Immature porcine knee cartilage lesions show good healing with or without autologous chondrocyte transplantation

OsteoArthritis and Cartilage (2006) 14, 1066e1074 ª 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2006.04.003

International Cartilage Repair Society

Immature porcine knee cartilage lesions show good healing with or without autologous chondrocyte transplantation A. I. Vasara M.D.*y, M. M. Hyttinen M.D.z, O. Pulliainen B.Sc.z, M. J. Lammi Ph.D.z, J. S. Jurvelin Ph.D.xk, L. Peterson M.D., Ph.D.{, A. Lindahl M.D., Ph.D.#, H. J. Helminen M.D., Ph.D.z and I. Kiviranta M.D., Ph.D.yy y Department of Orthopaedics, Helsinki University Hospital, Peijas Hospital, Vantaa, Finland z Institute of Biomedicine, Department of Anatomy, University of Kuopio, Kuopio, Finland x Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, Kuopio, Finland k Department of Applied Physics, University of Kuopio, Kuopio, Finland { Department of Orthopedics, Go¨teborg University, Sahlgrenska University Hospital, Gothenburg, Sweden # Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Go¨teborg University, Sahlgrenska University Hospital, Gothenburg, Sweden yy Department of Surgery, Division of Orthopaedics and Traumatology, Jyva¨skyla¨ Central Hospital, Jyva¨skyla¨, Finland Summary Objective: The purpose of this study was to find out how deep chondral lesions heal in growing animals spontaneously and after autologous chondrocyte transplantation. Methods: A 6 mm deep chondral lesion was created in the knee joints of 57 immature pigs and repaired with autologous chondrocyte transplantation covered with periosteum or muscle fascia, with periosteum only, or left untreated. After 3 and 12 months, the repair tissue was evaluated with International Cartilage Repair Society (ICRS) macroscopic grading, modified O’Driscoll histological scoring, and staining for collagen type II and hyaluronan, and with toluidine blue and safranin-O staining for glycosaminoglycans. The repair tissue structure was also examined with quantitative polarized light microscopy and indentation analysis of the cartilage stiffness. Results: The ICRS grading indicated nearly normal repair tissue in 65% (10/17) after the autologous chondrocyte transplantation and 86% (7/8) after no repair at 3 months. At 1 year, the repair tissue was nearly normal in all cases in the spontaneous repair group and in 38% (3/8) in the chondrocyte transplantation group. In most cases, the cartilage repair tissue stained intensely for glycosaminoglycans and collagen type II indicating repair tissue with true constituents of articular cartilage. There was a statistical difference in the total histological scores at 3 months (P ¼ 0.028) with the best repair in the spontaneous repair group. A marked subchondral bone reaction, staining with toluidine blue and collagen type II, was seen in 65% of all animals. Conclusions: The spontaneous repair ability of full thickness cartilage defects of immature pigs is significant and periosteum or autologous chondrocytes do not bring any additional benefits to the repair. ª 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Cartilage repair, Autologous chondrocyte transplantation, Periosteum, Animal experiment, Pig, Spontaneous repair, Subchondral bone.

to maturity4e6. Also the superficial cartilage wounds in fetal lambs have been shown to regenerate without scarring7. A previous study showed that chondral lesions do not heal in immature or mature dogs, but osteochondral lesions heal similarly in both age groups8. Especially deep chondral defects heal poorly in mature rabbits9e11, goats12, and dogs13e17. Autologous chondrocyte transplantation is a surgical method for cartilage repair18. The results have been good in adults and adolescents and the method is gaining wider acceptance19,20. The animal experiments of autologous chondrocyte transplantation have been performed with either mature animals10,12,14,21 or with immature22 animals but without a control group of spontaneous healing. The aim of the present study was to find out how deep chondral lesions heal in growing animals spontaneously and after

Introduction It is well known that mature cartilage has poor capacity to heal1. Osteochondral lesions can show some repair with fibrocartilage that has durability and biomechanical properties inferior to native articular cartilage2,3. Normally, the repair capacity decreases with age. It is possible that immature cartilage has greater intrinsic capacity to heal but the results have been contradictory. Osteochondral lesions of immature rabbits heal well and maintain the repair tissue *Address correspondence and reprint requests to: Anna Irmeli Vasara, M.D., Helsinki University Hospital, Peijas Hospital, Department of Orthopaedics, Vilkenintie 16 A, FIN-00640 Helsinki, Finland. Tel: 358-40-5261552; Fax: 358-9-62274750; E-mail: anna.vasara@fimnet.fi Received 27 October 2005; revision accepted 4 April 2006.

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Osteoarthritis and Cartilage Vol. 14, No. 10 autologous chondrocyte transplantation. We also wanted to investigate the significance of the periosteum to the repair and compare it to fascia as cover material.

Materials and methods In this study, 57 skeletally immature pigs (90e100 kg) aged 8e9 months were used. The animals were divided into six groups (Table I); (1) repair with autologous chondrocyte transplantation with periosteum (ACT-P-3) (because of the large individual variation in this group, we performed the same procedure for an additional group of eight pigs to exclude any technical reasons for the variability; this is why the number of animals in this group is larger than in the other groups); (2) repair with periosteum alone (Periosteum-3); (3) repair with autologous chondrocyte transplantation with muscle fascia (ACT-F-3); (4) spontaneous repair (Empty-3); (5) repair with autologous chondrocyte transplantation with periosteum (ACT-P-12); (6) spontaneous repair (Empty-12). The abbreviations show the 3 and 12 months follow-up times used. The pigs were housed at the National Laboratory Animal Center, Kuopio, Finland, in individual pens with floor area of 2
2 m. The experimental design was approved by the local Animal Care and Use Committee. The pigs in the ACT groups were operated twice. First one knee was operated for the cartilage biopsy and 4 weeks later the other knee for the cartilage repair procedure. There were three animals with both operations done on the same knee, however, the results from these animals appeared not to differ from the others. These animals did not have reactive synovitis sometimes seen after recurrent operations in the same joint. For the groups with no chondrocyte transplantation, only one operation was performed.

arthrotomy. An approximately 300 mg cartilage biopsy was taken from the margin of the lateral facet of the patellar groove of the femur to sterile 0.9% NaCl solution. The wound was then closed in layers. The cartilage sample was sent for chondrocyte culture to the Sahlgrenska University Hospital, Gothenburg, Sweden. In all groups, a 6 mm diameter cartilage lesion was created on the upper part of the lateral facet of the patellar groove of the femur [Fig. 1(A)] with a dermal biopsy punch (Stiefel Laboratories Ltd, Sligo, Ireland) without penetration of the subchondral bone. This was done through a lateral arthrotomy on an unoperated knee. The cartilage was carefully excised down to the cartilageebone interface with a knife and a curette while avoiding damage and bleeding of the subchondral bone. Then, in groups ACT-P-3, Periosteum3, and ACT-P-12 an 8 mm (diameter) circular periosteal flap was taken with a biopsy punch from the anteromedial surface of the tibia. The flap was then sutured over the lesion with the cambium layer facing the subchondral bone using 6-0 resorbable suture material (Vicryl, Ethicon Inc.) and fibrin glue (Tisseel Duo Quick, Immuno AG, Vienna, Austria) to seal the seams [Fig. 1(B)]. Through an opening in the seam the precultured chondrocytes (3
106 cells in 100 ml of implantation medium) were injected underneath the periosteal flap. The opening was closed with sutures and sealed with fibrin glue. In group ACT-F-3, the 8 mm fascia flap was taken from the quadriceps muscle fascia through the arthrotomy wound and fixed as described. In groups Empty-3 and Empty-12 the lesions were left untreated. The wound was closed in layers. No immobilization was used postoperatively. Flunixin meglumin 2.2 mg/kg i.m. (Finadyne 50 mg/ml,

ANESTHESIA

Before operation, 0.05 mg/kg atropine (Atropin 1 mg/ml, Leiras Oy, Finland) and 8 mg/kg azaperone were given intramuscularly (i.m.). For induction of anesthesia propofol 3 mg/kg (Rapinovet 10 mg/ml, ScheringePlough A/S, Denmark) was used intravenously (i.v.) and continued with 15 mg/kg/h infusion for the duration of the operation. N2O and oxygen were used for inhalation anesthesia. Cefuroxime (Kefurion, Orion Pharma, Finland) was given preoperatively (3000 mg i.v) for infection prophylaxis and continued for 4 days with a daily dose of 4500 mg. OPERATIVE PROCEDURES

First operation for groups ACT-P-3, ACT-F-3, and ACT-P12 was a cartilage biopsy performed through a lateral Table I The procedures and follow-up times in different groups Group

ACT-P-3 ACT-F-3 Periosteum-3 Empty-3 ACT-P-12 Empty-12

Number of Transplanted samples in chondrocytes histological analysis 14 6

Yes Yes

7 7 6 6

No No Yes No

Cover

Follow-up time

Periosteum Muscle fascia Periosteum No Periosteum No

3 months 3 months 3 months 3 months 12 months 12 months

Fig. 1. A 6 mm (diameter) full thickness chondral lesion was made with a biopsy punch and then carefully curetted down to subchondral bone (A), a periosteum patch sutured over the lesion (B), and the same lesion 3 months after autologous chondrocyte transplantation (C). A microscopic image of a fresh chondral lesion showing microscopic penetrations through the subchondral bone plate (D).

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A. I. Vasara et al.: Repair ability of porcine knee cartilage lesions

ScheringePlough A/S, Denmark) was given for 3 days for analgesia. CELL CULTURE

The cartilage biopsies were sent in sterile sodium chloride to Sahlgrenska University Hospital, Gothenburg, Sweden within 24 h of harvesting. The cartilage was then minced and digested with collagenase overnight at 37(C. The isolated cells were resuspended in Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (DMEM/F12) medium (6000 cells/cm2) supplemented with gentamicin sulfate (50 mg/ml), amphotericin B (2 mg/ml), ascorbic acid (50 mg/ ml), L-glutamine (Gibco-BRL) and 10% fetal calf serum. The cells were cultured in monolayer culture until enough cells were obtained. Before the implantation the cells were counted, the viability was tested with trypan blue and 3
106 cells/animal were diluted in 100 ml of Ham’s F12 medium supplemented with gentamicin sulphate (50 mg/ml), amphotericin B (2 mg/ml) and 20% fetal calf serum. The diluted cells (100 ml, concentration 30
106 cells/ml) were sent for implantation in 1 ml syringes. MACROSCOPIC EVALUATION AND CARTILAGE INDENTATION

After 3 or 12 months, the animals were sedated with atropine 0.05 mg/kg and azaperone 8.0 mg/kg and euthanized with T-61 4 ml/50 kg (Hoechst Roussel Vet GmbH, Germany). After euthanizing the animals, both knees were x-rayed and the radiographs were evaluated for fractures, bony defects and osteophytes. The knee joints were opened, the lesions were photographed, the macroscopic findings, such as the repair of the lesion, and the cartilage surfaces were evaluated and any other joint or synovial pathology recorded. The repair tissue was macroscopically evaluated using the International Cartilage Repair Society (ICRS) macroscopic grading (www.cartilage.org)23. The repair tissue, adjacent cartilage and the control cartilage from the other knee were studied with a commercial arthroscopic indentation instrument (Artscan 200, Artscan Oy, Helsinki, Finland)24. Earlier, the indenter force by which the tissue resists the instantaneous indentation has been shown to provide a solid indicator of cartilage dynamic stiffness24. In this study, we used a novel configuration of the instrument, optimized for the measurement of thin laboratory animal cartilage25. As shown earlier26, the use of a small spherical-ended indenter reduces the effect of finite tissue thickness on the indentation response. During the indentation measurement, instantaneous compressions (3 N) were applied on to cartilage by pressing the tip of the instrument against the cartilage and the mean value of two largest indenter forces was chosen to represent the cartilage dynamic stiffness. The measurement was repeated two times and the final stiffness value was obtained as a mean.

SAMPLE COLLECTION

The cartilage samples were prepared by sawing slices perpendicular to the articular surface in the middle of the lesions, using a dentist’s drill equipped with two cutting discs separated by a 2-mm-thick spacer. The samples were fixed for 48 h in 4% formaldehyde in 0.07 M sodium phosphate buffer, pH 7.0, at 4(C. Then the slices were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) and 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 14 days at room temperature27.

HISTOLOGICAL ANALYSIS

The samples were stained for metachromacy of cartilaginous tissue with toluidine blue and for proteoglycans with safranin-O27. Hyaluronan was stained using a specific, biotinylated hyaluronan binding probe (bHABC) and avidinebiotineperoxidase system (ABC, Vector Laboratories, CA, USA)28. Immunohistochemical recognition of type II collagen was made with mouse monoclonal E8 antibody29. The sections were pretreated with testicular hyaluronidase (2 mg/ml in Phosphate buffered saline (PBS), pH 5, for 1 h at 37(C) and pronase (1 mg/ml in PBS, pH 7.3, for 30 min at 37(C) before staining with Envision kit according to the manufacturer’s instructions (DakoCytomation, Glostrup, Denmark). The histological scoring was made from safranin-O stained sections by three independent observers with the modified O’Driscoll score30,31. Unstained sections were cut for quantitative polarized microscopy32. QUANTITATIVE POLARIZED LIGHT MICROSCOPY

The degree of collagen fibril parallelism, i.e., anisotropy was measured with quantitative polarized light microscopy33. Measurements were made in 12 horizontal fractions from cartilage or repair tissue surface to subchondral interface. Three randomly selected histological sections were analyzed from each site. The mean parallelism of collagen network was calculated for each fraction and was compared to the control tissue. From the same data, the thickness of the cartilage and repair tissue were measured. STATISTICAL METHODS

The statistical differences were calculated using analysis of variance between groups (ANOVA) with general scores (Monte Carlo P-value) and permutation test with Hochberg adjusted P-values. A P-value below 0.05 was considered to be significant.

Results After 3 months’ follow-up at the age of 11e12 months the pigs were skeletally immature. The open epiphyseal lines could be seen in the radiographs and articular cartilage did not show calcified cartilage in the histological sections. Also the collagen organization of the cartilage remained immature type and arch-like formations typical to adult cartilage could not be seen. After 12 months’ follow-up the cartilage was of more mature type, but no calcified cartilage could be detected. The epiphyseal lines were partly open and the skeletal maturity was not yet reached. Osteoarthritic changes were seen in the patellofemoral joint in six cases, two in ACT-P-3 (11%), one in ACT-F-3 (16%), one in Empty-3 (13%), two in ACT-P-12 (25%). All except the one in ACT-F-3 were excluded from the histological analysis as the lesions site could not be definitively identified. There were no signs of patellofemoral osteoarthritis in the control knees. Eighty percent of the lesions were at least 75% filled and 92% of the lesions were at least 50% filled with repair tissue [Figs. 1(C), 2(D) and 3(D)]. Overgrowth was seen in two cases in group ACT-F-3, in one case in groups ACT-P-3, Periosteum-3, and Empty-3, but in none of the longer follow-up groups. The ICRS macroscopic grading indicated nearly normal repair tissue in 65% (10/ 17) of ACT-P-3, 83% (5/6) in ACT-F-3, 43% (3/7) in Periosteum-3, and 86% (7/8) in Empty-3. At 1 year, the repair

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Fig. 2. Spontaneous repair of a 6 mm wide full thickness chondral lesion showing good repair (A). Histological stainings with toluidine blue (B), hyaluronan binding probe (C), and collagen type II (D). Note the marked subchondral bone reaction. Scale bar 2 mm. A quantitative polarized light image demonstrating the birefringency of the repair tissue shows the random organization of the collagen fibers (E). Scale bar 0.2 mm.

Fig. 3. A 6 mm wide full thickness chondral lesion repaired with autologous chondrocytes covered with muscle fascia showing good macroscopic repair (A). Histological stainings with toluidine blue (B), hyaluronan binding probe (C), and collagen type II (D) show that the repair is mostly fibrous tissue. Scale bar 2 mm. A quantitative polarized light image demonstrating the birefringency of the repair tissue shows the random organization of the collagen fibers (E). Scale bar 0.2 mm.

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A. I. Vasara et al.: Repair ability of porcine knee cartilage lesions Table II Modified O’Driscoll histological scores

Group

N

ACT-P-3 14 ACT-F-3 6 Periost-3 7 Empty-3 7 ACT-P-12 6 Empty-12 6

Tissue Matrix Structural Clustering Formation Archit. of morphology staining integrity (max 3) of tidemark the surface (max 3) (max 3) (max 4) (max 4) (max 3) 2 1 2 2 1 2

2 2 2 3 1 2

2 2 2 3 2 3

2 2 2 2 2 2

tissue was nearly normal in all cases in Empty-12 group and in 38% (3/8) in the chondrocyte transplantation group (ACTP-12). Two animals were lost during follow-up due to lameness and pleuritis, respectively, and three animals due to complications in anesthesia. In the final analysis, there were histological samples from 46 pigs. In six cases (three animals from ACT-P-3, two from ACT-P-12, one from Empty-3), the histology was excluded from the analysis as the site of the original lesion could not be definitively identified due to degenerative changes (n ¼ 5) in the joint or technical problems in the sample collection (n ¼ 1). The final number of analyzed histological samples in each group can be seen in Table I and the median histological scores in Table II. In the groups ACT-P-3, ACT-F-3, Periosteum-3, and Empty-3 all lesions were at least 75% filled with repair tissue. There was a statistical difference in the total histological scores at 3 months (P ¼ 0.028) and this difference was localized between the Empty-3 and ACT-F-3 groups (P ¼ 0.038) (Fig. 4). At 3 months, the histological scores differed significantly in the categories of tissue morphology (P ¼ 0.0267) and surface architecture (P ¼ 0.0032) (Table II). In group Empty-3, the matrix staining was normal or nearly normal in six out of seven cases (Fig. 2). In all other groups the matrix staining ranged from slight to moderate. The tissue morphology was best in groups ACT-P-3,

Fig. 4. The modified O’Driscoll histological scores of the defects in each group showing the total score of each specimen. Empty (spontaneous repair), ACT-P (autologous chondrocyte transplantation with periosteum), Periost (autologous periosteum transplantation without cells), and ACT-F (autologous chondrocyte transplantation with muscle fascia).

0 0 0 0 0 0

2 2 2 2 2 1

Filling of Lateral Basal Inflamm the defect integr. integr. (max 4) (max 4) (max 2) (max 3) 4 3 4 4 4 3

2 2 2 2 2 1

3 3 3 3 3 3

4 4 4 4 4 4

Total score (max 32) (minemax) 22 20 21 24 20 22

(16e26) (13e23) (18e26) (21e26) (12e24) (17e24)

Empty-3 and Empty-12. Periosteal covering over the empty lesion site did not show any benefit for the repair. There were a few cases where predominantly true hyaline cartilage could be seen, but mostly the repair tissue was of mixed type with areas of both fibrous and hyaline cartilage, or in some cases completely fibrous tissue. In Empty-12 four animals showed mostly hyaline type of repair. In most cases, the cartilage repair tissue stained intensely for glycosaminoglycans and collagen type II indicating repair tissue with true constituents of articular cartilage. The cell type differed from rounded chondrocytes to fibroblastlike cells that were more abundant in the nonmetachromatic areas poor of glycosaminoglycans. The structural integrity ranged from no organization to some columnar organization being the best in the spontaneous repair groups. In all cases, the surfaces exhibited slight to moderate irregularity. The basal integration was good in nearly all cases. The lateral integration ranged from partial to total integration, and the marginal area often contained chondrocyte clusters. No tidemark or calcified cartilage could be seen in any of the histological samples. Also no inflammatory reactions were seen in the histological specimens. The repair in the fascia group (ACT-F-3) had the lowest scores (Fig. 3). In these animals, there were large subchondral reactions and the repair tissue did not stain well for glycosaminoglycans or collagen type II. Three cases in groups ACT-P-12 and Empty-12 had lesions less than 75% filled. There was no statistical difference in the total histological scores between Empty-3 and Empty12 or between the 12 months follow-up groups (Fig. 4). After the follow-up of 12 months, some cases in both spontaneous repair and ACT groups showed lower histological score values than after 3 months’ follow-up. In other cases, the repair tissue collagen was more organized and resembled native cartilage more closely than at 3 months. In these cases, the columnar organization of rounded chondrocytes in the deep layer as well as the more horizontal organization of the surface layer could also be seen. A marked subchondral bone reaction was seen in 65% of all pigs (Figs. 2 and 3). The repair tissue seemed to spread downwards through the subchondral bone plate and there was often strong cartilage-like staining with toluidine blue [Figs. 2(A) and 3(A)] and type II collagen [Figs. 2(C) and 3(C)] in that area. The hyaluronan staining was strong in the subchondral part of the lesion and in fibrous repair tissue [Fig. 3(B)], while in cartilaginous tissue the staining was low because cartilage glycosaminoglycans mask hyaluronan and hinder the binding of the probe. This staining pattern had, however, disappeared in the 12 months follow-up group. At that time the reactive area had turned to bone, although the organization of the bone was not normal. In all repair sites, the quantitative polarized microscopy showed collagen structure that was random in all directions

Osteoarthritis and Cartilage Vol. 14, No. 10 [Figs. 2(E) and 3(E)] with no zonal architecture that is typical to collagen organization in normal cartilage. The degree of parallelism of collagen network in repair tissue showed exceptionally high variance in repair tissue both at 3 months and 1 year time points postoperatively. The coefficient of variation was very high and ranging between 22% and 47%. The accuracy of the parallelism estimates was not improved even when the number of random sections was finally increased to five per each anatomical site. The results suggest extremely large variation in the collagen structures along the z-dimension (‘depth’) of the tissue block. Very likely this is due to momentarily changing course of collagen fibers in the repair tissue. Since no statistical strength could be obtained with this study design, parallelism data is not shown. Polarized light imaging was used to measure the thickness of the adjacent cartilage, repair tissue, and control cartilage. The repair tissue was thinner at 12 month follow-up than at 3 months. This can partly be due to the remodeling of the subchondral bone. The cartilage indentation analysis showed that the repair tissue was softer than adjacent cartilage both at 3 and 12 months after the repair (Fig. 5). The adjacent cartilage and control knee cartilage indentation measurements showed no differences during the follow-up period.

Discussion In immature pigs we found good spontaneous repair of cartilage lesions with no additional benefits from periosteal coverage or autologous chondrocytes. It is possible that the immature cartilage has a better intrinsic capacity to repair6. There are age-dependent changes in cartilage matrix constituents such as the increased amount of collagen crosslinking and reduced aggrecan synthesis at older age but their meaning is unknown34e36. Active reorganization of collagen network between 4 and 21 months may also increase repair capacity porcine articular cartilage37. In humans, articular cartilage also undergoes significant changes in adolescence as the volume of articular cartilage increases during the rapid growth phase38. On the other hand, the immature cartilage is more vulnerable to load-induced injury39.

Fig. 5. The indentation stiffness of the repair tissue and the adjacent cartilage. All repair tissues were softer than the adjacent cartilage.

1071 It has been shown that osteochondral defects in mature goats do not heal40, but there are several studies showing good repair of osteochondral lesions in immature animals4e6,8,41. In young animals, the high level of Transforming growth factor-b1 (TGF-b1) and the increase of TGF-b1 after trauma may enhance the healing capacity42. Superficial cartilage wounds have been shown to heal without scarring in fetal lambs7. However, Tew et al.43 found no maturation-dependent difference in the intrinsic repair of cartilage wounds in vitro. A recent study showed better results with autologous chondrocyte transplantation, than with periosteum alone, in immature mini-pigs, but included no control group without repair22. In another study using adult mini-pigs, the spontaneous repair of 4 mm diameter deep chondral lesions was poor both after 4 and 24 weeks30. In a study with adult dogs, similar lesion showed tissue filling from 38% to 57% being mostly fibrous and fibrocartilage tissue44. In mature sheep, the deep chondral lesions showed poor fibrous repairs45. Improved healing of cartilage lesions has been seen after microfracturing the subchondral bone plate in sheep45 and in horses46. It seems that pure chondral lesions do not heal, but the communication between the repair tissue and the subchondral bone plate can facilitate chondral repair. Young age seems to improve both healing and remodeling of the repair tissue. The fact that animals in this study were still growing most likely increases the capacity for repair of cartilage lesions. Cartilage injuries are common lesions after acute trauma to the immature knee47. It is well accepted that autologous chondrocyte transplantation is currently not a procedure for elderly and osteoarthritic patients, because in these patients the chondrocytes may not have the capacity to produce durable repair tissue and other treatment options are available. In contrast, it may be, that the growing adolescents and children have even capacity to spontaneous cartilage repair or at least they might have better success with cartilage repair than adults. To date we have limited knowledge of the spontaneous cartilage repair in children. A previous case report suggests good healing of a large fixed chondral fragment48. A recent study showed excellent results of autologous chondrocyte transplantation in adolescent athletes20. Other resurfacing procedures such as microfracture and autologous osteochondral transplantation also show better results in young patients as well49,50. There is no calcified cartilage layer in immature pigs. We found it possible to debride cartilage without damaging the subchondral bone plate macroscopically. Slight bleeding from the bottom revealed, however, opening of the subchondral bone plate trabeculae. We examined the technique by making histological sections of fresh lesions ex vivo [Fig. 1(D)] and found that the subchondral bone plate was mostly intact but microscopic openings could not be avoided. As the subchondral bone plate was soft and there was no calcified cartilage, it is possible that the bone marrow contributed significantly to the repair. This might explain why we found good spontaneous repair in immature animals with no additional benefit from the autologous chondrocytes. Possibly a larger lesion would not have been able to heal. The critical size of the lesion may be larger in immature animals. In the present study, most of the repair tissues obtained contained mostly fibrocartilage. For hyaline cartilage we included tissue that not only stained for proteoglycans and type II collagen, but also had spherical chondrocytic cells in lacunae and matrix with hyaline-like appearance in the histological sections. Many of the macroscopically good repairs did not fill the criteria of hyaline-like appearance of

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the matrix and were graded as fibrocartilage and results from quantitative analysis of collagen network parallelism support this idea. It has been stated that intact calcified cartilage would be essential for hyaline type of repair51. It is possible that in this study the bleeding and the cells derived from the subchondral bone contributed to the repair and induced more fibrocartilagenous repair. There were four repairs representing hyaline repair out of six histological sections in the 12 months spontaneous repair group. This might also indicate maturation of the more fibrocartilagenous graft towards hyaline cartilage at 12 months. The ICRS macroscopic grading showed different results than the histological grading. The macroscopic grading does not take into account the overgrowth of the graft that was quite massive in some cases. Also the total degeneration of the patellofemoral joint seen in six cases seemed unrelated to the method of repair but was more likely caused by mechanical factors, such as surface defects and malposition of the patella after operation. The ICRS grading is designed for arthroscopic evaluation of human cartilage repair procedures and not for this kind of animal experiments and it seems that in this study, the results on ICRS grading represented the amount of lesion filling but less the quality of the repair. Indentation analysis revealed that the repair tissue did not show normal cartilage mechanical properties at 3 or 12 months. In humans we have shown that the repair tissue reached an average of 62% of the normal cartilage stiffness at 1 year, but in certain cases the stiffness was similar to that of normal cartilage52. The results of the present study are consistent with those of the previous animal experiments where good cartilage repair of osteochondral lesions did not reach the normal mechanical properties of articular cartilage5,6. This can be due to the fact that organization of the collagen network, especially in the superficial layer, significantly contributes to cartilage stiffness53. In the present study, the repair tissue did not restore the normal threedimensional collagen organization of articular cartilage. The primary time point for all groups was set at 3 months. After the excellent spontaneous repair results were seen at 3 months, we started the 12 months follow-up from ACT-P and Empty groups to evaluate longer-term results. While designing this prolongation the authors made the conclusion that longer follow-up for the groups with periosteum and ACT with fascia would bring no additional benefit. The importance of periosteum in chondrocyte transplantation is commonly accepted10,54, and according to some reports periosteal patch alone may have a significant role in the cartilage repair process55e57. However, the role of the periosteum in chondrocyte transplantation has been unclear. The fascial covering (ACT-F-3) showed histologically inferior repair tissue compared to other groups. The reason for this is unknown, but it indicates that the method of lesion coverage is significant for the repair. As new methods of chondrocyte transplantation without periosteal coverage are introduced the effects of the matrix or coverage should be addressed58,59. The delamination of the periosteum has been shown to be a problem in animals with thin cartilage such as goats12,60. The cartilage thickness in pigs was similar to cartilage thickness in humans, where early delamination of the periosteum has not been a problem. Definitive conclusions about delamination of the grafts cannot be made at 3 months when strong spontaneous repair is present. There was a fibrous layer on the surface of the repair tissue that could be seen in all groups irrespective of treatment and also in the untreated group.

The subchondral reaction has also been seen in other animal experiments12e14,61. One explanation for the deep subchondral cartilage type reaction could be the growth of the surrounding bone with slower growth underneath the lesion. Another explanation may be a reparative process in the subchondral bone after surgical trauma or excessive loading of the debrided defect. Our earlier study with mature goats showed similar reaction12. The present study demonstrates that the type of reaction is not specific for animal species or the age of the animals. Neither is it induced by the transplanted chondrocytes as it is seen in the spontaneous repair groups as well. Therefore, the most likely explanations are related to mechanical conditions of the repair tissue. Controlled partial weight bearing as used in clinical setting is not possible in animal experiments. In this study, no knee immobilization was applied. For the first few days the animals showed limping. The weight of the animals was large considering the size of their joints and it is possible that they are more susceptible to degenerative processes of the joints or trabecular fatigue fractures. This subchondral bone reaction resembled cartilage at 3 months but had turned to bone at 12 months so it may be part of natural healing process. The quality and importance of this reaction warrant further studies. In this study, some grafts deteriorated by 12 months but some showed good repair tissue that was more articular cartilage like than at 3 months. The reason for this dual faith of the grafts is not known. This seems to be similar to the clinical situation where some grafts deteriorate early, but those that last up to 12 months seem to be durable even for longer periods19. It seems clear that the graft as well as the subchondral bone go through significant changes during several months after cartilage repair and it has been suggested that subchondral bone plate advancement could cause the deterioration of the repair tissue62. In conclusion, the spontaneous repair ability of full thickness cartilage defects of immature pigs is significant and periosteum or autologous chondrocytes do not bring any additional benefits to the repair. The animals used in many cartilage repair experiments are adolescent animals which may confuse the results as is shown in this study. Choosing the animal model and a relevant age of the animals should be emphasized when planning animal experiments.

Acknowledgments We are grateful to Dr Raija Tammi, M.D., Ph.D., (University of Kuopio, Department of Anatomy, Kuopio, Finland) for kindly providing the bHABC probe, Jani Hirvonen M.Sc., B.Eng. (Department of Applied Physics, University of Kuopio) for assisting in biomechanical measurements, and Hannu Kautiainen, B.A., (Rheumatism Foundation Hospital, Heinola, Finland) for statistical analysis. This study has been financially supported by National Technology Agency (TEKES, project 40714/01, Finland), Academy of Finland (project no. 54054), Kuopio and Jyva¨skyla¨ Central Hospital (TEVO, Finland), and Finnish Medical Foundation.

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