组织工程软骨与柚皮苷联合修复兔膝关节软骨缺损的组织学证实

背景：目前临床上虽有多种方法用于治疗软骨缺损,但没有从根本上解决关节软骨缺损修复问题。 目的：通过组织学研究进一步评价柚皮苷结合组织工程软骨修复兔关节软骨缺损的效果。 方法：取兔骨髓间充质干细胞体外增殖后,复合于改建后的脱细胞真皮基质载体上,制成组织工程软骨,植入到兔膝关节软骨缺损,并以柚皮苷汤灌胃,于 4,8周后分别对修复组织进行苏木精-伊红、Masson三色染色、甲苯胺蓝染色、Ⅱ型胶原染色、Ⅹ型胶原染色等组织学检查。 结果与结论：术后8周, 柚皮苷结合干细胞复合体组缺损处修复组织变成乳白色,半透明光滑组织,缺损修复组织与周围正常软骨已基本难区分,表面光滑。组织学检查发现修复缺损处基本为新生软骨填充。结果证实,柚皮苷结合组织工程软骨能提高家兔膝关节软骨缺损的修复质量。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix

A co-cross-linked synthetic extracellular matrix (sECM) composed of chemically modified hyaluronic acid and gelatin was used as a cell delivery vehicle for osteochondral defect repair in a rabbit model. A full-thickness defect was created in the patellar groove of the femoral articular cartilage in each of 2 rabbit joints, and 4 experimental groups were assigned (12 rabbits/group): untreated control, autologous mesenchymal stem cells (MSCs) only, sECM only, and MSCs + sECM. The sECM hydrogels were allowed to cross-link in the defect in situ. Rabbits were sacrificed at 4, 8, and 12 weeks post-surgery, and cartilage repair was evaluated and scored. In the controls, defects were filled with fibrous tissue. In the MSC-only group, hyaline-like cartilage filled the peripheral area of the defect, but the center was filled with fibrous tissue. In the sECM-only group, hyaline cartilage with zonal architecture filled the defect at 12 weeks, but an interface between repaired and adjacent host cartilage was evident. In the MSCs + sECM group, defects were completely filled with elastic, firm, translucent cartilage at 12 weeks and showed superior integration of the repair tissue with the normal cartilage. The sECM delivers and retains MSCs, and the injectable cell-seeded sECM could be delivered arthroscopically in the clinic.     

In vivo cultivation of human articular chondrocytes in a nude mouse-based contained defect organ culture model

The nude mouse model is an established method to cultivate and investigate tissue engineered cartilage analogues under in vivo conditions. One limitation of this common approach is the lack of appropriate surrounding articular tissues. Thus the bonding capacity of cartilage repair tissue cannot be evaluated. Widely applied surgical techniques in cartilage repair such as conventional and three-dimensional autologous chondrocyte implantation (ACI) based on a collagen gel matrix cannot be included into nude mouse studies, since their application require a contained defect. The aim of this study is to apply an organ culture defect model for the in vivo cultivation of different cell-matrix-constructs.Cartilage defects were created on osteochondral specimens which had been harvested from 10 human knee joints during total knee replacement. Autologous chondrocytes were isolated from the cartilage samples and cultivated in monolayer until passage 2. On each osteochondral block defects were treated either by conventional ACI or a collagen gel seeded with autologous chondrocytes, including a defect left empty as a control. The samples were implanted into the subcutaneous pouches of nude mice and cultivated for six weeks. After retrieval, the specimens were examined histologically, immunohistochemically and by cell morphology quantification.In both, ACI and collagen gel based defect treatment, a repair tissue was formed, which filled the defect and bonded to the adjacent tissues. The repair tissue was immature with low production of collagen type II. In both groups redifferentiation of chondrocytes remained incomplete. Different appearances of interface zones between the repair tissue and the adjacent cartilage were found.The presented contained defect organ culture model offers the possibility to directly compare different types of clinically applied biologic cartilage repair techniques using human articular tissues in a nude mouse model.     

多孔丝素蛋白/羟基磷灰石复合脂肪间充质干细胞修复兔关节软骨及软骨下骨缺损

背景：丝素蛋白/羟基磷灰石是细胞立体培养的良好支架,是临床常用的骨缺损修复材料,具有良好的生物相容性。脂肪干细胞具有向骨及软骨细胞分化的潜能,适合骨软骨缺损修复。 目的：观察转化生长因子β1和胰岛素样生长因子1联合成软骨诱导脂肪干细胞与丝素蛋白/羟基磷灰石复合后修复兔关节软骨及软骨下骨缺损的效果。 方法：取新西兰大白兔56只,2只用于传代培养脂肪间充质干细胞,以3×109 L-1浓度接种到丝素蛋白/羟基磷灰石。其余54只新西兰大白兔,在股骨髁间制备软骨缺损模型,随机分为细胞复合材料组、单纯材料组和空白对照组,细胞复合材料组植入复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石;单纯材料组植入丝素蛋白/羟基磷灰石;空白对照组不作任何植入。从大体、影像学、组织学观察比较缺损的修复情况。 结果与结论：12周时大体观察、CT、磁共振和组织学检查细胞材料复合组软骨及软骨下骨缺损区完全被软骨组织修复,修复组织与周围软骨色泽相近,支架材料基本吸收,未见明显退变和白细胞浸润,所有标本均未见丝素蛋白残留。单纯材料组缺损区缩小、部分修复,且呈纤维软骨样修复。空白对照组缺损无明显修复。提示复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石修复兔关节软骨及软骨下骨缺损能力优于单纯丝素蛋白/羟基磷灰石材料。丝素蛋白/羟基磷灰石复合脂肪间充质干细胞可形成透明软骨修复动物膝关节全层软骨缺损,重建关节的解剖结构和功能,可作为新型骨软骨组织工程支架。     

Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge

Articular cartilage has limited capacity for repair. In the present study, tissue-engineered two-phase composite material was used for the repair of osteochondral defects in young adult rabbit knee. This composite material is composed of an injectable calcium phosphate (ICP) and a hyaluronan (HA) derivate of either ACP or HYAFF 11 sponge. The osteochondral defect, 3 mm in diameter and 3 mm deep, was created in the weight-bearing region of the medial femoral condyle. The bone portion of the defect was first filled with ICP to a level approximately 1 mm below the articular surface. HA sponge (3 mm in diameter and 1-1.2 mm thick), with or without loading of autologous bone marrow-derived progenitor cells (MPCs), was then inserted into the defect on top of the ICP as it hardened. Animals were allowed free cage activity postoperatively, and killed 4 or 12 weeks (for the HYAFF 11 sponge group) after the surgery. At 4 weeks, histological examination showed that the defect was filled up to 90-100% of its depth. Whitish repair tissue on the top appeared to be integrated with the surrounding articular cartilage. Four distinct zones of repair tissue were identified: a superficial layer, a chondroid tissue layer, an interface between HA sponge and ICP, and the ICP material. Evidence of extensive osteoclastic and osteoblastic activities was observed in the bone tissue surrounding the defect edge and in ICP material. By 12 weeks, the zonal features of the repair tissue became more distinct; chondrocytes were arranged in a columnar array, and a calcified layer of cartilage was formed beneath the chondroid tissue in some specimens. The healing tissue of the HA sponge material loaded with MPCs had higher cellular density and better integration with the surrounding cartilage than HA sponge material not loaded with MPCs. This study suggests that using a two-phase composite graft may hold potential for the repair of osteochondral defects by providing mechanical support that mimicks subchondral bone, while also providing a chondrogenic scaffold for the top cartilage repair.     

Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.     

Repair of articular cartilage defect by cell transplantation

Full-thickness articular cartilage defects are a major clinical problem; however, at present there is no treatment that is widely accepted to regeneratively repair these lesions. The current therapeutic approach is to drill or abrade the base of the defect to expose the bone marrow with its cells and growth factors. This usually results in a repaired tissue of fibrocartilage that functions poorly in the loaded joint environment. Recently, autologous cultured chondrocyte transplantation and mosaic plasty were explored. We can repair small articular cartilage defects using these methods, although their effectiveness is still controversial. We have reported that transplantation of allogeneic chondrocytes embedded in collagen gels or allogeneic chondrocytes cultured in collagen gels could repair articular cartilage defect in a rabbit model. We also reported that autologous culture-expanded bone marrow mesenchymal cell transplantation could repair articular cartilage defect in a rabbit model. This procedure offers expedient clinical use, given that autologous bone marrow cells are easily obtained and can be culture-expanded. We transplanted autologous culture-expanded bone marrow cells into the cartilage defect of the osteoarthritic knee joint on 11 patients at the time of high tibial osteotomy. As early as 6.8 weeks after transplantation, the defect was covered with white soft tissue, in which slight metachromasia was histologically observed. Thirty-three weeks after transplantation, the repaired tissue had hardened. Histologically, repaired tissues showed stronger metachromasia and a partial hyaline cartilage-like appearance. This procedure may prove a promising method by which to repair articular cartilage defects.     

Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold)

The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.     

羟基丁酸-羟基辛酸共聚体/胶原骨软骨一体化支架修复膝关节软骨损伤

BACKGROUND: Due to the complex physiological characteristics of the osteochondral tissue, the clinical repair of knee cartilage injury often has dissatisfied outcomes. Tissue engineering methods and tools provide a new idea for osteochondral repair. OBJECTIVE: To observe the effect of poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold on the repair of articular cartilage injury in a rabbit. METHODS: The poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold was prepared by solvent casting/particle leaching method. Then, seed cells were isolated and cultured on the scaffold. Twenty-four healthy New Zealand white rabbits, 4 weeks of age, were used for the study. Under balanced anesthesia, an articular cartilage defect (4.5 mm in diameter, 5 mm in depth) was created on the rabbit’s femoral condyle using a bone drill. After modeling, rabbits were randomized into three groups and given direct suture in blank group, pure scaffold implantation in control group and implantation of the scaffold-cell complex in experimental group. Femoral condyle of each rabbit was taken out for gross and histological observations at 8, 20 weeks after surgery. RESULTS AND CONCLUSION: At 8 weeks after surgery, transparent film-covered defects and small/irregular cells were found in the experimental group; the defects were filled with fibrous tissues in the control group; while there was no repair in the blank group. Until the 20th week, the defects were covered with hyaline cartilage-like tissues, accompanied by regular cell arrangement in the experimental group; in the control group, the defects were covered with white membranous tissues, and many chondrocytes were found at the basement and edge; in the blank group, some newborn tissues were visible at the defect region. These findings suggest that the poly (hydroxybutyrate-co- hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold carrying seed cells contributes to articular cartilage repair.     

Nasal chondrocytes and fibrin sealant for cartilage tissue engineering

Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage.     

Cartilage repair in transplanted scaffold-free chondrocyte sheets using a minipig model

Lacking a blood supply and having a low cellular density, articular cartilage has a minimal ability for self-repair. Therefore, wide-ranging cartilage damage rarely resolves spontaneously. Cartilage damage is typically treated by chondrocyte transplantation, mosaicplasty or microfracture. Recent advances in tissue engineering have prompted research on techniques to repair articular cartilage damage using a variety of transplanted cells. We studied the repair and regeneration of cartilage damage using layered chondrocyte sheets prepared on a temperature-responsive culture dish. We previously reported achieving robust tissue repair when covering only the surface layer with layered chondrocyte sheets when researching partial-thickness defects in the articular cartilage of domestic rabbits. The present study was an experiment on the repair and regeneration of articular cartilage in a minipig model of full-thickness defects. Good safranin-O staining and integration with surrounding tissues was achieved in animals transplanted with layered chondrocyte sheets. However, tissue having poor safranin-O staining-not noted in the domestic rabbit experiments-was identified in some of the animals, and the subchondral bone was poorly repaired in these. Thus, although layered chondrocyte sheets facilitate articular cartilage repair, further investigations into appropriate animal models and culture and transplant conditions are required.     

Repair of osteochondral defects in rabbits with ectopically produced cartilage

Cartilage has poor regenerative capacity. Donor site morbidity and interference with joint homeostasis should be considered when applying the autologous chondrocyte transplantation technique. The use of ectopically produced cartilage, derived from periosteum, might be a novel method to heal cartilage defects. Ectopic cartilage was produced by dissecting a piece of periosteum from the tibia of rabbits. After 14 days the reactive tissue at the dissection site was harvested and a graft was cored out and press-fit implanted in an osteochondral defect in the medial condyle of the femur with or without addition of hyaluronan. After 3 weeks and 3 months the repair reaction was evaluated by histology. Thionine- and collagen type II-stained sections were evaluated for graft viability, ingrowth of the graft, and joint surface repair. Empty defects remained empty 3 weeks after implantation, ectopic cartilage filled the defect to the level of the surrounding cartilage. Histologically, the grafts were viable, consisting mainly of cartilage, and showed a variable pattern of ingrowth. Three months after implantation empty defects with or without hyaluronan were filled primarily with fibrocartilaginous tissue. Defects treated with ectopic cartilage contained mixtures of fibrocartilaginous and hyaline cartilage. Sometimes a tidemark was observed in the new articular cartilage and the orientation of the cells resembled that of healthy articular cartilage. Subchondral bone repair was excellent. The modified O'Driscoll scores for empty defects without and with hyaluronan were 12.7 +/- 6.4 and 15.3 +/- 3.2; for treated defects scores were better (15.4 +/- 3.9 and 18.2 +/- 2.9). In this conceptual study the use of ectopic cartilage derived from periosteum appears to be a promising novel method for joint surface repair in rabbits.     

Role of insulin like growth factor-I in repair response in immature cartilage

OBJECTIVE: The purpose of this study was to investigate the effects of exogenous local Insulin like growth factor-I (IGF-I) on the repair of full-thickness articular cartilage defects in immature rabbits. DESIGN: Thirty-six skeletally immature New Zealand rabbits between 6 and 8 weeks old were used. A single defect, 3.5-mm-wide by 4-mm-deep full-thickness articular cartilage defect in the medial femoral condyle, was created. The defect was either filled with a collagen sponge or with a collagen sponge impregnated with 5 mug of recombinant IGF-I. The animals were sacrificed at 4, 8 or 12 weeks, and the repair tissue was examined macroscopically and histologically. Repair tissue was also examined immunohistochemically for the presence of type-I collagen, type-II collagen and PCNA at all weeks. RESULTS: Newly formed tissue in all of the defects in the IGF-I group had the gross, histological and histochemical appearance of a smooth, intact hyaline articular cartilage. The average total scores on the histological grading scale were significantly better (p<0.05) for the defects treated with recombinant IGF-I at all time points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of the repair cartilage in the IGF-I treated defects. The control groups demonstrated minimum staining with type-II collagen antibody. CONCLUSIONS: These findings suggest that repair of full-thickness immature cartilage defects can be enhanced by recombinant IGF-I.     

The use of polylactic acid matrix and periosteal grafts for the reconstruction of rabbit knee articular defects.

In order to find a material that would improve cartilage repair, we investigated the use of porous polylactic acid matrix (PLA) with and without periosteal grafts in large articular defects in the medial femoral condyles of 18 New Zealand white rabbit knees. The right knee defect was filled with PLA, the left defect was filled with PLA and a periosteal graft. All animals were killed at 12 weeks. PLA allowed for the de novo growth of neocartilage at the articular surface in all specimens and appeared to serve as a scaffolding for cell migration and matrix formation. Histologically, small amounts of PLA remained under the neocartilage with the majority being replaced by bone. PLA was a suitable carrier for periosteal grafts with a high graft survival rate (89%) and proliferation of a neocartilage which was thicker and more closely resembled articular cartilage than PLA alone knees. Biochemically, there was more type II collagen in the grafted knees (83%) than in the PLA alone knees (65%). Biomechanical tests of the neocartilage included equilibrium displacement, aggregate modulus, and apparent permeability. These tests were not statistically different between PLA alone and grafted knees. Comparison to normal cartilage indicated that the neocartilage was less stiff but had similar permeability. A consistent repair of the articular defects was achieved with and without periosteal grafts resulting in a tissue that closely resembled hyaline articular cartilage.     

Tissue engineering-based cartilage repair with allogenous chondrocytes and gelatin-chondroitin-hyaluronan tri-copolymer scaffold: a porcine model assessed at 18, 24, and 36 weeks

We previously showed that cartilage tissue can be engineered in vitro with porcine chondrocytes and gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer which mimic natural cartilage matrix for use as a scaffold. In this animal study, 15 miniature pigs were used in a randomized control study to compare tissue engineering with allogenous chondrocytes, autogenous osteochondral (OC) transplantation, and spontaneous repair for OC articular defects. In another study, 6 pigs were used as external controls in which full thickness (FT) and OC defects were either allowed to heal spontaneously or were filled with scaffold alone. After exclusion of cases with infection and secondary arthritis, the best results were obtained with autogenous OC transplantation, except that integration into host cartilage was poor. The results for the tissue engineering-treated group were satisfactory, the repair tissue being hyaline cartilage and/or fibrocartilage. Spontaneous healing and filling with scaffold alone did not result in good repair. With OC defects, the subchondral bone plate was not restored by cartilage tissue engineering. These results show that tri-copolymer can be used in in vivo cartilage tissue engineering for the treatment of FT articular defects.     

Effects of growth factors on heparin-carrying polystyrene-coated atelocollagen scaffold for articular cartilage tissue engineering

The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here, we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal extracellular environment using an HCPS we originally developed, and an atelocollagen honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1 within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS generated an extracellular environment similar to that of heparan sulfate proteoglycan within the scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.     

组织工程软骨种子细胞的定向诱导分化

背景：软骨修复一直是骨科治疗难题和研究热点。随着组织工程学的发展,近年来应用种子细胞诱导分化为软骨细胞,构建组织工程软骨修复软骨缺损的思路倍受关注,并已取得一定成功。 目的：讨论组织工程软骨的种子细胞选择、成软骨定向诱导分化的培养条件,尤其是细胞因子、诱导方法、培养方式等因素的作用。 方法：计算机检索中国期刊全文数据库及PubMed 数据库2000-01/2010-09相关文章,检索词为“软骨形成,软骨缺损,刺激因子,软骨修复,组织工程,chondrification, cartilage defect; stimulating factor,cartilage repair,tissue engineering”。语种限定为：中文与英文。纳入与软骨形成、诱导分化、组织工程支架方面的基础和临床研究,排除内容陈旧及重复研究。计算机初检得到155篇文献,根据纳入排除标准,最终纳入41篇。 结果与结论：种子细胞在特定细胞因子作用下可定向分化为软骨细胞。通过选择合适的种子细胞及细胞因子定向诱导分化构建组织工程软骨,能够为成功治愈关节软骨缺损提供新思路。软骨定向分化因子转化生长因子β、碱性成纤维细胞生长因子、骨生成蛋白、胰岛素样生长因子、生长激素等调控诱导可通过介质添加物、基因转染、不同培养方式等几种方式促进向软骨方向转化。相信随着软骨定向诱导分化研究的深入,为临床上运用软骨组织工程学来解决关节软骨缺损修复这一难题提供了有效依据,有广泛的临床应用前景。     

Effects of basic fibroblast growth factor on the repair of large osteochondral defects of articular cartilage in rabbits: dose-response effects and long-term outcomes

Articular cartilage possesses a limited capacity for self-renewal. The regenerated tissue often resembles fibrocartilage-like tissue rather than hyaline cartilage, and degeneration of the articular surface eventually occurs. The purpose of this study was to investigate the effect of basic fibroblast growth factor (bFGF) on the healing of full-thickness articular cartilage defects. bFGF (0, 10, 50, 100, 250, 500, or 1000 ng) was mixed with collagen gel and implanted into full-thickness articular cartilage defects drilled into rabbit knees. The repaired tissue was examined grossly and histologically, and was evaluated with the use of a grading scale at 4, 12, 24, and 50 weeks. At 4 weeks, treatment with 100 ng of bFGF had greatly stimulated cartilage repair both grossly and histologically in comparison with untreated defects (those filled with plain collagen gel). The average total scores on the histological grading scale were significantly better for the defects treated with bFGF than for the untreated defects. These improvements were evident as long as 50 weeks postoperatively, although slight deterioration was noted in the repaired cartilage. Immunohistochemical staining for type II collagen showed that this cartilage-specific collagen was diffusely distributed in the repaired tissue at 50 weeks. These findings suggest that bFGF may be a practical and important candidate for use in cartilage repair.     

Use of a biphasic graft constructed with chondrocytes overlying a beta-tricalcium phosphate block in the treatment of rabbit osteochondral defects

To evaluate the ability of a biphasic construct to repair osteochondral defects in articular cartilage, plugs made of chondrocytes in collagen gel overlying a resorbable porous beta-tricalcium phosphate (TCP) block were implanted into defects in rabbit knees. The repair tissue was evaluated at 8, 12, and 30 weeks. Eight weeks after implantation of the biphasic construct, histologic examination showed hyaline-like cartilage formation that was positive for safranin O and type II collagen. At 12 weeks, most of the beta-TCP was replaced by bone, with a small amount remaining in the underlying cartilage. In the cell-seeded layer, the newly formed middle and deep cartilage adjacent to the subchondral bone stained with safranin O, but no staining was observed in the superficial layer. In addition, cell morphology was distinctly different from the deep levels of the reparative cartilage, with hypertrophic cells at the bottom of the cartilaginous layer. At 30 weeks, beta-TCP had completely resorbed and a tidemark was observed in some areas. In contrast, controls (defects filled with a beta-TCP block alone) showed no cartilage formation but instead had subchondral bone formation. These findings indicate that beta-TCP-supported chondrocytes in collagen gel can partially repair isolated articular cartilage osteochondral defects.     

The synergistic effects of microfracture, perforated decalcified cortical bone matrix and adenovirus-bone morphogenetic protein-4 in cartilage defect repair

We reported a technique for articular cartilage repair, consisting of microfracture, a biomaterial scaffold of perforated decalcified cortical bone matrix (DCBM) and adenovirus-bone morphogenetic protein-4 (Ad-BMP4) gene therapy. In the present study, we evaluated its effects on the quality and quantity for induction of articular cartilage regeneration. Full-thickness defects were created in the articular cartilage of the trochlear groove of rabbits. Four groups were assigned: Ad-BMP4/perforated DCBM composite (group I); perforated DCBM alone without Ad-BMP4 (group II); DCBM without perforated (group III) and microfracture alone (group IV). Animals were sacrificed 6, 12 and 24 weeks postoperation. The harvested tissues were analyzed by magnetic resonance image, scanning electron microscope, histological examination and immunohistochemistry. Group I showed vigorous and rapid repair leading to regeneration of hyaline articular cartilage at 6 weeks and to complete repair of articular cartilage and subchondral bone at 12 weeks. Groups II and III completely repaired the defect with hyaline cartilage at 24 weeks, but group II was more rapid than group III in the regeneration of repair tissue. In group IV the defects were concave and filled with fibrous tissue at 24 weeks. These findings demonstrated that this composite biotechnology can rapidly repair large areas of cartilage defect with regeneration of native hyaline articular cartilage.