Treatment of Articular Cartilage Defects with Microfracture And

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Treatment of Articular Cartilage Defects With Microfracture and Autologous Matrix-Induced Chondrogenesis Leads to Extensive Subchondral Bone Cyst Formation in a Sheep Model

Aswin Beck,*y DVM, DACVS-LA, David J. Murphy,y BVSc, Dip VCS, MS, MANZCVS, DACVS, Richard Carey-Smith,z BSc(Hons), MBBS, MRCS(Eng), FRCS(Tr&Orth), FRACS(Ortho), FAOrthA, David J. Wood,z BSc, MBBS, MS, FRCS, FRACS, FAOrthA, and Ming H. Zheng,*§ MBBS, DM, PhD, FRCP, ARCPA Investigation performed at Murdoch University, Murdoch, Australia, and University of Western Australia, Crawley, Australia

Background: Microfracture and the autologous matrix-induced chondrogenesis (AMIC) technique are popular for the treatment of articular cartilage defects. However, breaching of the subchondral bone plate could compromise the subchondral bone structure. Hypothesis: Microfracture and AMIC will cause deleterious effects on the subchondral bone structure. Study Design: Controlled laboratory study. Methods: A total of 36 sheep received an 8-mm-diameter cartilage defect in the left medial femoral condyle. Control animals (n = 12) received no further treatment, and the rest received 5 microfracture holes either with a type I/III collagen scaffold implanted (n = 12; AMIC group) or without the collagen scaffold (n = 12; microfracture group). Macroscopic infill of defects, histology, and histomorphometry of the subchondral bone were performed at 13 and 26 weeks postoperatively, and micro–computed tomography (CT) was also performed at 26 weeks postoperatively. Results: Microfracture and AMIC resulted in subchondral bone cyst formation in 5 of 12 (42%) and 11 of 12 (92%) specimens at 13 and 26 weeks, respectively. Subchondral bone changes induced by microfracture and AMIC were characterized by an increased percentage of bone volume, increased trabecular thickness, and a decreased trabecular separation, and extended beyond the area below the defect. High numbers of osteoclasts were observed at the cyst periphery, and all cysts communicated with the microfracture holes. Cartilage repair tissue was of poor quality and quantity at both time points and rarely reached the tidemark at 13 weeks. Conclusion: Microfracture technique caused bone cyst formation and induced severe pathology of the subchondral bone in a sheep model. Clinical Relevance: The potential of microfracture technique to induce subchondral bone pathology should be considered. Keywords: microfracture; AMIC; subchondral bone pathology; subchondral bone cyst

Symptomatic articular cartilage defects are one of the most mosaicplasty,9 corrective osteotomy,27 cartilage resurfacing common knee injuries, arising from acute trauma, overuse, techniques and tissue engineering techniques using combi- ligamentous instability, malalignment, meniscectomy, or nations of autologous cells (chondrocytes38 and mesenchy- osteochondritis dissecans.53 Surgical treatment options mal stem cells47), bioscaffolds,28 and growth factors.48 include bone marrow–stimulating techniques such as abra- Currently, microfracture and the autologous matrix- sion arthroplasty26,46 and microfracture,21,49 osteochondral induced chondrogenesis (AMIC) technique, an improved microfracture technique by implantation of a collagen scaffold with the aim of creating a biological chamber within the cartilage lesion,13-15,18,20,37 are popular techniques of car- The American Journal of Sports Medicine, Vol. 44, No. 10 DOI: 10.1177/0363546516652619 tilage repair by virtue of being cost-effective, one-step proce- 20 Ó 2016 The Author(s) dures that can be performed arthroscopically. Steadman

2629 2630 Beck et al The American Journal of Sports Medicine et al49 showed that microfracture achieved significant maintained with 2% isoflurane mixed in 100% oxygen after improvement in Lysholm and Tegner scores in 80% of endotracheal intubation. Additional analgesia consisted of patients in an 11-year follow-up study. A systematic review transdermal fentanyl patches (2 mg/kg/h), epidural anes- by Mithoefer et al37 of 28 studies and 3122 patients treated thesia (100 mg lidocaine, 0.3 mg buprenorphine), and car- with microfracture showed an improvement in knee function. profen (4 mg/kg). The left stifle joints were surgically However, microfracture technique could achieve only an approached via a lateral parapatellar arthrotomy as effective short-term outcome, and variable results after 24 described previously.1 Briefly, a skin incision was made months were reported. parallel and lateral to the patellar ligament, and dissection An increasing body of evidence suggests that articular continued until the joint capsule and patellar ligament cartilage and subchondral bone are considered a single func- were exposed. The lateral femoropatellar ligament was tional unit that is essential for joint function.31,50 While transected and the patella reflected medially. The joint importance of subchondral bone integrity in the cause and was visually inspected for any sign of cartilage damage surgical management of osteoarthritis has been well docu- or preexisting osteoarthritis. Standardized 8-mm-diameter mented,22,40 the focus of articular cartilage repair techni- full-thickness cartilage defects extending into the calcified ques has predominantly been placed on the cartilaginous cartilage layer were created on the weightbearing surface component, with very little attention to the investigation of the medial femoral condyle using a disposable skin of subchondral bone integrity after cartilage repair. For biopsy punch and a No. 15 scalpel. The joint capsule, lat- example, both microfracture and AMIC techniques comprise eral femoropatellar ligament, fascia, subcutaneous tissue, perforation of the subchondral bone plate to facilitate hem- and skin were closed in layers. Animals were housed in orrhage and thereby the passage of mesenchymal stem cells, individual pens for 2 weeks postoperatively to restrict their platelets, fat, and growth factors from the bone marrow into movement before being turning out into small paddocks. the chondral defects. Although these techniques can induce formation of functional fibrocartilage within the defect,17,42 they may lead to the damage of the subchondral bone plate. Treatment Groups In clinical cases, an increased failure rate of autologous We evaluated 36 defects in 36 sheep at endpoints of 13 and chondrocyte implantation has been observed after marrow 36,38 26 weeks. The animals were divided into 3 treatment stimulation techniques, as the latter has been associ- groups as follows: ated with alterations in the subchondral bone.36 Considering that microfracture and AMIC compromise Control group (n = 12): Defects in the control group the subchondral bone integrity in the repair of articular received no further treatment other than the cartilage cartilage but little is known about their effect on the sub- defect. chondral bone structure, the aim of our study was to inves- Microfracture group (n = 12): Defects received 5 microfrac- tigate the effect of microfracture and AMIC on the ture perforations, introduced using a chondro-pick, subchondral bone structure in a sheep model. We hypoth- until subchondral bleeding was observed. esized that the microfracture technique would cause sub- AMIC group (n = 12): A collagen scaffold (Celgrow type I/ chondral bone pathology and degenerative changes. III porcine collagen membrane; Orthocell) was trimmed to size and implanted after the microfracture procedure, with the porous layer facing the bone sur- METHODS face and fixed using commercial fibrin glue (Tisseel; Animals Baxter Healthcare Pty Ltd). The patella was then repo- sitioned and the leg flexed 5 times. After dislocation of A total of 36 mature female sheep (Merino/Border Leices- the patella again, the integrity of the inserted implant ter), 5 to 7 years of age with a mean body weight (6SD) was confirmed by means of visual inspection. of 71.2 6 10.6 kg, were included. The study was approved by the institutional ethical committee. The sheep were Ex Vivo Analysis assessed to be healthy based on clinical examination, and all were free of lameness. The animals were euthanized after 13 (n = 18) or 26 weeks (n = 18) with an intravenous overdose of pentobarbitone Surgery and evaluations performed, as outlined in Figure 1. The left stifle was opened and evaluated for adverse events. Sheep were anesthetized by means of intravenous injection Defects were photographed, and the percentage defect of ketamine (4 mg/kg) and diazepam (0.2 mg/kg) and infill was calculated using a semi-automatic threshold-

§ Address correspondence to Ming-Hao Zheng, MBBS, DM, PhD, FRCP, ARCPA, Centre for Orthopaedic Research (M508), School of Surgery, The Uni- versity of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia (email: [email protected]). *Centre for Orthopaedic Research (M508), School of Surgery, University of Western Australia, Crawley, Western Australia, Australia. ySchool of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, Australia. zPerth Orthopaedic Institute, University of Western Australia, Nedlands, Western Australia, Australia. One or more of the authors has declared the following potential conflict of interest or source of funding: The study was funded by Orthocell. D.J.W. is a paid speaker for DePuy and receives research support from Zimmer; M.H.Z. receives shares from Orthocell. AJSM Vol. 44, No. 10, 2016 Subchondral Bone Cyst: Microfracture and AMIC 2631

Histomorphometry was performed using Bioquant Osteo Histomorphometry software (Bioquant Osteo). The region of interest was drawn manually, included the subarticular spongiosa immediately below the cartilage defects, and measured 8 3 8 mm. Subchondral bone cysts were excluded. Parameters of interest were bone volume fraction (BV/TV [percentages]), the bone surface–to–bone volume ratio as an index of structure complexity (BS/BV [in mm–1]), trabecular thickness (Tb.Th [mm]), and trabecular separation (Tb.Sp [mm]) and were calculated automati- cally after semiautomatic selection of bone. Micro-CT. The specimens of the 26-week groups were scanned in a micro-CT scanner (Skyscan 1172; Skyscan). As the changes of the subchondral bone at 13 weeks may still be at the fracture healing and modeling stage4,8,41 and may not reflect the final stage when the cartilage repair is completed, CT scanning was not performed at Figure 1. Ex vivo analysis. AMIC, autologous matrix-induced this time point. The micro-CT scanner was operated at chondrogenesis; micro-CT, micro–computed tomography; a setting of 50 kV and 800 mA, with an isotropic voxel microfx, microfracture. size of 20.7 mm. Images were reconstructed and analyzed using the manufacturer’s software NRecon and CTAna- based approach using commercial software (ImageJ). The lyzer, respectively (SkyScan). If subchondral bone cysts distal femur was removed and placed in 10% neutral buffered were present, their borders were manually contoured and formalin. Forty-eight hours later, specimens were further the cyst volume calculated. For evaluation of trabecular dissected in a standardized manner. The medial femoral con- bone microarchitecture, 2 volumes of interest (VOIs) dyle was removed at a level 10 mm below the cartilage sur- were selected based on a modified protocol described by face and trimmed to 20 3 20–mm blocks with the cartilage Orth et al41 and were contoured manually. Standardized defect central. This block was then split in a sagittal plane dimensions of 8 mm (sagittal plane) and 3.9 mm (frontal to create 2 equal-sized blocks (10 3 20 mm). The medial plane, 190 slices) were set for all VOIs. Total depth half of the 26-week group was used for micro–computed (apical-basal orientation) of the VOI did not exceed tomography (CT) scanning before the decalcification process. 8 mm. The subarticular spongiosa–defect (SAS-defect) Histology and Histomorphometry. The samples were was located strictly basally to the cartilage defect within decalcified, embedded in paraffin, and sectioned to 5-mm- trabecular bone with exclusion of the subchondral bone thick slices. Sections were stained with hematoxylin and plate. The subarticular spongiosa–medial (SAS-medial) eosin, safranin O/fast green, and toluidine blue stain and ana- was placed in an area distant from the defect, neighboring lyzed with a bright-field light microscope. Per defect, 2 sec- and medial to the defect VOI. Subchondral bone cysts were tions through different microfracture holes were evaluated excluded for the analysis of microarchitecture parameters. using a modified O’Driscoll histological scoring system as pre- Semiautomated segmentation within the VOI was done viously described.7 Repair tissue had a higher cell density using a fixed lower threshold of 89 of the maximal gray than intact cartilage; therefore, alterations incellularity value (0-255) to accurately segment bone mineralized tis- were changed into hypercellularity instead of hypocellularity sue from bone marrow. The same parameters of interest (normal cellularity, 3; slight hypercellularity, 2; moderate as for histomorphometry (BV/TV, BS/BV, Tb.Th, and hypercellularity, 1; severe hypercellularity, 0).19 Because tis- Tb.Sp) were calculated. sue consisted largely of undifferentiated mesenchyme, the The frequency of subchondral bone pathology (cyst for- parameter ‘‘chondrocyte clustering’’ within the graft was not mation, erosion of the subchondral bone plate, intrale- included in the analysis. The maximum possible score (nor- sional osteophyte formation, and upward migration of the mal cartilage) was therefore 25. Due to the absence of newly subchondral bone plate) was evaluated. formed tissue, scoring was not possible in the control groups. Qualitative analysis of the bone marrow and subchondral tra- Statistical Analysis becular bone structure was performed using criteria described by Zanetti et al.54 Bone marrow edema was defined Results are expressed as mean 6 SD. The macroscopic tis- as presence of swollen fat cells surrounded by eosinophilic sue infill data were not normally distributed, and therefore staining, marrow fibrosis as replacement of fat cells by fibrous nonparametric analysis was performed (Kruskal Wallis tissue, and marrow necrosis as formation of foam cells and test). The effect of treatment on O’Driscoll histology scores presence of swollen fat cells with loss of nuclei. Trabecular was evaluated using the Mann-Whitney U nonparametric bone structure was evaluated for pathology, consisting of test. Micro-CT data and histomorphometry data were nor- necrosis (characterized as lossofnuclei),sclerosis,and mally distributed, and an independent-samples t test was repeated remodeling (characterized by bone formation with performed. Correlations between macroscopic tissue infill, reversal lines and bone resorption with increased osteoclastic histological scores, micro-CT indices, and cyst size were activity). tested via Spearman rank correlation coefficients (r). 2632 Beck et al The American Journal of Sports Medicine

TABLE 1 Histological O’Driscoll Scores at 13 and 26 Weeksa

13 Weeks 26 Weeks O’Driscoll Score AMIC Microfx AMIC Microfx

Chondrocyte cell morphology (0-4 points) 2 (0-3) 2 (0-4) 2 (0-4) 3 (0-4) Matrix staining (0-3 points) 1 (1-1) 1 (0-2) 1 (1-2) 1 (1-2) Surface regularity (0-3 points) 2 (0-2) 0 (0-3) 2 (1-3) 2 (0-3) Structural integrity (0-2 points) 1 (0-2) 0 (0-2) 2 (0-2) 2 (0-2) Thickness (0-2 points) 0 (0-0) 0 (0-1) 0 (0-1) 0 (0-2) Bonding with adjacent cartilage (0-2 points) 0 (0-1) 0 (0-0) 1 (0-2) 1 (0-2) Normal cellularity (0-3 points) 1 (1-2) 1 (0-2) 1 (0-2) 1 (0-2) Free from degeneration in adjacent cartilage (0-3 points) 1 (1-1) 1 (1-2) 1.5 (1-2) 2 (1-2) Subchondral bone health (0-3 points) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-2) Total score (maximum 25 points) 7.5 (3-10) 6 (3-13) 11 (6-16) 12.5 (5-20)

aData are reported as median (range). AMIC, autologous matrix-induced chondrogenesis; Microfx, microfracture.

Differences were considered significant at P .05. All cal- groups nearly doubled by 26 weeks, but no significant dif- culations were performed with SPSS (version 22.0; IBM ferences existed between AMIC (68% 6 7%) and microfrac- Corp). ture (63% 6 25%) groups. Differences between both treatment groups and the control group at 26 weeks were significant (P .02). RESULTS Microscopic Evaluation Surgeries O’Driscoll Scoring. The results of the O’Driscoll scoring All surgeries were performed without complications, and only are summarized in Table 1. The median total score for the 1collagenscaffolddislodgeduponflexionofthelimbintra- AMIC and microfracture groups 13 weeks after surgery operatively. A new scaffold was subsequently implanted. All was 7.5 (range, 3-10) and 6 (range, 3-13), respectively, and animals recovered well from surgery. Twenty-one sheep was not significantly different between groups (P = .8). developed seromas of the surgical sites, which resolved spon- The median total score increased to 11 (range, 6-16) and taneously within 2 weeks postoperatively, except 1 that was 12.5 (range, 5-20) for AMIC and microfracture, respectively, drained via needle aspiration.Onesheepdevelopedaleft at 26 weeks. Differences between treatments were not sig- hindlimb lameness after surgery, which improved within 2 nificant (P = .5), but the median total scores were signifi- weeks. cantly higher at 26 weeks compared with 13 weeks (P = .01). Newly formed tissue at 13 weeks consisted predominantly of incompletely differentiated mesenchyme. At 26 Ex Vivo Analysis weeks, there was a slight improvement in the cell morphology. Differences between groups or time points were not sig- Macroscopic Evaluation nificant. The integrity of the tissue at 13 weeks was poor, No adverse reactions or evidence of inflammation and arthri- although improved by 26 weeks. The thickness of the graft tis was present within the stifle joints and the overlying soft did not reach the level of the subchondral bone plate at 13 tissues. The macroscopic infill of the cartilage defects was weeks, except for 1 sample, and rarely reached 50% of the incomplete and characterized by marginal repair tissue normal cartilage thickness at 26 weeks. As the tissue at from the perimeter of the lesion toward the center for all 13 weeks never reached the level of the subchondral bone groups and nodule-like repair tissue at the level of the micro- plate, bonding with adjacent cartilage was nonexistent. By fracture holes in both treatment groups. Repair tissue in the 26 weeks, bonding with adjacent cartilage occurred often microfracture holes at 13 weeks rarely reached above the at one end (Table 1). Most grafts were moderately hypercel- level of the subchondral bone. One defect in the AMIC group lular at 13 weeks and remained so over time (Table 1). The (26 weeks) contained minimal tissue infill, and the subchon- subchondral bone health was poor in all, except for 1 speci- dral bone was discolored with a gelatinous appearance. men, at both time points due to cyst formation and fibrosis. At 13 weeks, the average tissue infill in the AMIC group Overall, the cartilage defect was characterized by a lack (36% 6 8%) was not significantly different from that of the of sufficient infill and tissue formation at both time points. microfracture group (33% 6 5%). Both treatment groups Tissue consisted predominantly of a mixture of vascular- contained a significantly higher percentage infill compared ized dense fibrous tissue at 13 weeks and progressed to with that of the control group (20.9%; P \ .01). The amount more fibrocartilaginous tissue at 26 weeks. There was no of tissue infill in the control group increased to 33% 6 12% evidence of cartilage formation originating from the at 26 weeks. The percentage tissue infill in both treatment periphery of the defect. Representative images of the AJSM Vol. 44, No. 10, 2016 Subchondral Bone Cyst: Microfracture and AMIC 2633

Figure 2. Representative image of cartilage defect area treated with AMIC at 13 weeks (top: hematoxylin and eosin [H&E] stain; bottom: safranin O/fast green stain; scale bar = 2 mm). Newly formed tissue within the microfracture holes (middle and right holes) reaches to the level of the tidemark and is interrupted. Tissue within the left hole reaches around 50% of the cartilage thickness and is not bonded with the surrounding cartilage. A thin layer of undifferentiated tissue (asterisk) connects the repair tissue of the left and middle fracture hole. There is no safranin O stain uptake in the newly formed tissue in the 3 microfracture holes and reduced uptake in the surrounding cartilage. Inset 1: Repair tissue within the microfracture hole is characterized by a mixture of vascularized dense connective tissue. There is some bridging of repair tissue with the base of the surrounding cartilage but no integration more superficially, resulting in cleft formation (asterisk). Inset 2: Repair tissue within the microfracture hole is characterized by a mixture of vascularized dense connective tissue. There is no evidence of cartilage formation, and the surface is interrupted. Inset 3: Repair tissue within the microfracture hole is characterized by a mixture of vascularized dense connective tissue. There is no evidence of cartilage formation, and the surface is interrupted. Inset 4: Chondrocyte cluster formation and matrix flow are present at the periphery of the defect. A remnant of the collagen scaffold (arrow) is attached and integrated with the cartilage on the periphery of the defect. All insets: H&E stain, scale bar = 200 mm. AMIC, autologous matrix-induced chondrogenesis. 2634 Beck et al The American Journal of Sports Medicine

Figure 3. Representative image of cartilage defect area treated with microfracture at 26 weeks (top: safranin O/fast green stain; bottom: hematoxylin and eosin [H&E] stain; scale bar = 2 mm). Newly formed tissue remains depressed within the microfracture holes. There is more safranin O stain uptake within the repair tissue compared with tissue at 13 weeks, although differences in intensity and distribution exist within and between microfracture holes. The left microfracture hole is filled with glucosaminogly- can-rich cartilaginous tissue, although the tissue does not reach the level of the tidemark. The right hole contains a mixture of predominantly fibrous and some cartilaginous tissue. Inset 1 (safranin O/fast green stain): Repair tissue within the left microfracture hole consists of chondrocytes. Endochondral ossification is present in the deeper zone. Inset 2 (H&E stain): Repair tissue consists of predominantly fibrous tissue with debris on the surface. Inset 3: Repair tissue consists of predominantly fibrous tissue with some cartilaginous tissue in the deeper zone. A fragment of calcified cartilage is present within the debris on the surface (H&E stain). Inset 4: At the periphery of the cartilage defect, there is no sign of cartilage formation. Adhesion of the superficial cartilage layer to residual cartilage within the defect is present. All insets: scale bar = 200 mm. AJSM Vol. 44, No. 10, 2016 Subchondral Bone Cyst: Microfracture and AMIC 2635

Figure 4. Repair tissue does not reach the surface and consists of undifferentiated and fibrous tissue (hematoxylin and eosin [H&E] stain; scale bar = 3 mm). Trabecular resorption is ongoing, leading to enlargement and cyst formation of the cavity. Inset 1: Bone marrow edema is present in the subchondral bone surrounding the enlarging microfracture defect. Inset 2: Minimal endochondral ossification is observed at the edges of the repair tissue in the subchondral area. Inset 3: Repair tissue consisting of hypervascular undifferentiated tissue. Inset 4: High numbers of osteoclasts (arrows) and resorption lacunae are observed at the margins of the enlarging subchondral defect. All insets: H&E stain; scale bar = 200 mm. surface defect area of specimens treated with the micro- specimens (Figure 4). In these cases, the spaces were fracture technique are shown in Figures 2 and 3. entirely filled with graft material with minimal endochondral ossification present at the periphery (Figure 4, inset Qualitative Histological and Histopathological Observations 2). The majority of subchondral bone cysts were filled At 13 Weeks. Repair tissue within the microfracture with highly vascular dense fibrous tissue with high num- holes was highly vascular (Figure 4, inset 3), and hemor- bers of osteoclasts and resorption lacunae at the cyst rhage was present in some samples. Reparative tissue periphery (Figure 4, inset 4). Trabecular changes consisted was mainly localized in the trabecular area and rarely of mild sclerosis in the treatment groups, and there was reached the level of the subchondral bone plate (Figure minimal reversal line formation. 4). No signs of inflammation or reactive foreign-body, At 26 Weeks. Repair tissue within the microfracture giant-cell reactions due to the implantation of the collagen holes that did not communicate with subchondral bone membrane were observed in the AMIC group. Mild bone cysts and protruded above the cement line more or less marrow edema was present in the majority of the micro- had a triangular shape, with the base toward the articular fracture-treated specimens (Figure 4, inset 1) and was surface, and contained mainly fibrocartilage. Endochon- seen in half of the control specimens. Bone marrow fibrosis dral ossification was evident at the periphery of the bone was also frequently present in both treatment groups and defect. When there was communication between the micro- varied from mild to extensive. Subchondral bone cyst for- fracture holes and a subchondral bone cyst, tissue within mation was present in 3 of 6 AMIC and 2 of 6 microfracture was often depressed, did not protrude above the cement specimens (total cyst prevalence after penetration of the line, and often consisted of undifferentiated mesenchyme subchondral bone: 5/12 [42%]). Enlargement and progres- toward the base and more fibrous tissue at the surface. sion of the microfracture hole deeper in the subchondral The quantity and quality of the repair tissue over cysts bone were noted in 2 of 6 AMIC and 2 of 6 microfracture appeared similar to the tissue at 13 weeks, although 2636 Beck et al The American Journal of Sports Medicine

Figure 5. Subchondral bone cyst communicating with the microfracture hole (hematoxylin and eosin [H&E] stain; scale bar = 3 mm). The trabecular bone surrounding the cyst is sclerotic. Inset 1: Endochondral ossification is observed at the edges of the repair tissue in the subchondral area. Inset 2: High numbers of osteoclasts (arrows) and resorption lacunae are observed at the margins of the subchondral bone cyst. Inset 3: The periphery of the cysts consists of dense fibrous tissue, and high numbers of osteoclasts (arrows) are observed. Inset 4: Dense fibrous tissue within the bone cyst. All insets: H&E stain; scale bar = 200 mm.

remodeling, with frequent observation of reversal lines in more endochondral ossification was evident at the periph- all treatment samples (Figure 6, inset 1). Trabecular bone ery of the bone defect at 26 weeks (Figure 5, inset 1), and surrounding the cysts was thickened (Figures 5 and 6) repair tissue displayed less vasculature as compared with and consisted of woven bone. Some trabecular sclerosis the 13-week samples. and bone remodeling were present in the control group. Subchondral bone cysts were present in 11 of 12 sam- Osteoclastic activity was rarely observed in this group. ples (92%) (Figure 5). The periphery of these subchondral bone cystic lesions often consisted of fibrous tissue, often Histomorphometry more or less organized in bundles parallel to the surrounding trabeculae (Figure 5, inset 3, and Figure 6, inset 2). Histomorphometry results for the subarticular spongiosa High numbers of osteoclasts and resorption (Howship) lacu- immediately below the defect for condyles treated with nae were frequently present at the cyst periphery (Figure 5, AMIC and microfracture alone were not significantly differ- insets 2 and 3, and Figure 6, inset 2). Cystic lesions were ent from each other. Therefore, both groups were combined often filled with fibrous tissue (Figure 5, inset 4), and to evaluate the effect of the microfracture technique on the some contained necrotic bone fragments. Bone marrow subarticular spongiosa microarchitecture. The histomorph- edema, necrosis, and fibrosis were present in all treatment ometry results are summarized in Figure 7. groups and were moderate to extensive. Mild bone marrow BV/TV. The average BV/TV in the control groups at 13 edema, but no necrosis or fibrosis, was present in control and 26 weeks was similar (47% 6 7% and 43% 6 11%, samples. Trabecular bone abnormalities were characterized respectively) (Figure 7A). Microfracture technique increased by moderate to severe necrosis, sclerosis, and repeat bone the ratio at both time points, although this difference was AJSM Vol. 44, No. 10, 2016 Subchondral Bone Cyst: Microfracture and AMIC 2637

Figure 6. Subchondral bone cyst surrounded by sclerotic trabecular bone (hematoxylin and eosin [H&E] stain; scale bar = 2 mm). Inset 1: Reversal lines within the trabecular bone indicating bone remodeling (arrows). Inset 2: The cyst lining consists of dense fibrous tissue, and high numbers of osteoclasts (arrows) are observed. All insets: H&E stain; scale bar = 200 mm. significant only at 26 weeks (65% 6 8%; P \ .001). The BV/ Tb.Th and Tb.Sp. The average Tb.Th at 13 weeks for the TV in the microfracture-treated group was significantly microfracture-treated and control groups was 0.53 6 higher at 26 weeks compared with 13 weeks (P =.025),indi- 0.24 mm and 0.57 6 0.20 mm, respectively, and the differ- cating that the subchondral bone architecture progressively ence was not significant. For both groups, a significant became denser over time. increase was noted over time (1.0 6 0.22 mm and 0.90 6 BS/BV. The BS/BV ratio was not significantly altered 0.22 mm, respectively), but there was no significant differ- by application of the microfracture technique, as differen- ence between the microfracture-treated group and controls ces between both groups were not significantly different at at 26 weeks (Figure 7C). The average Tb.Sp at both time both time points (Figure 7B). A significant decrease in the points was significantly lower in the microfracture-treated ratio and therefore in the complexity occurred between 13 groups compared with the control groups. Interestingly, and 26 weeks in both groups. The average relative decrease a significant increase in Tb.Sp was seen in the control group was higher in the microfracture-treated group (52%) com- between 13 and 26 weeks (Figure 7D). The BV/TV in the pared with the control group (38%). control group remained relatively constant over time, 2638 Beck et al The American Journal of Sports Medicine

Figure 7. Histomorphometry. (A) Bone volume fraction (BV/TV), (B) bone surface–to–bone volume ratio (BS/BV), (C) trabecular thickness (Tb.Th), and (D) trabecular separation (Tb.Sp) at 13 and 26 weeks. Significant differences and respective P values are indicated. mfx, microfracture; AMIC, autologous matrix-induced chondrogenesis. indicating that no net bone formation or resorption occurred 10% (P = .007). The increase in BV/TV extended to a similar over time. As biomechanical forces within the epiphysis are extent into the subarticular spongiosa distant to the defect not or minimally altered by creation of a cartilage defect in the microfracture-treated defects, and there were no sig- without destroying the subchondral bone plate, Tb.Th may nificant differences between SAS-defect and SAS-medial be compensated by increased Tb.Sp. values for each group. In microfracture-treated animals, an increase in rela- BS/BV. The BS/BV ratio in the control group was 7.4 6 tive bone volume was due to Tb.Th associated with 1.2 mm–1 immediately below and 7.6 6 1.1 mm–1 distant to a decrease in Tb.Sp, likely induced by altered biomechan- the cartilage defect (Figure 9B). Microfracture treatment ical forces due to cyst formation and exacerbated over decreased the ratio within the spongiosa below the defect time. (6.2 6 1.8 mm–1), although this decrease was not significant. A significant decrease in the ratio was seen in the spongiosa Micro-CT distant to the cartilage defect (4.6 6 1.5 mm–1; P \ .001). Representative 2- and 3-dimensional images of AMIC, Similar to the BV/TV, the changes in BS/BV ratio extended microfracture, and control samples are illustrated in Fig- beyond the area below the defect, with no significant differ- ure 8. Similar to histomorphometry, micro-CT indices for ences between SAS-defect and SAS-medial. the subarticular spongiosa immediately below the defect Tb.Th and Tb.Sp. The Tb.Th within the subarticular (SAS-defect) for condyles treated with AMIC and micro- spongiosa was 0.42 6 0.05 mm below and 0.40 6 fracture alone were not significantly different from each 0.04 mm distant to the defect. Microfracture technique other, and both groups were combined to evaluate the increased the trabecular thickness significantly below effect of the microfracture technique on the subarticular (0.53 6 0.11 mm; P = .031) and distant to the defect (0.66 spongiosa microarchitecture. The results for the bone mor- 6 0.19 mm; P = .004). Conversely, application of microfrac- phometric parameters are summarized in Figure 9. ture technique resulted in a significant decrease in Tb.Sp BV/TV. The mean BV/TV of the subarticular spongiosa below and distant to the defect (Figure 9D). Changes in the control group was 55% 6 9% (Figure 9A). Microfrac- extended to a similar extent beyond the volume below ture technique significantly increased the BV/TV to 71% 6 the defect. AJSM Vol. 44, No. 10, 2016 Subchondral Bone Cyst: Microfracture and AMIC 2639

Figure 8. Representative micro–computed tomography slices of the different groups at 26 weeks. The upper row represents 2- dimensional slices and the lower row 3-dimensional reconstructions of the subchondral bone structure. There is subchondral bone cyst formation and extensive trabecular thickening around the cyst in AMIC and microfracture groups compared with the control cartilage defects. AMIC, autologous matrix-induced chondrogenesis; microfx, microfracture.

The results of the micro-CT analysis were largely in DISCUSSION agreement with the findings of the histomorphometry, and we can thus conclude that microfracture alters the subartic- The results of this preclinical study show that AMIC and ular spongiosa structure. Alterations were characterized by microfracture technique facilitate repair of a surgically cre- a significant increase in BV/TV and Tb.Th, and a significant ated articular cartilage defect more effectively than does decrease in Tb.Sp, leading to an overall less complex bone no treatment, but induced severe pathology of the subchon- structure, mainly in the spongiosa distant to the cartilage dral bone. Although microfracture resulted in better infill defect. All changes in trabecular bone architecture extended compared with no treatment, defect infill remained mini- beyond the volume below the affected cartilage surface. mal and repair tissue was of poor quality.16,24 Subchondral bone pathology was present in 100% of sheep While AMIC and microfracture repair the articular carti- receiving microfracture technique 26 weeks after surgery. lage defects by encouraging the ingrowth of the cartilaginous Eleven of 12 samples (92%) treated with microfracture tech- tissue matrix via endochondral ossification, they also can nique and none in the control group developed subchondral damage the subchondral bone structures by inducing the for- cystic lesions. The 1 sheep that did not develop a cyst devel- mation of bone cysts and other subchondral bone pathology. oped upward migration of the subchondral bone plate in the By means of histomorphometry and micro-CT assessment, defect and belonged to the microfracture group. The average we showed that subchondral bone cysts were communicating cyst volume at 26 weeks was 119 mm3 (range, 14-237 mm3), with the surgically created microfracture holes. Histopatho- and there was no significant difference between AMIC and logic characterization of bone cysts showed typical features microfracture. Erosion of the subchondral bone plate was of inflammatory and mechanically associated changes, present in 6 of 12 defects (50%) treated with microfracture including osteoclastic bone resorption, dense fibrous tissue technique (5 AMIC and 1 microfracture). Intralesional osteo- infill of cysts, and fractures through the subchondral bone phyte formation was observed in 2 sheep treated with AMIC plate. The associated subchondral bone pathology included and 1 control sheep (3/18 sheep; 17%), and upward migration an increase in the percentage bone volume, trabecular thick- of the subchondral bone was observed in 1 sheep treated with ening, and decreased complexity of the trabecular pattern microfracture and 1 control sheep (2/18 sheep; 11%). There that extended beyond the region below the cartilage defect was no correlation between cyst size, macroscopic defect infill, and subchondral bone cyst formation. histological O’Driscoll scores, and micro-CT indices. The lack There are a number of other studies that have used of correlations does not further elucidate the effect of the a similar medial femoral condyle model in sheep, but their observed subchondral bone changes on the cartilage repair focus is generally on the repair of articular cartilage. For tissue. However, the microscopic observation of similar quan- example, Dorotka et al12 evaluated the addition of a trilay- tity (depressed within microfracture hole) and quality of ered type I, II, and III collagen scaffold to microfractured repair tissue at 13 weeks and 26 weeks when the hole com- cartilage defects and concluded that this technique did municated with a subchondral bone cyst supports arrested not enhance the repair at 4 and 12 months. Breinan development of the repair tissue as a consequence of cyst for- et al4 created cartilage lesions in the trochlear grooves of mation and expansion. dogs and found an increased defect infill in AMIC using 2640 Beck et al The American Journal of Sports Medicine

Figure 9. Micro–computed tomography. (A) Bone volume fraction (BV/TV), (B) bone surface–to–bone volume ratio (BS/BV), (C) trabecular thickness (Tb.Th), and (D) trabecular separation (Tb.Sp) of the subarticular spongiosa below the cartilage defect and medial to the defect at 13 and 26 weeks. Significant differences and respective P values are indicated. mfx, microfracture; AMIC, autologous matrix-induced chondrogenesis. a type II collagen matrix compared with microfracture Marchand et al,35 however, also observed thicker trabecu- alone after 15 weeks. Tissue was predominantly fibrocarti- lae in a rabbit model when more residual drill holes were laginous with less than 2% hyaline cartilage, and the present. Trabecular thickening possibly occurs as compen- authors concluded that AMIC may be beneficial for treat- sation for the failure to repair the drill holes. Micro-archi- ment of cartilage defects.4 We also found predominantly tectural alterations in subchondral bone comparable with fibrocartilaginous tissue in this sheep model if healing that of the present study have been reported in osteoar- was not compromised by cyst formation. Tissue connecting thritic femoral heads of individuals in areas with full- with subchondral bone cysts consisted mainly of undiffer- thickness cartilage loss,6 and therefore we interpreted entiated mesenchyme and was often depressed within the our findings as a degenerative change in the subchondral microfracture hole. This indicates that cyst development spongiosa rather than weakening of the microarchitecture. compromises or at least delays cartilage repair. Conversely, Patel et al44 observed a decrease in BV/TV and Orth et al41 used a similar micro-CT approach to evalu- Tb.Th and an increase in Tb.Sp in advanced osteoarthritis ate the effect of subchondral drilling on subchondral bone in human knees compared with normal knees and stated microarchitecture in the medial femoral condyles of sheep that it is possible that differences in studies of osteoar- and showed, similar to our study, significant alterations in thritic trabecular bone may be due to the severity of bone morphometric parameters were present at the 6- arthrosis. The defects in our study were larger (50 mm2) month time point. However, alterations in morphometric compared with the defects in the study by Orth et al41 parameters induced by microfracture technique were in (32 mm2), and instead of microdrilling, we used a surgical the opposite direction, as Orth et al reported a decrease awl for penetration of the subchondral bone plate. These in BV/TV and Tb.Th and an increase in BS/BV and Tb.Sp differences could account for some variability in results. and concluded that microfracture technique only weakens Microfracture and the AMIC technique in the present the entire microarchitecture of subchondral bone. study induced a high prevalence of subchondral bone cyst AJSM Vol. 44, No. 10, 2016 Subchondral Bone Cyst: Microfracture and AMIC 2641 formation (92%). In a similar preclinical model, Orth et al41 bone alterations. Both translational data and clinical evi- observed bone cyst formation 6 months after subchondral dence support the importance of the subchondral bone in drilling in 63% of specimens, and Hoemann et al24 observed osteochondral healing,40 and we believe that future studies bone cyst formation 6 months after microfracture technique should include evaluation of repair of the entire osteochon- in 74% of specimens. Frisbie et al16 reported subchondral dral unit, not merely the articular cartilage. Similarly, bone cysts in 38% of horses treated with microfracture 12 Outerbridge classification on evaluation of articular carti- months postoperatively, as well as elevation of the subchon- lage damage via arthroscopy is not sufficient to grade the dral bone into the defect if the calcified cartilage layer was extent of osteochondral pathology.5 removed before microfracturing. Another study did not There were a number of other limitations in this study. report cyst formation but other subchondral bone pathology We opted to use skeletally mature sheep because we consisting of subchondral bone damage at 4 months and ele- believed that they would be more representative of the vation of the subchondral bone into the defect in all speci- patient population in need of treatment. However, mature mens at 12 months in sheep.12 Taken together, it is clear age has been associated with a reduced osteogenic poten- that microfracture and the AMIC technique can induce sub- tial of stem cells,11,34 which could have contributed to the chondral bone pathology. The mechanism of development of observed bone pathology/bone cyst formation. bone cysts after microfracture and AMIC technique is We opted to debride the cartilage defects extending into unclear but might be due to a combination of increased the calcified layer as this could be achieved most consistently synovial fluid pressure and cytokine-induced osteoclast- and without inadvertently abrading the subchondral bone mediated bone resorption. Landells29 hypothesized that plate. Removing the calcified cartilage layer would expose subchondral bone cysts arose from intrusion of synovial blood vessels at the cement line,33 which could influence fluid into the bone at the joint surface by demonstrating the healing within the defect. The contribution and role of communication between subchondral bone cysts and the the calcified layer in cartilage healing and articular cartilage articular surface via fissures or fractured trabeculae in oste- repair techniques are unclear, and it remains debatable oarthritic hip joints. This is then followed by a rim of scle- whether this layer should be removed or not. Breinan et al4 rotic tissue due to displacement of trabeculae and new reported more reparative tissue with exposure of the sub- bone formation in response to strain.29 Recently, Cox chondral bone, although of inferior quality compared with et al10 demonstrated via a computational model that fluid the more hyaline tissue formed over an intact calcified layer, pressure resulted in an irregularly shaped cavity, which and stated that an intact calcified layer is likely a critical fac- became rounded and obtained a sclerotic bone rim after tor. Similarly, in a horse model, Frisbie et al16 showed that removal of the pressurized fluid. Li et al30 also reported removal of this layer increased the overall repair tissue; how- more sclerosis of the trabecular bone surrounding bone cysts ever, this did not result in improved histologic character, in osteoarthritic hip specimens compared with the subchon- matrix proteins, or mRNA expression. In addition, removal dral bone distant to cysts. Although not evaluated objec- of this layer resulted in an increase in the level of the sub- tively, the morphology of the cysts in the present study chondral bone into the defect space, an observation recog- was irregular, supporting the role of pressured fluid in the nized in early osteoarthritis.32 Despite evidence of this development of these cysts. We observed abnormally higher degenerative change, the authors recommended removal of numbers of osteoclasts and Howship lacunae at the periph- the calcified cartilage layer for debridement of clinical ery of the cystic lesions in our study. This osteoclastic bone lesions.16 More recently, Hoemann et al25 reported that com- resorption is suspected to be activated via the migration of plete removal of the calcified layer in a rabbit trochlear model cytokines including interleukin (IL)–1, IL-6, tumor necrosis resulted in severe subchondral bone resorption and fibrous factor alpha, and RANKL from the synovial fluid into the repair, whereas debridement into the calcified layer allowed subchondral bone.2,23,51,52 for hyaline repair tissue formation. Similar to our study, Surgical approach via an arthrotomy, as well as the cre- newly formed tissue did not integrate well with the defect ation of an osteochondral defect and trauma to the sub- base when calcified cartilage was present.25 chondral bone, could result in inflammation with Animals were allowed immediate postoperative weight- upregulation of pro-inflammatory cytokines. Cytokines in bearing, but exercise was restricted by individual housing the joint fluid or within the cystic lesions were not mea- for 2 weeks. Although implant integrity was confirmed sured in our study but would have been useful to further intraoperatively, retention in the postoperative period support this theory. could not be confirmed. It is widely accepted that mechanical Despite the striking similarities between bone cyst for- stimulation is necessary for cartilage repair. Rodrigo et al45 mation seen in advanced osteoarthritis and that seen after advocated continuous passive motion after microfracture microfracture technique in preclinical models, the clinical technique. Palmoski et al,43 however, showed that joint load- significance of their presence in these animal models has ing, not motion, was important to the biological and func- been questioned.16,24 Their presence is often considered tional properties of normal articular cartilage. Recently, it to be secondary to the immediate weightbearing of the ani- has been recognized that dynamic compression and shear mal after surgery. Studies that consider them ‘‘incidental’’ are necessary to induce chondrogenesis of mesenchymal typically evaluate the quality and composition of the newly stem cells.39 It is clear that some loading is required postoper- formed cartilage and find significant improvement with atively, and it seems intuitive that excessive loading would be the ‘‘new’’ treatment. It is therefore plausible that these detrimental, but it remains unclear what loading is consid- studies underestimate the influence of the subchondral ered ‘‘excessive.’’ It is possible that the inability to fully control 2642 Beck et al The American Journal of Sports Medicine postoperative loading and locomotion affected the severity of REFERENCES subchondral bone changes and, thus, that a more controlled environment may have reduced this severity. Interestingly, 1. 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