The menisci are semilunar fibrocartilaginous structures attached to the tibial plateau that play important roles in knee function. They distribute stresses over a broad area of articular cartilage, absorb shocks during dynamic loading, and assist in lubrication of the joint. These functions enhance the ability of articular cartilage to provide a smooth, nearly frictionless articulation and to distribute loads evenly to the underlying bone of the femur and tibia. Damage to, or removal of, the menisci alters static load transmission across the knee joint1,6,8,11 and is associated with radiographic and arthroscopic evidence of degeneration of the articular cartilage of the knee10,12-14,16,18,19,26,27,29,32. The experimental and clinical finding that loss of meniscal tissue and function results in degenerative arthritis of the knee has led to efforts to repair and, when possible, to preserve damaged or torn menisci. Currently, however, there are few strategies available to replace menisci that have been irreversibly damaged or that have been operatively removed. Milachowski et al.22 were the first, to our knowledge, to replace a human meniscus with a cadaveric meniscus. The results of similar strategies have been reported by several groups3,17,30,31; however, it has been unclear, in these studies of humans, whether meniscal transplants prevent arthritis and whether they cause a rejection phenomenon. The operative techniques for fixation of the meniscal allografts have usually involved bone blocks attached to each end of the meniscus and have not been simple.
The aims of the present study, therefore, were to establish a simple operative method for meniscal transplantation in a large-animal model and to determine whether meniscal transplantation provides protection of the articular surfaces, whether meniscal allografts have the same protective effect as meniscal autogenous grafts, and whether there is any rejection phenomenon associated with meniscal allografts.
*Although none of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article, benefits have been or will be received, but are directed solely to a research fund, foundation, educational institution, or other nonprofit organization with which one or more of the authors is associated. The study was supported by St. George Private Hospital/Health Care of Australia and St. George Hospital/South Sydney Area Health Service. Johnson and Johnson/Mitek Medical supplied the suture anchors and suture materials, and Synthes supplied the screw implants for the experiment.
†Department of Orthopaedic Surgery, St. George Hospital Campus, University of New South Wales, Kogarah, Sydney, New South Wales 2217, Australia. E-mail address for Dr. Murrell: murrellg@ori.org.au.
‡Division of Comparative Medicine, School of Medicine, State University of New York, Buffalo, Room 118, Biological Education Building, Buffalo, New York 14214.
§Douglass Hanly Moir Pathology, Sydney, 95 Epping Road, North Ryde, New South Wales 2113, Australia.
Experimental Design
Twenty-eight Merino Border-Leister second-generation crossbred wether sheep (Talawa, Lithgow, Australia), weighing between forty and fifty kilograms, were used in this study. The study was approved by the Animal Care and Ethics Committee of our institution, and we followed the regulations of our university, the Animal Research Act and Regulation of the State, and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes guidelines. The twenty-eight sheep were randomly divided into four groups. The first group (four sheep) was treated with a sham operation consisting of a medial arthrotomy without violation of the meniscus. The second group was treated with a medial meniscectomy, with complete removal of the medial meniscus. The third group was treated with an autogenous graft; the medial meniscus was removed and immediately reimplanted. The fourth group was treated with an allograft; the medial meniscus was removed and replaced by a fresh meniscus from another sheep. The second, third, and fourth groups consisted of eight sheep each.
Operative Technique
The operations were performed with the sheep under general anesthesia, induced intravenously with ten milligrams of thiopental per kilogram of body weight and maintained with 1 to 4 percent (volume per volume) halothane (Fluothane) in oxygen after the animals were intubated. With the animal supine, the right lower limb was shaved, scrubbed with 4 percent (weight per volume) chlorhexidine gluconate (Pre-Scrub II; SmartCare, Phoenix, Arizona), then scrubbed with 10 percent (weight per volume) povidone-iodine (Betadine), and isolated with Impervious Split Sheets (Surgical Products, Kimberly-Clark, Roswell, Georgia). A single dose of one gram of cephalothin sodium (Keflin) was administered intravenously preoperatively.
A medial arthrotomy of the stifle joint (knee) was performed with use of a vertical incision. To achieve full access to the medial compartment of the knee, the medial collateral ligament was detached from the femoral insertion with a 1.5-centimeter-diameter predrilled and tapped bone block. The joint capsule was incised parallel to the anterior and posterior edges of the medial collateral ligament and along its femoral insertion three centimeters anterior and three centimeters posterior to the medial collateral ligament. In the group treated with the sham operation, the medial collateral ligament was reattached with a thirty by four-millimeter partially threaded cancellous-bone screw (Synthes; Rosebery, New South Wales, Australia); the joint capsule, retinaculum, and subcutaneous tissue were closed anatomically with use of 3-0 absorbable Vicryl (polyglactin) sutures; and the skin was closed with 2-0 absorbable PDS-II (polydioxanone) sutures (Johnson and Johnson Medical, North Ryde, New South Wales, Australia).
In the groups treated with a meniscectomy or transplantation, the joint capsule was dissected from the periphery of the meniscus, the attachments of the anterior and posterior horns were transected, and the meniscus was removed in one piece. In the group treated with the meniscectomy, the meniscus was not replaced and the joint was closed as it was after the sham operations. In the group treated with the autogenous graft, the meniscus was replaced as will be described. In the group treated with the allograft, the removed meniscus was transferred to serum-free tissue-culture medium (DMEM; Gibco, Life Technologies, Melbourne, Australia) and maintained at room temperature for forty-five minutes. The meniscus was then transplanted into a recipient animal. In both groups treated with a meniscal transplant, three Mitek GII suture anchors (Johnson and Johnson Medical) loaded with number-1 Ethibond sutures were inserted at the insertion sites of the anterior and posterior horns and at the medial edge of the medial tibial condyle at the margin of the articular cartilage; the meniscal transplant was secured to the tibial plateau with these sutures (Figs. 1-A and 1-B).
The wound margins were infiltrated with 0.5 percent bupivacaine (Marcaine), and 0.3 milligram of buprenorphine hydrochloride (Temgesic) was administered intramuscularly before extubation was performed. The wound was dressed with ten by ten-centimeter paraffin gauze (Jelanet; Smith and Nephew, Clayton, New South Wales, Australia), and the limb was wrapped with two layers of cotton wool and two layers of elastic bandage (Elastoplast; Smith and Nephew).
Postoperatively, the sheep were closely monitored for signs of distress and pain, and an intramuscular injection of buprenorphine was repeated every twelve hours for two days, or longer when needed. The animals were allowed to bear weight immediately and to continue to recover in 1.8-square-meter holding pens (one animal in each pen). On the fourth postoperative day and for the next four postoperative weeks, the animals were kept in pairs in temperature-controlled rooms with twelve-hour light-dark-cycle lighting. For the remaining sixteen weeks of the study, the sheep were allowed to exercise freely in an open 600-square-meter yard during the day and were penned at night. At sixteen weeks after the operation, the animals were killed with the intravenous administration of 160 milligrams of pentobarbital (Lethobarb) per kilogram of body weight.
Macroscopic Evaluation
After the animals were killed, the knee joints were examined for range of motion and for stability (with use of the drawer test at 90 degrees of flexion and with varus and valgus stress tests at 20 degrees of flexion). The joints were then dissected, with the femur separated from the tibia and the menisci left attached to the tibial plateaus. The integrity and size of the meniscus and the articular cartilage surfaces of the medial femoral and tibial condyles were evaluated macroscopically by individuals who were blinded to the experimental group. The macroscopic damage to the cartilage of the medial tibial and femoral condyles was graded as 0 points if the cartilage was normal, 1 point if there was focal softening, 2 points if there was surface fibrillation, 3 points if there were fissures and clefts, 4 points if there was less than one square centimeter of erosion of the cartilage, and 5 points if there was more than one square centimeter of erosion of the cartilage; 1 point was added if osteophytes were present.
Photographs of the tibial plateaus, with and without the menisci in place, and of the exposed femoral condyles were made with a scale as a reference, with use of a Canon EOS camera (Canon, Tokyo, Japan). The camera had a variable-focal-length lens of twenty-eight to eighty millimeters and a three-centimeter extension tube with a film plane-subject distance of twenty centimeters. The photographs were made with use of 3200K color balanced tungsten-filament indoor lights (Philips Lighting, Eindhoven, The Netherlands) oriented at a 45-degree angle to the subject, an 80B filter, and 100 ISO color print film (Fuji Reala; Fuji Photo Film, Tokyo, Japan). After ten by fifteen-centimeter prints had been developed, the area of the tibial condyle uncovered by meniscal or scar tissue, the area of the meniscus and the scar tissue, and the area of damaged cartilage on both the medial tibial and the medial femoral condyle were digitized and calculated with a Sigma Scan pad (Jandel Scientific, San Rafael, California).
Histological Evaluation
Tissue samples for histological evaluation were collected from the synovial membrane, the meniscus, the tibia, and the femur. Synovial membrane was obtained immediately superior to the anterior horn of the medial meniscus. Coronal sections were obtained from the junction of the middle and posterior thirds of the medial meniscus. Coronal osteochondral slices (five to six millimeters wide) were obtained from the central areas of the medial femoral and tibial condyles. The tissue samples were fixed in 10 percent (volume per weight) neutral buffered formalin at room temperature for twenty-four hours. The osteochondral components were decalcified in Rapid Decalcifier (R.D.O. Apex Engineering Product, Plainfield, Illinois) until just pliable and slightly gritty on sectioning (four to five hours). The synovial, meniscal, and decalcified osteochondral components were embedded in paraffin, and four-micrometer-thick sections were cut in transverse and longitudinal orientations for the meniscal samples and in transverse orientation for the osteochondral samples; they were then stained with hematoxylin and eosin and toluidine blue.
Light microscopic assessment of specimens of the synovial membrane, meniscus, and osteochondral fragments was performed with both polarized and nonpolarized light by one observer who was blinded to the experimental groups. Assessment of the synovial membrane included noting the configuration of the surface of the synovial membrane and the presence or absence of synoviocyte hyperplasia. The presence or absence of a subsynovial inflammatory infiltrate of lymphoid and plasma cells was noted and, if one was present, its distribution (diffuse or focal) and its severity (mild [less than twenty cells in a group], moderate [twenty to 100 cells in a group], or severe [more than 100 cells with lymphoid follicle formation) were documented.
Changes in the articular cartilage and subchondral bone on the medial femoral and tibial condyles were assessed with use of the histological-histochemical grading system established by Mankin et al.20. The structure of the cartilage was graded as 0 points if it was normal, 1 point if there were surface irregularities, 2 points if there were pannus and surface irregularities, 3 points if there were clefts to the transitional zone, 4 points if there were clefts to the radial zone, 5 points if there were clefts to the calcified zone, and 6 points if there was complete disorganization. The nature of the chondrocytes was graded as 0 points if they were normal, 1 point if there was diffuse hypercellularity, 2 points if there was cloning, and 3 points if there was hypocellularity. Proteoglycan staining was graded as 0 points if it was normal, 1 point if it was slightly reduced, 2 points if it was moderately reduced, 3 points if it was severely reduced, and 4 points if no stain was noted. The integrity of the tidemark was graded as 0 points if it was intact and 1 point if it was crossed by blood vessels.
The menisci were evaluated in terms of their contour and collagen distribution on polarized light microscopy and in terms of the cellularity, the nature of the connective-tissue cells, and the presence or absence of fibrinoid degeneration of the collagen. The meniscal configuration was assessed as to whether it was smooth or irregular, either diffusely or focally.
Statistical Analysis
All values are expressed as the mean and the standard error of the mean. Data were compared with use of unpaired two-way Student t tests and the Mann-Whitney rank-sum test. P values of less than 0.05 were considered to be significant. The correlation between the size of the area of damaged cartilage and the size of the area of the medial tibial condyle that was uncovered was calculated with use of a Spearman rank-order correlation. Histological findings in the synovial membrane and menisci were compared with use of chi-square analyses.
Every animal recovered from the operation. There were no infections or other complications. At four months postoperatively, all knees were stable to anteroposterior, varus, and valgus testing and all had a full range of motion (0 to 140 degrees of flexion).
Macroscopic Evaluation
The menisci in the knees that had been treated with the sham operation had a shiny-white color and a smooth appearance (Fig. 2, A). In the group treated with the meniscectomy, repair tissue consisting of thin and soft peripheral scar tissue partially covered the area of the resected meniscus on the medial tibial condyle (Fig. 2, B). All of the transplanted menisci had healed to the surrounding capsule. The macroscopic appearance and size of both the meniscal autogenous grafts and the meniscal allografts were comparable with those in the group treated with the sham operation. The transplanted menisci were, however, less shiny than those in the group treated with the sham operation (Fig. 2, C and D).
The mean area (and standard error of the mean) of the cartilage on the medial tibial condyle that was uncovered was 74 ± 10 square millimeters in the group treated with the sham operation and 230 ± 20 square millimeters in the group treated with the meniscectomy (Fig. 3). Compared with the meniscectomies, the meniscal transplants decreased the amount of the medial tibial articular surface that was uncovered, to 146 ± 14 square millimeters in the group treated with the autogenous graft and to 153 ± 14 square millimeters in the group treated with the allograft; however, the meniscal transplants did not completely restore the size of the exposed area to that found in the group treated with the sham operation. The amount of the tibial articular surface that was exposed in the transplantation groups was midway between that found in the group treated with the meniscectomy and that found in the group treated with the sham operation (p < 0.01). There was no significant difference between the group treated with the autogenous graft and that treated with the allograft with respect to the amount of exposed cartilage on the tibial condyle.
The mean areas of the menisci after the sham operations (306 ± 17 square millimeters), the autogenous graft procedures (305 ± 11 square millimeters), and the allograft procedures (270 ± 17 square millimeters) were not significantly different from one another. The mean area of the peripheral scar tissue occupying the space of the original meniscus after the meniscectomies (174 ± 17 square millimeters) was almost half the mean areas of the menisci in the other three groups. This reduction was significant (p < 0.05).
No macroscopic damage was found in the articular cartilage of the medial compartment after the sham operations (Fig. 4, A). In contrast, the group treated with the meniscectomy had extensive destruction of the articular cartilage on the medial tibial and femoral condyles, changes that were reflected in the mean score of 6.5 ± 0.8 points for the macroscopic damage to the cartilage (Fig. 4, B). There was significantly less (p < 0.05) macroscopic evidence of joint destruction in the groups treated with the autogenous graft (mean score, 3.9 ± 0.7 points) and the allograft (mean score, 4.3 ± 0.6 points) (Fig. 4, C and D) compared with the group treated with the meniscectomy. However, there was no significant difference between the two transplantation groups with respect to the scores for macroscopic damage to the articular cartilage (Fig. 5).
The mean area of macroscopic damage to the articular cartilage was minimum (2 ± 0.6 square millimeters) in the group treated with the sham operation. It was significantly greater (p < 0.01) in the group treated with the meniscectomy (33 ± 6 square millimeters). The mean area of macroscopic damage was reduced by approximately 50 percent in both transplantation groups when compared with the group treated with the meniscectomy (p < 0.05). There was no significant difference with respect to the area of macroscopic damage to the articular cartilage between the group treated with the autogenous graft (17 ± 4 square millimeters) and that treated with the allograft (15 ± 5 square millimeters) (Fig. 6).
The data from all groups were combined, and linear regression analysis was performed in order to determine if there was an association between the amount of medial tibial cartilage that was uncovered and the area of macroscopic damage to the articular cartilage in the medial compartment (Fig. 7). This analysis confirmed a good correlation between the two parameters (r = 0.74).
Histological Evaluation
In all groups, including the one treated with the sham operation, the synovial membrane had a villous configuration with mild synoviocyte hyperplasia. A focal mild subsynovial inflammatory infiltrate of lymphocytes and plasma cells (approximately twenty cells in each group) was noted in two of the eight knees treated with an autogenous graft and in four of the eight treated with an allograft.
Use of the histological system of Mankin et al.20 to grade the damage to the articular cartilage on the medial tibial and femoral condyles demonstrated the maintenance of normal cartilage in the group treated with the sham operation (mean score, 2 ± 0.4 points) and significantly greater damage (p < 0.001) in the groups treated with the meniscectomy and the meniscal transplants. There were no significant differences among the groups treated with the meniscectomy (mean score, 19 ± 2 points), the autogenous graft (mean score, 15 ± 2 points), and the allograft (mean score, 19 ± 2 points) with respect to the microscopic destruction of cartilage.
The histological characteristics of the meniscus remained normal in all knees treated with the sham operation. Mild surface irregularity was found in two of the eight meniscal autogenous grafts, and mild surface fibrillation was noted in six of the eight meniscal allografts. Mild focal fibrinoid degeneration was detected in one meniscal autogenous graft; in contrast, all of the allografts had clear areas of fibrinoid degeneration. On polarized-light microscopy, collagen bundles were distributed in a predominantly circumferential manner with fewer radial fibers in all menisci in all groups. The connective-tissue cells were single, evenly distributed, and slightly round, giving them a chondroid appearance in all of the knees treated with the sham operation. Focal clustering or cloning of chondroid cells was found in four of the eight meniscal autogenous grafts (Fig. 8-A). In contrast, there was diffuse, easily recognizable clustering or cloning of the chondroid cells in all of the allografts (Fig. 8-B). Fibrosis was evident at the peripheral margin of all of the meniscal transplants but was absent in the menisci from the knees treated with the sham operation. A focal mild inflammatory infiltrate that was composed predominantly of plasma cells and lymphocytes was found in the peripheral third of two of the eight meniscal autogenous grafts and in four of the eight meniscal allografts (Fig. 8-C). Compared with the group treated with the sham operation, there was a significant increase in surface irregularity (p < 0.01), fibrinoid degeneration (p < 0.001), and cloning of chondroid cells (p < 0.001) in the allografts but not in the autogenous grafts (Table I).
The overall aim of meniscal transplantation is to protect the articular cartilage from the degeneration that occurs after meniscectomy. To our knowledge, this is the first report that clearly demonstrated, in large laboratory animals, that meniscal transplants, both autogenous and allogenic, have a protective effect on the articular cartilage, as determined by macroscopic grading of the cartilage and by measurement of the areas of damaged cartilage in the medial compartment of the sheep knee. The effects of meniscal allografts with respect to the protection of cartilage were similar to those of meniscal autogenous grafts at four months after transplantation in this model.
Medial meniscectomy in animals is frequently used as a model for osteoarthritis of the knee, and the degree of degeneration that we observed in the articular cartilage in our group treated with meniscectomy was consistent with the findings of other studies2,4,7,15,21,23,28. The group treated with the sham operation, in which no damage to the cartilage was seen, served as a reliable negative control. The groups treated with the meniscal transplants had a 34 and 40 percent reduction in the score for macroscopic damage to the cartilage and an approximately 50 percent reduction in the area of damaged cartilage compared with the group treated with the meniscectomy. The correlation between the area of cartilage damage and the area of the medial tibial condyle that was exposed is also consistent with a protective effect of the meniscal transplants.
In studies of meniscal allografts in dogs, Arnoczky et al.2 and Mikic et al.21 reported some degree of protection of the cartilage by the grafts. However, they did not grade their observations or compare the results with those after meniscectomy. Edwards et al.9 evaluated osteoarthritic changes in sheep knees radiographically and found no difference between the results of meniscal transplants and those of meniscectomy at twenty-one months. They concluded that meniscal autogenous grafts and allografts did not protect cartilage. Using a rabbit model, Cummins et al.7 found that meniscal allografts offered some protection to the articular cartilage, as evaluated histologically. In our sheep model, we found no difference between the group treated with the meniscectomy and those treated with a transplant when the most damaged cartilage areas were graded histologically.
From a biomechanical viewpoint, the sizing, positioning, and fixation of a meniscal graft seem to be crucial. Recent studies of human cadavera demonstrated that secure, isometric fixation of the anterior and posterior horns of the meniscal transplant was essential to reduce contact pressure and to increase the contact area in the knee joint5,25. Current techniques for meniscal transplantation involve the use of bone blocks or sutures through tibial tunnels to secure both ends of the meniscal transplant4,9,15,22. While we did not specifically address graft fixation in the present study, we did find that using suture anchors provided a simple and quick means to secure the ends of the meniscal graft onto the tibia, and there was uniformly satisfactory healing of the transplanted menisci to the joint capsules.
It is interesting to note that in our study even the meniscal autogenous grafts did not provide complete protection to the articular cartilage. This was not due to the effect of operative trauma, as the group treated with the sham operation (which included detachment of the medial collateral ligament, a medial arthrotomy, and exposure of the entire medial meniscus) did not have any cartilage damage. As the shape and size of the autogenous grafts were ideal and the sizes of the allografts and the autogenous grafts at four months after transplantation were comparable with those of the menisci in the group treated with the sham operation, we hypothesized that the damage to the articular cartilage observed in the transplantation groups was secondary to nonisometric positioning and tensioning and to rigidity of fixation of the grafts.
The most remarkable difference between the autogenous grafts and the allografts was in their histological appearance. The autogenous grafts had a normal or nearly normal histological appearance. In contrast, there was obvious fibrinoid degeneration, areas of hypocellularity, and cloning of meniscal cells in all of the allografts. These findings are consistent with those reported in menisci during the first six months after transplantation in other studies2,15,21,24.
It is possible that the perivascular plasma-cell and lymphocyte infiltrations at the peripheral portion of the meniscal allografts were cellular markers of an immune response or of rejection; however, they were observed only in a few grafts and they were not significantly more frequent or severe than the infiltrations in the autogenous grafts. This observation is similar to the findings in studies of meniscal allografts in dogs and mice4,15,21,24, and they suggest that rejection phenomena directed against allogenic meniscal tissue in dogs, mice, and sheep are limited, if present at all. Whether the fibrinoid degeneration and the cloning of meniscal cells in the meniscal allografts affect the ultimate long-term outcome of transplantation of a meniscal allograft remains an interesting unanswered question. This study did not address the issue of fresh compared with stored tissue.
In summary, the results of our study of a sheep model suggest that meniscal transplantation after meniscectomy provides considerable and significant, but not complete, protection from macroscopic damage to the articular cartilage on the medial aspects of the tibia and femur. The protective effects of meniscal allografts on the articular cartilage were comparable with those of the meniscal autogenous grafts. There was no significant cellular immune response to the allografts. If these results are applicable to humans, they would support the use of meniscal transplantation, but it may be necessary to pay special attention to the isometricity of meniscal grafts.
Note: The authors thank Mr. Mark Bromley, Dr. Ashish Diwan, Mr. Daniel Jang, Dr. Andrea Kruller, Dr. Jianhao Lin, Mr. John Pachas, Dr. Minoo Patel, Mr. Grant Taggart, Dr. Dong Wang, Dr. Min Wang, Dr. Ai-Qun Wei, and Mr. Kevin Woodford for their technical assistance.