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.     

胶原酶消化分离关节软骨种子细胞的优化获取

目的 探讨Ⅱ型胶原酶法消化分离关节软骨种子细胞的优化获取，以期保证获取关节软骨种子细胞的最佳生物活性。方法 采用不同浓度及不同消化时间的关节软骨细胞收获量、细胞存活率及细胞体外培养的生物活性为观察指标，探讨消化酶浓度及消化时间对于种子细胞获取量及细胞活性的影响。结果 种子细胞获取率与消化浓度无显著的线性相关关系；细胞活性在超过2．5h消化时间后显著减弱，部分细胞出现易老化现象；在0．15％消化酶浓度消化2．5h内所获取的种子细胞可达到满意的种子细胞获取率及良好的种子细胞生物活性。结论 适当浓度的胶原酶，在相应的时间内消化分离关节软骨种子细胞，或获取数量多及活性高的关节软骨组织工程种子细胞。     

负重关节与非负重关节软骨基因表达差异的初步研究

关节软骨覆盖于关节表面,具有较好的润滑性能且富有弹性,在吸收负重载荷的过程中起到重要作用。本团队收集了新鲜尸体的膝、肩关节软骨并进行转录组测序,发现负重关节与非负重关节软骨之间的基因表达存在差异。     

Comparison of gene expression patterns in articular cartilage and dedifferentiated articular chondrocytes

During monolayer culture, articular chondrocytes dedifferentiate into fibroblast‐like cells. The mechanisms underlying this process are poorly understood. We sought to further characterize dedifferentiation by identifying an extended panel of genes that distinguish articular cartilage from dedifferentiated chondrocytes. Thirty‐nine candidate marker‐genes were identified from previous studies on articular‐cartilage gene‐expression. Real‐time PCR was used to evaluate the mRNA levels for these candidates in calf articular cartilage and dedifferentiated articular chondrocytes. Twenty‐two of the candidate marker genes exhibited at least a two‐fold difference in gene expression in the two cell types. Twelve of these genes had at least a ten‐fold difference in gene expression. Tenascin C (TNC), type I collagen (COL1A1), and hypoxia‐inducible factor 1 alpha (HIF1α) showed the highest relative expression levels in dedifferentiated chonodrocytes. Type II collagen (COL2A1), type XI collagen (COL11A2), and superficial zone protein (SZP) showed the highest relative expression levels in articular cartilage. In contrast to previous findings, fibromodulin mRNA, and protein levels were higher in dedifferentiated chondrocytes. Compared to smaller subsets of markers, this panel of 12 highly differentially expressed genes may more precisely distinguish articular cartilage from dedifferentiated chondrocytes. Since many of the genes up‐regulated in dedifferentiated chondrocytes are also expressed during cartilage development, dedifferentiated chondrocytes may possess features of cartilage precursor cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:234–245, 2012     

Cryopreservation of intact human articular cartilage.

Damaged articular cartilage (AC) impairs joint function and many treatment techniques are being investigated to determine their long term results. Successful cryopreservation of AC can provide a reliable source of intact matrix with viable chondrocytes to maintain the cartilage over long periods of time. This study investigated the application of an established cryopreservation protocol to determine the recovery of intact chondrocytes from human AC. Ten millimeter diameter osteochondral dowels were harvested from two human donors. The cryopreservation protocol was performed and the samples were rapidly warmed from varying experimental holding temperatures (-10, -20, -30, -40 degrees C), with and without plunging into liquid nitrogen, using 1 M dimethyl sulfoxide as cryoprotectant. The cartilage was stained with membrane integrity dyes and viewed under fluorescence microscopy. The percent of intact chondrocytes was compared to fresh controls. Low recovery of intact chondrocytes was recorded from all temperature levels with and without cryoprotectant. The results of this experiment demonstrated that the cryopreservation procedure used to achieve moderate success with intact sheep AC was not successful with intact human AC and further investigation is required.     

A novel exogenous concentration-gradient collagen scaffold augments full-thickness articular cartilage repair

OBJECTIVES: A collagen scaffold has been long used in order to enhance the regeneration of articular cartilage. In the present study, we investigate the effectiveness of a concentration-gradient (CG) collagen that is designed to recruit efficiently the mesenchymal stem cells (MSCs) to the central region of the full-thickness cartilage defects via haptotaxis. METHODS: The present study used Cellmatrix((R)) (0.3% type I collagen; Nitta gelatin, Osaka, Japan) as the collagen material. We prepared 33%CG collagen gel and 50%CG collagen gel. No gradient collagen gel served as negative control. Full-thickness cartilage defects were created at the patella groove of the rabbit knee, to which the three different collagen gels were transplanted. Bromodeoxyuridine (BrdU) positive, proliferating cells were enumerated and localized, whereas the histological grading score for cartilage regeneration was counted. The expression of type I and type II collagens was evaluated by immunohistochemistry. We also confirmed that the MSCs migrate toward the collagen substrate of higher concentration in a stringently in vitro haptotactic manner. RESULTS: Enumeration of the BrdU-positive cells demonstrated that 33%CG collagen gel recruited a significantly larger number of proliferating cells to the central region of the cartilage defect. The histological grading score for the regenerated cartilage treated with 33%CG collagen gel was superior to the other groups. CONCLUSIONS: CG collagen scaffold recruits effectively the MSCs to the center of full-thickness cartilage defect and enhances regeneration of the full-thickness cartilage defect.     

生物力学在关节软骨修复中的作用

活动关节软骨是无血管的透明软骨,损伤后修复困难。传统的修复方式以手术为主,但修复的软骨组织常常无法满足透明软骨的结构条件。软骨组织工程是修复关节软骨的又一途径,在过去几十年,研究者们除了关注"细胞、支架、生长因子"3要素,也开始关注力学条件对构建组织工程软骨的作用。活动关节有复杂的力学性能,关节软骨、软骨基质和其中的细胞都受到不同强度、频率和不同方向的力学刺激,从而影响其功能和结构。在构建组织工程软骨的过程中,添加了力学刺激对软骨细胞的功能、间充质细胞的分化都有重要作用。何种力学条件最有利于构建具有类似天然透明软骨结构和功能的组织工程软骨是该研究领域的热点。     

A review of the differences between normal and osteoarthritis articular cartilage in human knee and ankle joints

BackgroundOsteoarthritis (OA) is the most common joint disease yet its pathophysiology is still poorly understood. It is more prevalent in some lower limb joints than others; in particular the knee is more commonly affected than the ankle. Research into articular cartilage and OA has primarily focussed on using animal models. However, it is apparent that articular cartilage differs between species, so more research is concentrating on human cartilage.ObjectiveThis paper reviews recent studies that have been undertaken to elucidate the reasons for this, and to discover if the findings would alter the conception that articular cartilage is not capable of repair.MethodPrimary research papers into human knee and ankle cartilage published since 1997 have been reviewed.ResultsDifferences in the structure, metabolism, physical properties and response to trauma have been found, implying that ankle cartilage may be more resistant to damage.ConclusionsMore research is needed before definitive conclusions can be reached, but the findings so far suggest that OA should not be accepted as the inevitable outcome of joint injury and individuals and practitioners, such as podiatrists, may be able to use simple measures to prevent or delay its onset.     

Factors influencing changes in articular cartilage following hemiarthroplasty in sheep.

This study examined the relationship between acetabular cartilage properties after hemiarthroplasty surgery and surgical variables including femoral head size and position. Nineteen sheep received unilateral hip arthroplasties and were euthanized one year post-operatively to harvest the femora and acetabula. Cartilage histology, biochemistry and material properties were determined from samples located in the superior load-bearing region. Femoral head size mismatch, leg length difference, anterior-posterior and medial lateral offset and anteversion were measured. In the acetabulum. substantial cartilage degradation occurred with widespread librillation and significant changes in the biochemical and material properties compared to the intact contralateral joint. Regression analyses on the surgical variables explained 75-80% of the changes in tissue biochemistry but did not explain the material changes. Head size mismatch and leg length difference were the most significant contributors of the five variables examined and therefore may be critical to successful outcome in hemiarthroplasty.     

组织工程软骨对兔膝关节胫骨平台浅层全关节软骨缺损的修复作用

目的探讨组织工程软骨对兔膝关节胫骨平台外侧髁浅层全关节软骨缺损的修复作用，并检测其修复组织的软骨类型。方法取2周龄乳兔软骨细胞体外培养传代至第3代后，与人胎盘Ⅰ型胶原蛋白海绵复合后植入成年兔的胫骨平台外侧髁完全软骨缺损区，并设立空白对照组，分别于术后4、12、24周取材。观察修复效果。运用天狼星红染色检测Ⅰ型胶原，Ⅱ型胶原单克隆抗体检测Ⅱ型胶原的表达。结果实验组术后4周缺损表面未见明显的新生软骨形成，组织学切片上少数几个呈上皮样生长的软骨细胞，苏木素-伊红（HE）染色胞浆呈深蓝色，周围软骨基质染色成浅蓝色；12周，缺损表面有少量的新生软骨形成，组织学切片上可见软骨细胞呈边缘不规则的，小蜂窝状结构，软骨细胞周围有软骨陷窝形成；24周，缺损表面的新生软骨较4、12周的新生软骨明显，表面光滑，且与周围组织结合紧密，但仍存在部分缺损尚未修复，组织学切片上可见软骨细胞呈边缘不规则的，多层细胞的蜂窝状结构，软骨细胞周围有软骨陷窝形成，并分泌甲苯胺蓝异染的软骨基质。而对照组则均未见明显的修复；随着术后时间的延长，Ⅰ型胶原的表达呈逐渐减弱的趋势，而Ⅱ型胶原的表达呈逐渐增强的趋势。结论该方法制成的组织工程软骨对兔胫骨平台外侧髁全层软骨缺损有修复作用，运用该方法不能完全修复兔胫骨平台外侧髁软骨完全缺损；形成的新生软骨为透明软骨样组织。     

组织工程化软骨修复兔膝关节软骨缺损的实验研究

目的 评估同种异体组织工程软骨修复兔膝关节全层软骨缺损的有效性。方法分离收集成年新西兰大白兔软骨细胞进行体外培养。建立双侧兔膝关节软骨缺损模型，用去端肽胶原（atelocollagen）凝胶与所培养的异体兔关节软骨细胞共同植入兔膝关节软骨缺损处，并设对照组。分别于手术后4周、8周观察大体标本以及组织学修复结果，并进行Wakitan的评分，评估此方法的有效性。结果大体观察结果表明，与对照组相比，实验组缺损处由软骨组织修复而对照组缺损处由纤维样组织填充。组织学观察可以见到实验组关节软骨缺损处有密集的软骨细胞而对照组关节缺损处只有纤维细胞无软骨细胞。结论通过短期观察表明以同种异体软骨细胞-去端肽胶原复合物修复全层软骨缺损的方法是有效可行的，为其进一步临床应用提供了参考。     

Rapid phenotypic changes in passaged articular chondrocyte subpopulations.

Articular chondrocytes are often expanded in vitro and then used to assist in healing articular cartilage defects. We investigated the extent of dedifferentiation in monolayer-passaged, zonal articular chondrocytes by using quantitative, real-time PCR. The relative gene expressions for collagen type I and II, aggrecan, and superficial zone protein were analyzed for relevant passage numbers (P0-P4) to determine how the expansion of chondrocytes affects the expression of cartilage extracellular matrix proteins. Results reveal that dramatic changes occur as early as first passage. Furthermore, these changes are shown to persist even when the expanded cells are encapsulated in 3D, alginate beads. Successful tissue engineering and autologous cell transplantation procedures rely heavily on having a cell source that expresses the chondrocytic phenotype. The results of this study suggest that major problems exist at the front-end of cartilage regeneration efforts.     

Microinjury to the synovial membrane may cause disaggregation of proteoglycans in rabbit knee joint articular cartilage

Proteoglycans (PGs) isolated from articular cartilage (AC) of mature rabbits subjected to two or more consecutive intraarticular (IA) injections of sterile saline 24 h apart showed an aggregation defect in the presence of excess hyaluronic acid (HA). Although the PG contents of experimental and control cartilages were indistinguishable, a higher proportion of PGs were extractable from the 3 X IA tissues, as assessed by uronic acid analysis. Proteoglycans from experimental and control cartilages when examined by Sepharose CL-2B chromatography showed two subunit populations, the smaller (KAV = 0.70) containing more ketatan sulphate than the larger (KAV = 0.31). Cultures of AC from IA joints released more 35SO4-labelled PGs into the media over 72 h than control tissues and consisted mainly of PG degradation products although 20% could aggregate in the presence of HA. Examination of PG aggregation 2 weeks after 2 X IA or 3 X IA injections showed that the defect initiated was still present; however, cartilage of immature rabbits was not affected by the 2 X IA procedure.     

纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞修复兔膝关节软骨缺损的实验研究

目的：探讨纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞作为软骨组织工程支架的可行性及有效性,并为后续研究可注射性材料做基础。方法：体外分离培养软骨细胞后接种到纤维蛋白凝胶和脱钙骨基质支架材料体外培养4周,然后植入兔膝关节软骨缺损区继续培养4、8、12周后取材,分别行大体、组织学、Ⅱ型胶原免疫组织化学观察。并进行Wakitani评分,观察其体内修复关节缺损效果。结果：大体观察4周后,实验组软骨缺损区可有乳白色组织修复,12周可修复完全,并无明显凹凸感。光镜下8周可见大量软骨细胞修复,并在TB染色下见Ⅱ型胶原比4周时明显增多。12周时软骨陷窝结构形成,细胞形态排列及Ⅱ型胶原与正常软骨组织相近。结论：纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞可以作为软骨组织工程支架材料,能够用于再生修复软骨的缺损。并为构建可注射性修复材料提供途径。     

Bupivacaine and saline effects on articular cartilage

Isotonic saline solution causes acute inhibition of 35SO4 incorporation into intact articular cartilage slices in vitro, but there is no evidence of ultrastructural damage to chondrocytes following treatment with bupivacaine or saline solution in vivo. Also, there is recovery of 35SO4 incorporation by 1-3 days following in vivo intraarticular administration of bupivacaine in saline solution to young pigs and adult dogs. There does not appear to be any contraindication to the use of intraarticular bupivacaine based on these findings.     

微粒骨膜-三维支架修复大面积关节软骨缺损

目的 探讨微粒骨膜-三维支架修复大面积关节软骨缺损的有效性和可行性.方法 于兔股骨滑车关节面制作直径4.5 mm深达软骨下骨板的全层软骨缺损模型,缺损处随机行自体微粒骨膜-纤维蛋白混凝物、单纯纤维蛋白"浇铸"移植.分别于术后3 h、4 d及1、2、4、8、12、24周取材,行大体观察、苏木素.伊红(HE)、Masson及藏红花(safranin-0)染色组织学检查,并进行组织学评分半定量分析.结果 微粒骨膜.三维支架制备简便.微粒骨膜被均匀种植于纤维蛋白三维支架中,可随意"浇铸"充填骨软骨缺损,移植物不易脱落,手术1次完成.术后微粒骨膜在缺损空间内全方位迅速增殖、分化、分泌基质完成缺损骨软骨修复.新生软骨具有与周围正常软骨基本一致的厚度、细胞形态及排列、基质胶原及蛋白多糖染色,且与周边软骨及软骨下骨结合良好.术后4、8、12及24周,两组组织学评分差异有统计学意义(P<0.05).结论 该方法能简单高效地构建工程化组织复合体,随意浇铸充填软骨缺损,完成较大面积关节软骨缺损的生物性修复.     

人胎关节软骨细胞体外培养的生物学特性

目的 研究软骨组织工程中传代软骨细胞与原代的差异。方法 用５个月人胎关节软骨分离培养细胞,观察细胞存活率、贴壁率、生长曲线和组织形态学的改变。结果 ⑴软骨块在４℃下,３ｄ内细胞存活率可达９３．４％～９７．６％。⑵原代细胞为圆形或三角形;第４代有一半转变成梭形,到第６代全部变为长梭形。⑶传代细胞贴壁时间（２～３ｈ）短于原代（４～７ｈ）。玻璃瓶内贴壁率传代细胞为７８．７％～８５．５％,原代８．８％。⑷     

Gene expression profile of mechanically impacted bovine articular cartilage explants.

Traumatic injury to a joint can initiate cartilage degradation. Blunt trauma increases matrix damage and decreases proteoglycan synthesis in in vitro models. Few studies have investigated gene expression of articular cartilage (AC) following mechanical loading. Recent advances in microarray technology allow analysis of a number of genes, and may elucidate pathways of AC degradation. In the present study, we used a bovine cDNA microarray to determine how acute trauma of cartilage explants in the absence of underlying bone alters gene expression. Results indicate that at least 19 genes were differentially expressed at 3 h after trauma. Fourteen genes were up-regulated and five genes were down-regulated relative to control explants. The up-regulated genes included cytokine and chemokine receptors, enzymes, and molecules involved in signal transduction. Genes of adhesion molecules and apoptosis were down-regulated. The results of this study highlight the potential benefits of using a bovine cDNA microarray to study cartilage metabolism.     

A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat

The basic molecular characteristics of intervertebral disc cells are still poorly defined. This study compared the phenotypes of nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) cells using rat coccygeal discs and AC from both young and aged animals and a combination of microarray, real-time RT-PCR and immunohistochemistry. Microarray analysis identified 63 genes with at least a fivefold difference in fluorescence intensity between the NP and AF cells and 41 genes with a fivefold or greater difference comparing NP cells and articular chondrocytes. In young rats, the relative mRNA levels, assessed by real-time RT-PCR, of annexin A3, glypican 3 (gpc3), keratin 19 (k19) and pleiotrophin (ptn) were significantly higher in NP compared to AF and AC samples. Furthermore, vimentin (vim) mRNA was higher in NP versus AC, and expression levels of cartilage oligomeric matrix protein (comp) and matrix gla protein (mgp) were lower in NP versus AC. Higher NP levels of comp and mgp mRNA and higher AF levels of gpc3, k19, mgp and ptn mRNA were found in aged compared to young tissue. However, the large differences between NP and AC expression of gpc3 and k19 were obvious even in the aged animals. Furthermore, the differences in expression levels of gpc3 and k19 were also evident at the protein level, with intense immunostaining for both proteins in NP and non-existent immunoreaction in AF and AC. Future studies using different species are required to evaluate whether the expression of these molecules can be used to characterize NP cells and distinguish them from other chondrocyte-like cells.