Bone Marrow Stimulation of the Medial Femoral Produces Inferior and Repair Compared to the Trochlea in a Rabbit Surgical Model

Hongmei Chen,1 Anik Chevrier,1 Caroline D. Hoemann,1 Jun Sun,2 Genevieve Picard,1 Michael D. Buschmann1

1Department of Chemical Engineering and Institute of Biomedical Engineering, E´cole Polytechnique de Montre´al, PO Box 6079, Station Centre- ville, Montre´al, Quebec, Canada H3C 3A7, 2Piramal Healthcare (Canada), BioSyntech Canada Inc., Laval, Quebec, Canada Received 15 September 2012; accepted 6 June 2013 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22422

ABSTRACT: The influence of the location of cartilage lesions on cartilage repair outcome is incompletely understood. This study compared cartilage and bone repair in medial femoral condylar (MFC) versus femoral trochlear (TR) defects 3 months after stimulation in mature rabbits. Intact femurs from adult rabbits served as controls. Results from quantitative histomorphom- etry and histological scoring showed that bone marrow stimulation produced inferior soft tissue repair in MFC versus TR defects, as indicated by significantly lower % Fill (p ¼ 0.03), a significant increase in collagen type I immunostaining (p < 0.00001) and lower O’Driscoll scores (p < 0.05). 3D micro-CT analysis showed that repaired TR defects regained normal un-operated values of bone volume fraction, trabecular thickness, and trabecular number, whereas in MFC defects the repaired bone architecture appeared immature and less dense compared to intact un-operated MFC controls (p < 0.0001). Severe medial meniscal damage was found in 28% of operated animals and was strongly correlated with (i) low cartilage defect fill, (ii) incomplete bone repair in MFC, and (iii) with a more posterior defect placement in the weight-bearing region. We conclude that the location of cartilage lesions influences cartilage repair, with better outcome in TR versus MFC defects in rabbits. Meniscal degeneration is associated with cartilage damage. ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Keywords: cartilage repair; medial femoral condyle; trochlea; bone marrow stimulation; meniscus degeneration

Articular cartilage lesions are a prevalent pathology more fill and better tissue integration but with a more found in about 65% of knee arthroscopies.1–3 The fibrous character was reported in TR versus MFC medial femoral condyle (MFC) is the most commonly defects.15 Less hyaline repair in TR versus MFC defects affected area accounting for 34–58% of chondral inju- was also found in sheep after ACI.14 Thus studies to ries and is the predominant location for severe grade- date suggest that the location of cartilage defects can IV lesions exposing subchondral bone.1,3 The trochlea exert an important influence on outcome, however no (TR) as primary lesion site occurs less frequently, in clear trend of specific locations which give rise consis- 6–8% of cases,2 and is usually associated with other tently to better or worse repair have been identified. patellofemoral pathologies.4 Previous cartilage repair Moreover, the underlying factors which create such clinical research and animal studies have indicated location dependency, for example, bone structure, the some influence of lesion location on repair outcome,5–7 number/characteristics of bone-resident progenitors, or yet there are discrepancies in literature: Steadman8 load bearing conditions, are also unknown. reported good results in all compartments with chon- The purpose of this study was to compare cartilage dral lesions after microfracture (MFX) and no associa- and bone repair in MFC versus TR defects after bone tion between location of lesion and functional marrow stimulation in adult rabbits at 3 months post- improvement. A prospective cohort study using MFX operative. We also sought to determine whether differ- for Outerbridge grade-III or -IV lesions of MFC, lateral ent bone marrow stimulation techniques would have femoral condyle (LFC) or TR did not reveal an effect of differential effects on defect repair in MFC. Finally defect location on the outcome scores.9 In other the association of meniscus degeneration with carti- studies, MFX was reported more successful in treating lage repair was also investigated. condylar versus patellofemoral lesions,10,11 producing significantly greater KOOS improvement in MFC MATERIALS AND METHODS compared to LFC defects at 3-years follow-up.12 Carti- Study Design and Rabbit Surgical Model of Bone Marrow lage repair studies have been performed in animal Stimulation models with defects in different anatomical loca- The research protocol was reviewed and approved by an tions.13–15 At 6-months of repair after MFX in sheep, institutional ethics committee for animal research. Sixteen skeletally mature (9- to 10-month-old) female New Zealand White rabbits underwent sequential bilateral arthrotomies Grant sponsor: Canadian Institutes of Health Research; with a medial parapatellar incision. Full thickness cartilage Grant sponsor: Natural Sciences and Engineering Research defects were created in each knee in the central trochlear Council of Canada; Grant sponsor: Biomedical Science and groove (TR) by manual curettage and by burring in the MFC Technology Research Group/Groupe de recherche en sciences et due to higher subchondral . The average dimen- technologies biome´dicales; Grant sponsor: Piramal Healthcare Canada. sion of TR and MFC defects were 4.0 mm 4.5 mm and Correspondence to: Michael D. Buschmann (T: 514-340-4711 ext. 3.7 mm 3.9 mm, respectively, based on the available 4931; F: 514-340-2980; E-mail: [email protected]) surface. Since the degree of flexion of the rabbit knee was # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. not fixed at surgery, the region of exposed condyle and the

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2013 1 2 CHEN ET AL. location of MFC defects varied along the anterior–posterior (ROI) defined in Figure 1. Subchondral bone morphometric axis of the condyle and thus were quantified at sacrifice. The indices, including bone volume fraction (% BV/TV), bone defects were completely debrided of the articular and calci- surface density (BS/TV), trabecular thickness (Tb.Th), tra- fied cartilage to expose subchondral bone with visible punc- becular number (Tb.N), and connectivity density (Conn.Dn), tuate bleeding. Four subchondral perforations were made on were quantified using a global thresholding procedure in each TR defect with customized surgical tools described CTAn software (version 1.9.3.0, Skyscan, Kontich, Belgium). previously.16 Two perforations were made on each MFC defect since it was too small for four holes. This hole Histoprocessing, Histology, Immunohistochemistry and placement resulted in 9–17% of the surface area in TR or Histomorphometry MFC defects being perforated. Animals were randomly Fixed samples underwent HCl decalcification and OCT assigned into Group 1 (N ¼ 8) with drill holes of 6 mm deep embedding. Transverse sections were collected systematically (DRL6/G1) in left knees and of 2 mm deep (DRL2/G1) in from three distinct levels at the locations of the original right knees, or Group 2 (N ¼ 8) being drilled (DRL2/G2) in distal and proximal holes (approximated by pictures taken at left knees and microfractured (MFX2/G2) in right knees, surgery), and midway between the holes in all defects, and both to 2 mm depth. Constant irrigation with cooled sterile stained with Safranin-O (Saf-O)/Fast Green, collagen type I Ringer lactate solution was applied.16 Animals were allowed and collagen type II. O’Driscoll histological scoring19 was immediate ambulation in cages after recovery from anesthe- carried out on blinded Saf-O stained sections using a sia, and were sacrificed at 3 months post-operation, a time modified scoring system,20 and the results verified by a point when end stage soft tissue repair in TR defects was blinded second reader. The percentage of the projected defect observed in a previous study.17 Additional four adult rabbits volume that was filled with repair tissue (% Fill) and the (12 months old) receiving no surgical intervention served as percentage of all nonmineralized repair tissue in defects intact controls. staining positively for Saf-O (% Saf-O), collagen type II (% Col2), and collagen type I (% Col1) were quantified (Northern Gross Examination, Micro-CT Imaging and Analysis Eclipse Version 8.0) as previously described.17 Intact (un- At necropsy, were evaluated by two observers and operated) specimens (N ¼ 8 knees) were also processed to photo-documented. Collected femur ends were fixed and obtain the thickness of articular cartilage, the calcified micro-CT scanned (Skyscan x-ray microtomography 1172, cartilage layer and the subchondral bone plate from digitized Kontich, Belgium, at a 10 mm/pixel resolution). The micro- images of Saf-O stained sections. The analysis was performed CT image stacks from MFC samples were reconstructed and using NDP view software (Version 1.2.25, Hamamatsu repositioned as previously described for TR.18 3D micro-CT Photonics, Hamamatsu City, Japan) with five measurements analysis was carried out by applying a region of interest per section and three sections per specimen.

Figure 1. 3D region of interest (ROI) used in quantitative micro-CT analysis, defined as 3 mm (W) 3 mm (L) 2 mm (H) with its top surface coinciding with the bone surface in intact controls (A and B), and the projected bone surface in defects (C and D). Arrows indicate peripheral osteophyte formation.

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Scoring of Meniscus Degeneration and Osteophytes the knee. The higher the score, the more anterior was the The severity of medial meniscal damage was graded initially created defect on the MFC. (Fig. 2A–F). A score of five represented a healthy meniscus, whereas a score of 0 indicates a severely degenerated, Statistical Analysis fragmented meniscus. Peripheral osteophyte formation were Statistical analyses were performed with Statistica (version encoded as 0 if present at both the medial and lateral sides 10.0, Statsoft, Inc., Tulsa, OK). Numerical data were pre- of the joints, 1 if present at the medial side only, and 2 if no sented as mean standard deviation. The effect of defect osteophyte was observed. The placement of MFC cartilage location (TR vs. MFC) was analyzed using the General defects was characterized by two investigators on photo- Linear Model (GLM) with location and animal taken as graphs taken at necropsy, and rated as 4, anterior (e.g., predictors. The effects of treatment (DRL6 vs. DRL2 and Fig. 2G); 3, mid-anterior; 2, mid; 1, mid-posterior; and 0, DRL2 vs. MFX2) on soft tissue repair and subchondral bone posterior (e.g., Fig. 2H), according to the ICRS grid map of repair in MFC defects were evaluated by GLM with treat-

Figure 2. Representative photographs showing levels (0–5) of damaged medial menisci at 3 months after bone marrow stimulation on cartilage defects in the MFC (A–F). Arrows show medial menisci that were normal (score 5 in A), intact but slightly rough surface (score 4 in B), intact with fibrillated and rough surface (score 3 in C), split (score 2 in D), largely damaged (score 1 in E), and completely fragmented (score 0 in F). G and H are photographs taken at necropsy to show different anterior versus posterior placement of the defect on MFC.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2013 4 CHEN ET AL. ment and animal taken as predictors. Histomorphometric parameters of % Fill, % Saf-O, % Col2, and % Col1 were analyzed together as an aggregate indicator of overall quantity and quality of repair cartilage17 by GLM with two repeated-measure variables—the histomorphometric param- eter and the section level (through distal holes, through proximal holes and between holes). The subchondral bone morphometric data from treated defects were compared to those from intact controls using one-way ANOVA. O’Driscoll histological scores in TR and MFC defects were compared with the nonparametric Wilcoxon matched pairs test, as was the effect of bone marrow stimulation techniques on menis- cus scores. Correlations between meniscal integrity with defect repair features including % Fill, O’Driscoll sum score, and % BV/TV, the presence of osteophytes, and the anterior versus posterior placement of MFC defects were analyzed by calculating the Pearson correlation coefficients (r). p values <0.05 were considered statistically significant.

RESULTS Intact TR and MFC From Un-Operated Rabbits Had Distinct Articular Cartilage and Subchondral Bone Structure We analyzed TR and MFC from intact skeletally mature rabbit knees by histomorphometry and micro- CT, and found structural differences (Fig. 3). The articular cartilage layer was two-fold thicker in MFC than in TR (252 58 mm vs. 129 33 mm), as was the calcified cartilage layer (120 34 mm vs. 67 30 mm). The subchondral bone plate was also thicker in MFC versus TR (383 157 mm vs. 259 161 mm. Fig. 3G). The epiphyseal line in rabbit TR was about 3–4 mm from the articular surface such that our 6 mm deep drill holes penetrated through it and reached the metaphyseal bone marrow (Fig. 3D) 16; in contrast, it was inaccessible by deep perforation in the MFC (Fig. 3A). Quantitative micro- CT analysis on the 3D ROIs (Fig. 1A and B) showed significant differences between intact TR and MFC. The subchondral bone density was higher and the trabeculae were thicker, less numerous, and less Figure 3. Structure of MFC (A–C) and TR (D–F) in intact connected in MFC versus TR (p < 0.05, Fig. 4). rabbits, and thickness of articular cartilage (AC), calcified cartilage (CC) and subchondral bone plate (BP) in MFC and TR (G). Arrows show the epiphysial line in TR, below which the Bone Marrow Stimulation Produced Inferior Cartilage metaphyseal bone marrow () is accessible by 6 mm deep Repair in MFC Versus TR Defects perforations (indicated by the red dash lines). At 3 months post-operative, MFC defects showed lower quality repair compared to TR (Figs. 5 and 6). Six out of 32 MFC defects had depressed repair tissue with very little fill (e.g., Figs. 5E, 6I and 6K) while no TR and % Col1 in Fig. 7) were analyzed together as defects were depressed. Quantitative histomorphome- repeated-measure variables for an aggregate indicator try revealed a significant decrease in % Fill in the of overall repair quantity and quality. The O’Driscoll chondral zone of MFC versus TR defects (p ¼ 0.031, sum score was significantly lower in MFC versus TR Fig. 7), consistent with lower O’Driscoll scores in MFC defects (p ¼ 0.004), as were the subcategories of Saf-O for repair tissue thickness (p ¼ 0.026, data not shown). staining of the matrix, absence of hypocellularity and Both MFC and TR defects were equally positive for of clustering, as well as adjacent articular Col2 staining in the repair tissue matrix (% Col2 cartilage health (p < 0.05, data not shown). The differ- 80%, Fig. 7). However, Col1 was more widespread in ent surgical treatments (DRL6 vs. DRL2 and DRL2 vs. MFC versus TR defects (% Col1: 51.9% vs. 13.4%, MFX2) did not produce different cartilage repair out- p < 0.00001) (Fig. 7). Improvement in tissue repair comes in the MFC (data not shown) unlike the quality in TR versus MFC was significant (p ¼ 0.002) improvement seen with deeper drilling (6 mm vs. when the four parameters (% Fill, % Saf-O, % Col2, 2 mm) published previously for TR.17

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Figure 4. Subchondral bone structure by 3D micro-CT in the ROIs of Figure 1. Significant differences were observed comparing all five subchondral bone parameters (A–E) in MFC defects to intact MFC (p < 0.00008, one-way ANOVA). In contrast, TR repair bone had % BV/TV and average trabecular thickness (Tb.Th) similar to those from intact TR, but higher BS/TV and connectivity density (Conn.Dn) (p < 0.006). Significant differences (p < 0.05) were found in all bone morphometric indices comparing intact TR to intact MFC, except for % BV/TV. †p < 0.05 comparing intact TR to intact MFC controls.

Figure 5. Representative Safranin-O/Fast Green staining of cartilage defects from Group 1 animals, treated with 6 mm deep drill (DRL6/G1, left panel) in left knees or 2 mm shallow drill (DRL2/G2, right panel) in right knees, showing the best (A–D), median (E–H), and worst (I–L) cases of 3 months tissue repair in MFC (A, E, I, C, G, & K) and TR (B, F, J, D, H, & L) according to the sum O’Driscoll score. Bar ¼ 1 mm.

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Figure 6. Representative Safranin-O/Fast Green staining of cartilage defects from Group 2 animals, treated with drilling (DRL2/G2, left panel) in left knees or microfracture (MFX2/G2, right panel) in right knees, both to 2 mm depth, showing the best (A–D), median (E–H), and worst (I–L) cases of 3 months tissue repair in MFC (A, E, I, C, G, & K) and TR (B, F, J, D, H, & L) according to the sum O’Driscoll score. Note that MFC defects shown in I and K had no or very little soft repair tissue. Bar ¼ 1 mm.

Bone Marrow Stimulation Led to Worse Subchondral Bone significantly lower bone volume fraction than that of Repair in MFC Versus TR Defects intact MFC controls (45.8% vs. 63.4%, p < 0.00004. The repair of subchondral bone was still on-going at Fig. 4A). Additional bone morphometric features in 3 months post-operative, and bone structure under- MFC defects were significantly different from those in neath the defects displayed clear differences compared intact MFC, with thinner but more numerous trabecu- to intact controls. Quantitative micro-CT analysis lae, and with greater bone surface density and connec- revealed that subchondral bone in MFC defects had a tivity density (Fig. 4B–E). In contrast, the subchondral bone volume density was restored in TR defects, with similar trabecular thickness and trabecular number values as in intact TR (p > 0.2), although the repaired bone still had a higher level of remodeling and connec- tivity compared to the native TR (Fig. 4B and E).

Medial Meniscus Degeneration Was Correlated to a More Posterior Placement of Cartilage Defects and to Reduced Quality of Repair in the MFC Damage to the medial meniscus was found in 53% of all operated knees at necropsy while the lateral meniscus, patella and tibial plateau were macroscop- ically normal. Most meniscal damage was minor (roughened surface, score 3) with meniscal integrity upheld in most knees (e.g., Fig. 2A–C). But 9 out of 32 Figure 7. Histomorphometric analyses of soft tissue repair (28%) menisci showed severe degeneration (Fig. 2D–F) 3 months following bone marrow stimulation in a rabbit model. and four were completely fragmented. No association % Fill refers to percent projected defect volume filled with repair tissue; % Saf-O, % Col2, and % Col1 refers to percent all was detected between meniscus damage and the nonmineralized repair tissue in defects stained positively. Mar- specific bone marrow stimulation technique (DRL6, row stimulation produced more fill (p ¼ 0.03) and lower type I collagen content (p < 0.00001) in TR versus MFC defects. DRL2, or MFX2). However, meniscus damage was Improvement in tissue repair in TR versus MFC was significant significantly correlated with lower % Fill, lower (p ¼ 0.002) when four parameters (% Fill, % Saf-O, % Col2, and % Col1) were analyzed together for an aggregate indicator of O’Driscoll sum score, and lower % BV/TV in MFC overall repair quantity and quality. defects (Table 1). Peripheral osteophyte formation

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Table 1. Pearson Correlation Coefficients (r) of Meniscal Integrity With Repair Features and Anterior Placement of MFC Defects, and Peripheral Osteophyte Formation

Sum O’Driscoll Anterior MFC Absence of % Fill Score % BV/TV Defect Placement Osteophytes r (p-value) 0.504 (0.003) 0.587 (<0.001) 0.403 (0.025) 0.650 (<0.001) 0.548 (0.001)

(Fig. 1C and D) was also noted in 81% of the operated drilling in TR may potentially recruit a greater knees, either at the medial site only (58%), or at both number of cells and a variety of cell types from the medial and lateral sides (42%), the latter being deep marrow stroma, resulting in improved cartilage significantly associated with meniscus damage (Ta- repair.17 Nonetheless, the superiority of TR versus ble 1). We quantified that 40.6% of the MFC defects MFC repair was not solely due to access to deep were created more anterior, 37.5% more posterior, and marrow stroma, since significant effects of defect 22% in the mid region of the condyle along the location were always detected on aggregate histo- anterior–posterior axis. We found that a more posteri- morphometry features, % Col1 and % BV/TV even or initial placement of the MFC defect was strongly when all eight deep drill samples were excluded from and significantly correlated with meniscal degenera- the statistical analysis. tion (Table 1). Improved repair in TR versus MFC may also be due to a greater chondrogenic potential of progenitor cells DISCUSSION in subchondral bone for TR versus MFC, and is This study compared cartilage and bone repair in consistent with our observations in a related study of MFC versus TR defects after bone marrow stimulation greater occurring in TR versus MFC and found inferior repair in MFC at 3 months post- defects at 3 weeks post-operative in mature rabbits.25 operative. We found less fill in MFC versus TR defects, Additionally, there may be other features that poten- consistent with the previous finding in sheep after tially affect the repair in MFC versus TR, including MFX15; yet in contrast to this sheep model, we found a subchondral vascularization,21 remodeling,20,26 local more fibrous character of the repair tissue in MFC in inflammation, debridement of a curved condylar rabbits. The location dependency seen in different versus concave trochlear surface, and biomechanical species or between human and animals suggested that factors including damaged menisci. some underlying factors such as bone structure, the We found that meniscus degeneration seen in some number or characteristics of bone-resident progenitors, rabbits after marrow stimulation (Fig. 2) was correlat- and load bearing conditions may be responsible for ed to poor cartilage repair and to posterior placement location dependent outcomes. Unlike the convex MFC, of the MFC defects (Table 1). To our knowledge, TR could provide a shielding effect for the fibrin blood meniscus degeneration has not been reported in an clot and initial granulation repair tissue in the animal model for cartilage repair, although medial debrided lesion, which has been shown to be a central meniscus tears are known as one of the most common facet to subsequent defect repair.15,21 Since the rela- pathologies concomitant with articular lesions in tive compressive versus shear loading influences the humans.1–3 The defects placed more posteriorally in nature of the repair, the articular conformity of MFC directly oppose the medical meniscus, possibly tibiofemoral and patellofemoral compartment affects causing meniscus damage by contact force with the contact mechanics,22 and the MFC is weight-bearing defect. In this study, we used a rather aggressive while TR is partly weight-bearing in rabbits,23 the model, with defects created bilaterally and concurrent- observed degenerative changes including loss of gly- ly in TR and MFC (four defects per animal), and the cosaminoglycan, hypocellularity and cell clustering at defects occupied a large portion of the surface area in the rims of MFC defects and fibrillation in the repair rabbit knees, about 65% width and 25% surface area, matrix may be related to unrestricted ambulation potentially promoting osteophyte formation at joint immediate post-operative and altered joint biomechan- margins, especially at the medial site of joint loading ics. (Fig. 1C and D). We previously reported that deeper drilling im- In conclusion, our study revealed that cartilage proved repair compared to shallower drilling in TR repair by bone marrow stimulation depends on the defects17,18; this effect, however, was not seen here for location of the cartilage defect whether placed on TR MFC defects. This could be related to inherent struc- versus MFC and more specifically anterior versus tural differences in TR versus MFC where our 6 mm posterior on the MFC. The underlying factors involved deep drill holes reached the metaphyseal bone marrow in this differential repair outcome that require further in TR,16 but not in MFC (Fig. 3A and D). It was study include subchondral bone structure, chondro- previously suggested that different cell types may genic potential of trabecular bone-resident precursors, reside in specific regions of the marrow,24 and deep as well as geometric and biomechanical factors.

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