Repairing articular cartilage defects with tissueengineering cartilage in rabbits

Objective: To investigate the effect of cancellous bone matrix gelatin ( BMG ) engineered with allogeneic chondrocytes in repairing articular cartilage defects in rabbits. Methods: Chondrocytes were seeded onto three-dimensional cancellous BMG and cultured in vitro for 12 days to prepare BMG-chondrocyte complexes. Under anesthesia with 2.5% pentobarbital sodium (1ml/kg body weight), articular cartilage defects were made on the right knee joints of 38 healthy New Zealand white rabbits (regardless of sex, aged 4-5 months and weighing 2. 5-3 kg) and the defects were then treated with 2. 5% trypsin. Then BMG-chondrocyte complex ( Group A, n = 18 ), BMG (Group B, n = 10), and nothing (Group C, n = 10) were implanted into the cartilage defects, respectively. The repairing effects were assessed by macroscopic, histologic, transmission electron microscopic ( TEM ) observation, immunohistochemical examination and in situ hybridization detection, respectively, at 2, 4, 8, 12 and 24 weeks after operation. Results: Cancellous BMG was degraded within 8 weeks after operation. In Group A, lymphocyte infiltration was observed around the graft. At 24 weeks after operation, the cartilage defects were repaired by cartilage tissues and the articular cartilage and subchondral bone were soundly healed. Proteoglycan and type II collagen were detected in the matrix of the repaired tissues by Safranin-O staining and immunohistochemical staining, respectively. In situ hybridization proved gene expression of type II collagen in the cytoplasm of chondrocytes in the repaired tissues. TEM observation showed that chondrocytes and cartilage matrix in repaired tissues were almost same as those in the normal articular cartilage. In Group B, the defects were repaired by cartilage-fibrous tissues. In Group C, the defects were repaired only by fibrous tissues. Conclusions: Cancellous BMG can be regarded as the natural cell scaffolds for cartilage tissue engineering. Articular cartilage defects can be repaired by cancellous BMG engineered with allogeneic chondrocytes. The nature of repaired tissues is closest to the normal cartilage. Local administration of trypsin can promote the adherence of repaired tissues to host tissues. Transplantation of allogeneic chondrocytes has immunogenicity, but the immune reaction is weak.     

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。     

软骨代谢标志物对骨关节炎软骨改变的反映

目的研究血清中蛋白聚糖和Ⅱ型胶原水平与骨关节炎严重程度间的关系,及对手术后软骨代谢改变的反映。方法研究对象包括65例膝骨关节炎患者及22名正常人。45例患者分别行不同方式手术治疗,术后半年复查。正常人及患者术前和复查时抽取静脉血,摄下肢负重位X线片。应用酶联免疫吸附方法检测血清样本中蛋白聚糖及Ⅱ型胶原的水平。结果骨关节炎患者血清中蛋白聚糖及Ⅱ型胶原水平均显著高于正常对照组,差异有显著性意义(P<0.05)。血清中蛋白聚糖和Ⅱ型胶原水平在轻型骨关节炎组升高,在关节明显狭窄组水平最高,而在严重狭窄组明显降低。术后半年,全膝关节置换术组血清中蛋白聚糖水平较术前明显下降(P<0.05);截骨术组血清中Ⅱ型胶原水平较术前明显下降(P<0.05)。结论血清中蛋白聚糖和Ⅱ型胶原的水平与骨关节炎软骨的破坏程度、软骨细胞的合成反应及软骨的总量有关。蛋白聚糖和Ⅱ型胶原水平是反映软骨代谢改变的较敏感的指标。     

耳软骨整体再造鼻侧软骨的可行性分析

目的：以耳软骨为供区,探索整体再造鼻侧软骨的方法。方法：20具（40侧）尸体标本,取下耳软骨40枚,鼻侧软骨40枚。CT扫描后重建三维图像,测量鼻侧软骨各解剖分区的相关数据。设计耳软骨3个供区可整体移植再造同侧鼻侧软骨,测量相关的形态数据。结果：耳软骨3个供区的相关形态数据大于同侧鼻侧软骨的相应数据。结论：耳甲腔、耳屏及两者之间连接的峡部（CVIT区域）,三角窝、耳轮及两者的连接部（TFH区域）,耳甲艇、耳轮及两者之间的连接部分（CBH区域）可整体取下,修整后整体再造同侧鼻侧软骨。     

同种异体骨软骨移植修复关节软骨缺损的研究进展

同种异体骨软骨移植修复膝关节软骨和骨软骨缺损适用于大面积骨软骨缺损的修复,且不受缺损形状和面积的限制,在临床上已取得令人鼓舞的结果。现对国内外同种异体骨软骨移植修复关节软骨缺损的研究进展作一综述。     

Articular cartilage restoration with costal cartilage previously fused with bone

A novel procedure was developed for restoration of an articular cartilage defect using an autologous costal cartilage prepared with iliac bone, and the durability in vivo of this biologic construct was examined. First, an osteochondral complex was prepared (successful preparation, 67 of 80). Cancellous bone blocks isolated from the ilium of male Japanese White rabbits aged 5 months were implanted onto the surface of the costal cartilage before being tied by a pair of 3-0 silk thread sutures that were looped around the costal cartilage from behind. Second, 3 months later, the bone-attached costal cartilage was harvested and implanted into a full-thickness cartilage defect induced in a trochlear groove of the femur. All of the grafts were fixed to the recipient, maintaining its cartilage structure until 6 months (n = 28) and 12 months (n = 12) after implantation. However, when the costal cartilage without any bony portion was implanted into a similarly induced defect, 42% (10 of 24) were detached from the recipient before 12 months after implantation. The nontreated defect did not heal spontaneously to a satisfactory level (n = 12). These findings suggest that an osteochondral fragment, prepared by grafting cancellous bone onto costal cartilage, can be used for articular cartilage restoration.     

Articular cartilage injuries

The acute and repetitive impact and torsional joint loading that occurs during participation in sports can damage articular surfaces causing pain, joint dysfunction, and effusions. In some instances, this articular surface damage leads to progressive joint degeneration. Three classes of chondral and osteochondral injuries can be identified based on the type of tissue damage and the repair response: (1) damage to the joint surface that does not cause visible mechanical disruption of the articular surface, but does cause chondral damage and may cause subchondral bone injury; (2) mechanical disruption of the articular surface limited to articular cartilage; and (3) mechanical disruption of articular cartilage and subchondral bone. In most instances, joints can repair damage that does not disrupt the articular surface if they are protected from additional injury. Mechanical disruption of articular cartilage stimulates chondrocyte synthetic activity, but it rarely results in repair of the injury. Disruption of subchondral bone stimulates chondral and bony repair, but it rarely restores an articular surface that duplicates the biologic and mechanical properties of normal articular cartilage. In selected patients, surgeons have used operative treatments including penetrating subchondral bone, soft tissue grafts, and cell transplants and osteochondral autografts and allografts to restore articular surfaces after chondral injuries. Experimental studies indicate that use of artificial matrices and growth factors also may promote formation of a new joint surface. However, an operative treatment of an articular surface injury that will benefit patients must not just provide a new joint surface, it must produce better long-term joint function than would be expected if the injury was left untreated or treated by irrigation and debridement alone. Therefore, before selecting a treatment for a patient with an articular cartilage injury, the surgeon should define the type of injury and understand its likely natural history.     

Cryopreservation of cartilage

Summary We have investigated the viability and function of cells in cartilage slices after various methods of preservation, and have examined the viability of cells by measuring the incorporation of Na₂ ³⁵SO₄ at different concentrations, temperatures and times of exposure to cryopreservatives. We have assessed the viability of cells when exposed to prefreezing temperatures, and after preservation at –80° C. The best survival rate was found to be with a concentration of cryopreservatives of approximately 10%, with pre-freeze exposure for four hours at 4° C. In the stage cooling technique, the best initial cooling was at –30° C for 30 minutes, followed by rapid cooling of the cartilage to –80° C. The best survival rate for cryopreserved cartilage in 10% DMSO was, on average, 19% in intact slices and 34% when holes had been made in the slices. Proteoglycan synthesis after thawing appeared normal, and proteoglycan labelled after 48 hours in culture also showed a normal pattern.

---

Résumé Nous avons étudié la viabilité et la fonction cellulaire de coupes de cartilage après diverses méthodes de conservation et nous avons évalué la viabilité des cellules en mesurant l'absorption du Na₂ ³⁵SO₄ à différentes concentrations, températures et durées d'exposition aux cryo-préservateurs. Nous avons évalué la viabilité des cellules exposées aux températures de pré-congélation et après conservation à –80° C. Nous avons trouvé que le meilleur pourcentage de survie était obtenu avec une concentration de cryo-préservateurs d'environ 10%, avec une pré-congélation de 4 heures à 4° C. Dans la technique de refroidissement par étapes, le meilleur refroidissement initial est de –30° C pendant 30 minutes, suivi d'un refroidissement rapide du cartilage jusqu'a –80° C. Le meilleur taux de survie pour le cartilage cryo-préservé dans 10% de DMSO a été, en moyenne, de 19% dans les coupes intactes et de 34% quand des perforations ont été pratiquées dans les coupes. La synthèse du protéoglycan après décongélation paraît normale et le protéoglycan marqué, après une culture de 48 heures, s'avère également normal.

     

Transplanted tissue-engineered cartilage

OBJECTIVE: To evaluate the feasibility of transplanting sculpted autogenous tissue-engineered cartilage (TEC) with the hope that it will retain precise 3-dimensional morphologic features after transplantation. Transplanted TEC is described in terms of the gross morphologic and histologic characteristics in contrast to pretransplanted TEC. METHODS: Synthetic scaffolds of a polyglycolic acid and poly-l-lactic acid polymer, coated with chondrocytes derived from rabbit auricular cartilage in concentrations ranging from 2.7 x 10(6)/mL to 6 x 10(7)/mL, were incubated in vivo on the dorsum of a rabbit for 8 weeks and then retrieved. The resultant TEC specimens were then sculpted into defined shapes and transplanted into a different location in the same rabbit, where they were allowed to incubate for another 8 weeks. The specimens were then retrieved and compared with the TEC before transplantation according to size, weight, and histomorphometric analysis. RESULTS: Thirteen chondrocyte-laden templates were successfully engineered to develop TEC. In each case, they were sculpted and transplanted to a different site in the same rabbit. Eight weeks after transplantation, all sculpted TEC specimens lost their original 3-dimensional morphologic features and experienced a significant decrease in mass. Histologically, the staining intensity of both hemotoxylin-eosin and safranin O was dramatically reduced following transplantation. In addition, there was a reduction in chondrocyte viability. Two consistent histologic findings were a foreign-body reaction to the synthetic polymer and ongoing cellular activity directed toward the formation of bone. CONCLUSIONS: Transplanting autogenous TEC does not allow the preservation of precise morphologic features that are needed for clinical implantation. The osteogenic progression and foreign-body reaction must also be controlled.     

Articular cartilage biology

Articular cartilage is a complex tissue maintained by chondrocytes, which undergo metabolic changes as a result of aging, disease, and injury. These changes may hinder tissue maintenance and repair, resulting in accelerated loss of articular surface and leading to end-stage arthritis. Researchers are investigating both normal and pathologic cellular and molecular processes as well as the development of chondroprotective agents to improve the metabolic function of articular cartilage. Current research is helping to clarify the mechanisms by which a variety of agents, such as glucosamine, chondroitin sulfate, hyaluronic acid, green tea, glucocorticoids, and nonsteroidal anti-inflammatory drugs, can modify the symptoms and course of osteoarthritis. Also under investigation are methods of stimulating repair or replacing damaged cartilage, such as matrix metalloproteinase inhibitors, gene therapy, growth factors, cytokine inhibitors, and artificial cartilage substitutes. Tissue engineering, the combining of artificial matrices with cells and growth factors or genes, offers great potential for improving patient care.     

微小切口断层肋软骨切取与雕刻技术

目的:研究适合鼻部整形的肋软骨切取和雕刻新技术。方法:选择340例适合用肋软骨移植进行鼻整形的患者。采用25~30mm长的唇型微小切口,用弧型切骨刀切取断层肋软骨。对肋软骨毛料进行精细雕刻处理,制作鼻整形所需的各种移植物。结果:340例患者经微小切口切取断层肋软骨块全部顺利完成。用新式雕刻法制备出6类肋软骨移植物,用于鼻整形全部获得成功。1年后随访158例,胸部切口均达到无瘢痕或少瘢痕愈合效果。未发现有鼻部肋软骨移植物有变形或明显吸收现象。结论:经微小切口采集断层肋软骨和软骨精细雕刻法是患者容易接受的鼻整形新技术,可以提高鼻整形质量。