Cartilage repair: generations of autologous chondrocyte transplantation

Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation.     

Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?: a morphological and molecular study of OCD fragments

Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in “bundles” and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae close to mineralized “islands” in the cartilage and hypertrophic chondrocytes containing accumulated matrix proteins. Immunostaining revealed: (1) that types I, II, VI and X collagens and aggrecans were deposited intracellulary and (2) co‐localization within the islands of types I, II, X collagens and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non‐conclusive, only single nucleotide polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification.     

应力环境对组织培养法保存的人骨软骨移植物的影响

目的 研究应力环境对普通组织培养基体外保存的骨软骨移植物的保存质量的影响,从而改进组织培养法保存软骨的效果.方法用摇床作为动态加载装置对体外保存的骨软骨移植物进行持续周期性应力加载,以模拟体内关节软骨的应力环境.摇床在37℃的孵箱中工作,频率设置为1 Hz.在储存2周时,用Western bloting的方法对关节软骨中的肌动蛋白β-actin进行半定量分析,用透射电镜观察胶原的超微结构,并与对照组比较.结果与静态环境下保存相比,动态加载环境使关节软骨细胞外基质中β-actin的表达水平明显提高,并能更好地保护基质中胶原成分的超微结构.结论应力环境有利于保存人同种异体移植物软骨细胞和胶原的超微结构.动态力学加载可提供一种更好的库存人同种异体骨软骨移植物的方法.

Abstract：

Objective To study the effect of the loading environment on the chondrocytes and the collagen ultrastructure at articular cartilage of the osteochondral allograft graft preserved by tissue culture method so as to improve the preservation effect. Methods A kind of shaking table was used as a dynamic loading device for storage of the osteochondral allograft to simulate the mechanical environment of normal articular cartilage in vivo. The device worked in an incubator at 3711 and the frequency was 1Hz. Western blotting was used for analysis of β-actin at the articular cartilage of the graft for investigating the effects of stress condition on the chondrocyte function. Transmission electron microscopy was used to observe the collagen ultrastructure at the articular cartilage of the graft two weeks after storage and compared with the control group. Results The dynamic loading condition could significantly increase the expression of β-actin and keep the ultrastructure of the collagen fibril in articular cartilage compared with the static condition. Conclusions The loading condition benefits the chondrocyte function and collagen ultrastructure of the articular cartilage in human osteochondral allograft. The dynamic mechanical loading may provide a better method in preservation of the human osteochondral allograft.     

Three-year clinical outcome after chondrocyte transplantation using a hyaluronan matrix for cartilage repair

Repair of articular cartilage represents a significant clinical problem and although various new techniques - including the use of autologous chondrocytes - have been developed within the last century the clinical efficacy of these procedures is still discussed controversially. Although autologous chondrocyte transplantation (ACT) has been widely used with success, it has several inherent limitations, including its invasive nature and problems related to the use of the periosteal flap. To overcome these problems autologous chondrocytes transplantation combined with the use of biodegradable scaffolds has received wide attention. Among these, a hyaluronan-based scaffold has been found useful for inducing hyaline cartilage regeneration. In the present study, we have investigated the mid-term efficacy and safety of Hyalograft C grafts in a group of 36 patients undergoing surgery for chronic cartilage lesions of the knee. Clinical Outcome was assessed prospectively before and at 12, 24, and 36 months after surgery. No major adverse events have been reported during the 3-year follow-up. Significant improvements of the evaluated scores were observed (P < 0.02) at 1 year and a continued increase of clinical performance was evident at 2 and 3 years follow-up. Patients under 30 years of age with single lesions showed statistically significant improvements at all follow-up visits compared to those over 30 with multiple defects (P < 0.01). Hyalograft C compares favorably with classic ACT and is particularly indicated in younger patients with single lesions. The graft can be implanted through a miniarthrotomy and needs no additional fixation with sutures except optional fibrin gluing at the defect borders. These results suggest that Hyalograft C is a valid alternative to ACT.     

脐带全层源间充质干细胞分离培养及向成软骨细胞分化的实验研究

目的从人脐带全层中分离培养间充质干细胞(MSCs),并进行成软骨诱导分化,为组织工程软骨和软骨损伤后修复提供种子细胞。方法采用胶原酶消化法从脐带全层中分离培养间充质干细胞,显微镜下观察细胞形态,细胞计数法绘制细胞生长曲线,采用流式细胞仪检测细胞周期及细胞表型,采用微团细胞培养在软骨诱导液中向软骨细胞分化,阿尔辛蓝及甲苯胺蓝染色检测细胞分化情况,RT-PCR法检测诱导后细胞表达聚集蛋白聚糖(ACAN)基因情况。结果人脐带全层来源的MSCs呈成纤维样形态漩涡状贴壁生长,细胞高表达HLA-I类分子、CD73、CD90、CD166及CD105,不表达CD34、CD45、CD14、CD31、CD80、CD86及HLA-DR。细胞诱导分化21d后,阿尔辛兰及甲苯胺蓝染色阳性;RT-PCR检测诱导后的细胞表达ACAN,而对照组无表达。结论人脐带全层为成体MSCs提供一种新而方便的来源,人脐带间充质干细胞(hucMSCs)体外培养能够向软骨细胞分化。     

软骨细胞在微载体中的培养和快速扩增

目的：探索在短期内获得大量成活率高、分化良好的兔关节软骨细胞的方法,方法：应用胰蛋白酶、胶酶消化的方法从新生新西兰兔关节软骨处分离、培养软骨细胞,并将获得的软骨细胞在旋转生物反应应（RCCS）内应用Cytodex-3微载体进行培养。应用倒置显微镜和扫描电镜对微载体表面的软骨细胞进行动态观察,并对收获的软骨细胞进行Ⅰ、Ⅱ型胶原的细胞免疫化学染色分析。结果：并节软骨细胞可快速贴附于Cytodex-3微载体表面,细胞伸展后生长加速,到培养后期,细胞密度可达最初接种的20倍。在微载体上收获的软骨细胞Ⅰ型胶原的免疫细胞化学染色呈阴性,Ⅱ型胶原染色则呈强阳性。结论：微载体细胞培养技术是一种简便、快速的体外细胞扩增方法,可为构体建组织工程化人工软骨提供大量软骨软件。     

Autologe Chondrozytentransplantation zur Behandlung von Knorpeldefekten des Kniegelenks

BACKGROUND: Currently the use of autologous chondrocytes as a cartilage-repair procedure for the repair of injured articular cartilage of the knee joint, is recommended. METHODS: This review presents the technique of autologous chondrocyte transplantation (ACT) and their modifications as matrix-associated autologous chondrocyte transplantation (MACT). Beside the surgical procedure the experimental and clinical results are discussed. Furthermore the major complications and the indication guidelines are presented. RESULTS: Articular cartilage in adults has a poor ability to self-repair after a substantial injury. Surgical therapeutic efforts in treating cartilage defects have focused on bringing new cells capable of chondrogenesis into the lesions. With ACT good to excellent clinical results are seen in isolated posttraumatic lesions of the knee joint in the younger patient with the formation of hyaline-like repair tissue. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. The current limitations include osteoarthritic defects and higher patient age. CONCLUSION: With the right indication and operative technique ACT is an effective and save option for the treatment of large full thickness cartilage defect of the knee joint.     

Chondrogenesis in a hyaluronic acid scaffold: comparison between chondrocytes and MSC from bone marrow and adipose tissue

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3 weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.     

不同细胞移植修复兔全层关节软骨缺损的比较研究

目的 :比较软骨细胞、骨髓基质细胞及成纤维细胞对全层关节软骨缺损的修复作用。材料和方法 :取幼兔的软骨细胞、骨髓基质细胞及成纤维细胞 ,共 3种有生成软骨潜力的细胞进行体外分离培养 ;以聚乳酸 (PLA)为载体 ,将培养的原代细胞植入PLA支架上 ,形成细胞 -PLA复合物。于 2 8只成年新西兰大白兔的股骨滑车关节面上造成直径 4 5mm、深 3 0mm的全层关节软骨缺损 ,将 3种细胞 -PLA复合物分别植入关节软骨缺损处。植入细胞 -PLA复合物为实验组 ,单纯植入PLA支架为对照组。术后 6周、12周观察缺损修复情况及新生组织类型。结果 :软骨细胞移植组为软骨样组织修复 ,分界明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨部分重建 ;细胞排列紊乱。骨髓基质细胞移植组为软骨样组织修复 ,分界不明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨重建良好 ,软骨下潮线恢复 ;细胞排列趋于正常。成纤维细胞移植组为纤维组织修复 ,甲苯胺兰及Ⅱ型胶原染色阴性 ;软骨下潮线消失。对照组为纤维组织修复。结论 :软骨细胞、骨髓基质细胞移植修复软骨缺损明显优于成纤维细胞及对照组。骨髓基质细胞与软骨细胞移植组的修复结果无统计学差异 ,但骨髓基质细胞修复组织的细胞排列有序 ,软骨下骨重建良好 ,与周围组织融合密切 ,更接近正?     

转染转化生长因子-β3对前软骨干细胞增殖及向成软骨方向分化的影响

目的 探讨稳定表达重组转化生长因子-β3(hTGF-β3)对前软骨干细胞(precartilaginous stem cells,PSCs)增殖及向成软骨方向定向分化的诱导作用.方法 免疫磁珠法分离纯化得到新生大鼠PSCs后,用线性化的聚乙烯亚胺转染pcDNA3.1(+)-hTGF-β3到体外单层培养的PSCs中,抗生素筛选使其稳定表达.采用四甲基偶氮唑盐比色法(MTT法)、流式细胞术(FCM)测定转染对PSCs增殖和DNA合成的影响,实时定量RT-PCR、免疫组化及Western blot方法比较转染后目的基因和软骨特异性标志物表达的情况.结果 hTGF-β3在分离纯化的PSCs中稳定表达.与未转染组细胞相比,转染后细胞DNA合成增多,增殖速度加快.PCR和免疫组化学结果表明,转染后软骨标志物基因上调明显,分泌软骨多糖基质及Ⅱ型胶原蛋白也明显增加.结论 通过hTGF-β3基因强化的PSCs可以稳定表达高效的hTGF-β3蛋白,从而促进PSCs的增殖并向成软骨方向分化,为软骨缺损的高效修复提供了新的思路.     

聚乙醇酸负载同种异体软骨细胞移植修复兔关节软骨缺损

目的：应用聚乙醇酸（ＰＧＡ）负载的兔软骨细胞培养移植修复同种异体关节软骨缺损．方法：应用在生物体内可降解吸收、纤维状多孔态的ＰＧＡ作为支架行兔软骨细胞培养．培养１４天后，软骨细胞在ＰＧＡ提供的三维空间中大量分裂、增殖并合成大量软骨基质，形成ＰＧＡ－软骨细胞复合体，然后利用该复合体移植修复同种异体兔膝关节全层软骨缺损，对侧膝关节作对照．术后行大体、组织学、电镜动态观察及修复组织厚度测定．结果：ＰＧＡ在术后８周完全降解吸收，实验侧与对照侧修复组织的厚度有显著性差异（Ｐ＜０．０１）；术后１６周在实验侧可见典型的软骨组织，电镜下为成熟的软骨细胞，而对照侧为纤维组织修复．结论：应用ＰＧＡ－软骨细胞复合体移植，可修复同种异体的兔关节软骨缺损，为临床治疗关节软骨缺损奠定了基础．     

Early postoperative adherence of matrix-induced autologous chondrocyte implantation for the treatment of full-thickness cartilage defects of the femoral condyle

Matrix-induced autologous chondrocyte implantation (MACI) is a tissue-engineering technique for the treatment of full-thickness articular cartilage defects and requires the use of a three-dimensional collagen type I–III membrane seeded with cultured autologous chondrocytes. The cell-scaffold construct is implanted in the debrided cartilage defect and fixed only with fibrin glue, with no periosteal cover or further surgical fixation. In a clinical pilot study, the MACI technique was used for the treatment of full-thickness, weight-bearing chondral defects of the femoral condyle in 16 patients. All patients were followed prospectively and the early postoperative attachment rate, 34.7 days (range: 22–47) after the scaffold implantation, was determined. With the use of high-resolution magnetic resonance imaging (MRI), the transplant was graded as completely attached, partially attached, or detached. In 14 of 16 patients (87.5%), a completely-attached graft was found, and the cartilage defect site was totally covered by the implanted scaffold and repair tissue. In one patient (6.25%), a partial attachment occurred with partial filling of the chondral defect. A complete detachment of the graft was found in one patient (6.25%), which resulted in an empty defect site with exposure of the subchondral bone. Interobserver variability for the MRI grading of the transplants showed substantial agreement (=0.775) and perfect agreement (_w=0.99). In conclusion, the implantation and fixation of a cell-scaffold construct in a deep cartilage defect of the femoral condyle with fibrin glue and with no further surgical fixation leads to a high attachment rate 34.7 days after the implantation, as determined with high resolution MRI.     

不同年龄阶段家兔髂软骨组织学特点

目的:观察比较不同年龄阶段新西兰大白兔髂软骨组织学形态异同。方法:选取1月龄、3月龄、4月龄、6月龄、12月龄的新西兰大白兔各3只。取其髂软骨及少量软骨下骨做HE染色、甲苯胺蓝染色及I型、II型胶原免疫组织化学染色;同时取1月龄、3月龄和12月龄组膝关节股骨滑车软骨做相同染色用于对比。结果:①幼年期(1月龄),髂软骨全部由透明软骨组成,随着年龄的增长,4月龄部分表层软骨细胞逐渐肥大、骨化,至成年期全层软骨细胞几乎完全骨化。甲苯胺蓝染色、II型胶原免疫组化染色均显示由幼年时的阳性逐渐减弱为成年时的弱阳性。I型胶原只在骨化区、软骨下骨及肌腱部位着色阳性。②随着年龄增长,关节软骨细胞密度逐渐下降,软骨层厚度逐渐变薄。细胞肥大及骨化发生在钙化层。结论:家兔幼年期髂软骨类似关节透明软骨,随着年龄增长髂软骨细胞逐渐肥大、骨化,至成年期仍有少量透明软骨细胞特有的II型胶原蛋白及蛋白多糖的合成。     

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies

Purpose

To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide^®) scaffold.

Methods

By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water–dioxane–PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT–PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide^® scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O’Driscoll score.

Results

In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide^® scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide^® scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells.

Conclusion

The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide^® scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide^® scaffold with cells.     

Cartilage repair with chondrocytes in fibrin hydrogel and MPEG polylactide scaffold: an in vivo study in goats

Polylactic acid polymers have been used extensively as biomaterials and have shown promising properties for cartilage tissue engineering. Numerous scaffold materials exist and the optimal scaffold needs to be identified. We have tried to assess the possibilities for cartilage repair by the use of two different scaffold techniques; autologous chondrocytes in a fibrin hydrogel and a novel MPEG-PLGA scaffold, where autologous chondrocytes are immobilized within the MPEG-PLGA scaffold by a fibrin hydrogel. Twenty adult goats were used for the study. A 6 mm circular full-thickness cartilage defect was created in both medial femoral condyles. The defects were randomized to the following four treatment groups. (1) Empty defect (control). (2) Subchondral drilling (control). (3) Fibrin hydrogel with autologous chondrocytes. (4) Fibrin hydrogel/chondrocyte solution in a MPEG-PLGA porous scaffold. Animals were followed for 4 month. Eight defects in each treatment group completed the study. ICRS macroscopic scoring (0-12). Indentation test was performed to assess stiffness of repair tissue. Histological analyses was performed using O'Driscoll and Pineda cartilage scores as well as percentage tissue filling of the defects. The MPEG-PLGA/chondrocytes scaffold was the superior treatment modality based on the macroscopic surface score, histological scores and defect filling. The mechanical test demonstrated no difference between treatment groups. The MPEG-PLGA/chondrocyte composite demonstrated significantly better cartilage repair response than empty defects, osteochondral drilling and fibrin hydrogel with chondrocytes. The novel MPEG-PLGA scaffold in combination with chondrocytes need further studies with respect to longer follow-up times.     

Distal realignment and patellar autologous chondrocyte implantation: mid-term results in a selected population

The aim of this prospective observational study was to assess the 3-year clinical outcome of distal realignment and membrane-seeded autologous chondrocyte implantation (MACI^®) in selected patients with patellofemoral malalignment and large, isolated, patellar cartilage lesions. Twelve patients (14 knees; 6 females, 6 males; mean age 31 years) with Fulkerson type II patellofemoral malalignment (lateralized and tilted patella) and Outerbridge grade III–IV isolated patellar cartilage lesions were treated. All had tibial tuberosity and trochlear sulcus >20 mm on a preoperative CT scan and a cartilage defect >3 cm². Patients with Outerbridge grade III–IV trochlear cartilage lesions, those with rheumatic, infective or neoplastic conditions, or ligament instability, diabetes or obesity and those aged >40 years were excluded. Follow-up was at 36 months. Patients were enrolled after diagnostic arthroscopy. Cartilage was harvested and sent for culture. After a mean period of 30 days (range 25–40) patients underwent transfer of the tibial tuberosity according to Fulkerson associated with a MACI procedure. Clinical assessment was performed with the Kujala, Lysholm, Tegner and Modified Cincinnati scores. The Patient Satisfaction Survey was administered at 36 months. Consistently improved knee function and activity levels were reflected by significantly increased Kujala, Lysholm, Tegner and Modified Cincinnati scores at 36 months. The significant clinical improvement support the value of associating distal realignment and autologous chondrocyte implantation in treating large, isolated, patellar cartilage lesions associated with patellofemoral malalignment.     

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

兔膝内侧副韧带修复及内侧半月板切除对膝骨关节病发病过程的影响

通过经典的建立膝骨关节病运动模型的方式，设计了在切除内侧半月板的情况下，将内侧副韧带（ＭＣＬ）切断和切断后再缝合恢复张力的两组动物模型。观察术后５、１０、１５、２０天，关节软骨的组织学、组织化学及扫描电镜下形态学方面的变化，旨在探讨切除内恻半月板时，恢复ＭＣＬ的张力对膝骨关节病（ＯＡ）发病过程的影响。实验结果显示，ＭＣＬ切断无张力组和ＭＣＬ缝合有张力组于术后５天，在光镜下，关节软骨即出现某此变化。包括敕骨细胞排列紊乱，增生层有软骨细胞簇集现象、族集细胞的周围甲苯胺兰深染。增生层和肥大层一些软骨陷窝有细胞消失现象，其周围淡染。术后５、１０天扫描电镜观察，两实验组关节软骨表面未见异常改变。术后１５、２０天，光镜下，两实验组关节软骨退行性改变进一步加重，扫描电镜观察，两实验组软骨表面正常结构消失、胶原纤维断裂，软骨显微骨折、出现部分软骨细胞裸露及细胞消失现象。韧带无张力组及有张力组，在软骨退行性变化方面未见明显程度上的差别，结果表明，只要兔半月板切除，无论ＭＣＬ张力存在与否，都不能阻止ＯＡ的产生，亦不能减轻其发生的程度。     

应用生物反应器体外培养骨软骨复合体修复犬关节软骨损伤

目的 探讨应用灌注型生物反应器体外培养骨软骨复合体修复关节软骨损伤的可行性. 方法 体外分离犬骨髓间充质干细胞(MSCs),流式细胞仪鉴定.经过生长因子诱导生成软骨细胞和成骨细胞后,免疫组化及碱性磷酸酶等染色鉴定.将软骨细胞、成骨细胞接种到三维多孔β-磷酸三钙(β-TCP)陶瓷支架材料上,置于灌注型生物反应器中复合培养21 d,构建骨软骨复合体,扫描电镜观察细胞在支架材料上的黏附、伸展和增殖情况,并模仿马赛克骨软骨移植术用塑形良好的骨软骨复合体修复犬软骨缺损,切片染色观察其修复情况. 结果 扫描电镜显示软骨细胞、成骨细胞在β-TCP支架上的黏附、伸展和增殖良好,实验组缺损区软骨厚度与正常软骨组织接近.实验组与阴性时照组比较,差异有统计学意义(q=12.337 0,P＜0.01);与空白对照组比较,差异有统计学意义(q=31.5393,P＜0.01). 结论 灌注型生物反应器使软骨和成骨细胞在三维载体内存活并增殖,提高细胞在载体内的复合效率.     

雌激素对软骨细胞胶原表型表达的影响

目的 :观察雌激素对体外培养兔关节软骨细胞胶原表型表达的影响。方法 :体外培养雌兔关节软骨细胞 ,随机分为A、B两组 ,A组中加入 1 7β -雌二醇 0mol/L、1 0 - 6 mol/L、1 0 - 7mol/L、1 0 - 8mol/L、1 0 - 9mol/L、1 0 - 10 mol/L、1 0 - 11mol/L、1 0 - 12 mol/L干预 72小时 ;B组先用 1 0ng/mlIL - 1 β干预 2 4小时 ,随后分别加入 0mol/L、1 0 - 6 mol/L～ 1 0 - 12 mol/L 1 7β -雌二醇作用 72小时。采用RT-PCR方法观察软骨细胞Ⅰ、Ⅱ、Ⅲ型胶原表达。结果 :1 0 - 6 mol/L雌二醇或单用IL - 1 β均抑制正常软骨细胞Ⅱ型胶原mRNA表达 ,低浓度雌二醇 (1 0 - 11、1 0 - 12 mol/L)能够对抗IL - 1 β的抑制作用 ;所有软骨细胞均未表达Ⅲ型胶原mRNA ;几乎所有浓度雌二醇 (1 0 - 12 mol/L除外 )均诱导软骨细胞表达Ⅰ型胶原。结论 :雌激素对关节软骨细胞胶原表型表达的调控作用随其浓度变化而不同。对变性软骨细胞而言 ,低于生理浓度的雌激素 (1 0 - 12 mol/L)对维持其胶原表型最适宜。