Molecular Characterization of Articular Cartilage from Young Adults with Femoroacetabular Impingement

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1 1457 COPYRIGHT Ó 2013 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED Molecular Characterization of Articular Cartilage from Young Aults with Femoroacetabular Impingement Shingo Hashimoto, MD, PhD, Muhamma Farooq Rai, PhD, Corey S. Gill, MD, Zhiqi Zhang, MD, Lina J. Sanell, PhD, an John C. Clohisy, MD Investigation performe at Washington University School of Meicine at Barnes-Jewish Hospital, St. Louis, Missouri Backgroun: Femoroacetabular impingement is a frequent cause of hip pain an may lea to seconary osteoarthritis, yet little is known about the molecular events linking mechanical hip impingement an articular cartilage egeneration. The first goal of this stuy was to quantify the expression of inflammatory cytokine an chemokine, matrix-egraing, an extracellular matrix genes in articular cartilage harveste from control hips an hips with femoroacetabular impingement an en-stage osteoarthritis. The secon goal was to analyze the relative expression of these genes in articular cartilage harveste at various stages of osteoarthritis. Methos: Cartilage samples were obtaine from thirty-two hips unergoing hip preservation surgery for femoroacetabular impingement or hip arthroplasty. Three control cartilage samples were also analyze. Specimens were grae intraoperatively with regar to the severity of cartilage amage, the raiographic osteoarthritis grae was recore, an quantitative RT-PCR (real-time polymerase chain reaction) was performe to etermine relative gene expression. Results: Except for interleukin-1b (IL-1b) an CXCL2, the mrna (messenger RNA) expression of all other chemokine (IL-8, CXCL1, CXCL3, CXCL6, CCL3, an CCL3L1), matrix-egraing (matrix metalloproteinase [MMP]-13 an ADAMTS-4), an structural matrix (COL2A1 [collagen, type II, alpha] an ACAN [aggregan]) genes was higher overall in cartilage from hips with femoroacetabular impingement compare with hips with osteoarthritis an normal controls. The ifferences reache significance (p 0.05) for seven of these ten quantifie genes, with CXCL3, CXCL6, an COL2A1 being elevate in the femoroacetabular impingement group compare with only the control group an IL-8, CCL3L1, ADAMTS-4, an ACAN being elevate compare with both the osteoarthritis an control groups. When samples were groupe accoring to the stage of the egenerative cascae, mrna expression was relatively higher in one of the two mile stages of femoroacetabular impingement (chonromalacia or cleavage/thinning), with the ifference reaching significance for IL-8, CXCL2, CXCL3, CCL3L1, an ACAN. ACAN expression was iminishe in hips with osteoarthritis compare with femoroacetabular impingement but elevate compare with the control articular cartilage. Conclusions: Articular cartilage from the impingement zone of hips with femoroacetabular impingement (an particularly those hips in the cleavage/thinning stage) expresse higher levels of certain inflammatory, anabolic, an catabolic genes, representing a heightene metabolic state. Clinical Relevance: The articular cartilage from the impingement zone of hips with femoroacetabular impingement was metabolically hyperactive, supporting the concept that such impingement is a structural precursor to hip osteoarthritis. Disclosure: One or more of the authors receive payments or services, either irectly or inirectly (i.e., via his or her institution), from a thir party in support of an aspect of this work. In aition, one or more of the authors, or his or her institution, has ha a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomeical arena that coul be perceive to influence or have the potential to influence what is written in this work. No author has ha any other relationships, or has engage in any other activities, that coul be perceive to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitte by authors are always provie with the online version of the article. A commentary by Darryl D. D Lima, MD, PhD, is linke to the online version of this article at jbjs.org. J Bone Joint Surg Am. 2013;95:

2 1458 Recent avances in unerstaning structural hip isease have bolstere the theory of seconary osteoarthritis 1,2.In this moel of hip osteoarthritis pathophysiology, joint egeneration is seconary to an abnormal mechanical environment commonly cause by femoroacetabular impingement. This impingement results from a istinct morphologic abnormality of the acetabular rim (pincer eformity) an/or the femoral heaneck junction (cam eformity). These structural malformations prouce ynamic, repetitive abutment between the proximal aspect of the femur an the acetabular rim leaing to labrochonral issociation, articular cartilage etachment, an progressive joint egeneration 3-5. In this moel of seconary hip osteoarthritis, unerlying structural abnormalities of the acetabulum an proximal aspect of the femur causing femoroacetabular impingement meiate progressive osteoarthritis an eventually hip joint failure 1,4. Despite increasing knowlege regaring femoroacetabular impingement an seconary osteoarthritis, this theory remains controversial an the role of femoroacetabular impingement in the pathophysiology of osteoarthritis continues to be questione 6. Our unerstaning of the biology of osteoarthritis has expane markely in recent years. Current information inicates that articular cartilage egeneration, characteristic of osteoarthritis, is meiate by several istinct molecular meiators such as cytokines, chemokines, an metalloproteinases 7 that are release by intra-articular an periarticular soft tissues (Table I). Cytokines are cell-signaling proteins prouce by chonrocytes an synovial cells within the hip joint that act to stimulate inflammation an regulate extracellular matrix homeostasis 7. Interleukin-1b (IL-1b) is a cytokine that strongly increases inflammation an is thought to play a pivotal role in both early an late-stage osteoarthritis 8. IL-1b causes articular cartilage estruction in part by stimulating prouction of MMP (matrix metalloproteinase)-13 an ADAMTS (a isintegrin an metalloproteinase with thrombosponin motifs)-4, the enzymes responsible for cleavage of the major structural proteins in the cartilage matrix (type-ii collagen an aggrecan, respectively) Aitionally, more recent investigations have implicate certain chemoattractive cytokines (chemokines) as potential meiators of articular cartilage egeneration s represent a large family of structurally relate inflammatory an immune system meiators that may have important roles in normal articular cartilage physiology an TABLE I Caniate Genes Important in the Potential Biological Link Between Femoroacetabular Impingement an Seconary Osteoarthritis NCBI* Accession No. Gene Name Symbol Alias Forwar Primer Reverse Primer Metabolic Role NM_ Interleukin-1 beta IL-1b (59-TCCAGGAGAATGACCTGAGC-39) (59-GTGATCGTACAGGTGCATCG-39) Inflammation NM_ Interleukin-8 IL-8 (59-GAAGGTGCAGTTTTGCCAAG-39) (59-TGTGGTCCACTCTCAATCCTC-39) Inflammation NM_ (C-X-C) motif ligan 1 CXCL1 GRO-a (59-GGGAATTCACCCCAAGAAC-39) (59-GATGCAGGATTGAGGCAAG-39) Inflammation NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ (C-X-C) motif ligan 2 (C-X-C) motif ligan 3 (C-X-C) motif ligan 6 (C-C) motif ligan 3 (C-C) motif ligan 3-like 1 Matrix metalloproteinase 13 A isintegrin an metalloproteinase with thrombosponin motifs 4 Collagen, type II, alpha CXCL2 GRO-b (59-TGCAGGGAATTCACCTCAAG-39) (59TCTTAACCATGGGCGATGC-39) Inflammation CXCL3 GRO-g (59-ACCGAAGTCATAGCCACACTC-39) (59-GGTGCTCCCCTTGTTCAGTA-39) Inflammation CXCL6 GCP-2 (59GTTTACGCGTTACGCTGAGAG-39) (59-ACTTCCACCTTGGAGCACTG-39) Inflammation CCL3 MIP-1a (59-GCAACCAGTTCTCTGCATCA-39) (59-TGGCTGCTCGTCTCAAAGTA-39) Inflammation CCL3L1 LD78b (59-GTCCTCTCTGCACCACTTGC-39) (59-GGAAGATGACACTGGGCTTG-39) Inflammation MMP-13 Collagenase 3 (59-TGGTCCAGGAGATGAAGACC-39) (59-TCCTCGGAGACTGGTAATGG-39) Degraation (catabolism) ADAMTS-4 (59-GGCTAAAGCGCTACCTGCTA-39) (59-GAGTCACCACCAAGCTGACA-39) Degraation (catabolism) COL2A1 (59-CCCAGAGGTGACAAAGGAGA-39) (59-CACCTTGGTCTCCAGAAGGA-39) Synthesis (anabolism) NM_ Aggrecan ACAN (59-GGCACTAGTCAACCCTTTGG-39) (59-CTGAACCCTGGTAACCCTGA-39) Synthesis (anabolism) NM_ Glyceralehye- 3-phosphate ehyrogenase GAPDH G3PDH (59-ACCCAGAAGACTGTGGATGG-39) (59-GAGGCAGGGATGATGTTCTG-39) Housekeeping *NCBI = National Center for Biotechnology Information.

3 1459 TABLE II Source of Cartilage Samples Control FAI* OA* Hips (no.) Mean age (range) (yr) 28 (15 to 44) 24.1 (13 to 37) 52.7 (37 to 73) Sex 3 female 17 male, 8 female 5 male, 2 female Tissue source Surgical specimen (DDH control ) Surgical specimen Surgical specimen *FAI = femoroacetabular impingement, an OA = osteoarthritis. DDH = evelopmental ysplasia of hip. The cartilage was harveste to prevent seconary FAI after acetabular reorientation 26 an was use as a control tissue as there were no signs of tissue egeneration at the time of surgery. isease states 16,17. prouction is upregulate by proinflammatory molecules such as IL-1b an tumor necrosis factor alpha (TNF-a), an chemokines may play a role in osteoarthritis by recruiting inflammatory cells to injure cartilage, by irectly stimulating inflammation an prouction of egraative enzymes such as MMP-13, or by stimulating the eath of chonrocytes through apoptosis 16,18,19. Despite our improve unerstaning of hip pathomechanics an osteoarthritis pathobiology, the cellular an molecular links between the pathologic mechanical environment an the metabolic alterations of articular cartilage in hip osteoarthritis are not unerstoo. An improve unerstaning of these biologic cascaes will facilitate future isease staging an therapeutic strategies for pre-arthritic an early arthritic hip isease. Nevertheless, characterization of the cellular an molecular events that meiate articular cartilage egeneration remains problematic because of the current limitations of animal moels of hip osteoarthritis, the questionable relevance of in vitro osteoarthritis moels, an the inherent limitations in obtaining an stuying human cartilage tissues from pre-arthritic an/or early arthritic hips. Over the past ecae, there has been an increase utilization of hip joint preservation proceures esigne to surgically normalize or improve the mechanical environment of pre-arthritic an early arthritic hips 20,21. A common component of hip joint preservation proceures for femoroacetabular impingement is resection of a prominent anterolateral femoral hea-neck junction to relieve mechanical impingement The articular cartilage harveste from this tissue provies unique biologic specimens for the analysis of metabolic activity an gene expression in articular chonrocytes. We quantifie mrna (messenger RNA) expression of genes that are potentially important in the evelopment of seconary osteoarthritis in hips with femoroacetabular impingement. The first goal of this stuy was to quantify the expression of genes for inflammatory cytokines an chemokines (IL-1b, IL-8,CXCL1,CXCL2,CXCL3,CXCL6,CCL3,anCCL3L1), egraative enzymes (MMP-13 an ADAMTS-4), an major structural proteins in the cartilage extracellular matrix (COL2A1 an ACAN) (see Table I for efinitions) in articular cartilage harveste from normal hips an hips with femoroacetabular impingement an en-stage osteoarthritis. The secon goal was to analyze the relative expression of these genes in articular cartilage harveste at various stages of the osteoarthritic cascae. Materials an Methos Patients an Cartilage Samples We analyze articular cartilage tissues from thirty-two patients (thirty-two hips) unergoing hip preservation surgery for femoroacetabular impingement, total hip replacement, or hip resurfacing (Table II). All tissues were obtaine from the anterolateral femoral hea-neck junction in the area of mechanical impingement. For comparison, articular cartilage samples without signs of tissue egeneration (control samples) were obtaine from patients unergoing hip preservation surgery involving acetabular reorientation for evelopmental ysplasia of the hip. The anterolateral hea-neck junction (which was without signs of articular cartilage egeneration) was then remove from the latter patients to prevent potential seconary femoroacetabular impingement 26. The three patients (all female) from whom a control cartilage sample was obtaine for comparison ha a mean age of twenty-eight years (range, fifteen to forty-four years). Each patient signe a research consent form for the stuy, which was approve by the university s institutional review boar. All surgical proceures were performe by a single surgeon (J.C.C.) from 2009 to Twenty-five of the thirty-two non-control patients (an hips) were treate with hip preservation surgery an forme the primary stuy group. All of these hips were treate for symptomatic femoroacetabular impingement with open femoral hea-neck osteochonroplasty; arthroscopic proceures performeuring the stuy perio to treat femoroacetabular impingement were exclue because of the ifficulties encountere in tissue harvesting an processing. The mean age of these eight female an seventeen male patients was 24.1 years (range, thirteen to thirty-seven years). All patients ha a clinical iagnosis of femoroacetabular impingement as etermine by one of the authors (J.C.C.). All ha groin pain, restricte hip internal rotation in flexion, an a positive anterior impingement test. All were evaluate with preoperative raiographs mae accoring to a previously publishe protocol 27 an were foun to have structural abnormalities consistent with cam or combine cam an pincer impingement. Aitionally, the raiographic assessment of the osteoarthritis grae accoring to the Tönnis classification system 28 was recore. Twelve (48%) of the hips were Tönnis grae 0 (no raiographic evience of osteoarthritis), nine (36%) were grae 1 (sclerosis only), an four (16%) were grae 2 (moerate joint-space narrowing). None were Tönnis grae 3 (avance osteoarthritis with severe jointspace narrowing). All patients ha unergone prior unsuccessful nonsurgical treatment. The remaining seven non-control patients were treate with primary total hip replacement or hip resurfacing for en-stage osteoarthritis. The mean age of these two female an five male patients was 52.7 years (range, thirty-seven to seventy-three years). All ha a iagnosis of osteoarthritis with hip morphology consistent with femoroacetabular impingement an Tönnis grae-3 osteoarthritis on raiographs. Specimens were obtaine from the anterolateral femoral hea-neck junction of these hips in a fashion ientical to that in the hips with femoroacetabular impingement. These serve as a comparison group of biologic specimens representing en-stage hip osteoarthritis. At the time of surgery, the integrity of the articular cartilage at the femoral hea-neck junction was evaluate macroscopically an was classifie

4 1460 Fig. 1 Normalize mrna expression of selecte cytokine an chemokine, matrix-egraing, an structural matrix genes (efine in Table I) in articular cartilage samples obtaine from hips with femoroacetabular impingement (FAI) an en-stage osteoarthritis (OA) compare with controls. Except for IL-1b an CXCL2, the expression of all genes was higher in hips with FAI compare with controls an hips with OA. Significant ifferences between two or more categories were observe for IL-8, CXCL3, CXCL6, CCL3L1, ADAMTS-4, COL2A1, an ACAN, inicating that the cartilage in hips with FAI was metabolically more active than the cartilage in hips with OA an controls. The ata are expresse as the mean an the stanar error of the mean relative to the mean expression of the control specimens. *P 0.05 compare with hips with FAI. ^P 0.05 between controls an hips with OA. with use of the system of Beck et al. 3. This system inclues criteria to evaluate the macroscopic appearance of the articular cartilage, integrity of the cartilage surface, an fixation of the cartilage to the unerlying subchonral bone (see Appenix). This system was use by the senior surgeon (J.C.C.) to classify all samples into one of five categories: normal, chonromalacia, eboning, cleavage an/or thinning, or efect. The most severe area of isease in each specimen was use for the final graing. After the cartilage was harveste from the anterolateral impingement zone of the femoral hea-neck junction, RNA was extracte from the sample an quantitative RT-PCR (real-time polymerase chain reaction) was performe to evaluate gene expression in the articular cartilage. The genes selecte for evaluation inclue genes for cytokines, chemokines, egraative enzymes, an extracellular matrix proteins thought to play a role in osteoarthritis or joint egraation (Table I). Isolation of RNA an Quantitative RT-PCR The cartilage tissues were immerse promptly in TRIzol reagent (Invitrogen, Carlsba, California) on reception to avoi potential RNA egraation. In aition, all RNA preparation was carrie out uner RNase-free conitions. Isolation of total RNA an quantitative RT-PCR were carrie out as escribe previously, with slight moifications 29,30. Briefly, total RNA was first extracte from the cartilage with TRIzol reagent accoring to the protocol recommene by the manufacturer. After the RNA extraction, RNA clean-up was performe with use of an RNeasy Mini Kit (Qiagen, Valencia, California). Total RNA was reverse-transcribe with SuperScript II reverse transcriptase (Invitrogen) to synthesize first-stran complementary DNA (cdna). Using this cdna, quantitative RT-PCR was performe with 20 ml of reaction mixture containing SYBR Green PCR Master Mix (Applie Biosystems, Foster City, California) an primers on a 7500 Fast Real-Time PCR system (Applie Biosystems). Primers 29,30 for quantitative RT-PCR were selecte for each gene (Table I), an the issociation curve was etermine. The primer esign parameters inclue a primer size of 18 to 21 bp, a prouct size of 80 to 150 bp, a primer annealing temperature of 59 to 61 C, an a primer GC content of 45% to 55%. Results were normalize to the glyceralehye-3-phosphate ehyrogenase (GAPDH) level. The three control articular cartilage samples an seven en-stage osteoarthritis samples were analyze for comparison. The comparative C t (threshol cycles) metho was use to evaluate the expression level of each

5 1461 Fig. 2 Normalize mrna expression of selecte cytokine an chemokine, matrix-egraing, an structural matrix genes (efine in Table I) in articular cartilage samples obtaine from hips with femoroacetabular impingement (FAI) classifie intraoperatively accoring to the Beck criteria (normal, chonromalacia, cleavage/thinning, anefect) an hips with en-stage osteoarthritis (OA) compare with controls. The mrna expression in the FAI cleavage/thinning category was higher than those in the other FAI categories, OA, an normal control cartilage except in the cases of CXCL1, CXCL6, an MMP-13, for which the expression in the FAI cleavage/thinning category remaine relatively similar to that in the chonromalacia category. The mrna expression levels of IL-8, CXCL2, CXCL3, CCL3L1, an ACAN were significantly higher in the FAI cleavage/thinning category than in the other categories inicate by asterisks, inicating that cartilage at the cleavage/thinning stage expresse more inflammatory an catabolic meiators. None of the other groups iffere significantly. The ata are expresse as the mean an the stanar error of the mean relative to the mean expression of the control specimens. A p value of 0.05 was consiere significant. *P **P < ***P < target gene relative to the level in the controls. All graphs epict the expression level in each non-control patient group ivie by that in the controls. Statistical Analysis Data are expresse as the mean an the stanar error of the mean unless otherwise inicate. The nonparametric Mann-Whitney U test was use for comparisons of normally istributeata among the groups. Analysis of variance (ANOVA) followe by the Tukey honestly significant ifference post hoc test were use for multiple comparisons. Differences in gene expression were consiere significant at a p value of Source of Funing The stuy was fune by NIH (National Institutes of Health) grants R , R01AR050847, R02AR058978, an P30AR M.F.R. is supporte by a Ruth L. Kirschstein National Research Service Awar Fellowship (T32- AR060719) from the NIH. Results Expression of cytokine, chemokine, egraative enzyme, an matrix genes was evaluate in three istinct groups of articular cartilage specimens. The genes analyze are shown in Table I an the characteristics of the tissue sources are summarizeintableii.geneexpressioninthetwenty-fivehips with a clinical iagnosis of femoroacetabular impingement was compare with gene expression in the three control samples of cartilage from hips without osteoarthritis as well as the seven samples obtaine from hips with en-stage osteoarthritis

6 1462 at the time of total hip arthroplasty or hip resurfacing (Fig. 1). Expression of all inflammatory cytokines an chemokines except IL-1b an CXCL2 was elevate in the samples from the hips with femoroacetabular impingement compare with the control samples. The ifference was significant (p 0.05) for IL-8, CXCL3, CXCL6, an CCL3L1, an significant ifferences were also observe for ADAMTS-4, COL2A1, an ACAN. Expression of certain of these chemokine (IL-8 an CCL3L1), matrix-egraing (ADAMTS-4), an extracellular matrix (ACAN) genes was also higher in cartilage from hips with femoroacetabular impingement compare with en-stage osteoarthritis. In aition, the gene expression ifferences between the control cartilage an the cartilage from hips with en-stage osteoarthritis were variable an not significant except in the case of ACAN, which was significantly upregulate in the osteoarthritis group. Articular cartilage amage observe intraoperatively in hips with femoroacetabular impingement an osteoarthritis was grae in orer to compare expression of cytokine, chemokine, egraative enzyme, an matrix genes in articular cartilage from ifferent stages of the osteoarthritic cascae. Accoring to the Beck criteria, three (12%) of the hips with femoroacetabular impingement were classifie as showing a normal appearance; thirteen (52%), chonromalacia; zero (0%), eboning; five (20%), cleavage/thinning; an four (16%), a full-thickness efect (see Appenix). The raiographic osteoarthritis scoring was grae 0 (no evience of osteoarthritis) in twelve (48%) of these hips, grae 1 (sclerosis only) in nine (36%), an grae 2 (moerate joint space narrowing) in four (16%). All samples from hips with en-stage osteoarthritis were classifie as showing a full-thickness efect, an the raiographic scoring was grae 3 (severe joint space narrowing) in all of these hips. Analysis of gene expression accoring to the intraoperative classification of articular cartilage amage (Fig. 2) reveale that the expression of certain chemokine (IL-8, CXCL2, CXCL3, an CCL3L1) an extracellular matrix (ACAN) genes was significantly higher in the cleavage/thinning stage. Gene expression was generally lower in the control cartilage than in the other groups except in the case of IL-1b, which was expresse at a relatively similar level in the control cartilage an cartilage at the cleavage/thinning stage. Discussion Femoroacetabular impingement is a common cause of hip pain in aolescents an young aults 31, an it has been implicate as an important etiologic factor in seconary hip osteoarthritis. In recent years, a better unerstaning of the clinical presentation an structural characteristics of this conition has le to increase utilization of surgical proceures esigne to preserve the hip joint with the aim of relieving symptoms, enhancing function, an improving the mechanical environment of the hip joint. Although an increasing boy of literature suggests that femoroacetabular impingement plays a role in the evelopment of seconary osteoarthritis 3,4,31, there remains substantial controversy regaring this topic 6,32. Aitionally, the impact of femoroacetabular impingement on articular cartilage an joint biology at the cellular an molecular level is poorly unerstoo. In orer to better unerstan the biological link between femoroacetabular impingement an osteoarthritis, we compare the expression of cytokine, chemokine, egraative enzyme, an cartilage matrix genes in articular cartilage from hips with femoroacetabular impingement, normal hips, an hips with osteoarthritis. Furthermore, we compare gene expression in the articular cartilage from hips in various stages of the osteoarthritic cascae (as etermine by intraoperative morphologic graing). To our knowlege, this is the first report of metabolic activity levels in the articular cartilage of human subjects with femoroacetabular impingement. Articular cartilage obtaine from the impingement zone (anterolateral hea-neck junction) of hips with femoroacetabular impingement expresse markely elevate levels of most chemokines anegraative enzymes, but not of the proinflammatory cytokine IL-1b, compare with normal articular cartilage (Fig. 1). Cartilage specimens from hips with femoroacetabular impingement also expresse significantly higher levels of certain chemokines an other markers (IL-8, CCL3L1, ADAMTS-4, an ACAN) compare with articular cartilage from hips with en-stage osteoarthritis. In the comparison among ifferent stages of articular cartilage egraation, the cleavage/thinning stage was the most metabolically active as inicate by our panel of target genes. Importantly, there was a tren towarecrease expression of matrix protein genes (COL2A1 an ACAN) in en-stage osteoarthritis compare with femoroacetabular impingement, although this ecrease was significant only for ACAN. The early pathophysiology of osteoarthritis is poorly unerstoo, an very limite information exists regaring the biologic cascae that meiates osteoarthritis in the human hip. Nevertheless, previous work suggests that early changes after injury to articular cartilage inclue hypertrophy, collagen eformation, proteoglycan epletion, an mil inflammation These events are reversible, as chonrocytes can egrae amage molecules an increase matrix prouction 36. Thus, both anabolism an catabolism are increase in early osteoarthritis, with the balance moving towar catabolism with isease progression 33. These previous observations are consistent with the ata from the hips with femoroacetabular impingement an osteoarthritis in the present stuy (Fig. 1). The samples from hips with femoroacetabular impingement emonstrate higher metabolic activity involving inflammatory chemokine (IL-8 an CCL3L1), matrix-egraing (ADAMTS-4), an extracellular matrix (ACAN) genes compare with hips with en-stage osteoarthritis. The ecrease in matrix protein gene expression in hips with en-stage osteoarthritis may inicate a loss of anabolic activity an an imbalance favoring catabolism. Conventional iagnosis an treatment of pre-arthritic, early arthritic, an avance arthritic conitions is highly epenent on patient symptoms, physical examination, an raiographic evaluation 20. It is important to note that the

7 1463 majority (84%) of the hips with femoroacetabular impingement in the present stuy ha no or only early raiographic signs of osteoarthritis (Tönnis grae 0 or 1), yet the alterations in articular cartilage metabolic activity were profoun. This fining unerscores the concept that the biology of the osteoarthritic cascae far precees raiographic evience of isease. Consequently, alternative methos of iagnosis anisease staging are being investigate. Biologic markers from bloo, urine, an synovial flui are consiere potential caniates for future iagnostic anisease staging strategies 37,38. In the present stuy, articular cartilage from hips with femoroacetabular impingement emonstrate a molecular signature compare with normal cartilage. Several of the chemokines that were highly expresse in cartilage from hips with femoroacetabular impingement were most markely elevate when the stage of articular cartilage egeneration was classifie as chonromalacia or cleavage/thinning. These morphologic stages of articular cartilage egeneration commonly precee raiographic osteoarthritic changes, suggesting that specific cytokine an chemokine gene expression levels may have potential in characterizing the early (pre-arthritic) molecular changes that are occurring in articular cartilage. These new finings provie a basis for pursuing istinct chemokines as caniate biomarkers for the iagnosis an staging of pre-osteoarthritic an early osteoarthritic isorers 39. Another important aspect of this stuy was the availability of control hip cartilage samples from age-matche subjects without osteoarthritis who were unergoing hip surgery. These samples provie baseline ata for comparison with tissue from hips with femoroacetabular impingement an osteoarthritis. Nevertheless, the stuy has limitations. First, although we ientifie local alterations in articular cartilage gene expression in femoroacetabular impingement that may meiate the osteoarthritic cascae, we have not ientifie the specific molecular an/or mechanistic role of each factor in the pathophysiology of femoroacetabular impingement, as such stuies were beyon the scope of this report. Future investigations will focus on the mechanisms of the osteoarthritic cascae. Secon, we measure gene expression in only one specific area of articular cartilage (the anterolateral femoral hea-neck junction); the expression an molecular characteristics of articular cartilage in other areas of the hip were not etermine. This limitation coul not be overcome because the surgical goal of preserving the joint in patients with femoroacetabular impingement exclue the possibility of harvesting tissues from other regions. Therefore, the impact of femoroacetabular impingement pathomechanics on articular cartilage away from the impingement zone remains unclear. In conclusion, these finings provie novel information regaring the pathophysiology of femoroacetabular impingement an the molecular basis of human hip osteoarthritis. Specifically, we emonstrate the feasibility of analyzing gene expression in articular cartilage samples obtaine from the impingement zone at the time of joint preservation surgery. Analysis of these tissues suggests that the mechanical isease of femoroacetabular impingement causes localize articular cartilage alterations that are consistent with early osteoarthritic egeneration. Specifically, articular cartilage in the femoroacetabular impingement zone ha high metabolic activity, both catabolic an anabolic, that commonly precee raiographic evience of osteoarthritis. Appenix A table showing the Beck criteria for intraoperative graing of articular cartilage lesions is available with the online version of this article as a ata supplement at jbjs.org. n Shingo Hashimoto, MD, PhD Muhamma Farooq Rai, PhD Corey S. Gill, MD Zhiqi Zhang, MD Lina J. Sanell, PhD John C. Clohisy, MD Department of Orthopaeic Surgery, Washington University School of Meicine at Barnes-Jewish Hospital, 660 South Eucli Avenue, Box 8233, St. Louis, MO aress for S. Hashimoto: hashimotos@wuosis.wustl.eu. aress for M.F. Rai: raim@wustl.eu. aress for C.S. Gill: gillc@wuosis.wustl.eu. aress for Z. Zhang: zhzhqi@mail2.sysu.eu.cn. aress for L.J. Sanell: sanelll@wuosis.wustl.eu. aress for J.C. Clohisy: clohisyj@wuosis.wustl.eu References 1. Clohisy JC, Dobson MA, Robison JF, Warth LC, Zheng J, Liu SS, Yehyawi TM, Callaghan JJ. Raiographic structural abnormalities associate with premature, natural hip-joint failure. J Bone Joint Surg Am May;93(Suppl 2): Murphy SB, Ganz R, Müller ME. 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