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.     

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.     

Experimental study on the repair of full thickness articular cartilage defects: effects of varying periods of continuous passive motion, cage activity, and immobilization

In order to clarify the dose/response characteristics of continuous passive motion (CPM), the repair response of full thickness articular cartilage defects was studied in a rabbit model. The following combinations of CPM and immobilization (Imm) were utilized: CPM, 24 h/day; CPM, 8 h/day and Imm, 16 h/day; CPM, 2 h/day and Imm 22 h/day; Imm 24 h/day; and normal cage activity. These regimens were used only in the initial week and then all rabbits were permitted to move freely in their cages, except for a sixth Imm-CPM group that was kept immobilized in the initial week and then CPM 24 h/day for another week. The CPM 24 h/day and the CPM 8 h/day groups (groups 1 and 4, respectively) showed better repair than the other groups, i.e., better surface congruity, larger positive Safranin-O staining area, and greater number of chondrocytes in the repair tissue. The CPM 2 h/day group (group 3), however, showed only slightly better repair than the Imm group (group 4). The CPM following immobilization was not effective to overcome the harmful effect of immobilization. We conclude that in the present model, CPM for 8 or 24 h/day is effective for adequate cartilage repair even with some component of immobilization. Its application should be at least 8 h/day. On the contrary, if CPM is delayed for a week following immobilization, the effect of CPM on cartilage will be reduced.     

The potential for regeneration of articular cartilage in defects created by chondral shaving and subchondral abrasion. An experimental investigation in rabbits

Animal models for chondral shaving and subchondral abrasion were created to resolve the controversy about the nature of the repair tissue after these procedures and to determine the effect of continuous passive motion on the quality of the repair tissue. Chondral shaving was performed on the patella in forty adolescent rabbits, and subchondral abrasion was performed on the patella in another forty rabbits. In both procedures, a three-millimeter-diameter defect was created. After the operation, twenty animals from each group were allowed intermittent active motion; the remainder were treated by continuous passive motion for two weeks, followed by intermittent active motion. Half of the animals from each group were killed at four weeks and the other half, at twelve weeks. There was no evidence of repair tissue in the defects at either four or twelve weeks after chondral shaving, regardless of the postoperative treatment. The remaining underlying cartilage, however, had degenerated. After abrasion of subchondral bone, the defects in animals that were treated with only intermittent active motion healed at twelve weeks, although the quality of the repair tissue varied. All ten of the animals that were treated with continuous passive motion, however, had mature, hyaline-like cartilage as the predominant repair tissue at twelve weeks, compared with six of the ten animals that were treated with intermittent active motion (p less than 0.05). We concluded that, in this model, partial-thickness defects created by chondral shaving do not heal; rather, the remaining underlying cartilage degenerates. Full-thickness defects created by subchondral abrasion can heal by regeneration of hyaline-like cartilage. Such healing is enhanced by continuous passive motion for two weeks postoperatively.     

Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A follow-up report at one year

An autogenous graft of tibial periosteum was sutured (with its cambium layer facing into the joint) to the base of a five by ten-millimeter full-thickness defect in the patellar groove of each of forty-five adolescent rabbits. The rabbits were randomly treated postoperatively by either four weeks of immobilization in a cast, intermittent active motion in a cage, or two weeks of continuous passive motion. One year postoperatively, the regenerated tissue from each rabbit was analyzed macroscopically, histologically, histochemically, and biochemically. Gross degenerative changes were seen in 57 per cent of the rabbits that had been immobilized in a cast, in 73 per cent of the rabbits that had been allowed intermittent active motion, and in 22 per cent of the rabbits that had been subjected to continuous passive motion (p less than 0.05). Out of a possible score of 7.0 points for the nature of the regenerated tissue, the scores for the three groups were: immobilization in a cast, 4.1 points; intermittent active motion, 4.0 points; and continuous passive motion, 5.9 points (p greater than 0.05). Out of a possible perfect combined score of 10.0 points for the structural characteristics of the regenerated tissue, the cast-immobilization group scored 3.8 points; the intermittent active-motion group, 2.5 points; and the continuous passive-motion group, 6.4 points (p less than 0.001). The total scores for freedom from cellular changes of degeneration, a perfect score being 5.0 points, were: immobilization in a cast, 2.4 points; intermittent active motion, 2.3 points; and continuous passive motion, 3.9 points (p less than 0.01). Degenerative changes in the adjacent cartilage, which were noted in 42 and 46 per cent of the knees in the immobilization and intermittent active-motion groups, respectively, were not found in the knees that had been subjected to continuous passive motion (p less than 0.05). The total indices, which were derived by combining the scores for all categories (maximum, 24.0 points), revealed that the index for the continuous passive-motion group was significantly better than the index for either of the other two groups: immobilization in a cast, 12.9 points; intermittent active motion, 11.2 points; and continuous passive motion, 19.2 points (p less than 0.0005).(ABSTRACT TRUNCATED AT 400 WORDS)     

Neochondrogenesis in free intraarticular periosteal autografts in an immobilized and paralyzed limb. An experimental investigation in the rabbit

The purpose of this investigation was to determine whether neochondrogenesis can be induced in free intraarticular autografts of periosteum in the complete absence of motion. In 17 adolescent rabbits, a rectangular graft of periosteum was elevated from the medial aspect of each proximal tibia and folded back on itself so that its deep (cambium) layer was facing outward on both sides. The grafts were transplanted into both ipsilateral knee joints that had been paralyzed by section of the femoral and sciatic nerves, and a cast was applied to one hind limb to provide immobilization. The opposite knee joint of each animal was then placed in the continuous passive motion (CPM) apparatus. At the time of death (21 days postoperatively), some degree of neochondrogenesis was evident in 69% of the grafts in the group with casts and in 100% of the grafts in the CPM group. Hyaline cartilage was the predominant tissue in 13% of the grafts in the group with casts compared to 63% of the grafts in the CPM group. Although this investigation has confirmed the chondrogenic potential of free periosteal grafts in a synovial fluid environment (and the significantly stimulating effect of CPM), the results have also demonstrated that at least some hyaline cartilage can be formed by periosteal grafts even with paralysis of the limb plus immobilization of the joint (presumably complete immobilization). Thus, other factors capable of stimulating neochondrogenesis warrant investigation.     

Periosteal neochondrogenesis for biologically resurfacing joints: its cellular origin

The purpose of this study was to determine if the hyaline-like cartilage produced in major full-thickness defects of a joint surface, treated by a free periosteal graft and subjected to continuous passive motion (CPM), originated exclusively from the progenitor cells of the cambium of the graft. Free periosteal grafts were raised from the tibia of both hind legs of eight male New Zealand rabbits and transplanted into full-thickness defects across the entire width of the patellar groove of 15 female rabbits. Postoperatively, CPM was instituted and the animals were sacrificed after 3 weeks. Cells from the regenerated tissue were grown from tissue explants and their karyotypes determined. In 33% of the rabbits, all cells contained a Y (male) chromosome, indicating that regenerated tissue originated exclusively from the progenitor cell of the periosteal graft. Karyotypes of the cells from the other 67% were mosaics (both female and male); thus, their cellular origin was from both the periosteal allograft and the pluripotential mesenchymal cells in the subchondral tissues.     

骨膜覆盖对骨髓基质细胞移植修复软骨缺损的影响

目的 :了解在软骨缺损修复过程中 ,骨膜覆盖对移植细胞存留的影响及与早期病理结果相关性以及核酸荧光染料标记法用于追踪移植细胞的可行性。方法 :将体外培养MSC用核酸荧光染料标记后 ,复合胶原海绵植入兔关节软骨全层缺损中 ,一组用骨膜覆盖 ,另一组不加覆盖。分别于 2周、 6周切取修复组织标本 ,2周标本胶原酶消化流式细胞仪检测 ,6周标本做光镜与电镜组织形态学观察。结果 :标记细胞荧光强度仍可被荧光镜和流式细胞仪所检测 ,骨膜覆盖组荧光标记率明显高于非骨膜覆盖组 ,差异有显著性 ,组织学观察也好于对照组。结论 :核酸荧光染料标记法可以作为短期追踪移植细胞的方法。骨膜覆盖能够提高种子细胞在缺损处的存留率 ,影响早期修复组织的病理结果。     

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。     

自体骨膜游离移植修复髋膝关节软骨大面积缺损的远期观察

目的观察自体骨膜游离移植修复髋、膝关节软骨大面积缺损的远期临床效果. 方法在动物实验基础上,于1987年2月～1996年8月,采用自体骨膜游离移植修复52例关节软骨大面积缺损的患者,其中有完整随访资料的37例中,先天性髋关节脱位16例,创伤性髋关节炎6例,股骨头低毒性感染1例,强直性脊柱炎2例,膝关节内骨折6例,骨性关节炎4例,创伤感染后膝关节僵直2例.术前有髋关节脱位者行松解牵引术,术中切除病变的软骨组织达出血骨组织;从胫骨前内侧切取大于软骨缺损10%～15%的骨膜,生发层朝向关节腔,缝合固定在软骨缺损表面;双侧移植骨膜的关节间放置硅胶膜;术后进行CPM练习,逐渐增加活动范围,活动时间为4～6周;出院后6个月内避免负重,可主动练习关节活动. 结果术后37例获7～15年随访,平均10.5年.功能评价标准按有无关节疼痛、关节活动角度、日常生活能力和X线片测量关节间隙宽度进行评价,结果:优11例,良18例,差8例. 结论游离骨膜移植修复关节软骨缺损,经远期随访应用于临床是可行的.     

基因修饰的组织工程骨联合带血管蒂骨膜移植修复长段骨缺损的研究

目的评价骨形态发生蛋白2（bone morphogenetic protein2,BMP-2）基因修饰的组织工程骨联合带血管蒂骨膜移植修复长段骨缺损的效果。方法分离培养兔骨髓基质干细胞,经BMP-2基因转染后复合异种骨支架体外构建基因修饰的组织工程骨（gene modified tissue engineering bone,GMB）。建立兔双侧桡骨缺损（长2.5cm）模型,采用5种方法修复。A组：GMB＋带血管蒂骨膜移植;B组：GMB＋血管束植入;C组：GMB＋游离骨膜移植;D组：GMB;E组：单纯支架。于术后第4、8、12周行X线、组织学、生物力学测定和微血管墨汁灌注等观察血管形成及成骨情况。结果①A组血运建立快,第8周时即可修复骨缺损,其修复机制包括膜内成骨和软骨成骨两种机制;②B组血管束发出分支向移植骨内长入,但中心区成骨缓慢,第12周时骨缺损得到完全修复;③C组第4周时游离骨膜成活并发出微小血管,第8周时形成薄层外骨痂,第12周时骨缺损基本修复;④D组在BMP-2基因诱导下成骨速度和质量优于E组,可在第12周时使骨缺损部分修复,但中心区呈＂空心＂现象;而E组第12周时形成骨不连,缺损区内被纤维组织填充。结论带血管蒂骨膜与BMP-2基因修饰的组织工程骨联合移植,既提供了血运又提供了骨膜成骨细胞,同时具有良好的骨生成、骨诱导和骨引导作用,是治疗节段性骨缺损较为理想的方法。     

Biodegradable guide for bone regeneration. Polyurethane membranes tested in rabbit radius defects.

Segmental, 1-cm osteoperiosteal defects were produced in both radii of 10 rabbits. One defect was covered with a biodegradable polyurethane membrane formed as a tube. The contralateral defect served as an untreated control. Healing was analyzed by radiographic and histologic studies after 5 weeks. Nine out of 10 control defects displayed nonunion, whereas membrane-treated defects consistently healed by forming callus external to the membrane fusing the bone fragments. Loose connective tissue was predominant in the bone gap underneath the membrane. Presumably, the membrane has served as a scaffold for regenerating periosteum.     

Biological resurfacing of full-thickness defects in patellar articular cartilage of the rabbit. Investigation of autogenous periosteal grafts subjected to continuous passive motion.

We compared the effects of continuous passive motion with those of intermittent active motion on the results of the resurfacing with autogenous periosteal grafts of full-thickness defects on the articular surface of rabbit patellae. Of 45 rabbits with defects, 30 received grafts. Fifteen of these had continuous passive motion for two weeks and intermittent active motion for four weeks; the other 15 had intermittent active motion for six weeks. In 15 the defects were not grafted (control group) and they had intermittent active motion for six weeks. Ten more rabbits had a sham operation. Six weeks after surgery, the results were assessed by the gross appearance, histology, histochemistry, immunohistochemistry and electron microscopy. By all assessments the quality of neochondrogenesis produced by periosteal grafts was superior to that in ungrafted defects (p less than 0.05) and the results in continuous passive motion treated animals were superior to those in intermittent active motion treated animals (p less than 0.05). The periosteal grafts produced hyaline cartilage containing type II collagen but the organisation of its fibres was irregular.     

自体软骨细胞团块植入修复兔陈旧性关节软骨缺损的实验研究

目的 观察自体软骨细胞团块植入对兔关节软骨缺损的修复作用. 方法 24只成年新西兰大白兔48侧膝关节,随机分为三组(n=16)并制备双膝关节股骨滑车软骨缺损模型.空白对照组无特殊处理,骨膜移植组将骨膜覆盖缺损并缝合于缺损两侧的股骨髁上,实验组将自体软骨细胞团块植入缺损中.术后3、6个月分别取材(n=8),进行大体和组织学观察,修复组织行Wakitani评分并进行比较. 结果实验组共成功取材11个缺损关节,9个为透明软骨修复,2个因植入细胞生长状态差未修复;骨膜移植组修复组织为纤维软骨或纤维组织,修复组织薄,基质异染弱;空白对照组仅有少量纤维组织填充缺损底部.修复组织Wakitani评分:实验组3.82分,骨膜移植组6.71分,空白对照组9.23分,差异有统计学意义(F=5.96,P=0.00). 结论自体软骨细胞团块植入能较好修复关节软骨缺损,修复的质量与植入细胞的质量有关.     

Autoarthroplasty of knee cartilage defects by osteoperiosteal grafts

Five fresh osteochondral fractures of the knee, which could not be fixed because of extensive fragmentation, were treated by excision of the fragments and reconstruction of the joint surface defect by an autogenous osteoperiosteal graft. The procedure was also used for joint surface reconstruction in sclerotic osteochondritis of the femoral condyle (nine knees) and grave patellofemoral chondromalacia (three knees). Plaster cast immobilization for 3 weeks was used in the two early cases. In all other cases, we employed a passive motion apparatus for 2 days postoperatively, followed by active mobilization in a knee brace with extension-flexion 30 to 90 degrees (femoral condyle reconstruction) or 0 to 45 degrees (patellar reconstruction). Gradual free movements were started 3 weeks postoperatively. The results after 1.5 to 6.5 years were satisfactory in all but one case. One arthroscopic removal of the loose graft was performed, as were two arthroscopic graft margin shavings. Three other reoperations were unrelated to the osteoperiosteal reconstruction. It appears that periosteal reconstruction should be considered in local osteochondral lesions, where excision of the injured cartilage is mandatory. The results were best in fresh trauma cases and younger people.     

Value of autologous chondrocyte transplantation in experimental cartilage defects reconstruction. Part III--Microscopic analysis of reconstructed cartilage thickness and integration with surrounding tissue]

Lack of cartilage vascularization is the reason of its low regenerative potential. The aim of this part of the study was microscopic evaluation of repair tissue thickness and its integration with surrounding cartilage, after autologous chondrocyte transplantation. MATERIAL AND METHODS: Repair of partial thickness cartilage defect (ICRS III(o) grade) on distal femur joint surface was evaluated (25 adolescent rabbits). Procedures were performed in two groups: I--autologous chondrocyte transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale. RESULTS: In group I, 8 weeks after the procedure most of defects were filled with the newly formed tissue almost completely. Regenerate thickness after 4 and 12 weeks usually exceeded 1/2 of surrounding cartilage. In group II, 8 weeks after the procedure regenerate thickness amounted to at least 1/2 of surrounding cartilage, but 4- and 12-week observation revealed the decreased repair tissue thickness. In group I, 4 weeks after the procedure regenerative tissue was well integrated with surrounding cartilage, and this trait still gradually increased with time. In group II, there was partial integration or no integration of repair tissue with surrounding cartilage. CONCLUSION: Obtained results indicate, that tissue formed after autologous chondrocyte transplantation with use of periosteal flap was better in its thickness and integration with surrounding cartilage, as compared to tissue formed after use of periosteum alone. Autologous chondrocyte transplantation can not guarantee complete filling of the cartilage defect with the graft tissue and full integration with surrounding cartilage, without three-dimensional scaffold application.     

The biologic concept of continuous passive motion of synovial joints. The first 18 years of basic research and its clinical application

The notoriously limited capacity of articular cartilage to heal or to regenerate plus the author's clinical observations and research on the deleterious effects of immobilization on joints led him to the biologic concept of continuous passive motion (CPM) of synovial joints in 1970. The hypothesis that CPM should stimulate pluripotential mesenchymal cells to differentiate into articular cartilage and should accelerate the healing of articular tissues has been validated by numerous scientific investigations of a variety of experimental models of the knee joint. These models have included full-thickness defects, intraarticular fractures, acute septic arthritis, partial thickness lacerations of the patellar tendon, semitendinosus tenodesis to replace the medial collateral ligament, autogeneic osteoperiosteal grafts in major defects, free autogeneic periosteal grafts, and periosteal allografts. In 1978, the author collaborated with Saringer, an engineer, to develop CPM devices for humans. CPM is clinically indicated following such procedures as open reduction of fractures, arthrolysis for posttraumatic arthritis, synovectomy, drainage of septic arthritis, release of joint contractures, total arthroplasty, tendon repair, and ligament reconstruction. Clinically, CPM is an important stimulus to joint regeneration processes.     

Traumatic heterotopic bone formation in the quadriceps muscle: No progression by continuous passive motion in rabbits

One hind limb in each of 20 New Zealand White rabbits was immobilized for 3 weeks together with daily forcible manipulation to induce heterotopic bone formation in the quadriceps muscle. the rabbits were then divided equally into a control group and a group treated with continuous passive motion (CPM). the effect of CPM on the development of heterotopic bone formation was assessed by radiographs of the femur and by histology. Treatment with CPM did not lead to increased heterotopic bone formation, as compared to the control group.     

Effect of gradual weight‐bearing on regenerated articular cartilage after joint distraction and motion in a rabbit model

The purpose of this study was to clarify the effect of gradual weight bearing (GWB) on regenerating cartilage. We developed a novel external fixation device (EFD) with a controllable weight‐bearing system and continuous passive motion (CPM). A full‐thickness defect was created by resection of the entire articular surface of the tibial plateau after the EFD was fixed in the rabbit's left knee. In the GWB group (n = 6), GWB was started 6 weeks after surgery. In the CPM group (n = 6), CPM with EFD was applied in the same manner without GWB. The control group (n = 5) received only joint distraction. All rabbits were sacrificed 9 weeks after surgery. The central one‐third of the regenerated tissue was assessed and scored blindly using a grading scale modified from the International Cartilage Repair Society visual histological assessment scale. The areas stained by Safranin‐O and type II collagen antibody were measured, and the percentage of each area was calculated. There was no significant difference in the histological assessment scale among the groups. The percentage of the type II collagen‐positive area was significantly larger in the GWB group than in the CPM group. The present study suggests that optimal mechanical stress, such as GWB, may affect regeneration of cartilage, in vivo. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:600–606, 2010     

自体骨—骨膜移植修复骨软骨缺损：初步报告

根据骨膜具有再生关节软骨能力的特性，我们将取自于胫骨内侧骺端的骨一骨膜用于修复膝关节表面的骨软骨缺损。临床应用５例，包括剥脱性骨软骨炎、软骨下骨坏死及陈旧性髌骨骨折。经１６～２６个月随访，以膝关节功能评分法评价，移植手术获满意效果。摄片及磁共振影像证实缺损得以修复。此结果表明骨一骨膜移植是修复骨软骨缺损的有效方法。