We investigated the clinical, arthroscopic and

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1 Transplantation of cartilage-like tissue made by tissue engineering in the treatment of cartilage defects of the knee M. Ochi, Y. Uchio, K. Kawasaki, S. Wakitani, J. Iwasa From the Shimane Medical University, Izumo, Japan We investigated the clinical, arthroscopic and biomechanical outcome of transplanting autologous chondrocytes, cultured in atelocollagen gel, for the treatment of full-thickness defects of cartilage in 28 knees (26 patients) over a minimum period of 25 months. Transplantation eliminated locking of the knee and reduced pain and swelling in all patients. The mean Lysholm score improved significantly. Arthroscopic assessment indicated that 26 knees (93%) had a good or excellent outcome. There were few adverse features, except for marked hypertrophy of the graft in three knees, partial detachment of the periosteum in three and partial ossification of the graft in one. Biomechanical tests revealed that the transplants had acquired a hardness similar to that of the surrounding cartilage. We conclude that transplanting chondrocytes in a newly-formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee. J Bone Joint Surg [Br] 2002;84-B: Received 1 December 2000; Accepted after revision 30 May 2001 Hunter s statement 1 in 1743 that cartilage once destroyed, is not repaired has remained essentially true. Numerous attempts have been made to repair defects of cartilage which have been caused by trauma or osteochondritis dissecans, but the results have only reinforced his observation. These unsatisfactory results reflect the poor healing capacity of cartilage arising from its isolation from systemic regulation, and its lack of vessels and nerve supply. 2 None of the normal inflammatory and reparative processes M. Ochi, MD, PhD, Professor and Chairman Y. Uchio, MD, PhD, Assistant Professor K. Kawasaki, MD, Postgraduate J. Iwasa, MD, Postgraduate Department of Orthopaedics, Shimane Medical University, 89-1 Enya-cho, Izumo-shi, Shimane-ken , Japan. S. Wakitani, MD, PhD Department of Orthopaedic Surgery, Osaka-Minami National Hospital, 2-1, Kidohigashimati, Kawachinagano City, Osaka , Japan. Correspondence should be sent to Professor M. Ochi British Editorial Society of Bone and Joint Surgery X/02/ $2.00 is available for its repair. Furthermore, chondrocytes which are surrounded by an extracellular matrix cannot migrate to the site of injury from an intact healthy site, unlike most tissues. 3 Injuries which reach the subchondral bone may induce a systemic reaction and generate reparative tissue. Although type-ii collagen may be produced by the latter, the new tissue consists predominantly of type-i collagen, resulting in the formation of fibrocartilage. Type-I collagen does not have the biomechanical properties of articular cartilage. It cannot function as normal hyaline cartilage and eventually degenerates. 4 The techniques for repairing defects of cartilage have been partially successful, in that they may reduce pain and increase mobility. Until recently, however, there has been no well-established solution to this problem. Brittberg et al 5 introduced new cell technology, in which cultured chondrocytes were transplanted into defects, raising the expectations of a breakthrough in repairing damaged articular cartilage. In this technique, cartilage slices were first obtained by arthroscopy from an unloaded area of the femoral condyle. The chondrocytes were then multiplied under monolayer culture after enzymatic digestion, and grafted in suspension into a cartilaginous defect which had been covered with a flap of periosteum. The clinical results were satisfactory, and biopsies of the graft sites showed hyaline-like cartilage repair in 12 of 22 patients. Three theoretical issues about the culture and transplantation procedure are of concern. One is the maintenance of the chondrocyte phenotype during a prolonged monolayer culture. In such a culture, chondrocytes tend to assume a fibroblastic dedifferentiated morphology, lose their ability to form a matrix, and synthesise predominantly type-i collagen. 6 Dedifferentiated chondrocytes can reexpress the chondrocyte phenotype after only a limited number of passages. 7 Therefore it is unclear whether transplanted fibroblastic chondrocytes can re-express the phenotype after transplantation in suspension. The second concern is the risk that chondrocytes may leak from the site of the graft after resumption of load-bearing because they are transplanted in suspension and the third is whether they will be evenly distributed in the three-dimensional space of the defect. The transplanted chondrocytes may accumulate on one side of the defect, mainly as a result of gravity, and not be distributed evenly. This may lead to uneven regen- VOL. 84-B, NO. 4, MAY

2 572 M. OCHI, Y. UCHIO, K. KAWASAKI, S. WAKITANI, J. IWASA eration of cartilage after transplantation. If these concerns can be addressed, the technique should become more effective. We have used new tissue-engineering technology to create a cartilage-like tissue in a three-dimensional culture system in an attempt to address these concerns. Collagen, 8,9 fibrin glue, 10 alginate and agarose 16,17 have been repeatedly shown to maintain the chondrocyte phenotype in three-dimensional cultures. Furthermore, the newly-formed tissue acquires a firmness after prolonged cultivation in collagen I. 8 Chondrocytes can also be distributed uniformly in the three-dimensional space and synthesise an extracellular matrix, forming new cartilage-like tissue. In clinical applications, however, these culture materials raise problems of immunogenicity and safety. To resolve these issues we elected to use atelocollagen, from which telopeptides have been removed, in the chondrocyte culture. We chose atelocollagen because the antigenic determinants on the peptide chains of type-i collagen reside mainly in the telopeptide regions. 18,19 Furthermore, a collagen tube made of this material was successfully used as a nerve conduit in a neural gap, without problems of immunogenicity or inflammation. 20 Recently, the favourable effects of atelocollagen as a composite skin substitute, 21 a carrier for bone morphogenetic protein, 22 and as an artificial urinary tract 23 have been reported. Atelocollagen has been used clinically in plastic surgery and dermatology. Its use for human chondrocyte culture remains unclear and we therefore carried out several experimental studies before the clinical application. Our in vitro and in vivo experimental results supported the hypothesis that transplanting chondrocytes cultured in atelocollagen gel would be effective in repairing articular cartilage defects, not only in animals but also in man, by maintaining the chondrocyte phenotype, reducing the risk of leakage, and distributing grafted cells evenly throughout the grafted site. On the basis of these studies, in 1996, with the approval of the Ethics Committee of Shimane Medical University, we began transplanting cartilagelike tissue to repair cartilage defects in patients. We now present the clinical, arthroscopic, and biomechanical outcome of treating cartilage defects of the knee with transplanted chondrocytes cultured in atelocollagen gel with a minimum follow-up of two years. Patients and Methods We studied 26 patients (28 knees) with full-thickness defects of cartilage on the load-bearing surface of a femoral condyle or on a patellar facet (Outerbridge grade II to grade IV 27 ) and disabling symptoms such as locking, pain, swelling and retropatellar crepitus (Table I). All provided written, informed consent according to the format of the Ethics Committee. The causes of the osteochondral defect were trauma (16 knees), osteochondritis dissecans (9), osteoarthritis (2) and chondromalacia patellae (1). The lesions were on the medial femoral condyle in 12 knees, the lateral femoral condyle in 13 and the patella in two. One knee had lesions on the medial femoral condyle and patella. The mean size of the lesion was 3.0 cm 2 (0.7 to 16.0). Isolation and culture of chondrocytes. We established preoperatively that patients were not allergic to atelocollagen gel. With the patient under spinal anaesthesia, arthroscopy was carried out under tourniquet control. After observing the shape, size and location of the defect, specimens of cartilage weighing 300 to 500 mg were obtained through an anteromedial or anterolateral approach either from a detached cartilage fragment or from an unloaded area of either the medial or lateral femoral condyle. The specimens were minced and washed three times in sterile 0.9% sodium chloride supplemented with antibiotics (tobramycin 0.1mg/ml). Within two hours of collection, the chondrocytes were isolated by incubation with 0.25% trypsin in sterile saline at 37 C for 30 minutes and then 0.25% collagenase (CLAUS II, 247 g/mg; Worthington Biochemical, Freehold, New Jersey) in Ham s F-12 medium supplemented with 15% patient s serum, HEPES buffer (10 mmol/l), gentamicin sulphate (50 g/ml), and amphotericin B (10 g/ml) for four to six hours. The isolated cells were collected by centrifugation (1500 rpm) and washed three times with the culture medium. Eight volumes of atelocollagen solution (3% type-i collagen; Koken Co Ltd, Tokyo, Japan) were added to one volume of concentrated Ham s F-12 (10x) and one volume of 0.05N NaOH with 2.2% NaHCO 2 and 200 mm HEPES with gentle agitation at 0 C. The cells were mixed thoroughly in the collagenmedium mixture. The amount of this mixture was determined by the size of the cartilage defect according to the formula; defect area 0.3 ml. The mixture was then placed in a 60 mm diameter culture dish (Falcon; Becton-Dickinson, Oxnard, California) and completely gelated by incubation at 37 C for ten minutes before being overlaid with 6 ml of the culture medium. The mean number of cells per dish was ( to ). Cell cultures were incubated in 5% carbon dioxide and 95% air at 37 C and fed with fresh medium containing L-ascorbic acid (50 g/ml) every two days. The culture medium was tested for bacterial growth on blood agar plates every week. During the 24 hours before culture, antibiotics were omitted from the culture medium so that bacterial contamination, if present, would be more easily detected. The cultivation time was three to four weeks. By then the atelocollagen gel, including chondrocytes, had become opaque and had acquired a jelly-like hardness. Transplanting the atelocollagen gel. The gel was transplanted 21 to 26 days after harvest of the cartilage. Prophylactic antibiotics (cefazorin sodium, 1 g) were given intravenously each day during and for one week after surgery. Under spinal anaesthesia, a medial or lateral parapatellar arthrotomy was carried out under tourniquet control. The chondral lesion was debrided as far as normal surrounding cartilage and until subchondral bone was visible. The defect was covered by a sutured periosteal flap taken from the THE JOURNAL OF BONE AND JOINT SURGERY

3 TRANSPLANTATION OF CARTILAGE-LIKE TISSUE MADE BY TISSUE ENGINEERING IN THE TREATMENT OF CARTILAGE KNEE DEFECTS 573 Table I. Details of the 28 knees (26 patients) with cartilage defects treated with chondrocytes transplanted in atelocollagen gel Number of Duration of Age Site of previous symptoms Associated Follow-up Case Gender (yrs) Disease* lesion Side Cause of injury operations (yrs) surgery (mths) 1 M 14 OCD LFC R Gradual onset M 32 OCD LFC R Meniscal injury 1 15 Partial 57 (discoid meniscus) meniscectomy 3 M 13 OCD MFC R Soccer strain M 31 Trauma MFC R Fall ACL reconstruction 52 5 M 16 Chondromalacia Patella R Baseball strain 2 2 Maquet 52 patellae 6 M 16 OCD MFC R Soccer strain M 39 OCD MFC R Gradual onset F 20 OCD MFC R Gradual onset M 16 OCD MFC R Twist M 39 OCD MFC L Gradual onset F 34 Trauma LFC R Volleyball strain 1 3 ACL reconstruction F 23 Trauma LFC R Soccer strain ACL reconstruction M 15 OCD LFC L Occasionally M 14 Trauma LFC R Soccer strain M 29 Trauma MFC R Baseball strain 1 13 ACL reconstruction F 14 Trauma Patella R Twist ET, lateral release F 23 Trauma Patella, MFC R Traffic accident 2 1 ET, lateral release M 24 Trauma LFC L Twist F 41 OA MFC R Gradual onset M 37 Trauma LFC R Baseball strain F 24 Trauma LFC R Badminton strain 1 10 ACL reconstruction F 17 Trauma LFC L Volleyball strain 0 1 ACL reconstruction M 39 Trauma LFC R Volleyball strain 0 5 ACL reconstruction M 45 OA MFC R After infection F 38 Trauma LFC L Basketball strain 2 21 ACL reconstruction F 35 Trauma MFC L Twist 2 11 ACL reconstruction M 30 Trauma MFC R Soccer strain 1 24 ACL reconstruction F 22 Trauma LFC L Ski strain * OCD, osteochondritis dissecans; OA, osteoarthritis LFC, lateral femoral condyle; MFC, medial femoral condyle ET, Elmslie Trillat proximal medial tibia. The flap was shaped and sutured to the surrounding rim of normal cartilage with interrupted 5-0 nylon and loosely tied 4-0 vicryl sutures with the deep cambium layer facing the subchondral bone plate. After suturing half of the border of the flap, the chondrocyte-atelocollagen gel was placed in the defect, and the remaining border of the flap was sutured. The joint capsule, retinaculum, and skin were sutured in separate layers. The knee was supported by a light-weight brace for two weeks. If required, the anterior cruciate ligament (ACL) was reconstructed using hamstring tendons assisted by arthroscopy three weeks before transplantation, at the time of harvest of the cartilage. Two weeks after transplantation, continuous passive movement of the joint was begun. Partial weight-bearing was introduced three weeks after operation and was gradually increased to full weight-bearing with muscle training during the first eight weeks after surgery. Clinical assessment. The clinical outcome was graded as described by Lysholm and Gillquist 28 with zero being poor and 100 excellent. We report here the overall Lysholm score. For the ACL-reconstructed knee, the anterior laxity of the knee was examined under a 133 N force, anteriorly applied, with the knee flexed at 30 using the KT-2000 knee arthrometer (MED Metric Corporation, San Diego, California). Both knees were examined and the side-to-side difference was recorded as the laxity. Arthroscopic assessment. Arthroscopy was undertaken 6, 12, 18 and 24 months after surgery. The hardness of the graft was tested with a probing hook, and the gross appearance was considered to be biologically acceptable if the transplanted cartilaginous tissue was in contact with, as well as level with, the surrounding articular cartilage. The arthroscopic results were graded according to the assessment scale of cartilage repair developed by the International Cartilage Repair Society (ICRS). This 12-point scale awards up to four points each for the degree of repair of the defect, the degree of integration with the surrounding cartilage tissue, and macroscopic appearance. Grade I (12 points) is considered normal, grade II (8 to 11 points) nearly normal, grade III (4 to 7 points) abnormal, and grade IV (1 to 3 points) severely abnormal. Biomechanical assessment. Biomechanical tests were also carried out during postoperative arthroscopy. The stiffness of the grafts was assessed by an ultrasonic biosensor system (Axiom Co Ltd, Fukushima, Japan) which measured the degree of softness or hardness of materials. A change in resonance frequency is closely related to the stiffness of materials. The probe of the ultrasonic tactile sensor was pressed against the central and marginal areas of the graft and the surrounding normal cartilage. As a reference, gelatin was used to derive the relationship between the stiffness of material (y) and the change in ultrasonic resonance frequency (x) expressed by the for- VOL. 84-B, NO. 4, MAY 2002

4 574 M. OCHI, Y. UCHIO, K. KAWASAKI, S. WAKITANI, J. IWASA mula: y = x where r 2 = If the resonance frequencies of the reparative tissue and normal cartilage were and Hz, the stiffness values of each tissue were calculated to be 0.67 and g/mm, respectively by this equation. The ratio of stiffness in the reparative tissue to the surrounding normal cartilage was calculated and reported as a percentage of the native (control) cartilage. Histological examination, For ethical reasons, biopsy specimens were obtained from only two patients, a 34-yearold man and a 16-year-old boy, with osteochondritis dissecans of the medial femoral condyle. The man had reinjured the osteochondral lesion one year after transplantation and the graft had detached with part of the subchondral bone. We replaced the detached osteochondral fragment and sutured it to the surrounding rim of normal cartilage with interrupted 5-0 nylon and 4-0 vicryl sutures. A marginal area was trimmed and analysed histologically. The boy underwent retransplantation because of periosteal ossification of the graft six months after transplantation. Biopsy specimens of the fragment were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and cut into 8 m sections and stained with Toluidine Blue or Safranin-O Fast Green. For immunohistochemical analysis S-100 antibody (DAKO Immunoglobulin, Glostrup, Denmark) was used to detect the chondrocyte phenotype and mouse antihuman type-ii collagen antibody (Fuji Chemical Industry Ltd, Toyama, Japan) for type-ii collagen production. For electron-microscopic examination, specimens were fixed in 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1M phosphate-buffered saline. After post-fixation in 2% OsO 4, the specimens were dehydrated in ethanol, embedded in Epon 812, and sectioned with an MT-5000 microtome. Ultrathin sections (60 to 80 m) were stained with 25% uranyl acetate in methanol and 2% Reynold s lead citrate, and viewed with a CX-2000 transmission electron microscope (JEOL Co Ltd, Tokyo, Japan). Statistical analysis. All statistical analyses were done using the Statview 4.2 program (Abacus, Berkeley, California). The clinical and arthroscopic values (the Lysholm and ICRS scores) and the ratio of stiffness were shown as the mean ± standard deviation. The clinical and arthroscopic data were analysed statistically by paired Student t- tests to determine whether the values varied significantly between the preoperative and postoperative condition. The ratios of stiffness were analysed statistically by ANOVA in order to determine whether the ratio varied significantly either by site or the postoperative time period. P values of less than 0.05 were accepted as significant. Results The results of chondrocyte transplantation in the 25 knees with femoral and the three knees with patellar defects are summarised in Table II. None of the cell cultures contained bacteria or fungi, and none of the patients had infections of the knee after transplantation. Transplantation for a femoral condylar defect. Clini- Table II. Details of the lesions, transplants and outcome for the 28 knees (26 patients) treated with chondrocytes transplanted in atelocollagen gel Graft stiffness Graft stiffness Size of lesion Cell number Lysholm score Arthroscopic (centre) at 2 years (margin) at 2 years Case (cm 2 ) ( 10 6 ) Harvest site* Preop Postop grade (ICRS) (% of control) (% of control) Free body NWA NWA Free body NWA NWA NWA + free body NWA + DC NWA + DC NWA + free body NWA + DC NWA + DC NWA + free body NWA NWA NWA + DC NWA + DC NWA NWA NWA NWA + DC NWA NWA NWA NWA + DC NWA + DC NWA + DC NWA + DC * NWA, non-weight bearing area; DC, detached cartilage THE JOURNAL OF BONE AND JOINT SURGERY

5 TRANSPLANTATION OF CARTILAGE-LIKE TISSUE MADE BY TISSUE ENGINEERING IN THE TREATMENT OF CARTILAGE KNEE DEFECTS 575 Fig. 1a Fig. 1b Case 3. Transplantation of autologous chondrocytes embedded in atelocollagen gel in a 13-year-old boy. Arthroscopy showing a) a cartilage defect in the medial femoral condyle before transplantation, and b) two years after transplantation. At the final followup (54 months) he was asymptomatic with a range of flexion from 0 to 145. He could play soccer comfortably. cally, in all patients, pain, swelling and crepitus were reduced, and locking of the knee resolved. Two years after surgery, clinical assessment showed that 22 of 25 knees (88%) had excellent results, scoring more than 90 points on the Lysholm scale, and the patients had returned to normal activities. Three knees had crepitus and occasional discomfort. The preoperative scores were significantly lower than the postoperative scores (70.6 ± 11.6 v 96.7 ± 4.7 points, p < 0.001). Two years after reconstruction of the ACL, the side-to-side difference in anterior laxity was 1.2 ± 2.3 mm (-3.0 to +5.0). All patients, except one woman (case 12), had stable knees (side-to-side difference 3.0 mm) and could return to their previous sporting activities. One patient (case 12), with a side-to-side difference of 5 mm, was able to play soccer without any disability. Arthroscopy, undertaken six months after surgery, showed that the transplants were congruous with the surrounding articular surface. They were white and slightly fibrillated, but soft in both the central and marginal areas. One year after transplantation, the surface had become smooth, and the marginal areas slightly firmer, whereas the central areas remained soft. At two years after surgery, the transplant had acquired a hyaline-like smooth surface and was as firm as the surrounding normal cartilage (Fig. 1). Arthroscopic assessment indicated that 26 knees (93%) had good or excellent outcomes (ICRS grade 1 or 2). No infection was detected. There were three knees with marked hypertrophy of the graft, three with partial detachment of the sutured periosteum, and one with partial ossification of the graft. Biomechanical tests showed that the transplants were as firm as the surrounding cartilage at one and two years after operation (centre, 91.5 ± 15.7% at one year and 89.0 ± 23.3% at two years; margin, 92.0 ± 21.7% at one year and ± 15.2% at two years). There was no significant correlation between the stiffness of the grafted sites and the time after transplantation, although there was a tendency for the grafted site to become more stiff with the passage of time. Transplantation for patellar defects. The clinical results for the three patients who had transplantation for a patellar defect were excellent (Table II). All returned to normal activities such as running, jumping or twisting. Arthroscopic assessment showed that the surface of the graft was smooth and slightly soft in all knees, although one showed periosteal detachment. The transplants acquired nearly the same stiffness as intact, normal cartilage. Histological examination. When stained with Toluidine Blue, the transplanted cells appeared round and had a metachromatic territorial matrix (Fig. 2a). The border between the grafted chondrocyte-gel complex and the subchondral bone of the defect was obscure, indicating a steady integration of the grafted tissue into the adjacent subchondral bone. Transmission electron micrographs showed that round-cell processes extended into the adjacent pericellular matrix (Fig. 2b). Immunohistochemical examinations indicated that transplanted cells stained positive with S-100 protein and the pericellular matrix with type-ii collagen, suggesting a similarity to normal hyaline cartilage (Figs 2c and 2d). Discussion We obtained good to excellent results when transplanting cartilage-like tissue made by tissue-engineering techniques into cartilage defects of the knee. Autologous chondrocyte transplantation appears to provide smooth hyaline cartilage. Grande et al 29 reported that 82% of the total area of cartilage was reconstituted in rabbits which received autologous chondrocyte transplants in vitro. Peterson et al 30 and Brittberg et al 31 showed that in the rabbit autologous chondrocyte transplantation was more effective in replacing hyaline cartilage VOL. 84-B, NO. 4, MAY 2002

6 576 M. OCHI, Y. UCHIO, K. KAWASAKI, S. WAKITANI, J. IWASA Fig. 2a Fig. 2b Fig. 2c Fig. 2d Photomicrographs of a detached fragment obtained one year after transplantation in a 34-year-old man who had osteochondritis dissecans of the medial femoral condyle and who then reinjured his knee. Figure 2a. Toluidine Blue ( 67). Figure 2b. Transmission electron micrograph ( 2333). Figures 2c and 2d. Immunohistochemical staining with c) anti-s-100 antibody ( 267) and d) anti-type-ii collagen antibody ( 267). than periosteum alone. Based on these results, Brittberg et al 5 used this method for repairing defects in the knee and reported good clinical results. Their results are supported by those of Minas and Nehrer. 32 The recent follow-up report of Peterson et al 33 also confirms the good outcome after autologous chondrocyte transplantation which was shown in their initial study. 5 The clinical results were good to excellent in 89 of 101 (88%) patients and biopsies of 37 graft sites showed repair by hyaline-like cartilage. By contrast, Breinan et al 34 found no significant differences in histological and histomorphometric variables for the reparative tissue in the defects of three groups in an animal model: chondrocyte injection with a periosteal flap, periosteal flap alone or none. Katsube et al 26 found that sites reconstructed by transplanted chondrocytes cultured in collagen gel had significantly better histological scores than those reconstructed by cultured chondrocytes in suspension, at six months after surgery. In addition, Peterson et al 33 detected fibrous tissue and mixed fibrous and hyaline cartilage in six of 21 patients with femoral lesions. Transplanting cells into an area where the number of cells available for repair is limited seems to be justifiable. Based on observations which suggest that injections of cells alone are insufficient to form hyaline cartilage, several issues must be considered. The first is the stability of the phenotype of cultured chondrocytes during prolonged culture. In a monolayer culture, chondrocytes tend to assume a fibroblastic, dedifferentiated morphology, lose the ability to accumulate matrix, and predominantly synthesise type-i collagen. 6 Our previous in vitro study in which human articular chondrocytes were used also showed that chondrocytes in a monolayer culture differentiated into fibroblast-like cells and synthesised chondroitin-4-sulphatedominant proteoglycans. 25 By contrast, chondrocytes cultured in atelocollagen gel maintained the chondrocyte phenotype and synthesised a chondroitin-6-sulphate-rich matrix. In addition, Coon and Cahn 35 reported that dedifferentiated chondrocytes, during four successive clonal passages in monolayer culture, re-expressed chondrocyte phenotypes when they were in contact with normal THE JOURNAL OF BONE AND JOINT SURGERY

7 TRANSPLANTATION OF CARTILAGE-LIKE TISSUE MADE BY TISSUE ENGINEERING IN THE TREATMENT OF CARTILAGE KNEE DEFECTS 577 chondrocytes or were in the presence of unknown highmolecular-weight molecules. Dedifferentiated chondrocytes only re-express the differentiated chondrocyte phenotype after a limited number of passages. 7 These data suggest that cultivation in atelocollagen gel is effective in maintaining the chondrocyte phenotype. Transplantation of chondrocytes with the chondrocyte phenotype may form hyaline cartilage better than that of dedifferentiated chondrocytes. Secondly, transplanted chondrocytes may leak from the grafted site through the sutures over the periosteum after load-bearing and decrease the number of chondrocytes available to form hyaline cartilage. Grande et al 29 reported that about 8% of cells leaked from the graft. Furthermore, one month after transplantation Katsube et al 26 showed that almost all the chondrocytes embedded within an atelocollagen gel were evenly distributed within the defect, but that they were almost no chondrocytes in a suspension culture group. These data suggest that transplanting a cartilage-like tissue gel reduces the risk of leakage and distributes the chondrocytes more evenly. The third issue is the possible uneven distribution of cells in the grafted space because of gravity or knee movement. This could lead to the uneven formation of cartilage, even if large numbers of cells do not leak from the grafted site. Transplantation of collagen gel has the advantage of a more uniform distribution of chondrocytes. 26 Incomplete integration between the cartilage-like tissue and the subchondral bone interferes with the attachment of the graft. 36 In our patients, in whom the grafted tissue was detached with part of the subchondral bone, and in the in vivo experimental specimens of Katsube et al, 26 there was good integration between the graft and subchondral bone with atelocollagen gel. Thus, several improvements over those of Brittberg et al 5 may reflect the good or excellent outcome in our study when compared with those of Peterson et al, 30 although our clinical and arthroscopic assessments were different. A marked hypertrophic response at the grafted site was noted in three of our 28 patients as well as in 26 of the 53 patients described by Peterson et al. 30 It is still unclear whether this was caused by hypertrophy of the periosteum, the graft, or both. Peterson et al 30 used a suspension, not gel, and speculated that chemical factors from the periosteum promote maturation of grafted chondrocytes and that too vigorous a response may induce hypertrophy. We agree with their theory because many unpredictable factors, such as cells and cytokines within periosteum, can induce chondrogenesis. 37 In a chronological MRI study on the grafted site, hypertrophy in our series appeared to be induced mainly by hypertrophy of the periosteal flap. Although the exact biological role of the grafted periosteum is unclear, viable periosteum should be replaced with non-viable periosteum, or an absorbable artificial membrane. Should an appropriate, biological, inert membrane become available, it may be better simply to cover the cartilage defect with the material after transplanting the cell-matrix complexes. Isogai et al 38 showed experimentally that tissue-engineering techniques can create whole joint structures with selective placement of periosteum, chondrocytes, and tenocytes into a biodegradable synthetic polymer scaffold. Their technique is currently unsuitable for clinical application because it does not create a vascular network and is subject to immunological rejection. We expect, however, that future tissue-engineering techniques will proceed along these lines. 39 We have used tissue engineering and the transplantation of cartilage-like tissue to repair cartilage defects in man. We have attempted to improve the surgical procedure of Brittberg et al 5 with a modification for cartilage repair with hyaline cartilage. We now need to improve our technique in order to develop a cartilage-like tissue of higher quality with a larger number of chondrocytes and a richer extracellular matrix. We may need to include cytokines or chemical factors capable of genetic induction with chondrocytes cultured in atelocollagen gel. 40,41 We anticipate that large bony defects secondary to a fracture, a cyst or the collapse of osteonecrosis, may be repaired in the future by more advanced techniques of tissue-engineering. We conclude that transplanting chondrocytes in an atelocollagen gel shows much promise in repairing full-thickness cartilage defects of the knee. Longer follow-up and comparison with other techniques of chondral repair are necessary in order to confirm this. Randomised trials may also need to be undertaken to establish whether the retained phenotype, reduced leakage, and more homogeneous distribution of cells in the defect provided by atelocollagen gel have clinical advantages over transplanting cells in solution. Although none of the authors have 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 non-profit institution with which one or more of the authors is associated. References 1. Newman AP. Articular cartilage repair. Am J Sports Med 1998;26: Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg [Am] 1982;64-A: Buckwalter JA, Mankin HJ. Articular cartilage: Part I: Tissue design and chondrocyte-matrix interactions. J Bone Joint Surg [Am]. 1997; 79-A: Furukawa T, Eyre DR, Koide S, Glimcher MJ. Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee. J Bone Joint Surg [Am] 1980;62-A: Brittberg M, Lindahl A, Nilsson A, et al. 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