Limited Integrative Repair Capacity of Native Cartilage Autografts Within Cartilage Defects in a Sheep Model Kolja Gelse,1 Dominic Riedel,1 Milena Pachowsky,1 Friedrich F. Hennig,1 Siegfried Trattnig,2 Go¨tz H. Welsch1,2 1 Department of Orthopaedic Trauma Surgery, University Hospital Erlangen, Krankenhausstr. 12 91054, Erlangen, Germany, 2Department of Radiology, MR Center, Medical University of Vienna, Lazarettgasse 14, Vienna, Austria
Received 17 July 2014; accepted 20 October 2014 Published online 3 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22773
ABSTRACT: The purpose of this study was to investigate integration and cellular outgrowth of native cartilage autografts transplanted into articular cartilage defects. Native cartilage autografts were applied into chondral defects in the femoral condyle of adult sheep. Within the defects, the calcified cartilage layer was either left intact or perforated to induce bone marrow stimulation. Empty defects served as controls. The joints were analyzed after 6 and 26 weeks by macroscopic and histological analysis using the ICRS II Score and Modified O‘Driscoll Scores. Non-treated defects did not show any endogenous regenerative response and bone marrow stimulation induced fibrous repair tissue. Transplanted native cartilage grafts only insufficiently integrated with the defect borders. Cell death and loss of proteoglycans were present at the margins of the grafts at 6 weeks, which was only partially restored at 26 weeks. Significant cellular outgrowth from the grafts or defect borders could not be observed. Bonding of the grafts could be improved by additional bone marrow stimulation providing ingrowing cells that formed a fibrous interface predominantly composed of type I collagen. Transplanted native cartilage grafts remain as inert structures within cartilage defects and fail to induce integrative cartilage repair which rather demands additional cells provided by additional bone marrow stimulation. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:390–397, 2015. Keywords: cartilage repair; cartilage graft; bone marrow stimulation; progenitor cell; regeneration
The existence of progenitor cells within articular cartilage has been discussed in a number of recent studies.1–3 Particularly their capability to migrate and to grow out of the cartilage tissue would be one prerequisite for intrinsic cartilage regeneration.4,5 Indeed, cell outgrowth from cartilage tissue was demonstrated in vitro and in vivo.5–7 In rabbit and goat models, transplanted native cartilage fragments contributed to the formation of better repair tissue in treated cartilage defects than application of the scaffolds alone.6,8–10 Successful results following transplantation of autologous cartilage fragments or allogeneic particulated juvenile articular cartilage were also observed in large animal models11 and human studies.12,13 A variety of biodegradable scaffolds have been used for embedding the cartilage fragments. A three-dimensional scaffold seems to promote chondrocyte proliferation and outgrowth.14 These data encourage the establishment of one-stage cartilage repair procedures and would query the need for the two-stage autologous chondrocyte transplantation. So far, however, all of these promising results were observed in osteochondral defects or defects with completely removed calcified cartilage layer, which may allow the ingrowth of bone marrow-derived cells. Thus, it has still to be investigated to which extent the cells within the forming repair tissue originate from the transplanted cartilage graft or the bone marrow. Therefore, the aim of this study was to analyze and evaluate the role of native autologous cartilage grafts with respect to their integrative repair capacity (celluConflicts of interest: None. Grant sponsor: German Research Foundation; Grant number: WE 4881/1–1. Correspondence to: Kolja Gelse (T: 0049-9131-8533296; F: 00499131-8533300; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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lar outgrowth and integration) after transplantation into true chondral defects to provide a model with limited endogenous repair capacities. We addressed the medial femoral condyle of the adult sheep model, since this setting is characterized by a poor endogenous repair response similar to that of adult human joints.15 To rule out any ingrowth of bone marrow cells, the subchondral bone plate including the calcified cartilage layer was completely left intact. As an alternative treatment group, the calcified cartilage layer within the defects was perforated to induce bone marrow stimulation in order to monitor the influence of bone marrowderived cells.
MATERIALS AND METHODS Ten female adult merino sheep aged 3–4 years with body weights of 70–80 kg were used in this study. The animals were anesthetized and their knee joint capsule was opened by a medial parapatellar incision. The patella was displaced laterally to expose the medial femoral condyle. Each of the 10 animals was treated by two separate round 5 mm cartilage defects created into the main load-bearing zone of the medial femoral condyle using a sharp biopsy punch. The two lesions were clearly separated from each other by at least 5 mm of intact cartilage to avoid any cell migration or interaction between the two defects. The articular cartilage was carefully removed using a curette leaving the calcified cartilage layer and the subchondral bone plate intact.16 The preservation of the calcified cartilage layer was confirmed by complete absence of bleeding in the defect during surgery. Afterwards, the two prepared lesions in each of the animals were treated by two different approaches among the following four different treatment approaches: (1) No further treatment (empty cartilage lesion down to the tide-mark with exposed but completely intact calcified cartilage ¼ “control”); (2) Bone marrow stimulation (“BMS”) by treatment with five perforations using a custom-made micropick (each 1 mm in diameter and 3 mm deep) that were covered with fibrin glue (Beriplast,
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Aventis, Marburg, Germany). The perforations were applied through the preserved calcified cartilage layer, which stands in contrast to the original microfracturing method with the calcified cartilage layer being removed17 (Supplementary Figure); (3) Transplantation of a discoid articular cartilage autograft into the defect with an intact layer of calcified cartilage (Cartilage Autograft-Transplantation ¼ “CA”). The cartilaginous autograft was harvested from the very cranial zone of the femoral trochlea using a dermatome (0,8 mm thickness) and a 5 mm biopsy punch to generate a discoid graft that exactly fits into the defect; (4) Transplantation of a discoid articular cartilage autograft into the defect that had been treated by bone marrow stimulation with five perforations before (“CA þ BMS”)(Supplementary Figure). All grafts were additionally sealed with fibrin glue with a fibrinogen concentration of 90 mg/ml (Beriplast, Aventis Behring, Marburg, Germany) to increase initial stability and to avoid potential cell spread by initial bleeding in defects treated by bone marrow stimulation. Between the 10 animals, the two defects within each joint received two different treatment approaches that were arranged in an alternating manner to minimize spatial environmental differences. Following surgery, the animals were allowed to move freely in their stalls. The animals were first operated on their right knee joint. After 20 weeks, the animals were operated on their left knees according to the previous surgery on the right knee and were sacrificed 6 weeks later. This treatment scheme allowed two different follow-up periods with 6 weeks for the left knee joints and 26 weeks for the right knee joints. This scheme was chosen for ethical reasons to reduce the total number of animals and was approved by the Institutional and Governmental Review Boards. The knee joints were dissected and first assessed macroscopically followed by preparation for histological analysis as described below. Histological and Immunohistological Assessment The osteochondral specimens were fixed in 4% paraformaldehyde for at least 12 h, followed by decalcification in 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 months. After standard processing, the samples were embedded in paraffin. The specimens were scanned throughout the tissue blocks with serial transverse 5 mm sections and stained with toluidine blue to estimate the proteoglycan content. The morphological assessment of the different approaches was performed by three independent specialists according to the ICRS II Score18 and the O‘Driscoll Score.19 To determine the percentage of tight lateral and basal integration of the repair tissue with adjacent tissue and the percentage of filling of the defects, five parallel sections with a separation distance of 1 mm were captured using a microscope camera throughout each of the treated defects and further analyzed by a digital imaging programme. Tight integration was defined when direct contact of repair tissue to host tissue was present even after histological tissue processing. Immunohistochemical detection of type I and type II collagen was performed as described previously in detail.20 Briefly, deparaffinized sections were pretreated with 0.2% hyaluronidase for 60 min and subsequently with 0.2% pronase (Sigma–Aldrich) for 60 min. Sections were then exposed overnight to anti-human type I collagen antibodies (MP Biomedicals, Aurora, OH) diluted 1:200, or anti-human type
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II collagen antibodies (MP Biomedicals) diluted 1:500. After incubation with a biotinylated donkey anti-mouse secondary antibody (Dianova, Hamburg, Germany), a complex of streptavidine and biotinylated alkaline phosphatase was added. The sections were developed with fast red and counterstained with hematoxylin. Tunel Staining For the detection of in situ DNA breaks, the TUNEL reaction was applied using the In Situ Cell Death Detection Kit, AP (Roche, Mannheim, Germany). After deparaffinization, the sections were washed in PBS and digested with proteinase K (20 mg/ml; Boehringer, Ingelheim, Germany) for 15 min at 37 ˚C. After washing with PBS, sections were incubated with TdT-solution in reaction buffer at a volume ratio of 9:1 at 37 ˚C for 1 h followed by extensive washing. The converter AP antibody was added for 30 min at 37 ˚C. After washing, detection was performed by fast red staining and counterstaining with hematoxylin. Statistical Analysis All data are presented as mean SD. For the evaluation of morphological parameters non-parametric Kruskal–Wallis tests followed by post-hoc Dunn multiple comparison tests (Modified O’Driscoll Scores) or parametric analysis of variance (ANOVA) tests followed by Tukey–Kramer multiple comparison tests (ICRS II Score) were used to determine treatment-specific differences. Integration was assessed by analysis of variance followed by a Tukey–Kramer test. All statistical results were considered significant for p-values < 0.05.
RESULTS Filling of the defects was analyzed by gross observation (g.o.) and by histomorphological analysis (h.a.) at 6 weeks (n ¼ 5 each treatment) (Fig. 1) and 26 weeks (n ¼ 5 each treatment) (Fig. 2). Gross observation tended to slightly overestimate the filling of the defects compared to histological analysis, however, the differences were not significant. At 6 weeks, there was no relevant filling of nontreated control lesions despite of debris and remnants of fibrous tissue (21.0% 11,6 (g.o.); 10.0% 7.7 (h.a.)) (Fig. 1a–c). Filling of the defects was not increased at 26 weeks (16.3% 11.9 (g.o.); 8.8% 6.5 (h.a.))(Fig. 2a– c). Cellular ingrowth from the defect margins could not be observed at 6 weeks and only to a minimal extent at 26 weeks (Fig. 3a). During the observation period of 26 weeks, the layer of calcified cartilage and the underlying subchondral bone remained completely unaffected by the removal of the articular cartilage, which indicates complete lack of any endogenous repair response of induced cartilage defects. BMS induced the formation of fibrous tissue that hardly stained for proteoglycans at 6 weeks (Fig. 1d–f). At 26 weeks, chondrogenic differentiation of repair cells with formation of a fibrocartilaginous matrix was apparent, however, the tissue morphology was rather inhomogenous with fibrous tissue in the superficial layer and signs of endochondral ossification in deeper layers (Fig. 2d–f). The cells within the repair tissue JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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Figure 1. Repair tissues at 6 weeks after bone marrow stimulation by subchondral perforations (BMS), transplantation of native autologous cartilage grafts (CA), or combined treatment (CA þ BMS). The resulting repair tissues were examined histologically by toluidine blue staining and by gross observation (repair tissue of indicated treatment group is encircled). Representative sections of the average, best and worst repair tissues of each group are shown. Empty control defects show no relevant repair response (a–c). Defects treated by bone marrow stimulation (BMS) are partially filled with fibrous repair tissue (d–f). Transplanted native cartilage autografts (CA) are characterized by loss of proteoglycans (reduced toluidine blue staining) and by lack of basal integration (g–i). Native cartilage autografts transplanted into defects pretreated by bone marrow stimulation (CA þ BMS) also have a reduced content of proteoglycans and show partial basal bonding in the areas of initial perforations (j–l). Bar ¼ 1mm (histological section), bar ¼ 5 mm (macroscopic view).
were characterized by a spindle-shaped fibroblast-like phenotype in the superficial zone, round chondrocytelike phenotype in the middle zone, and a hypertrophic phenotype in the deepest zone. General filling of the defects following BMS was poor at 6 weeks (28.0% 14.7 (g.o.); 18.0% 11.2 (h.a.)) and was slightly increased at 26 weeks (33.3% 33.6 (g.o.); 41.7% 37.5 (h.a.)). Basal integration of the repair tissue was tight
in areas of the induced perforations and adjacent to the non-calcified articular cartilage, but insufficient in regions with remaining calcified cartilage (Fig. 2d–f). General integration of repair tissue following BMS was significantly better than that observed in control defects or following CA-treatment (Fig. 4). The subchondral bone plate and tide mark did not reconstitute during the observation period. Abnormal calcification
Figure 2. Repair tissues at 26 weeks after bone marrow stimulation by subchondral perforations (BMS), transplantation of native autologous cartilage grafts (CA), or combined treatment (CA þ BMS). The resulting repair tissues were examined histologically by toluidine blue staining and by gross observation (repair tissue of indicated treatment group is encircled). Representative sections of the average, best and worst repair tissues of each group are shown. Empty control defects lack any repair response (a–c). Defects treated by bone marrow stimulation (BMS) are partially filled with fibrocartilaginous repair tissue (d–f). Transplanted native cartilage autografts (CA) lack basal integration (g–h) or have been lost (i). Native cartilage autografts transplanted into defects pretreated by bone marrow stimulation (CA þ BMS) show tight lateral integration in most cases but basal bonding is limited to those areas of initial perforations (j–l). Bar ¼ 1mm (histological section), bar ¼ 5 mm (macroscopic view). JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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Figure 3. Features for integrative cartilage repair. Marginal ingrowth of repair cells into non-treated defects at 26 weeks (a). Reduced density of viable cells at the margins of implanted native cartilage grafts as indicated by empty lacunae (b) and apoptotic cells positive for TUNEL staining (c). Tight lateral integration but lack of basal integration of native cartilage grafts (*) with underlying calcified cartilage in CA þ BMS-treated defects (d). Lateral interface region is composed of fibrous tissue positive for type I collagen (e) and negative for type II collagen (f). Ingrowing cells induced by bone marrow stimulation form a pannus-like cell layer on transplanted native cartilage grafts at 26 weeks (g) that is negative for type II (h) but positive for type I collagen (i). Toluidin Blue Staining (a, b, d, g); TUNEL staining (c). Immunohistochemical staining for type I collagen (e, h) and type II collagen (f, i). Bars ¼ 100 mm (a, d–f), 50 mm (b, c, g–i).
Figure 4. Analysis of integration of repair tissues within cartilage defects. General integration (lateral and basal) of repair tissue induced by bone marrow stimulation (BMS) is significantly superior to that of transplanted native cartilage grafts (CA). Integration of native cartilage grafts is improved by additional bone marrow stimulation (CA þ BMS).
or excessive ossification within the repair tissue was apparent in some areas of the deeper zones at 26 weeks (Fig. 2e). Significant inflammation or vascular invasion was not apparent (Tables 1 and 2). The score for “overall assessment” of the BMS-treated defects increased from 6 to 26 weeks (Tables 1 and 2). The application of a native cartilage autograft (CA) into the defects was confronted with the problem of graft displacement. Only 60% of the grafts were retained within the defects at 6 and 26 weeks, which correlated with an incomplete general filling at 6 weeks (28.0% 29.2 (g.o.); 17.0% 13.6 (h.a.)) and 26 weeks (51.0% 39.7 (g.o.); 49.0% 39.4 (h.a.)). Apparently, integration of the native cartilage autograft was strongly dependent on the adjacent tissue type. There were no signs of basal integration of the native cartilage grafts with underlying calcified cartilage at 6 and 26 weeks (Figs. 1g–i and 2g–i), and only inconsistent lateral integration with adjacent non-calcified articular cartilage (Figs. 1g–i, 2g–i, and 4). Overall, general JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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2.5 4.3 5 6.1 8.75 12.4 17.5 20.4 2.5 2.5 12.5 21.6 73.8 42.6 72.5 29.4 95 8.7 82.5 30.3 95 8.7 1.25 2.2 13.8 9.6 3.8 4.1
1.0 2 6.0 3.7 6.0 5.8 15.0 27.5 5.0 4.5 2.0 2.4 80.0 24.4 90.0 20.0 100.0 0 100.0 0 100.0 0 6.0 4.9 4.0 3.7 6.0 7.3
9.0 4.0 14.0 9.7 16.0 12.4 53.0 33.7 18.0 12.8 54.0 27.2a 36.0 26.7 48.0 36.0 94.0 8.0 88.0 19.3 100.0 0.0 20.0 13.0 16.0 8.6 12.0 4.0
6 weeks (n ¼ 5)
4.0 0.0 3.0 0.0 4.0 0.0 5.0 0.0 4.0 0.0 3.0 0.0 1.0 1.7 1.25 1.6 3.16 1.3
4.0 0.0 3.0 0.0 4.0 0.0 4.8 0.4 4.0 0.0 3.0 0.0 0.0 0.0 0.2 0.4 2.85 1.7
3.6 0.4 1.2 0.7a 3.4 0.4 4.6 0.4 3.8 0.4 3.0 0.0 0.4 0.8 2.4 1.0 2.80 1.3
6 weeks (n ¼ 5)
Kruskal–Wallis Test followed by Dunn multiple comparison tests. a Statistical significant difference from “control defect” (for same treatment period) (p < 0.05) .bStatistical significant difference from “BMS” (for same treatment period) (p < 0.05). c Statistical significant difference from “CA” (for same treatment period) (p < 0.05).
Filling of defect Integration Matrix staining Cellular morphology Architecture within defect Architecture of surface Percentage of subchondral bone Formation of tidemark Total score
26 weeks (n ¼ 5)
6 weeks n ¼ 5)
Control defect
26 weeks (n ¼ 5)
2.7 1.6 0.8 0.7a 2.5 0.9 3.5 1.1 3.0 1.1 2.5 0.7 1.0 1.0 2.8 0.9 2.35 0.9
26 weeks (n ¼ 5)
30.0 23.1 44.2 33.1 47.5 32.4 77.5 14.1a 33.3 26.8 76.7 13.7a 24.2 26.2 51.7 24.9 96.7 3.7 83.3 32.9 97.5 2.5 37.5 31.5 50.0 30.4 42.5 28.5
BMS
BMS
Table 2. Assessment of Repair Tissue Using the Modified O’Driscoll Scores (MODS)
ANOVA followed by Tukey–Kramer post test. a Statistical significant difference from “control defect” (for same treatment period) (p < 0.05). b Statistical significant difference from “BMS” (for same treatment period) (p < 0.05). c Statistical significant difference from “CA” (for same treatment period) (p < 0.05).
Tissue morphology Matrix staining Cell morphology Chondrocyte clustering Surface architecture Basal integration Formation of tidemark Subchondral bone abnormalities/ marrow fibrosis Inflammation Abnormal calcification/ ossification Vascularization Surface/superficial assessment Mid/deep zone assessment Overall assessment
26 weeks (n ¼ 5)
6 weeks (n ¼ 5)
Control
Table 1. Assessment of Repair Tissue Using the ICRS II Score
3.6 0.4 2.8 0.4b 3.4 0.4 3.2 1.1 3.8 0.4 2.6 0.8 0.0 0.0 0.0 0.0 2.42 1.4
6 weeks (n ¼ 5)
CA
11.0 7.3a 14.0 9.7 24.0 28.5 34.0 16.2 23.0 30.5 2.0 2.4 b 96.0 3.7b 97.0 4.0 100.0 0.0 100.0 0.0 100.0 0.0 23.0 29.7 20.0 17.6 19.0 18.2
6 weeks (n ¼ 5)
CA
2.2 1.8 2.6 0.5b 2.6 1.3 3.2 1.8 2.8 1.4 2.2 1.0 0.2 0.4 0.4 0.5 1.98 1.0
26 weeks (n ¼ 5)
40.0 28.8 48.0 36.4 49.0 37.3 89.0 2.0a 37.0 37.2 22.0 27.1b 73.0 27.6 90.0 11.4 99.0 2.0 93.0 11.6 99.0 2.0 34.0 33.6 42.0 31.7 42.0 31.7
26 weeks (n ¼ 5)
61.7 17.7 a 74.3 21.2a 75.0 21.6a 67.5 25.1 68.3 29.8 a 58.3 25.4 37.5 17.2 55.0 26.2 95.0 4.1 94.2 10.9 95.0 7.6 70.8 24.5a 67.5 23.0a 67.5 23.0a
26 weeks (n ¼ 5)
3.2 0.7 2.0 0.6 2.6 0.8 3.0 0.6 3.8 0.4 2.8 0.4 0.0 0.0 a,c 3.4 0.8 2.60 1.1
6 weeks (n ¼ 5)
1.3 1.5 2.0 0.8 1.7 1.2 1.5 0.7a 2.2 1.0 1.7 1.1 0.5 1.1 3.2 0.7c 1.75 0.7
26 weeks (n ¼ 5)
CA þ BMS
41.0 21.0 54.0 28.5 51.0 28.7 58.0 24.8 30.0 28.6 31.0 20.0 a,c 12.0 9.3a,c 15.0 4.5 93.0 2.4 98.0 2.4 100.0 0.0 30.0 25.5 48.0 22.3 44.0 19.6
6 weeks (n ¼ 5)
CA þ BMS
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integration of transplanted native cartilage grafts was worse than integration of repair tissue induced by BMS (Fig. 4). The calcified layer was not penetrated and remained intact in this treatment group, and this status was sustained throughout the observation period without any changes in the subchondral architecture, abnormal calcification, or vascular invasion (Tables 1 and 2). A loss of proteoglycans within the matrix of the transplanted cartilage grafts was apparent at 6 weeks (Fig. 1g–i), which was partially restored at 26 weeks (Fig. 2g,h). All of the native cartilage autografts applied into defects with additional treatment by bone marrow stimulation (CA þ BMS) were retained within the defects throughout both observation periods. Partial delamination was observed in some cases, which resulted in incomplete filling at 6 weeks (48.0% 27.3 (g.o.); 43.0% 20.9 (h.a.)). At 26 weeks, filling was apparently increased (75.8% 22.8 (g.o.); 75.0% 20.4 (h.a.)). Similar to the defects treated by CA alone, a loss of proteoglycans within the matrix of the transplanted native cartilage grafts could be observed at 6 weeks (Fig. 1j–l). Signs of cell death as indicated by empty lacunae (Fig. 3b) and positive TUNEL staining (Fig. 3c) could be detected at the margins of the grafts. Viable chondrocytes sustained in the central parts of the grafts. At 26 weeks, the proteoglycan content of the grafts was at least partially restored (Fig. 2j,k). The simultaneous perforations of the subchondral bone plate coincided with cellular ingrowth from bone marrow spaces with formation a fibrous interface between graft and host cartilage (Figs. 1j,k and 3d). Lateral integration of the grafts was superior to transplanted grafts in defect with an intact subchondral bone plate. Cells within the interface region were spindle-shaped and formed a fibrous repair tissue layer that was positive for type I collagen (Fig. 3e) but negative for type II collagen (Fig. 3f). A similar pannus-like cell layer with a type I collagen-positive, but type II collagen-negative matrix could be observed at the surface of the grafts at 26 weeks (Fig. 3g–i). According to the ICRS II scoring system, CA þ BMS was the only treatment that induced significantly better repair at 26 weeks compared to non-treated control defects for the parameters “surface assessment,” “mid/deep zone assessment,” and “overall assessment” (p < 0.05) (Table 1).
DISCUSSION The central aim of this study was to evaluate if articular cartilage possesses intrinsic repair capacities mediated by the existence of cartilage-progenitor cells that have the capability to migrate and grow out of the tissue and to contribute to the formation of hyaline cartilage tissue.2,4,5,21,22 These cartilage-specific progenitor cells are supposed to be superior to other mesenchymal stem cells by stably retaining a nonhypertrophic chondrocytic phenotype.1,2 This capability stands in contrast to BMSCs that finally tend to
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undergo hypertrophy promoting inadvertant endochondral ossification within the repair tissue.23,24 In this animal model, the integrative repair of native articular cartilage grafts was generally insufficient. Cellular outgrowth from transplanted cartilage grafts could not be observed and the general poor repair of the defects can be ascribed to two major problems: transient graft degeneration and insufficient graft integration. The first major obstacle is based on degenerative changes of the grafts characterized by a loss of proteoglycans within their extracellular matrix. This phenomenon can be ascribed to two reasons. A transient postoperative inflammatory reaction may induce a catabolic response within the grafts. However, joint inflammation at 6 weeks postoperatively was only low to moderate as assessed by analysis of the synovial membrane. More important, we observed signs of cell death at the margins of the grafts, a phenomenon that not only interferes with graft integration but also contributes to graft degeneration. It has previously been shown that chondrocyte death is induced by the preparation of the grafts, while the amount of cell death depends on the method of dissection. Thus, the use of a trepine resulted in a loss of chondrocyte viability up to a depth of 150 mm25 whereas the use of sharp blades may reduce the extent of cell death up to a depth of 100 mm.26 These observations are consistent with previous reports of experimental wounding of cartilage.27,28 Despite using a sharp punch and dermatome for graft harvesting in this study, cell death could not be avoided within the margins of the grafts and at the defect borders in this study. During the ongoing observation period, the proteoglycan content was restored to some degree, which reflects the results of a recent study that demonstrated that cartilage has the capability to recover from hypocellularity following severe trauma by repopulation with chondrogenic progenitor cells.4 The second major problem of transplantation of native cartilage grafts is their poor integration with the host tissue, which resulted in loss of the graft at worst. Recent biomechanical studies demonstrated that cartilage-cartilage integration is often overestimated and mediated by a fibrous interlayer rather than by a truly interconnected collagen network.26 In addition to the cell death at the margin of the grafts and the defect border, the highly anti-adhesive properties of the initially intact cartilage matrix of the native grafts interferes with integration.25,29 Indeed, proteoglycans inhibit cartilage-cartilage integration30,31 and their removal by enzymatic treatment or by inflammatory cytokines was shown to improve integrative cartilage repair.32–34 Thus, the mature matrix and the native status of the transplanted cartilage grafts may interfere with integration with host cartilage. Furthermore, the absence of an additional protecting scaffold may expose the transplanted grafts to biomechanical shear forces which may result in micromotion of the JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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grafts that may additionally interfere with integration and bonding of the grafts. In this study, integration of cartilage grafts could be improved by additional pre-treatment by bone marrow stimulation (CA þ BMS). The ingrowing cells from the bone marrow obviously serve as a bonding kit by forming an interface of fibrous tissue composed of type I collagen. Indeed, deposition of collagen within the interface zone has been shown to be the major factor to promote adhesive strength in integrative cartilage repair.26,35 Interestingly, while integration of repair tissue with adjacent non-calcified cartilage is possible yet reduced, integration with the underlying calcified cartilage layer that was preserved in this study is virtually absent. Thus, calcification may mask epitopes that may otherwise mediate binding of cells or matrix molecules. The superior results of additional pre-treatment by bone marrow stimulation in this study reflects the success of previous repair approaches based on the transplantation of cartilage fragments that were applied into osteochondral defects or chondral defect with completely removed calcified cartilage layer.6,8–13 The difference in the poor outcome of our study and the successful repair of the mentioned previous studies may be ascribed to the different endogenous repair response induced by the creation of the defects themselves. In this sheep model, preservation of the calcified cartilage layer elicited virtually no endogenous repair response with no relevant cellular ingrowth from adjacent cartilage or bone marrow space. In contrast, removal of the calcified cartilage layer regularly results in some bleeding and micro-cracking of the subchondral bone plate and thereby initiating an endogenous repair response even without any additional graft application. The importance of removing the calcified cartilage layer for the formation of repair tissue by the original microfracturing technique has recently been demonstrated in a horse model.36 Thus, a significant portion of the cells within the forming repair tissue of recent successful repair approaches6,8–13 may not origin from the cartilage grafts themselves but rather from the bone marrow. Nevertheless, transplanted cartilage grafts may exert an immanent beneficial role by supporting chondrogenic differentiation of ingrowing mesenchymal repair cells, since cartilage tissue or chondrocytes were shown to exert significant pro-chondrogenic paracrine effects.37,38 There are some limitations and open questions of this study: First, one limitation of this study is the restricted follow-up period of 26 weeks. According to human MRI studies, the maturation of the repair tissue may further improve in the course of time.39 Since the sheep model closely reflects the limited repair response of human joints, it is worthwhile to investigate these cartilage repair concepts in the sheep model in long-term studies. Furthermore, the influence of the calcified cartilage layer and the role of different types of three-dimensional scaffolds embedJOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
ding the cartilage grafts should be investigated in more detail. In this respect, more durable scaffolds other than fibrin may provide better fixation of the grafts. A better protection from adverse shear forces would also allow to apply smaller (minced) cartilage fragments with a higher bioactive surface.6 The orientation of the grafts is another point that should be investigated in future studies. Since progenitor cells are considered to be in the superficial zone of articular cartilage, inverse orientation of the native cartilage autografts with the superficial zone facing downward might improve basal integration. In conclusion, intrinsic regeneration of cartilage defects with a preserved calcified cartilage layer is nearly absent and the integrative repair capacity of native cartilage autografts applied into such defects is also highly limited in the adult sheep model. Cellular outgrowth could not be observed and cell death, graft degeneration and anti-adhesive proteoglycans represent factors that interfere with the integration of native cartilage grafts. Additional ingrowing cells, example by bone marrow stimulating techniques, seem mandatory to mediate integration of the graft by providing repair cells and an interconnecting collagen network.
REFERENCES 1. Dowthwaite GP,Bishop JC, Redman SN, et al. 2004. The surface of articular cartilage contains a progenitor cell population. J Cell Sci 117:889–897. 2. Alsalameh S, Amin R, Gemba T, et al. 2004. Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage. Arthritis Rheum 50:1522–1532. 3. Grogan SP, Miyaki S, Asahara H, et al. 2009. Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis. Arthritis Res Ther 11:R85. 4. Seol D, McCabe DJ, Choe H, et al. 2012. Chondrogenic progenitor cells respond to cartilage injury. Arthritis Rheum 64:3626–3637. 5. Bos PK, Kops N, Verhaar JA, et al. 2008. Cellular origin of neocartilage formed at wound edges of articular cartilage in a tissue culture experiment. Osteoarthritis Cartilage 16:204–211. 6. Lu Y, Dhanaraj S, Wang Z, et al. 2006. Minced cartilage without cell culture serves as an effective intraoperative cell source for cartilage repair. J Orthop Res 24:1261–1270. 7. Morales TI. 2007. Chondrocyte moves: clever strategies. Osteoarthritis Cartilage 15:861–871. 8. Marmotti A, Bruzzone M, Bonasia DE, et al. 2012. Onestep osteochondral repair with cartilage fragments in a composite scaffold. Knee Surg Sports Traumatol Arthrosc 20:2590–2601. 9. Lind M, Larsen A. 2008. Equal cartilage repair response between autologous chondrocytes in a collagen scaffold and minced cartilage under a collagen scaffold: an in vivo study in goats. Connect Tissue Res 49:437–442. 10. Marmotti A, Bruzzone M, Bonasia DE, et al. 2013. Autologous cartilage fragments in a composite scaffold for one stage osteochondral repair in a goat model. Eur Cell Mater 26:15–31; discussion 31–12. 11. Frisbie DD, Lu Y, Kawcak CE, et al. 2009. In vivo evaluation of autologous cartilage fragment-loaded scaffolds implanted into equine articular defects and compared with autologous chondrocyte implantation. Am J Sports Med 37:71S–80S.
INTEGRATIVE REPAIR BY CARTILAGE AUTOGRAFTS 12. Cole BJ, Farr J, Winalski CS, et al. 2011. Outcomes after a single-stage procedure for cell-based cartilage repair: a prospective clinical safety trial with 2-year follow-up. Am J Sports Med 39:1170–1179. 13. Farr J, Tabet SK, Margerrison E, et al. 2014. Clinical, radiographic, and histological outcomes after cartilage repair with particulated juvenile articular cartilage: a 2-year prospective study. Am J Sports Med 42:1417–1425. 14. Sage A, Chang AA, Schumacher BL, et al. 2009. Cartilage outgrowth in fibrin scaffolds. American journal of rhinology & allergy 23:486–491. 15. Orth P, Meyer HL, Goebel L, et al. 2013. Improved repair of chondral and osteochondral defects in the ovine trochlea compared with the medial condyle. J Orthop Res 31:1772– 1779. 16. Mika J, Clanton TO, Pretzel D, et al. 2011. Surgical preparation for articular cartilage regeneration without penetration of the subchondral bone plate: in vitro and in vivo studies in humans and sheep. Am J Sports Med 39:624– 631. 17. Steadman JR, Rodkey WG, Briggs KK, et al. 1999. The microfracture technic in the management of complete cartilage defects in the knee joint. Orthopade 28:26–32. 18. Mainil-Varlet P, Van Damme B, Nesic D, et al. 2010. A new histology scoring system for the assessment of the quality of human cartilage repair: ICRS II. Am J Sports Med 38: 880–890. 19. O’Driscoll SW, Marx RG, Beaton DE, et al. 2001. Validation of a simple histological-histochemical cartilage scoring system. Tissue Eng 7:313–320. 20. Gelse K, Muhle C, Franke O, et al. 2008. Cell-based resurfacing of large cartilage defects: long-term evaluation of grafts from autologous transgene-activated periosteal cells in a porcine model of osteoarthritis. Arthritis Rheum 58: 475–488. 21. Williams R, Khan IM, Richardson K, et al. 2010. Identification and clonal characterisation of a progenitor cell subpopulation in normal human articular cartilage. PLoS ONE 5:e13246. 22. Koelling S, Kruegel J, Irmer M, et al. 2009. Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis. Cell Stem Cell 4:324– 335. 23. McCarthy HE, Bara JJ, Brakspear K, et al. 2011. The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse. Vet J 192:345–351. 24. Grogan SP, Barbero A, Diaz-Romero J, et al. 2007. Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity. Arthritis Rheum 56:586–595. 25. Archer CW, Redman S, Khan I, et al. 2006. Enhancing tissue integration in cartilage repair procedures. J Anat 209:481–493.
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26. Moretti M, Wendt D, Schaefer D, et al. 2005. Structural characterization and reliable biomechanical assessment of integrative cartilage repair. Journal of biomechanics 38: 1846–1854. 27. Hunziker EB, Quinn TM. 2003. Surgical removal of articular cartilage leads to loss of chondrocytes from cartilage bordering the wound edge. J Bone Joint Surg Am 85:85–92. 28. Tew SR, Kwan AP, Hann A, et al. 2000. The reactions of articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum 43:215–225. 29. Khan IM, Gilbert SJ, Singhrao SK, et al. 2008. Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review. Eur Cell Mater 16: 26–39. 30. Englert C, McGowan KB, Klein TJ, et al. 2005. Inhibition of integrative cartilage repair by proteoglycan 4 in synovial fluid. Arthritis Rheum 52:1091–1099. 31. Schaefer DB, Wendt D, Moretti M, et al. 2004. Lubricin reduces cartilage–cartilage integration. Biorheology 41:503–508. 32. Janssen LM, In der Maur CD, Bos PK, et al. 2006. Shortduration enzymatic treatment promotes integration of a cartilage graft in a defect. Ann Otol Rhinol Laryngol 115:461– 468. 33. Bos PK, DeGroot J, Budde M, et al. 2002. Specific enzymatic treatment of bovine and human articular cartilage: implications for integrative cartilage repair. Arthritis Rheum 46: 976–985. 34. Khan IM, Gonzalez LG, Francis L, et al. 2011. Interleukin1beta enhances cartilage-to-cartilage integration. Eur Cell Mater 22:190–201. 35. DiMicco MA, Sah RL. 2001. Integrative cartilage repair: adhesive strength is correlated with collagen deposition. J Orthop Res 19:1105–1112. 36. Frisbie DD, Morisset S, et al. 2006. Effects of calcified cartilage on healing of chondral defects treated with microfracture in horses. Ho CP 34:1824–1831. 37. Blanke M, Carl HD, Klinger P, et al. 2009. Transplanted chondrocytes inhibit endochondral ossification within cartilage repair tissue. Calcif Tissue Int 85:421–433. 38. Gelse K, Brem M, Klinger P, et al. 2009. Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue. J Orthop Res 27:1216– 1225. 39. Welsch GH, Mamisch TC, Marlovits S, et al. 2009. Quantitative T2 mapping during follow-up after matrix-associated autologous chondrocyte transplantation (MACT): full-thickness and zonal evaluation to visualize the maturation of cartilage repair tissue. J Orthop Res 27:957–963.
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Received 17 July 2014; accepted 20 October 2014 Published online 3 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22773
ABSTRACT: The purpose of this study was to investigate integration and cellular outgrowth of native cartilage autografts transplanted into articular cartilage defects. Native cartilage autografts were applied into chondral defects in the femoral condyle of adult sheep. Within the defects, the calcified cartilage layer was either left intact or perforated to induce bone marrow stimulation. Empty defects served as controls. The joints were analyzed after 6 and 26 weeks by macroscopic and histological analysis using the ICRS II Score and Modified O‘Driscoll Scores. Non-treated defects did not show any endogenous regenerative response and bone marrow stimulation induced fibrous repair tissue. Transplanted native cartilage grafts only insufficiently integrated with the defect borders. Cell death and loss of proteoglycans were present at the margins of the grafts at 6 weeks, which was only partially restored at 26 weeks. Significant cellular outgrowth from the grafts or defect borders could not be observed. Bonding of the grafts could be improved by additional bone marrow stimulation providing ingrowing cells that formed a fibrous interface predominantly composed of type I collagen. Transplanted native cartilage grafts remain as inert structures within cartilage defects and fail to induce integrative cartilage repair which rather demands additional cells provided by additional bone marrow stimulation. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:390–397, 2015. Keywords: cartilage repair; cartilage graft; bone marrow stimulation; progenitor cell; regeneration
The existence of progenitor cells within articular cartilage has been discussed in a number of recent studies.1–3 Particularly their capability to migrate and to grow out of the cartilage tissue would be one prerequisite for intrinsic cartilage regeneration.4,5 Indeed, cell outgrowth from cartilage tissue was demonstrated in vitro and in vivo.5–7 In rabbit and goat models, transplanted native cartilage fragments contributed to the formation of better repair tissue in treated cartilage defects than application of the scaffolds alone.6,8–10 Successful results following transplantation of autologous cartilage fragments or allogeneic particulated juvenile articular cartilage were also observed in large animal models11 and human studies.12,13 A variety of biodegradable scaffolds have been used for embedding the cartilage fragments. A three-dimensional scaffold seems to promote chondrocyte proliferation and outgrowth.14 These data encourage the establishment of one-stage cartilage repair procedures and would query the need for the two-stage autologous chondrocyte transplantation. So far, however, all of these promising results were observed in osteochondral defects or defects with completely removed calcified cartilage layer, which may allow the ingrowth of bone marrow-derived cells. Thus, it has still to be investigated to which extent the cells within the forming repair tissue originate from the transplanted cartilage graft or the bone marrow. Therefore, the aim of this study was to analyze and evaluate the role of native autologous cartilage grafts with respect to their integrative repair capacity (celluConflicts of interest: None. Grant sponsor: German Research Foundation; Grant number: WE 4881/1–1. Correspondence to: Kolja Gelse (T: 0049-9131-8533296; F: 00499131-8533300; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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lar outgrowth and integration) after transplantation into true chondral defects to provide a model with limited endogenous repair capacities. We addressed the medial femoral condyle of the adult sheep model, since this setting is characterized by a poor endogenous repair response similar to that of adult human joints.15 To rule out any ingrowth of bone marrow cells, the subchondral bone plate including the calcified cartilage layer was completely left intact. As an alternative treatment group, the calcified cartilage layer within the defects was perforated to induce bone marrow stimulation in order to monitor the influence of bone marrowderived cells.
MATERIALS AND METHODS Ten female adult merino sheep aged 3–4 years with body weights of 70–80 kg were used in this study. The animals were anesthetized and their knee joint capsule was opened by a medial parapatellar incision. The patella was displaced laterally to expose the medial femoral condyle. Each of the 10 animals was treated by two separate round 5 mm cartilage defects created into the main load-bearing zone of the medial femoral condyle using a sharp biopsy punch. The two lesions were clearly separated from each other by at least 5 mm of intact cartilage to avoid any cell migration or interaction between the two defects. The articular cartilage was carefully removed using a curette leaving the calcified cartilage layer and the subchondral bone plate intact.16 The preservation of the calcified cartilage layer was confirmed by complete absence of bleeding in the defect during surgery. Afterwards, the two prepared lesions in each of the animals were treated by two different approaches among the following four different treatment approaches: (1) No further treatment (empty cartilage lesion down to the tide-mark with exposed but completely intact calcified cartilage ¼ “control”); (2) Bone marrow stimulation (“BMS”) by treatment with five perforations using a custom-made micropick (each 1 mm in diameter and 3 mm deep) that were covered with fibrin glue (Beriplast,
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Aventis, Marburg, Germany). The perforations were applied through the preserved calcified cartilage layer, which stands in contrast to the original microfracturing method with the calcified cartilage layer being removed17 (Supplementary Figure); (3) Transplantation of a discoid articular cartilage autograft into the defect with an intact layer of calcified cartilage (Cartilage Autograft-Transplantation ¼ “CA”). The cartilaginous autograft was harvested from the very cranial zone of the femoral trochlea using a dermatome (0,8 mm thickness) and a 5 mm biopsy punch to generate a discoid graft that exactly fits into the defect; (4) Transplantation of a discoid articular cartilage autograft into the defect that had been treated by bone marrow stimulation with five perforations before (“CA þ BMS”)(Supplementary Figure). All grafts were additionally sealed with fibrin glue with a fibrinogen concentration of 90 mg/ml (Beriplast, Aventis Behring, Marburg, Germany) to increase initial stability and to avoid potential cell spread by initial bleeding in defects treated by bone marrow stimulation. Between the 10 animals, the two defects within each joint received two different treatment approaches that were arranged in an alternating manner to minimize spatial environmental differences. Following surgery, the animals were allowed to move freely in their stalls. The animals were first operated on their right knee joint. After 20 weeks, the animals were operated on their left knees according to the previous surgery on the right knee and were sacrificed 6 weeks later. This treatment scheme allowed two different follow-up periods with 6 weeks for the left knee joints and 26 weeks for the right knee joints. This scheme was chosen for ethical reasons to reduce the total number of animals and was approved by the Institutional and Governmental Review Boards. The knee joints were dissected and first assessed macroscopically followed by preparation for histological analysis as described below. Histological and Immunohistological Assessment The osteochondral specimens were fixed in 4% paraformaldehyde for at least 12 h, followed by decalcification in 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 months. After standard processing, the samples were embedded in paraffin. The specimens were scanned throughout the tissue blocks with serial transverse 5 mm sections and stained with toluidine blue to estimate the proteoglycan content. The morphological assessment of the different approaches was performed by three independent specialists according to the ICRS II Score18 and the O‘Driscoll Score.19 To determine the percentage of tight lateral and basal integration of the repair tissue with adjacent tissue and the percentage of filling of the defects, five parallel sections with a separation distance of 1 mm were captured using a microscope camera throughout each of the treated defects and further analyzed by a digital imaging programme. Tight integration was defined when direct contact of repair tissue to host tissue was present even after histological tissue processing. Immunohistochemical detection of type I and type II collagen was performed as described previously in detail.20 Briefly, deparaffinized sections were pretreated with 0.2% hyaluronidase for 60 min and subsequently with 0.2% pronase (Sigma–Aldrich) for 60 min. Sections were then exposed overnight to anti-human type I collagen antibodies (MP Biomedicals, Aurora, OH) diluted 1:200, or anti-human type
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II collagen antibodies (MP Biomedicals) diluted 1:500. After incubation with a biotinylated donkey anti-mouse secondary antibody (Dianova, Hamburg, Germany), a complex of streptavidine and biotinylated alkaline phosphatase was added. The sections were developed with fast red and counterstained with hematoxylin. Tunel Staining For the detection of in situ DNA breaks, the TUNEL reaction was applied using the In Situ Cell Death Detection Kit, AP (Roche, Mannheim, Germany). After deparaffinization, the sections were washed in PBS and digested with proteinase K (20 mg/ml; Boehringer, Ingelheim, Germany) for 15 min at 37 ˚C. After washing with PBS, sections were incubated with TdT-solution in reaction buffer at a volume ratio of 9:1 at 37 ˚C for 1 h followed by extensive washing. The converter AP antibody was added for 30 min at 37 ˚C. After washing, detection was performed by fast red staining and counterstaining with hematoxylin. Statistical Analysis All data are presented as mean SD. For the evaluation of morphological parameters non-parametric Kruskal–Wallis tests followed by post-hoc Dunn multiple comparison tests (Modified O’Driscoll Scores) or parametric analysis of variance (ANOVA) tests followed by Tukey–Kramer multiple comparison tests (ICRS II Score) were used to determine treatment-specific differences. Integration was assessed by analysis of variance followed by a Tukey–Kramer test. All statistical results were considered significant for p-values < 0.05.
RESULTS Filling of the defects was analyzed by gross observation (g.o.) and by histomorphological analysis (h.a.) at 6 weeks (n ¼ 5 each treatment) (Fig. 1) and 26 weeks (n ¼ 5 each treatment) (Fig. 2). Gross observation tended to slightly overestimate the filling of the defects compared to histological analysis, however, the differences were not significant. At 6 weeks, there was no relevant filling of nontreated control lesions despite of debris and remnants of fibrous tissue (21.0% 11,6 (g.o.); 10.0% 7.7 (h.a.)) (Fig. 1a–c). Filling of the defects was not increased at 26 weeks (16.3% 11.9 (g.o.); 8.8% 6.5 (h.a.))(Fig. 2a– c). Cellular ingrowth from the defect margins could not be observed at 6 weeks and only to a minimal extent at 26 weeks (Fig. 3a). During the observation period of 26 weeks, the layer of calcified cartilage and the underlying subchondral bone remained completely unaffected by the removal of the articular cartilage, which indicates complete lack of any endogenous repair response of induced cartilage defects. BMS induced the formation of fibrous tissue that hardly stained for proteoglycans at 6 weeks (Fig. 1d–f). At 26 weeks, chondrogenic differentiation of repair cells with formation of a fibrocartilaginous matrix was apparent, however, the tissue morphology was rather inhomogenous with fibrous tissue in the superficial layer and signs of endochondral ossification in deeper layers (Fig. 2d–f). The cells within the repair tissue JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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Figure 1. Repair tissues at 6 weeks after bone marrow stimulation by subchondral perforations (BMS), transplantation of native autologous cartilage grafts (CA), or combined treatment (CA þ BMS). The resulting repair tissues were examined histologically by toluidine blue staining and by gross observation (repair tissue of indicated treatment group is encircled). Representative sections of the average, best and worst repair tissues of each group are shown. Empty control defects show no relevant repair response (a–c). Defects treated by bone marrow stimulation (BMS) are partially filled with fibrous repair tissue (d–f). Transplanted native cartilage autografts (CA) are characterized by loss of proteoglycans (reduced toluidine blue staining) and by lack of basal integration (g–i). Native cartilage autografts transplanted into defects pretreated by bone marrow stimulation (CA þ BMS) also have a reduced content of proteoglycans and show partial basal bonding in the areas of initial perforations (j–l). Bar ¼ 1mm (histological section), bar ¼ 5 mm (macroscopic view).
were characterized by a spindle-shaped fibroblast-like phenotype in the superficial zone, round chondrocytelike phenotype in the middle zone, and a hypertrophic phenotype in the deepest zone. General filling of the defects following BMS was poor at 6 weeks (28.0% 14.7 (g.o.); 18.0% 11.2 (h.a.)) and was slightly increased at 26 weeks (33.3% 33.6 (g.o.); 41.7% 37.5 (h.a.)). Basal integration of the repair tissue was tight
in areas of the induced perforations and adjacent to the non-calcified articular cartilage, but insufficient in regions with remaining calcified cartilage (Fig. 2d–f). General integration of repair tissue following BMS was significantly better than that observed in control defects or following CA-treatment (Fig. 4). The subchondral bone plate and tide mark did not reconstitute during the observation period. Abnormal calcification
Figure 2. Repair tissues at 26 weeks after bone marrow stimulation by subchondral perforations (BMS), transplantation of native autologous cartilage grafts (CA), or combined treatment (CA þ BMS). The resulting repair tissues were examined histologically by toluidine blue staining and by gross observation (repair tissue of indicated treatment group is encircled). Representative sections of the average, best and worst repair tissues of each group are shown. Empty control defects lack any repair response (a–c). Defects treated by bone marrow stimulation (BMS) are partially filled with fibrocartilaginous repair tissue (d–f). Transplanted native cartilage autografts (CA) lack basal integration (g–h) or have been lost (i). Native cartilage autografts transplanted into defects pretreated by bone marrow stimulation (CA þ BMS) show tight lateral integration in most cases but basal bonding is limited to those areas of initial perforations (j–l). Bar ¼ 1mm (histological section), bar ¼ 5 mm (macroscopic view). JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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Figure 3. Features for integrative cartilage repair. Marginal ingrowth of repair cells into non-treated defects at 26 weeks (a). Reduced density of viable cells at the margins of implanted native cartilage grafts as indicated by empty lacunae (b) and apoptotic cells positive for TUNEL staining (c). Tight lateral integration but lack of basal integration of native cartilage grafts (*) with underlying calcified cartilage in CA þ BMS-treated defects (d). Lateral interface region is composed of fibrous tissue positive for type I collagen (e) and negative for type II collagen (f). Ingrowing cells induced by bone marrow stimulation form a pannus-like cell layer on transplanted native cartilage grafts at 26 weeks (g) that is negative for type II (h) but positive for type I collagen (i). Toluidin Blue Staining (a, b, d, g); TUNEL staining (c). Immunohistochemical staining for type I collagen (e, h) and type II collagen (f, i). Bars ¼ 100 mm (a, d–f), 50 mm (b, c, g–i).
Figure 4. Analysis of integration of repair tissues within cartilage defects. General integration (lateral and basal) of repair tissue induced by bone marrow stimulation (BMS) is significantly superior to that of transplanted native cartilage grafts (CA). Integration of native cartilage grafts is improved by additional bone marrow stimulation (CA þ BMS).
or excessive ossification within the repair tissue was apparent in some areas of the deeper zones at 26 weeks (Fig. 2e). Significant inflammation or vascular invasion was not apparent (Tables 1 and 2). The score for “overall assessment” of the BMS-treated defects increased from 6 to 26 weeks (Tables 1 and 2). The application of a native cartilage autograft (CA) into the defects was confronted with the problem of graft displacement. Only 60% of the grafts were retained within the defects at 6 and 26 weeks, which correlated with an incomplete general filling at 6 weeks (28.0% 29.2 (g.o.); 17.0% 13.6 (h.a.)) and 26 weeks (51.0% 39.7 (g.o.); 49.0% 39.4 (h.a.)). Apparently, integration of the native cartilage autograft was strongly dependent on the adjacent tissue type. There were no signs of basal integration of the native cartilage grafts with underlying calcified cartilage at 6 and 26 weeks (Figs. 1g–i and 2g–i), and only inconsistent lateral integration with adjacent non-calcified articular cartilage (Figs. 1g–i, 2g–i, and 4). Overall, general JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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2.5 4.3 5 6.1 8.75 12.4 17.5 20.4 2.5 2.5 12.5 21.6 73.8 42.6 72.5 29.4 95 8.7 82.5 30.3 95 8.7 1.25 2.2 13.8 9.6 3.8 4.1
1.0 2 6.0 3.7 6.0 5.8 15.0 27.5 5.0 4.5 2.0 2.4 80.0 24.4 90.0 20.0 100.0 0 100.0 0 100.0 0 6.0 4.9 4.0 3.7 6.0 7.3
9.0 4.0 14.0 9.7 16.0 12.4 53.0 33.7 18.0 12.8 54.0 27.2a 36.0 26.7 48.0 36.0 94.0 8.0 88.0 19.3 100.0 0.0 20.0 13.0 16.0 8.6 12.0 4.0
6 weeks (n ¼ 5)
4.0 0.0 3.0 0.0 4.0 0.0 5.0 0.0 4.0 0.0 3.0 0.0 1.0 1.7 1.25 1.6 3.16 1.3
4.0 0.0 3.0 0.0 4.0 0.0 4.8 0.4 4.0 0.0 3.0 0.0 0.0 0.0 0.2 0.4 2.85 1.7
3.6 0.4 1.2 0.7a 3.4 0.4 4.6 0.4 3.8 0.4 3.0 0.0 0.4 0.8 2.4 1.0 2.80 1.3
6 weeks (n ¼ 5)
Kruskal–Wallis Test followed by Dunn multiple comparison tests. a Statistical significant difference from “control defect” (for same treatment period) (p < 0.05) .bStatistical significant difference from “BMS” (for same treatment period) (p < 0.05). c Statistical significant difference from “CA” (for same treatment period) (p < 0.05).
Filling of defect Integration Matrix staining Cellular morphology Architecture within defect Architecture of surface Percentage of subchondral bone Formation of tidemark Total score
26 weeks (n ¼ 5)
6 weeks n ¼ 5)
Control defect
26 weeks (n ¼ 5)
2.7 1.6 0.8 0.7a 2.5 0.9 3.5 1.1 3.0 1.1 2.5 0.7 1.0 1.0 2.8 0.9 2.35 0.9
26 weeks (n ¼ 5)
30.0 23.1 44.2 33.1 47.5 32.4 77.5 14.1a 33.3 26.8 76.7 13.7a 24.2 26.2 51.7 24.9 96.7 3.7 83.3 32.9 97.5 2.5 37.5 31.5 50.0 30.4 42.5 28.5
BMS
BMS
Table 2. Assessment of Repair Tissue Using the Modified O’Driscoll Scores (MODS)
ANOVA followed by Tukey–Kramer post test. a Statistical significant difference from “control defect” (for same treatment period) (p < 0.05). b Statistical significant difference from “BMS” (for same treatment period) (p < 0.05). c Statistical significant difference from “CA” (for same treatment period) (p < 0.05).
Tissue morphology Matrix staining Cell morphology Chondrocyte clustering Surface architecture Basal integration Formation of tidemark Subchondral bone abnormalities/ marrow fibrosis Inflammation Abnormal calcification/ ossification Vascularization Surface/superficial assessment Mid/deep zone assessment Overall assessment
26 weeks (n ¼ 5)
6 weeks (n ¼ 5)
Control
Table 1. Assessment of Repair Tissue Using the ICRS II Score
3.6 0.4 2.8 0.4b 3.4 0.4 3.2 1.1 3.8 0.4 2.6 0.8 0.0 0.0 0.0 0.0 2.42 1.4
6 weeks (n ¼ 5)
CA
11.0 7.3a 14.0 9.7 24.0 28.5 34.0 16.2 23.0 30.5 2.0 2.4 b 96.0 3.7b 97.0 4.0 100.0 0.0 100.0 0.0 100.0 0.0 23.0 29.7 20.0 17.6 19.0 18.2
6 weeks (n ¼ 5)
CA
2.2 1.8 2.6 0.5b 2.6 1.3 3.2 1.8 2.8 1.4 2.2 1.0 0.2 0.4 0.4 0.5 1.98 1.0
26 weeks (n ¼ 5)
40.0 28.8 48.0 36.4 49.0 37.3 89.0 2.0a 37.0 37.2 22.0 27.1b 73.0 27.6 90.0 11.4 99.0 2.0 93.0 11.6 99.0 2.0 34.0 33.6 42.0 31.7 42.0 31.7
26 weeks (n ¼ 5)
61.7 17.7 a 74.3 21.2a 75.0 21.6a 67.5 25.1 68.3 29.8 a 58.3 25.4 37.5 17.2 55.0 26.2 95.0 4.1 94.2 10.9 95.0 7.6 70.8 24.5a 67.5 23.0a 67.5 23.0a
26 weeks (n ¼ 5)
3.2 0.7 2.0 0.6 2.6 0.8 3.0 0.6 3.8 0.4 2.8 0.4 0.0 0.0 a,c 3.4 0.8 2.60 1.1
6 weeks (n ¼ 5)
1.3 1.5 2.0 0.8 1.7 1.2 1.5 0.7a 2.2 1.0 1.7 1.1 0.5 1.1 3.2 0.7c 1.75 0.7
26 weeks (n ¼ 5)
CA þ BMS
41.0 21.0 54.0 28.5 51.0 28.7 58.0 24.8 30.0 28.6 31.0 20.0 a,c 12.0 9.3a,c 15.0 4.5 93.0 2.4 98.0 2.4 100.0 0.0 30.0 25.5 48.0 22.3 44.0 19.6
6 weeks (n ¼ 5)
CA þ BMS
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integration of transplanted native cartilage grafts was worse than integration of repair tissue induced by BMS (Fig. 4). The calcified layer was not penetrated and remained intact in this treatment group, and this status was sustained throughout the observation period without any changes in the subchondral architecture, abnormal calcification, or vascular invasion (Tables 1 and 2). A loss of proteoglycans within the matrix of the transplanted cartilage grafts was apparent at 6 weeks (Fig. 1g–i), which was partially restored at 26 weeks (Fig. 2g,h). All of the native cartilage autografts applied into defects with additional treatment by bone marrow stimulation (CA þ BMS) were retained within the defects throughout both observation periods. Partial delamination was observed in some cases, which resulted in incomplete filling at 6 weeks (48.0% 27.3 (g.o.); 43.0% 20.9 (h.a.)). At 26 weeks, filling was apparently increased (75.8% 22.8 (g.o.); 75.0% 20.4 (h.a.)). Similar to the defects treated by CA alone, a loss of proteoglycans within the matrix of the transplanted native cartilage grafts could be observed at 6 weeks (Fig. 1j–l). Signs of cell death as indicated by empty lacunae (Fig. 3b) and positive TUNEL staining (Fig. 3c) could be detected at the margins of the grafts. Viable chondrocytes sustained in the central parts of the grafts. At 26 weeks, the proteoglycan content of the grafts was at least partially restored (Fig. 2j,k). The simultaneous perforations of the subchondral bone plate coincided with cellular ingrowth from bone marrow spaces with formation a fibrous interface between graft and host cartilage (Figs. 1j,k and 3d). Lateral integration of the grafts was superior to transplanted grafts in defect with an intact subchondral bone plate. Cells within the interface region were spindle-shaped and formed a fibrous repair tissue layer that was positive for type I collagen (Fig. 3e) but negative for type II collagen (Fig. 3f). A similar pannus-like cell layer with a type I collagen-positive, but type II collagen-negative matrix could be observed at the surface of the grafts at 26 weeks (Fig. 3g–i). According to the ICRS II scoring system, CA þ BMS was the only treatment that induced significantly better repair at 26 weeks compared to non-treated control defects for the parameters “surface assessment,” “mid/deep zone assessment,” and “overall assessment” (p < 0.05) (Table 1).
DISCUSSION The central aim of this study was to evaluate if articular cartilage possesses intrinsic repair capacities mediated by the existence of cartilage-progenitor cells that have the capability to migrate and grow out of the tissue and to contribute to the formation of hyaline cartilage tissue.2,4,5,21,22 These cartilage-specific progenitor cells are supposed to be superior to other mesenchymal stem cells by stably retaining a nonhypertrophic chondrocytic phenotype.1,2 This capability stands in contrast to BMSCs that finally tend to
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undergo hypertrophy promoting inadvertant endochondral ossification within the repair tissue.23,24 In this animal model, the integrative repair of native articular cartilage grafts was generally insufficient. Cellular outgrowth from transplanted cartilage grafts could not be observed and the general poor repair of the defects can be ascribed to two major problems: transient graft degeneration and insufficient graft integration. The first major obstacle is based on degenerative changes of the grafts characterized by a loss of proteoglycans within their extracellular matrix. This phenomenon can be ascribed to two reasons. A transient postoperative inflammatory reaction may induce a catabolic response within the grafts. However, joint inflammation at 6 weeks postoperatively was only low to moderate as assessed by analysis of the synovial membrane. More important, we observed signs of cell death at the margins of the grafts, a phenomenon that not only interferes with graft integration but also contributes to graft degeneration. It has previously been shown that chondrocyte death is induced by the preparation of the grafts, while the amount of cell death depends on the method of dissection. Thus, the use of a trepine resulted in a loss of chondrocyte viability up to a depth of 150 mm25 whereas the use of sharp blades may reduce the extent of cell death up to a depth of 100 mm.26 These observations are consistent with previous reports of experimental wounding of cartilage.27,28 Despite using a sharp punch and dermatome for graft harvesting in this study, cell death could not be avoided within the margins of the grafts and at the defect borders in this study. During the ongoing observation period, the proteoglycan content was restored to some degree, which reflects the results of a recent study that demonstrated that cartilage has the capability to recover from hypocellularity following severe trauma by repopulation with chondrogenic progenitor cells.4 The second major problem of transplantation of native cartilage grafts is their poor integration with the host tissue, which resulted in loss of the graft at worst. Recent biomechanical studies demonstrated that cartilage-cartilage integration is often overestimated and mediated by a fibrous interlayer rather than by a truly interconnected collagen network.26 In addition to the cell death at the margin of the grafts and the defect border, the highly anti-adhesive properties of the initially intact cartilage matrix of the native grafts interferes with integration.25,29 Indeed, proteoglycans inhibit cartilage-cartilage integration30,31 and their removal by enzymatic treatment or by inflammatory cytokines was shown to improve integrative cartilage repair.32–34 Thus, the mature matrix and the native status of the transplanted cartilage grafts may interfere with integration with host cartilage. Furthermore, the absence of an additional protecting scaffold may expose the transplanted grafts to biomechanical shear forces which may result in micromotion of the JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
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grafts that may additionally interfere with integration and bonding of the grafts. In this study, integration of cartilage grafts could be improved by additional pre-treatment by bone marrow stimulation (CA þ BMS). The ingrowing cells from the bone marrow obviously serve as a bonding kit by forming an interface of fibrous tissue composed of type I collagen. Indeed, deposition of collagen within the interface zone has been shown to be the major factor to promote adhesive strength in integrative cartilage repair.26,35 Interestingly, while integration of repair tissue with adjacent non-calcified cartilage is possible yet reduced, integration with the underlying calcified cartilage layer that was preserved in this study is virtually absent. Thus, calcification may mask epitopes that may otherwise mediate binding of cells or matrix molecules. The superior results of additional pre-treatment by bone marrow stimulation in this study reflects the success of previous repair approaches based on the transplantation of cartilage fragments that were applied into osteochondral defects or chondral defect with completely removed calcified cartilage layer.6,8–13 The difference in the poor outcome of our study and the successful repair of the mentioned previous studies may be ascribed to the different endogenous repair response induced by the creation of the defects themselves. In this sheep model, preservation of the calcified cartilage layer elicited virtually no endogenous repair response with no relevant cellular ingrowth from adjacent cartilage or bone marrow space. In contrast, removal of the calcified cartilage layer regularly results in some bleeding and micro-cracking of the subchondral bone plate and thereby initiating an endogenous repair response even without any additional graft application. The importance of removing the calcified cartilage layer for the formation of repair tissue by the original microfracturing technique has recently been demonstrated in a horse model.36 Thus, a significant portion of the cells within the forming repair tissue of recent successful repair approaches6,8–13 may not origin from the cartilage grafts themselves but rather from the bone marrow. Nevertheless, transplanted cartilage grafts may exert an immanent beneficial role by supporting chondrogenic differentiation of ingrowing mesenchymal repair cells, since cartilage tissue or chondrocytes were shown to exert significant pro-chondrogenic paracrine effects.37,38 There are some limitations and open questions of this study: First, one limitation of this study is the restricted follow-up period of 26 weeks. According to human MRI studies, the maturation of the repair tissue may further improve in the course of time.39 Since the sheep model closely reflects the limited repair response of human joints, it is worthwhile to investigate these cartilage repair concepts in the sheep model in long-term studies. Furthermore, the influence of the calcified cartilage layer and the role of different types of three-dimensional scaffolds embedJOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2015
ding the cartilage grafts should be investigated in more detail. In this respect, more durable scaffolds other than fibrin may provide better fixation of the grafts. A better protection from adverse shear forces would also allow to apply smaller (minced) cartilage fragments with a higher bioactive surface.6 The orientation of the grafts is another point that should be investigated in future studies. Since progenitor cells are considered to be in the superficial zone of articular cartilage, inverse orientation of the native cartilage autografts with the superficial zone facing downward might improve basal integration. In conclusion, intrinsic regeneration of cartilage defects with a preserved calcified cartilage layer is nearly absent and the integrative repair capacity of native cartilage autografts applied into such defects is also highly limited in the adult sheep model. Cellular outgrowth could not be observed and cell death, graft degeneration and anti-adhesive proteoglycans represent factors that interfere with the integration of native cartilage grafts. Additional ingrowing cells, example by bone marrow stimulating techniques, seem mandatory to mediate integration of the graft by providing repair cells and an interconnecting collagen network.
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