程技术为损伤关节软骨的修复提供了全新的治疗方法。本文就关节软骨组织工程的种子细胞、生长因子、细胞外基质支架及修复关节软骨缺损的研究进展作一综述。     

鹿茸多肽诱导自体骨髓间质干细胞移植修复兔膝关节软骨缺损

目的探讨经鹿茸多肽(PAP)诱导的兔自体骨髓间质干细胞(MSCs)复合胶原膜(MCMG)对兔膝关节局部全层软骨缺损的修复作用。方法新西兰大白兔随机分成A,B,C3组,A组以PAP诱导后的自体MSCs和MCMG修复软骨缺损;B组以经转化生长因子β1(TGF-β1)诱导后的自体MSCs和MCMG修复软骨缺损;C组以自体MSCs和MCMG修复软骨缺损。分别于术后2、4和8周取材进行大体、组织学及免疫组织化学染色观察,根据关节软骨组织学计分标准进行评分。结果对术后8周大体及各阶段组织学形态评分结果显示:A组与B组修复差别无统计学意义(P>0.05),而A、B组明显优于C组(P<0.05)。结论经PAP诱导的兔自体MSCs/MCMG支架复合物可促进软骨缺损的快速修复,恢复软骨组织的结构和功能。     

Articular Cartilage Repair in the Knee Joint with Autologous Chondrocytes and Periosteal Graft

Objective Repair of articular cartilage defects of knee to restore a pain-free joint function. Indications Full-thickness chondral or osteochondral posttraumatic lesions and osteochondritis dissecans defects that have not been successfully repaired with methods such as debridement, drilling, and microfracturing. Contraindications Osteoarthritis. Rheumatoid arthritis. Surgical Technique During arthroscopy, the cartilage lesion is evaluated, and cartilage slices weighing 200-300 mg are harvested from the upper medial femoral condyle, a minor load bearing area. The chondrocytes are isolated enzymatically and grown in culture to increase the cell number during approximately 2 weeks. During the second operation, an arthrotomy is performed through a medial or lateral parapatellar approach. The defect is carefully debrided. A periosteal patch is obtained from proximal tibia, placed over the defect and sutured to the surrounding cartilage. The suture line is sealed with fibrin glue, and the chondrocytes are injected into the defect under the patch. Results Recently, Peterson has presented results in 213 patients with a follow-up between 2-10 years. He reported good to excellent results in 90% of 57 patients with single femoral condyle lesions, in 84% of 32 patients with osteochondritis dissecans and in 74% of 27 patients with femoral condyle lesions in combination with anterior cruciate ligament reconstruction. In 32 patients the patella was grafted and 22 improved, in twelve patients the trochlea was grafted and seven improved. and in 53 patients multiple lesions were grafted and 42 improved. Second-look arthroscopies were performed in 46 patients, 26 of them were biopsied; the transplanted tissues showed a hyaline-like appearance in 21 patients (80%).     

转壁反应器三维立体培养多样本骨髓间充质干细胞实验研究

目的探讨微载体三维立体培养自体骨髓间充质干细胞(MSC)方法。方法制备明胶多孔微载体,反应器内培养多样本自体MSC,比较其与普通培养的差异,研究生长曲线、粘附曲线、活力变化、糖消耗情况、蛋白合成以及碱性成纤维细胞生长因子影响。结果相转化法制备直径180～300um/孔径40～70um微载体。在合适的培养参数下,三维培养显著优于普通培养,细胞增殖迅速,糖消耗较快,蛋白产量无显著差异,碱性成纤维细胞生长因子可抑制三维培养的细胞传代老化。结论相转化法可有效制备多孔微载体,微载体三维立体培养自体MSC是获取组织工程种子细胞的有效方案。     

Living Donor Kidney and Autologous Stem Cell Transplantation for Primary Systemic Amyloidosis (AL) with Predominant Renal Involvement

Primary systemic amyloidosis (AL) is characterized by multiorgan deposition of monoclonal immunoglobulin light chain. Renal involvement is common and impaired kidney function is associated with reduced median survival. Autologous stem cell transplantation (SCT) for AL achieves superior response rates compared to chemotherapy alone but patients with end-stage renal disease (ESRD) may be excluded from consideration. A treatment approach consisting of living donor kidney transplantation (LDKTx) followed by autologous SCT was developed for AL with ESRD. Eight patients underwent LDKTx with immediate graft function. Two suffered unanticipated complications post-KTx and died 10 and 3 months later. Two cases of subclinical acute cellular rejection (ACR) and one case of clinical ACR occurred--all reversible with corticosteroid. Six patients had successful stem cell harvests performed and five of these underwent SCT with satisfactory trilineage engraftment. Renal function remained stable following SCT in four and was reduced in one due to infectious and bleeding complications. One patient, who has thus far elected not to undergo SCT, has proteinuria and histologic evidence of recurrent renal amyloidosis. This experience supports the feasibility of sequential living donor KTx and autologous SCT for carefully selected patients with ESRD due to AL.     

海藻酸钠载体培养成年兔软骨细胞的生物学性状研究

目的探索以海藻酸钠为载体的成年兔软骨细胞构建工程化软骨的可行性.方法取32周龄新西兰兔膝关节软骨,酶消化法得到高纯度软骨细胞,与海藻酸钠混合,种植密度为4×106/ml,通过硅胶盘模制成圆形柱状细胞盘(20ul/个),CaCl2溶液中凝胶化10min,DMEM/F12无血清培养基加20%FBS(fatal blood solution)于24孔培养板中培养.于2、4、6、8、10、12周取细胞盘,行HE、AB-PAS染色及免疫组化分析,测定细胞盘中蛋白多糖含量,并作投射电镜观察.结果成年软骨细胞在海藻酸钠中呈丛状或球状增殖,4周时达增殖高峰,盘中Ⅱ型胶原及蛋白多糖的含量随培养时间延长逐渐增加,无I型胶原产生.电镜观察软骨细胞超微结构无异常改变.结论成年兔软骨细胞在海藻酸钠中可良好生长增殖,海藻酸钠可保留软骨细胞分泌的基质,成功构建工程化软骨.     

Mid‐ to Long‐Term Clinical Outcomes of Cartilage Restoration of Knee Joint with Allogenic Next‐Generation Matrix‐Induced Autologous Chondrocyte Implantation (MACI)

ObjectiveCartilage defect is a common pathology still lacking a unified treating option. The purpose of this retrospective study is to evaluate the safety, efficacy, and clinical and radiological outcome of cartilage restoration of knee joint with allogenic next‐generation Matrix‐Induced Autologous Chondrocyte Implantation (MACI) for the first time, as well as the correlation between postoperative clinical and radiological outcomes and preoperative patient history and demographics.MethodsFrom July 2014 to August 2020, 15 patients who went through cartilage restoration with allogenic next‐generation MACI were included in this study. Patient demographics and PROM including the International Knee Documentation Committee (IKDC) subjective knee score, Lysholm score, Tegner Activity Scale (TAS), and Knee Injury and Osteoarthritis Outcome Score (KOOS) were obtained preoperatively, at 3, 6, 12 months postoperatively and the last follow‐up using an online questionnaire platform. MOCART 2.0 score was calculated at the last follow‐up. Analysis of variance (ANOVA) was used to compare PROM pre‐ and post‐operation, with two‐tailed p < 0.05 defined as statistical significant. Pearson correlation coefficient was used to evaluate correlation between the PROM and MOCART 2.0 score at the last follow‐up with patients demorgraphics.ResultsAll patients were followed for an average of 66.47 ± 24.15 months (range, 21–93). All patients were satisfied with the outcome of the surgery and no complication was reported at the end of the study. No significant improvement was observed until 1 year after the implantation, except for IKDC score at 6 months. All PROM showed significant improvement 1 year post‐op except for Lysholm score and TAS, which also increased significantly at the time of the last follow‐up. Pearson correlation coefficient showed that the size of the defect, before or after debridement, was significantly negatively correlated with final KOOS‐Pain (before debridement: r = −0.57, p < 0.05; after debridement: r = −0.54, p < 0.05) and KOOS‐Symptoms score (before debridement: r = −0.66, p < 0.05; after debridement: r = −0.67, p < 0.05). The MOCART 2.0 score was found significantly and negatively correlated with BMI (r = −0.60, p < 0.05), and significantly and positively correlated with Lysholm score (r = 0.70, p < 0.05).ConclusionThe next generation MACI with autologous chondrocyte and allogenic chondrocyte ECM scaffold could be used to treat focal articular cartilage defect in the knee joint safely and efficiently with lasting promising outcomes for more than 5 years. The size of the defects should be considered the most negatively correlated parameters influencing the postoperative clinical outcomes.