Allograft of cultured chondrocytes into articular cartilage defects in rabbits--experimental study of the repair of articular cartilage injuries

Articular cartilage defects were created by dill holes, 2 mm wide and 3 mm deep, through the articular cartilage into the subchondral bone in the patellar groove of the femur in mature rabbits. The defects received graft of cultured chondrocytes and the matrix obtained from the primary culture of chondrocytes isolated from the articular cartilage or auricular cartilage in immature rabbits. The isolated cells were cultured for 10 to 14 days. For graft, the cultured chondrocytes together with the matrix were detached from the culture chamber using rubber policemen and centrifuged. The repair of the grafted defects or defects without graft (control) was histologically studied 2 to 12 weeks after operation. The defects without the graft were progressively filled with fibrous tissue containing spindle shaped cells, fibers perpendicular to the surface, and matrix showing weak metachromasia with toluidin blue at 8 weeks. The defects received articular cartilage cell graft were occupied by new cartilage tissue consisting colonylike crumps of chondrocytes 2 weeks after operation. The crumps showed strong metachromasia with toluidin blue and strong stainability for safranin-O. By 4-8 weeks, the defects were filled with homogeneous cartilage. At 12 weeks, arrangement of the chondrocytes of the superficial layer of the new cartilage became columnar as seen in the normal articular cartilage. The defects received elastic cartilage cell graft were filled by reformed cartilage with chondrocytes surrounded by elastic fibers 2-12 weeks after operation. The results indicate that allograft of cultured chondrocytes with matrix into the articular cartilage defects accerated the repair process of the defects by formation of the new cartilage derived from the grafted chondrocytes.     

Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel

We attempted to repair full-thickness articular cartilage defects in rabbit knee joints with allogeneic cultured chondrocytes embedded in Atelocollagen gel. An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in the collagen gel and covered with periosteal flap (G group). In three other experimental groups, the same defects were transplanted with chondrocytes in monolayer culture with periosteal flap (M group), periosteal graft only (P group), or left empty (E group). At 4, 12, and 24 weeks after operation, the reparative tissue was analyzed macroscopically and histologically. At 4 weeks after operation, the surfaces of the reparative tissue were smooth, and the defects were filled with reparative tissues that resembled hyaline cartilage in all four groups. However, the reparative tissues degenerated gradually with time in the M, P, and E groups. In contrast, in the G group, the reparative tissue retained its thickness, and there was a steady integration of the grafted tissue into the adjacent normal cartilage at 24 weeks after operation. The results suggest that transplantation of allogeneic chondrocytes cultured in Atelocollagen gel is effective in repairing an articular cartilage defect.     

自体骨膜延迟游离移植修复成年后关节软骨缺损的实验研究

目的：研究年龄对自体骨膜游离移植修复关节软骨缺损的影响,探讨延迟游离移植能否提高成年后骨膜修复软骨能力。方法：选中国白兔,成年兔20只,幼兔10只,分3组。A组：成年兔左膝骨膜直接游离移植组;B组：成年兔右膝骨膜延迟游离移植组;C组：幼兔骨膜直接游离移植组,取骨膜或骨膜新生组织、行光镜、电镜组织学观察比较。结果：移植前B、C组骨膜厚度、细胞计数及细胞活跃程度均优于A组（均为P＜0．01）,移植后12周3组关节软骨缺损获得不同程度修复,C组优于A组（P＜0．01）及B组（P＜0．05）,B组优于A组（P＜0．01）。结论：自体骨膜局部剥离、原位激活,体内培养、延迟游离移植可提高成年骨膜成软骨能力,更好地修复成年后关节软骨缺损。     

The cellular origin of cartilage-like tissue after periosteal transplantation of full-thickness articular cartilage defects: an experimental study using transgenic rats expressing green fluorescent protein

BACKGROUND: Periosteal transplantation is commonly used for the treatment of articular cartilage defects. However, the cellular origin of the regenerated tissue after periosteal transplantation has not been well defined. The objective of this study was to investigate the cellular origin of the regenerated tissue after periosteal transplantation. METHOD: Free periosteum was harvested from the tibia of 10-week-old adolescent enhanced green fluorescent protein (GFP-) expressing transgenic Sprague Dawley (SD) rats and was transplanted to full-thickness articular cartilage defects of the patellar groove in normal 10-week-old adolescent SD rats. The periosteum was sutured to the defect with the cambium layer facing the joint cavity. 8 SD rats were killed at 4 weeks and 8 SD rats were killed at 8 weeks after surgery. The repaired tissue was assessed histologically and histochemically. GFP-positive cells derived from the donor periosteum could easily be detected in the repaired tissue by use of a fluorescent microscope. RESULTS: At both 4 and 8 weeks after transplantation, the entire area of the defects had been repaired, with the regenerated tissue being well stained histologically with safranin-O. Most cells in the whole area of the regenerated tissue were GFP-positive, indicating that very few of the cells were GFP-negative cells originating from the recipient rats. INTERPRETATION: This experiment demonstrates that most cells in regenerated tissue after periosteal transplantation using adolescent animals do not originate from recipient cells but from the periosteal cells of the donor.     

Potential healing benefit of an osteoperiosteal bone plug from the proximal tibia on a mosaicplasty donor-site defect in the knee

Introduction Autologous osteochondral transplantation is a popular treatment for articular cartilage lesions in the knee joint. The donor defect is commonly left empty and remains a matter of concern.Materials and methods In 20 knees of 10 goats, we created a standardised donor defect in the knee. In the control group the defects were left empty, whereas in two other groups a bone plug from the proximal tibia was press-fitted into the defect with or without a covering periosteal layer.Results Histological evaluation after 8 and 16 weeks showed that relatively rapid osteoclastic resorption of the bone plug occurred. Defects were mainly filled with fibrous tissue, and collapse of the adjacent bone and cartilage was visible, especially when the defects were left empty. Occasionally, some consolidation of the graft to the host bone could be detected, and in some samples periosteal chondrogenesis was present.Conclusion Our findings suggest that transplantation of a tibial bone plug with covering periosteum to the donor defect in a cartilage transplantation procedure does not have any additional value in an attempt to minimise the damage at the donor site. The observed resorption of the bone plug and the collapse of the adjacent joint margin remain a matter of concern with this technique.     

The cellular origin of cartilage-like tissue after periosteal transplantation of full-thickness articular cartilage defects: An experimental study using transgenic rats expressing green fluorescent protein

Background?Periosteal transplantation is commonly used for the treatment of articular cartilage defects. However, the cellular origin of the regenerated tissue after periosteal transplantation has not been well defined. The objective of this study was to investigate the cellular origin of the regenerated tissue after periosteal transplantation.

Method?Free periosteum was harvested from the tibia of 10-week-old adolescent enhanced green fluorescent protein (GFP-) expressing transgenic Sprague Dawley (SD) rats and was transplanted to full-thickness articular cartilage defects of the patellar groove in normal 10-week-old adolescent SD rats. The periosteum was sutured to the defect with the cambium layer facing the joint cavity. 8 SD rats were killed at 4 weeks and 8 SD rats were killed at 8 weeks after surgery. The repaired tissue was assessed histologically and histochemically. GFP-positive cells derived from the donor periosteum could easily be detected in the repaired tissue by use of a fluorescent microscope.

Results?At both 4 and 8 weeks after transplantation, the entire area of the defects had been repaired, with the regenerated tissue being well stained histologically with safranin-O. Most cells in the whole area of the regenerated tissue were GFP-positive, indicating that very few of the cells were GFP-negative cells originating from the recipient rats.

Interpretation?This experiment demonstrates that most cells in regenerated tissue after periosteal transplantation using adolescent animals do not originate from recipient cells but from the periosteal cells of the donor.

?     

冷冻保存胎兔颅骨骨膜修复关节软骨缺损的实验研究

为探讨关节软骨缺损的修复方法，采用家兔３０只，平均分成两组。在每个动物的双侧髌股关节股骨关节面作４ｍｍ×７ｍｍ全层软骨缺损。将经二步冷冻法保存的胎兔颅骨骨膜移植于一组动物一侧后肢作为实验组，另一侧用自体骨膜移植作为自体对照组。在另一组动物的一侧后肢用新鲜胎兔颅骨骨膜移植作为新鲜对照组，另一侧关节软骨缺损不修复作为空白对照组。术后肢体不作外固定。１６周后取材，经大体观察、组织学观察及氨基酸成分分析。结果发现，经冷冻保存的胎兔颅骨骨膜移植后能形成透明样软骨，与自体对照组无显著性差异（Ｐ＞０．０５），与新鲜对照组及空白对照组主要指标有显著性差异（Ｐ＜０．０５）。氨基酸分析示实验组新生物接近纤维软骨（Ｐ＞０．０５）。认为，冷冻胎兔颅骨骨膜移植为关节软骨缺损的修复提供了实验依据。     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

Cellular origin and evolution of neochondrogenesis in major full-thickness defects of a joint surface treated by free autogenous periosteal grafts and subjected to continuous passive motion in rabbits

A graft of periosteum from the tibia of 27 rabbits was incubated in vitro with tritiated thymidine for 24 hours and then transplanted into a full-thickness defect in the patellar groove. The rabbits were managed after the operation on continuous passive motion (CPM), and the joints excised at intervals of two to 21 days. After one week the cells had begun to synthesize glycosaminoglycan and by two weeks the tissue resembled immature hyaline cartilage. Thymidine-labeled cells were seen throughout the entire regenerated tissue. The cellular origin of the hyaline-like tissue that filled the defects was the progenitor cells of the periosteal graft.     

Transplantation of free tibial periosteal grafts for the repair of articular cartilage defect: An experimental study

Background:

Articular chondrocytes have got a long lifespan but rarely divides after maturity. Thus, an articular cartilage has a limited capacity for repair. Periosteal grafts have chondrogenic potential and have been used to repair defects in the articular cartilage. The purpose of the present study is to investigate the differentiation of free periosteal grafts in the patellofemoral joint where the cambium layer faces the subchondral bone and to investigate the applicability of periosteal grafts in the reconstruction of articular surfaces.

Materials and Methods:

The study was carried out over a period of 1 year on 25 adult, male Indian rabbits after obtaining permission from the institutional animal ethical committee. A full-thickness osteochondral defect was created by shaving off the whole articular cartilage of the patella of the left knee. The defect thus created was grafted with free periosteal graft. The patella of the right knee was taken as a control where no grafting was done after shaving off the articular cartilage. The first animal was used to study the normal histology of the patellar articular cartilage and periosteum obtained from the medial surface of tibial condyle. Rest 24 animals were subjected to patellectomy, 4 each at serial intervals of 2, 4, 8, 16, 32 and 48 weeks and the patellar articular surfaces were examined macroscopically and histologically.

Results:

The grafts got adherent to the underlying patellar articular surface at the end of 4 weeks. Microscopically, graft incorporation could be appreciated at 4 weeks. Mesenchymal cells of the cambium layer were seen differentiating into chondrocytes by the end of 4 weeks in four grafts (100%) and they were arranged in a haphazard manner. Till the end of 8 weeks, the cellular arrangement was mostly wooly. At 16 weeks, one graft (25%) had wooly arrangement of chondrocytes and three grafts (75%) had columnar formation of cells. Same percentage was maintained at 32 weeks. Four grafts (100%) at 48 weeks showed columnar orientation. The control side showed no changes over the shaved off articular surface in all the rabbits. One rabbit at 4 weeks had a dislocation of the patella on the control side. None of the rabbits developed any infection or wound dehiscence.

Conclusion:

Autologous periosteal graft transplantation can be a promising substitute for articular cartilaginous defects.     

Xeno-implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno-transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankin's score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Mason's trichrome. Pig cells and pig genetic material were detected in the neo-synthesized tissue by immunohistochemical detection of SLA-II-DQ and polymerase chain reaction analysis of the gene SLA-II-DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo-synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline-like extracellular matrix. The neo-synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft-vs.-host rejections or infiltration by immune cells. In the neo-synthesized tissue, pig genetic material was detected in 27 +/- 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo-synthesized cartilage. We conclude that the xeno-transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.     

Reconstruction of Articular Cartilage Defects with Free Periosteal Grafts: An Experimental Study

The chondrogenic potential of free autogenous periosteal grafts was studied histologically in 6-month-old rabbits. The grafts were taken from the tibia and transplanted to 7 × 14 mm large artificial defects of the femoral articular cartilage. The results revealed that the defects were repaired and filled after 4 weeks with a hyaline-like cartilage which was histologically similar to the cartilage adjacent to the transplant. The tissue maintained this morphology after 1 year of observation. In control animals where no periosteum was transplanted to the defect, no real cartilage was found. The tissue which partially filled the defect was a variable mixture of fibrous tissue and fibrocartilage.     

胶原蛋白水凝胶在软骨组织工程中的应用

由创伤或骨病所致的关节骨软骨损伤在临床中十分常见,其中软骨缺损者达40.31%。由于关节软骨自身修复能力低下,采用组织工程技术对关节软骨损伤进行修复是目前采用再生医学治疗关节软骨损伤的新方法。组织工程支架按照性状可分为预成型支架材料及水凝胶材料两大类。传统的预成型支架材料移植技术容易给缺损周边软骨带来继发损伤,也存在支架与缺损整合不紧密等问题。如何在避免二次损伤的基础上,应用理想的仿生材料复合种子细胞修复关节不规则软骨损伤将成为未来软骨损伤修复的主要问题。选取微创、仿生并且可以原位塑形的胶原蛋白水凝胶复合种子细胞修复关节软骨损伤为损伤关节软骨的修复带来了希望。本文结合国内外相关文献对目前胶原蛋白水凝胶在软骨组织工程中的应用做一综述。     

Articular cartilage defects in a rabbit model, retention rate of periosteal flap cover

BACKGROUND: A periosteal flap is frequently used in procedures involving repair of articular cartilage defects. Hypertrophy of the repair tissue, probably from a retained periosteum, is a clinical problem but not much is known about this issue. The objective of the present experimental study was to investigate the retention rate of periosteal flaps with respect to various postoperative mobilization regimes and the introduction of bone marrow elements underneath the flap. METHOD: We created a chondral lesion (diameter 4 mm) in both patellas of 18 New Zealand white rabbits. The subchondral bone was left intact in one knee. In the other, the bone plate was perforated, allowing bone marrow elements to enter the defect. All defects were covered with a periosteal flap, sutured and glued to the rim of the cartilage defect. Postoperatively, the rabbits were allocated to one of three groups: A. rehabilitation in cages for 4 days, then killed; B. rehabilitation in cages for 7 days, then free activity on the floor of a 10 m2 room until the end of the second week, then killed; C. rehabilitation in cages for 2 weeks, then killed. RESULTS: 16 of 23 periosteal flaps became detached within 2 weeks (one knee was excluded because of clinical signs of infection), with no difference in the retention rate with respect to mobilization regime or established access to bone marrow elements in the defect. The periosteum still served as a cover of the defect in 10 of 12 knees at day 4. This figure decreased to 7 of 23 knees at day 14. CONCLUSION: Our study is the first to document the retention rate of periosteal flaps in a rabbit model. The low retention rate observed may explain why periosteal hypertrophy is not reported in experimental studies where the periosteal flap is sutured to the cartilage rim.     

Value of osteo-chondral paste autologous transplantation in experimental cartilage defects reconstruction. Part IV--Microscopic analysis of cellularity and of traits of necrosis in the defect-filling tissue]

INTRODUCTION: Restricted potential of cartilage to regeneration following trauma induced search for new techniques of articular cartilage repair. Present study aimed at defining in experimental conditions of autologous osteochondral paste value by evaluation of cellularity and presence of necrosis in the defect-filling tissue. MATERIAL AND METHODS: Full thickness defect (IV(o)--ICRS scale) on distal rabbit femur joint surface was made. Three groups were specified: A--defect with paste graft (cartilage and contiguous bone collected from joint surface, crushed into homogenous paste; B--defect with the paste graft covered with periosteum; C--defect left unfilled. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale. RESULTS: In evaluation of cellularity in the developed repair tissue, in long-term observations best results were obtained in the group of osteochondral paste (A) and in the group of unfilled defect (C). Following 12 weeks of observation cellularity of the repair tissue in the groups amounted to around 95%, as compared to the surrounding healthy cartilage. In evaluation of necrotic traits in the newly developed tissue, their highest intensity after 4 weeks of observation was noted in the group with periosteum covered osteochondral paste (B), and lower but still significant in the group in which paste alone was applied (A). This resulted from massive dying out of the grafted bone elements, and in the case of group B, also of the periosteal cells in the covering flap and of certain number of chondrocytes composing the graft. The trait was more objectively evaluated in long-term observations, when necrotic foci involved only elements of the proper repair tissue. After 12 weeks of observation degenerative changes showed the least pronounced intensity in the group with the paste alone (A). Following 4 weeks the intensity was least pronounced in the group with the unfilled defect (C), i.a. due to the absence of graft with the decomposing bone elements. However, with elapsing time degenerative lesions intensified in the group, pointing to the low stability of the developed tissue. The results indicated that application of the osteochondral paste in treatment of cartilage defects yields a stable, highly cellular tissue, resistant to time and trauma.     

Healing of defects in canine articular cartilage: Distribution of nonvascular α-smooth muscle actin-containing cells

The objective of this study was to evaluate the types of tissue resulting from spontaneous healing of surgically created defects in adult canine articular cartilage up to 29 weeks postoperatively, with specific attention directed toward the presence and distribution of cells containing the contractile actin isoform, alpha-smooth muscle actin. Two 4-mm diameter defects were made in the trochlear groove to the depth of the tidemark in 20 adult mongrel dogs. The areal percentage of specific tissue types in the reparative material was determined histomorphometrically. Immunohistochemistry was employed to evaluate the percentage of alpha-smooth muscle actin-containing cells. The results showed that approximately 50% of the chondrocytes in the superficial zone of the uninvolved articular cartilage expressed alpha-smooth muscle actin. A significantly lower percentage of alpha-smooth muscle actin-positive chondrocytes appeared in the uninvolved deep zone. Notably, the deep zone adjacent to the defect contained a greater percentage of such cells than in the uninvolved deep zone. Also of interest was that a greater percentage of nonvascular cells in the hyaline cartilage and fibrocartilage of the reparative tissue contained alpha-smooth muscle actin-positive cells, compared to the fibrous tissue in the defects. The findings of this study revealed that canine articular cartilage has some potential for spontaneous regeneration, including integration with the calcified cartilage zone. By 29 weeks, up to 40% of an areal cross section of an untreated full-thickness chondral defect was found to fill with hyaline cartilage, with up to 19% judged histologically similar to articular cartilage. The results warrant further consideration of the role of alpha-smooth muscle actin in chondrocytes in normal articular cartilage and in reparative tissue.     

骨髓基质细胞修复兔关节软骨的实验研究

目的　研究骨髓基质细胞修复兔关节软骨的可行性。方法　取自体骨髓基质细胞体外培养扩增 ,聚乳酸吸附骨髓基质细胞植入兔膝关节软骨负重 (内髁 )和非负重区 (外髁 )缺损内 ,观察 4、 8、 12周后软骨缺损的修复情况 ,并对组织切片评分。结果　 8、 12周后 ,骨髓基质细胞在负重区缺损内可以形成透明软骨 ,组织评分接近正常软骨优于对照 ,非负重区内没有形成透明软骨。结论　骨髓基质细胞可以修复关节软骨缺损 ,摩擦和压应力是成软骨的重要条件。     

Articular cartilage defects in a rabbit model,retention rate of periosteal flap cover

Background A periosteal flap is frequently used in procedures involving repair of articular cartilage defects. Hypertrophy of the repair tissue, probably from a retained periosteum, is a clinical problem but not much is known about this issue. The objective of the present experimental study was to investigate the retention rate of periosteal flaps with respect to various postoperative mobilization regimes and the introduction of bone marrow elements underneath the flap.

Method We created a chondral lesion (diameter 4 mm) in both patellas of 18 New Zealand white rabbits. The subchondral bone was left intact in one knee. In the other, the bone plate was perforated, allowing bone marrow elements to enter the defect. All defects were covered with a periosteal flap, sutured and glued to the rim of the cartilage defect. Postoperatively, the rabbits were allocated to one of three groups: A. rehabilitation in cages for 4 days, then killed; B. rehabilitation in cages for 7 days, then free activity on the floor of a 10 m2 room until the end of the second week, then killed; C. rehabilitation in cages for 2 weeks, then killed.

Results 16 of 23 periosteal flaps became detached within 2 weeks (one knee was excluded because of clinical signs of infection), with no difference in the retention rate with respect to mobilization regime or established access to bone marrow elements in the defect. The periosteum still served as a cover of the defect in 10 of 12 knees at day 4. This figure decreased to 7 of 23 knees at day 14.

Conclusion Our study is the first to document the retention rate of periosteal flaps in a rabbit model. The low retention rate observed may explain why periosteal hypertrophy is not reported in experimental studies where the periosteal flap is sutured to the cartilage rim.     

The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. An experimental investigation in the rabbit

A rectangular graft of autogenous tibial periosteum was sutured (with its cambium layer facing into the joint) onto the base of a five by ten-millimeter full-thickness defect in the patellar groove of each of 143 adolescent and adult rabbits. The rabbits were managed postoperatively by either immobilization, intermittent active motion, continuous passive motion for two weeks, or continuous passive motion for four weeks. When the animals were killed four weeks postoperatively, the contour of the patellar groove had been restored in all of the rabbits in the group that had had four weeks of continuous passive motion, and the newly formed tissue in all of the defects in this group had the gross, histological, and histochemical appearance of smooth, intact hyaline articular cartilage. Histologically, the nature of the tissue that had formed, as well as its surface regularity, structural integrity, and bonding to the adjacent cartilage, were significantly better in the group that had had four weeks of continuous passive motion than in any of the other groups. The results were significantly worse when the orientation of the periosteal graft was reversed (that is, when it had been sutured into the defect with the cambium layer of the graft facing the subchondral bone rather than into the joint) or when no periosteal graft was used. Biochemical analyses revealed that, in the group that had had four weeks of continuous passive motion, the total hexosamine content, the levels of chondroitin sulphate and keratan sulphate, and the ratio of galactosamine to glucosamine were all comparable with the values for normal articular cartilage. In contrast, in the groups that were treated by immobilization, intermittent active motion, or two weeks of continuous passive motion, as well as in the adult rabbits, the content of the first three of these substances was significantly less than normal. In the groups that were treated by immobilization, intermittent active motion, or two weeks of continuous passive motion, 32 to 47 per cent of the total collagen was type II, while in the group that had had four weeks of continuous passive motion, 93 per cent of the total collagen was type II. These results demonstrate that, under the influence of continuous passive motion, free autogenous periosteal grafts can repair a large full-thickness defect in a joint surface by producing tissue that resembles articular cartilage grossly, histologically, and biochemically, and that contains predominantly type-II collagen.