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Knee injuries and their consequences Knee injuries and their consequences – the impact of impact Swärd, Per 2014 Link to publication Orthopaedics (Lund)]. Department of Orthopaedics, Lund University. Total number of authors: 1 Citation for published version (APA): Swärd, P. (2014). Knee injuries and their consequences – the impact of impact. 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LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Knee injuries and their consequences – the impact of impact Per Swärd DOCTORAL DISSERTATION by due permission of the Faculty of Medicine, Lund University, Sweden To be defended at Segerfalksalen, BMC, Sölvegatan 17, Lund, on 23 May 2014 at 1 pm Faculty opponent Professor Lars Engebretsen 1 Knee injuries and their consequences – the impact of impact Per Swärd 3 Cover illustration: Mouse knee joint. Bone, hyaline cartilage and meniscus visualised by toluidine blue staining (40 x magnification). Published with the kind permission of Dr. André Struglics. © Per Swärd Lund University, Faculty of Medicine Doctoral Dissertation Series 2014:60 ISBN 978-91-87651-86-1 ISSN 1652-8220 Printed in Sweden by Media-Tryck, Lund University Lund 2014 En del av Förpacknings- och Tidningsinsamlingen (FTI) 4 5 To Catrin 6 7 Contents Abstract 9 Populärvetenskaplig sammanfattning 11 Introduction 13 Preface 13 List of studies 14 Abbreviations 15 Background 17 The knee joint 17 The anterior cruciate ligament 17 Anterior cruciate ligament tears 19 Anterior cruciate ligament injury mechanism 20 Knee injury panorama 22 Osteoarthritis 25 Knee osteoarthritis 25 Post-traumatic knee osteoarthritis 26 Cartilage 27 Synovium 31 The meniscus 32 Bone 32 Inflammation 34 Objectives 39 Specific objectives 39 Subjects 41 Methods 47 Radiography of the knee (Studies I & II) 47 MRI of the knee (Studies I & IV) Alcian blue precipitation (Study III) Electrochemiluminescence (Study III) ELISA (Study IV) Western blot (Study V) Statistical methods Methodological considerations 49 49 50 50 50 51 53 Subjects 53 Imaging 54 Results 55 Radiological characteristics of post- and non-traumatic knee osteoarthritis (Study 1) Alignment and the risk of post-traumatic osteoarthritis (Study II) Cartilage and bone markers and inflammatory cytokines in the acute phase of knee injury (Studies III & IV) Osteochondral fractures and joint inflammation (Study IV) Protease activity in bovine cartilage explants co-incubated with joint capsule and/or mechanically injured (Study V) 55 56 57 58 60 Conclusions 63 Discussion and future perspectives 65 Post-traumatic and non-traumatic knee osteoarthritis The knee injury and the impact Mechanical impact and its role in osteoarthritis initiation after ACL injury Immunity vs biomechanics in driving the OA process after anterior cruciate ligament rupture Concluding remarks 65 66 67 68 73 Tack 75 References 77 8 Abstract Anterior cruciate ligament (ACL) injuries are common, severe knee injuries that result in a high risk of developing knee osteoarthritis (OA) in the affected individuals. As proof of high impact forces applied to cartilage and bone at the time of injury, traumatic bone marrow lesions and osteochondral fractures, located predominantly in the lateral tibiofemoral compartment, are commonly associated with an ACL injury. The subsequent risk of OA may be closely associated with the knee injury mechanism and the panorama of injuries in the knee sustained at the onset of injury. The purpose of this work was to acquire a better understanding of how the initial impact, related to the trauma mechanism of acute knee injuries, may influence acute and chronic knee pathology. In this work it was found that subjects with post-traumatic OA secondary to an ACL injury have more joint space narrowing and more osteophytes in the lateral compartment than in the medial compartment, compared with subjects with non traumatic OA. Furthermore, it was found that an acute knee injury is associated with instant and sustained synovial fluid biochemical alterations within the first month of knee injury, suggestive of increased cartilage turnover and severe joint inflammation. Those subjects who sustained an osteochondral fracture with disrupted cortical bone in association with the soft tissue knee injury had increased joint inflammation. In an in vitro bovine cartilage study, mechanical injury to cartilage increased the matrix metalloproteinase-induced cleavage of cartilage aggrecan. Moreover, findings from this model suggest that the aggrecan degradation may differ between cytokine-stimulated cartilage explants compared with cartilage explants mechanically injured and (or) co-incubated with joint capsule. Conclusively, the findings in this work underline the fact that the initial impact associated with an ACL appears to be important in terms of the risk of developing post-traumatic OA. In addition, this work emphasizes how the acute biological response to injury could be involved in cartilage degradation. A greater understanding of these processes could lead to the improved management of knee injured patients and possibly delay, or even prevent, OA development. 9 10 Populärvetenskaplig sammanfattning Främre korsbandsskador är vanliga, allvarliga och traumatiska knäskador. I kombination med associerade meniskskador och ändrad ledbelastning leder skadan på sikt till att en stor andel individer utvecklar artros i ung ålder. Detta benämns post-traumatisk artros. På kort sikt kan skadan leda till oförmåga att fortsätta sin aktivetet på samma nivå som tidigare. De kraftiga kompressionskrafter över brosk och ben då skenben och lårben kolliderar i skadeögonblicket leder till skador och celldöd i vävnaderna Detta bidrar sannolikt till risken för att utveckla post traumatisk artros. Hos fler än hälften av de skadade individerna kan man med MR visualisera en fraktur i kortikalt ben på skenben och/eller lårben. Skadorna som uppstår i samband med traumat är framförallt lokaliserade lateralt i knäleden och kan tillsammans med blödning i knäleden initiera ett inflammationssvar. Dessutom initieras en läkningsprocess som åtföljs av en ökad omsättning (nysyntes och nedbrytning) av olika broskproteiner. Det broskprotein som framförallt har undersökts under utförandet av detta avhandlingsarbete är aggrekan. Molekylen aggrekan består av ett protein på vilket det sitter ett stort antal sockerkedjor. Dessa är kraftigt negativt laddade och bidrar till broskets funktion genom att attrahera positiva motjoner, vilka genom diffusion drar till sig vatten. Detta leder till ett svullnadstryck och medför att brosket kan motstå de krafter som uppstår när man belastar knäleden. Under artrosprocessen bryts aggrekanet ned av olika enzym som finns i broskmiljön. Framförallt antas så kallade aggrekanaser och matrix metalloproteaser (MMP) ha stor betydelse. Dessa enzym klyver aggrekanmolekylen på olika ställen och genererar olika långa aggrekanfragment som kan mätas i ledväska och i broskmedium. Ett fragment som bildas efter klyvning av aggrekanaser och som analyseras i både arbete III och V är ARGS aggrekan. Ett fragment som bildas efter klyvning av MMP är FFGV-aggrecan (arbete V). Avhandlingen utgår från frågeställningen av vad ett vridvåld i knäleden (knäledsdistorsion) innebär i det akuta skedet och på längre sikt. I första delarbetet gjordes en jämförelse av lokalisationen av artrosförändringar mellan knäleder som har ett definierat trauma (främre korsbandsskada) mot de som inte har detta. Vi fann mer lateralt lokaliserad ledspringesänkning och osteofyter i knäleder där det funnits ett definierat trauma vilket indikerar att det initiala traumat har en viktig roll i den post-traumatiska artrosprocessen. Dessutom kan det vara en viktig 11 klinisk implikation, då det verkar som att efter en främre korsbandsskada utvecklas artros både medialt och lateralt i knäleden. Artros hos individer utan tidigare trauma är oftast lokaliserad medialt i knäleden. I andra delarbetet belyses vilken inverkan knäledens ställning har i samband med artrosutveckling efter en korsbandsskada. Vi fann att individer med varusställning (hjulbenthet) hade mer artros i sitt skadade knä jämfört med de med normal eller valgusställning (kobenthet). Även om skillnaderna mellan grupperna var stora, var de inte statistiskt säkerställda. Mer forskning på området för att utreda om varusställning ökar risken att utveckla artros efter en främre korsbandsskada är indicerad. Arbete III och IV utgår från mätning av ledvätskekoncentrationen av olika brosk-, ben- och inflammationsmarkörer. Vid jämförelse mellan individer med akut knäskada och knäfriska kontroller, visade sig ledvätskekoncentrationer i knäleden av total aggrekan och ARGS-aggrekan vara förhöjda från 1-3 dagar efter skadan upp till 23 dagar efter skadan. Ett annat broskprotein, COMP var förhöjt från 2-3 dagar efter skadan upp till 23 dagar efter skadan. Den nya kunskap som dessa fynd indikerar är att omsättningen av både aggrekan och COMP ökar nästan omedelbart efter skadan. Även flera pro-inflammatoriska proteiner och benassocierade proteiner återfanns i högre koncentrationer i knäleden hos de knäskadade individerna från 0-23 dagar efter knäskadan. I arbete IV undersöktes hur ovan nämnda biomarkörer relaterade till förekomst av MR-visualiserade osteokondrala frakturer där skenben och lårben kolliderade i samband med den främre korsbandsskadan. De inflammatoriska cytokinerna interleukin-8 och tumour necrosis factor-α var förhöjda i ledvätskan hos de individer som ådragit sig en osteokondral fraktur med åtföljande avbrott i det kortikala benet. Sådana frakturer skulle således kunna vara viktiga för det initiala inflammationssvaret på skadan, men även för risken att utveckla artros på lång sikt. Om så är fallet får utvärderas i framtida studier. I arbete V studerades effekterna på aggrekannedbrytning av ett trubbigt våld mot ungt kalvbrosk i laboratoriemiljö samt effekterna av att odla ungt kalvbrosk tillsammans med ledkapsel. Mekanisk skada av brosket ledde till ökad MMP aktivitet, medan en antydan till ökad aggrekanasaktivitet sågs i mekaniskt skadat brosk som odlades tillsammans med ledkapsel. I mekaniskt skadat brosk som behandlades med cytokin sågs mycket kraftig aggrekanasaktivitet, men ingen MMP-aktivitet. Denna avhandling belyser en del processer som skulle kunna ha betydelse för utvecklingen av artros efter en allvarlig knäskada och speciellt efter en främre korsbandsskada. Sammantaget stärker den antagandet att traumat över ben och brosk som sker i samband med att en främre korsbandsskada uppkommer kan ha stor betydelse för risken att utveckla post-traumatisk artros. 12 Introduction Preface Like many diseases, the manifest disease and pathogenesis of osteoarthritis (OA) are multidimensional. The dimensions of OA range from clinical symptoms and macroscopic features of disease through biomechanics to microscopic events at cell, extracellular matrix and molecular level. An increasing amount of knowledge is starting to bridge these dimensions, but there are still many large holes to fill, in order better to understand the disease. The main advantage of post-traumatic OA from the scientist’s perspective is that the time of disease onset is known and the progression of the disease is relatively rapid compared with non-traumatic OA. One of the major drawbacks from the patients’ perspective is that they are affected by the disease at a young age. During my time as a PhD student, I have tried better to understand why and through which processes a severe knee injury can lead to post-traumatic OA. This knowledge is crucial in order to understand the disease and to be able to construct viable treatment options and advice for subjects with severe knee injuries. The main hypothesis underlying the present thesis was that the impact on cartilage, bone and soft tissues of the knee, inflicted at the time of injury, plays an essential role when it comes to the future risk of developing post-traumatic OA. The strength of the present thesis is that, via the papers in the study, it links different clinical parameters and molecular mechanisms that may be important for post traumatic OA development. 13 List of studies I. II. III. IV. V. Swärd P, Kostogiannis I, Neuman P, Von Porat A, Boegard T, Roos H. Differences in the radiological characteristics between post-traumatic and non-traumatic knee osteoarthritis. Scand J Med Sci Sports. 2010;20(5):731-739. Swärd P, Fridén T, Boegard T, Kostogiannis I, Neuman P, Roos H. Association between varus alignment and post-traumatic osteoarthritis after anterior cruciate ligament injury. Knee Surg Sports Traumatol Arthrosc. 2013;21(9):2040-2047. Swärd P, Frobell R, Englund M, Roos H, Struglics A. Cartilage and bone markers and inflammatory cytokines are increased in synovial fluid in the acute phase of knee injury (hemarthrosis)--a cross-sectional analysis. Osteoarthritis Cartilage. 2012;20(11):1302-1308. Swärd P, Struglics A, Englund M, Roos H, Frobell R. Soft tissue knee injury with concomitant osteochondral fracture is associated with higher degree of acute joint inflammation. Am J Sports Med. Published online March 24, 2014. DOI: 10.1177/0363546514524924. Swärd P, Hansson M, Lohmander SL, Wang Y, Grodzinsky A, Struglics A. Evidence of increased protease activity in mechanically injured cartilage co-cultured with joint capsule. Manuscript. 14 Abbreviations ACL Anterior cruciate ligament ADAMTS A disintegrin and metalloproteinase with thrombospondin motifs BMI Body mass index BSP Bone sialoprotein C2C Newly formed epitope after cleavage of collagen at the type II collagen primary cleavage site COMP CRP Cartilage oligomeric matrix protein C-reactive protein CS Chondrotin sulphate DAMP Damage-associated molecular patterns ECM Extracellular matrix HA Hyaluronan KS Keratan sulphate MMP Matrix metalloproteinase MRI Magnetic resonance imaging OA Osteoarthritis OCL Osteocalcin OPN Osteopontin PF Patellofemoral sGAG Sulphated glycosaminoglycan SPARC TGF-β TIMP TNF-α secreted protein acidic and rich in cysteine Tumour growth factor-β Tissue inhibitor of matrix metalloprotease Tumour necrosis factor-α TF Tibiofemoral 15 16 Background The knee joint The knee joint is located between the two other joints of the lower limb; the hip and the ankle. The proximal end of the tibia and the distal end of the femur form the medial and lateral tibiofemoral compartments. The patella and the anterior part of the distal femur form the patellofemoral joint. Together, these joints form the knee joint. High demands are imposed on the knee joint and it has several functions which are essential for human beings to walk, run and jump. Primarily, it enables flexion-extension of the lower limb in the sagittal plane. In extension, full or close to full, the knee must be able to withstand the strong forces imposed on the knee by gravity. In flexion, rotation at the knee enables the leg to position the foot before placement. Small movements in the varus/valgus direction are also possible at the knee joint but only when the knee is flexed. Joint stability during movement is attained by the shape of the articular surfaces, the collateral and cruciate ligaments, the menisci and tendons and muscles crossing the knee joint (Figure 1) [1]. The anterior cruciate ligament The anterior cruciate ligament (ACL) has two functional bundles which connect the femur and the tibia; the anteromedial and the posterolateral bundles, named after their insertion sites on the tibia (Figure 2). These bundles bridge the posteromedial aspect of the lateral condyle and the medial tibial plateau where they insert next to and anterior to the tibial spines. The ACL has an intra-articular location, but it is separated from the synovial fluid by a synovial lining. The main blood supply originates from the femur and specifically from the central geniculate artery [2, 3]. Pacinian corpuscles, Golgi tendon organs and Ruffini endings are mechanoreceptors present in the ACL which contribute to the proprioceptive sense [4]. The extracellular matrix (ECM) of the ACL contains collagen types I, II, III and V, elastin and proteoglycans. The tensile properties of the ACL are mainly related to bundles of collagen type I and cross-linking of these [5, 6]. In the normal 17 ACL, fibroblasts reside along collagen bundles and are important for normal ligament turnover. Moreover, cells with progenitor potential are present [6]. The function of the ACL is to provide tibiofemoral joint stability in anterior-posterior translation and in internal-external rotation [7]. The ACL also restrains movements in the varus-valgus direction. The anteromedial and posterolateral bundles of the ACL act in synergy to stabilise the knee joint through its entire range of motion. In flexion, the anteriomedial bundle is tauter; in extension, the posterolateral bundle is tauter [3]. The combination of valgus and internal rotation of the tibia has been shown to increase ACL strain more than either motion alone [8]. Figure 1. Posterior view of the left knee showing the anterior and posterior cruciate ligaments and the menisci. The image which is from the 20th US edition of Gray's Anatomy of the Human Body was originally published in 1918 and has been transferred into the public domain. 18 Figure 2. Schematic drawing of the double-bundle ACL anatomy. AM, anteromedial bundle; PL, posterolateral bundle. Published with kind permission. © C. Kartus. Anterior cruciate ligament tears In Sweden, football (soccer) is the most common activity associated with ACL injury for both men and women. The second most common activity associated with ACL injury is downhill skiing for women and floorball for men (www.aclregister.nu). The incidence of ACL tears was shown to be 81 per 100,000 subjects aged between 10 and 64 years [9]. Based on these numbers, some 6,000 ACL injuries occur in the Swedish population every year, of which ~3,000 ACLs are surgically reconstructed. The indications for ACL reconstruction in Sweden are symptoms of instability and the failure of conservative treatment (www.aclregister.nu). In the USA, approximately 200,000 ACL reconstructions 19 are performed every year [10]. Women are known to be more susceptible to ACL rupture compared with men, and are injured at a younger age [11, 12]. Anatomic, neuromuscular and hormonal variations between men and women have been proposed to explain this observed difference [13-16]. From the individual’s perspective, the injury may, in the short term, lead to knee dysfunction and an inability to continue sports participation at the same level as pre-injury. In a recent review, it was summarised that, although normal to nearly normal knee function was regained in most ACL-reconstructed individuals, a relatively small number of individuals returned to their pre-injury activity level and competitive sports [17]. Psychological factors, including fear, lifestyle changes and personality, have an impact on why subjects do not return to their pre-injury sports activity after ACL reconstruction [18]. In professional sports, the return to play after ACL injury is much greater and Waldén et al. [12] reported that 94% of elite level football players returned to training within 10 months and that 89% participated in a match within 12 months after ACL reconstruction. An ACL tear also leads to a high risk of developing OA of the injured knee at a young age [19-22]. ACL re-injury/graft rupture and the high risk of contralateral ACL injury are other important issues that require consideration when advising an individual to return to sport [23]. In a reasonably sized study, in terms of the queries tested, it was reported that 4.5% sustained a graft rupture and 7.5% sustained a contralateral ACL injury during the five-year follow-up after ACL reconstruction. Importantly, 29% of individuals younger than 20 at the time of the first ACL injury sustained an ACL injury to either knee during follow-up [24]. Taken as a whole, the high risk of ACL injury, specifically in young women, and future complications related to the injury are regarded as one of the major problems in sports medicine [25]. Anterior cruciate ligament injury mechanism In the literature, the ACL injury mechanism is most often described as contact or non-contact. This definition may be misleading, as most ACL injuries that occur in contact sports are associated with a “non-contact” mechanism [26, 27]. A contact ACL injury mechanism typically occurs in American football, when the subject plants his foot and, at the same time, is tackled at the knee from the lateral side, resulting in a valgus collapse of the knee joint. The injury panorama of this injury mechanism often results in lesions of the ACL, medial collateral ligament and medial meniscus, referred to as the O’Donoghue triad [28]. Several mechanisms that may result in a non-contact ACL injury have been proposed. Most typically, the injury occurs as the athlete plants the foot with the knee in slight flexion and 20 during landing, side-cutting or deceleration. In this position, knee valgus motion, internal rotation of the tibia towards the femur or external rotation of the femur towards the tibia, combined with anterior translation of the tibia, ensue. This leads to high strain in and the rupture of the ACL [26, 29, 30]. This ACL injury mechanism, also known as the pivot shift injury, induces simultaneous lateral tibiofemoral compartment subluxation and combined compressive force, explaining the typical location of bone marrow lesions, on the posterolateral tibial plateau and the midportion of the lateral femoral condyle [2]. The impact site on the lateral femur is related to the degree of knee flexion at the time of injury. With the knee in a high degree of flexion or extension at the time of injury, the lateral tibia impacts the posterior or the anterior part of the lateral femur respectively [2]. Differences in the ACL injury mechanism between the sexes have been described. Investigating basketball players, Krosshaug et al. [31] reported that a valgus collapse in association with the injury was five times more common in women. A lower incidence of meniscal tears associated with the ACL injury has been indicated in women [32], which could be related to differences in injury mechanism between men and women. Furthermore, besides from showing that young age was associated with more traumatic bone marrow lesions on the lateral femoral condyle, it was in a study by Bisson et al. [33] demonstrated that male gender associated with mild traumatic bone marrow lesions on the lateral femoral condyle and tibial plateau. Male gender also associated with moderate and severe traumatic bone marrow lesions on the lateral femoral condyle. These findings may further highlight gender-specific differences in ACL injury mechanism and as a result, factors other than valgus collapse may be of greater importance for compressive injuries to soft tissues and bone associated with ACL injury. Even if they were not investigating bone-bruise patterns, Fridén et al. [27] proposed that the degree of compression between the tibia and femur was related to weight bearing or non-weight-bearing at the time of injury. A non-weight-bearing ACL injury mechanism, which typically occurs when skiing, was associated with larger numbers of intact menisci, indicating a lower degree of joint compression in this type of injury [27]. In a large-scale investigation of 525 subjects, younger age and not jumping at the time of ACL injury were associated with a bone bruise [34]. Jumping at the time of injury may indicate a non-weight-bearing ACL injury and these findings are thus in line with the hypothesis put forward by Fridén et al. [27]. 21 Knee injury panorama An acute knee injury with joint effusion and intra-articular bleeding (hemarthrosis) suggests significant intra-articular pathology. Anterior cruciate ligament tears, meniscal lesions and lesions of the medial collateral ligament are common after rotational knee injury [9]. In subjects (n=1,145) with an acute knee injury in whom MRI (magnetic resonance imaging) was performed a median of eight days after the trauma, 52% had sustained an ACL injury, 17% had transient patellar dislocation and 28% a medial collateral ligament tear. Among the ACL-injured subjects, 55% also sustained an associated meniscal tear [35]. These findings are in line with previous investigations of smaller study samples, regarding both the panorama of knee injuries [9] and the high prevalence of meniscal tears concomitant to the ACL tear [36, 37]. The majority of studies and the historical view indicate that lateral meniscal tears are more common than medial tears after ACL injury [36, 38-40]. However, recent studies have described a similar prevalence or even more medial than lateral meniscal tears in association with ACL injury [9, 37]. In a recent MRI-based study, the most common meniscal tear associated with the ACL injury was a longitudinal tear of the posterior horn of the medial meniscus [9]. The authors speculate that these tears may be difficult to detect during routine arthroscopy and could progress and produce symptoms in an unstable knee [9]. Isolated or multiple articular cartilage lesions are also a frequent finding after ACL injury [32, 41]. Posterolateral knee injury, which is relatively uncommon, is an important diagnosis which, if left untreated, can lead to severe knee disability [42, 43]. In almost all individuals, an acute knee injury also leads to a collision between the tibial plateau and the femoral condyle, as visualised using MRI by traumatic bone marrow lesions at the site of impact. These have been described as “fingerprints of injury mechanism”, or “kissing lesions” and have been detected in almost all subjects who have suffered an ACL tear [2, 44, 45]. Typically, the collision occurs in the lateral tibiofemoral compartment between the non-articular posterolateral tibial plateau and the articular midportion of the lateral femoral condyle [44, 46, 47]. Depending on the injury mechanism, other locations of traumatic bone marrow lesions may be present (see above). Traumatic bone marrow lesions in the medial tibiofemoral compartment have been ascribed to the contre-coup mechanism, but they are less common and are mostly associated with bone marrow lesions of the lateral compartment [48]. It should be noted that medial bone marrow lesions are commonly found in subjects with a combined ACL and posterolateral knee injury [42]. At the site of impact between tibia and femur, more than half of ACL-injured subjects also sustain a traumatic osteochondral fracture [39, 44]. These fractures 22 are prevalent in the lateral tibiofemoral compartment overlying the traumatic bone marrow lesions and present with or without the disruption of cortical bone (Figure 3). They indicate strong impact forces between the tibia and femur at the time of injury and correlate with the size of bone marrow lesions [39, 44] and the likelihood of associated meniscal tears [39]. The traumatic bone marrow lesions represent trabecular fractures, bleeding or oedema. In one study, human biopsy samples of cartilage and subchondral bone overlying MRI-detected bone bruises were obtained at a median of 4.5 weeks after ACL injury. Glycosaminoglycan loss and chondrocyte necrosis were observed in the overlying cartilage. In the subchondral bone, osteocyte necrosis was indicated by empty lacunae [49]. Figure 3. Magnetic resonance images of knees used in Study IV. Upper row: proton density T2 weighted sequence and bottom row: short-tau inversion recovery (STIR) sequence of knees with osteochondral fractures with disrupted cortical bone in the femur (yellow arrow) and without disrupted cortical bone in the femur (blue arrow). The STIR sequence images (bottom row) clearly show the surrounding post-traumatic bone marrow lesion of the osteochondral fracture above, indicating traumatic impact forces (white arrows). 23 24 Osteoarthritis Knee osteoarthritis Knee OA is more common in women and knee OA incidence and prevalence increase with increasing age. Knee OA may be isolated, or may be part of generalised OA, defined as affecting the hands and at least one large joint, or affecting three or more joints [50]. Obesity has been shown to be a highly important and modifiable risk factor for OA development and progression [51]. Adding to the complexity of the disease, the metabolic syndrome and two of its components, central obesity and hypertension, are also associated with the incidence of severe knee OA requiring total knee replacement, independent of body mass index (BMI) [52]. Systemically increased factors derived from visceral adipose tissue have been shown to increase the risk of hand OA and may also be involved in the pathogenesis of knee OA [53]. Today, knee OA is a leading cause of global disability. The ageing population and increasing rates of obesity worldwide forecast an increasing need for health care related to knee OA in the future [54]. Symptomatic knee OA affects almost 7% of the US population 50-84 years of age, of which approximately 50% are obese [55]. Petersson et al. [56] reported that 1.5% of individuals 35-54 years of age, living in a district in the southwest of Sweden, had symptomatic non-traumatic knee OA. The rates of radiographic knee OA are much higher. In the elderly (over the age of 60 to 75), the prevalence of radiographic OA was shown to be between 31-45% [57-59]. There may, furthermore, be ethnic differences regarding knee OA location. For example, in the Chinese, the lateral tibiofemoral compartment is affected more often than in Caucasians [60]. Importantly, OA has a large impact on quality of life and was estimated to reduce the remaining quality-adjusted life expectancy in persons with knee OA by 10-13%, with the higher rates applying to younger individuals with knee OA [55]. As indication of the increasing incidence of symptomatic knee OA, the numbers of total knee replacements performed in the USA more than doubled from 1999-2008. In younger individuals (45- to 64-year olds), the numbers more than tripled. In addition to increasing obesity rates, this increase may be related to wider indications of surgery and increasing numbers of severe knee injuries in young individuals [61]. An increased understanding of knee 25 OA risk factors, how best to treat symptoms and find treatments that can prevent or stop OA progression is needed. Apart from age, gender and obesity, also genetics, knee injuries and abnormal joint loading influence the risk of developing knee OA. Seven genetic variants associated with knee OA or total knee replacement have been identified [62] and 39% of the risk of developing knee OA has been attributed to genetic variation [63]. The possibility that abnormal, or overly high loads can lead to progressive cartilage degradation was indicated by the increased risk of knee OA in occupations where there is frequent heavy lifting, kneeling or squatting [64]. Moreover, malalignment has been shown to increase the risk of OA [65, 66] and elderly male former elite athletes engaged in non-impact sports have an increased knee OA prevalence after adjustment for previous knee injury, age, gender, BMI and occupational load [67]. Post-traumatic knee osteoarthritis As things stand, some 12% of the total OA burden has been ascribed to post traumatic OA [68]. It has been proposed that the incidence of post-traumatic OA is increasing in relation to increased numbers of individuals engaged in sports and over the last few decades, the increase in sports participation has been substantial. In the USA, the number of women participating in high-school sports has roughly doubled every decade [46]. Instability is as a major cause of disability after ACL injury [69]. This has influenced the treatment of ACL injuries and ACL reconstruction has been a preferred treatment, with the aim of re-establishing knee stability [70]. However, no differences in the long-term risk of OA development have been shown between ACL-injured subjects treated with or without primary ACL reconstruction in recent systematic literature analyses and from the early results of a randomised controlled trial [19, 21, 71, 72]. Associated injuries may be of greater significance for the long-term prognosis. In a review by Øiestad et al. [21], it was concluded that the prevalence of knee OA after an isolated ACL injury was 0-13%. The prevalence of knee OA after an ACL tear with an associated meniscal tear was 21-48% [21]. Differences in the classifications relating to the radiographic grading of OA between different studies have led to difficulties juxtaposing the current knowledge [21, 71]. In a recent meta-analysis only including studies using the Kellgren & Lawrence classification and with a minimum follow-up time of 10 years, it was shown that non-ACL-reconstructed knees had an increased relative risk of developing any grade of OA. However, the relative risk of progression to moderate or severe OA tended to be higher in ACL-reconstructed knees [71]. 26 Cartilage The proximal tibia, the distal femur and the patella are covered by a thin layer of hyaline cartilage, forming the articular surfaces of the knee joint. Normal cartilage is avascular, aneural, with no lymphatic vessels and the only cell type found in cartilage, the chondrocyte, obtains nutrition mainly from passive diffusion from synovial capillaries (see below). In normal articular knee cartilage, only 1-3% of the wet weight tissue consists of chondrocytes [51]. Some 70% of the tissue consists of water, whereas 20% of the wet weight is collagen (mainly type II; the fibril-forming collagen of cartilage) and approximately 5% is aggrecan [73]. The main function of cartilage is to distribute the load applied to the underlying bone and to allow movements of low friction at the knee joint [74]. These functional properties are preserved by the chondrocytes which maintain cartilage hemostasis by inducing proteolysis and the production of non-fibrillar collagens, proteoglycans and other non-collagenous molecules, as a reaction to biomechanical and biochemical stimuli [75]. The capacity of cartilage to absorb and distribute high loads at a specific site is related to the composition of cartilage ECM and specifically to the integrity of the fibrillar collagen and aggrecan networks that are present. Aggrecan forms large aggregates by binding to hyluronan and is substituted by negatively charged sulphated glycosaminoglycans (sGAGs) [76]. The high negative charge attracts counter-ions and, by diffusion, water is attracted to the aggrecan molecule. This results in a swelling pressure in cartilage which is retained by the tensile strength of the collagen fibril network and gives cartilage the ability to withstand compressive loads [76]. The fibrillar collagen and aggrecan networks, on the other hand, are dependent on other molecules present in the cartilage ECM, such as cartilage oligomeric matrix protein (COMP), non-fibrillar type IX collagen and members of the small leucin-rich repeat protein family. Important functions of these molecules include the regulation of collagen fibril formation and other collagen networks in the cartilage ECM. They also enable interactions between different ECM molecules and interactions with the chondrocytes [76-78]. Type II collagen has a long half-life (about 100 years) and its degradation is believed to be practically irreversible [79]. Several collagenases are able to cleave fibrillar type II collagen, of which the matrix metalloproteinase (MMP)-13 is believed to be the most important [51, 77]. Cleavage at the primary cleavage site in type II collagen by these collagenases generates two fragments, of which one can be detected by a neo-epitope antibody (against the newly formed epitope; named C2C). Interestingly, increased synovial fluid concentrations of C2C are associated with pre-radiographic cartilage lesions in the knee joint of ACL-injured subjects [80]. Aggrecan has a more rapid turnover. The cleavage of aggrecan can 27 be mediated by several proteolytic enzymes (Figure 4). The aggrecanases-1 and -2 (ADAMTS-4 and -5; a disintegrin and metalloproteinase with thrombospondin motifs), and, secondly, the MMPs are believed to be most important [81-84]. Another somewhat abundant protein in cartilage is COMP, which is believed to be important for the fibrillation of type II collagen in cartilage and for stabilising the collagen network in the adult cartilage. Its turnover is increased during the early progression of OA [77]. The turnover of COMP is also increased after an acute knee injury, as observed by increased synovial fluid concentrations from within one week of injury up to several years later [85]. HA G1 ADAMTS-4/5 G2 IGD G3 L MMP KS CS Figure 4. Aggrecan is bound to hyaluronan (HA); an interaction stabilised by the link protein (L). Aggrecan is composed of three globular domains; G1 (N-terminal side), G2 and G3, an inter globular domain (IGD), one region rich in keratan sulphate (KS) and two regions rich in chondroitin sulphate (CS1 and CS2). Several enzymes have been shown in vitro to cleave aggrecan in the IGD domain [81, 83, 84, 86]. Aggrecanase cleavage at the TEGE373↓374ARGS site and MMP cleavage at the IPEN341↓342FFGV site have been demonstrated in humans [81, 83, 84, 87-89]. Suggestive of rapid aggrecan turnover by increased aggrecanase activity, increased concentrations of ARGS-SELE and ARGS-CS1 fragments have been shown in the synovial fluid of subjects with acute knee injury. Moreover, increased aggrecanase cleavage in the aggrecan CS2 domain generates GRGT-G3, GLGS-G3 and AGEG-G3 fragments [88]. Figure reproduced with the kind permission of Dr. André Struglics. Cartilage can be divided into regions of different ECM organisation and molecular content, as well as chondrocyte organisation, shape and function throughout the depth of cartilage: the superficial (tangential), central (intermediate) and deep (radial) layers of cartilage [90]. The transitional zone between the cartilage and subchondral bone is called the calcified cartilage layer and it creates a barrier and 28 attachment site between cartilage and subchondral bone [91]. Detected differences in cell appearance and function between the different layers of cartilage include the following. (1) The number of chondrocytes per cartilage volume is higher in the superficial zone compared with deeper layers and the chondrocytes appear flattened and are aligned horizontally parallel to the joint surface [92, 93]. The superficial zone chondrocytes, furthermore, produce lubricin, as opposed to the deeper layers [94]. There is also a high content of collagen fibres running parallel to the cartilage surface, although there may be differences between weight- and non-weight-bearing regions (see below) [7]. (2) In the central layer, the chondrocytes are rounder and the proteoglycan content is higher compared with the superficial or deep zones [92, 95]. (3) In the deep layer, the collagen fibres are thicker and round chondrocytes are aligned in columns [90, 92]. In addition to these depth-related differences, the cartilage ECM also differs depending on the proximity to the chondrocytes. With an increasing distance from the cells, ECM is classified as pericellular, territorial and interterritorial, with apparent differences in ECM organisation and function [77]. Mesenchymal progenitor cells, which could have the potential to regenerate focal cartilage defects, have been found in cartilage [96]. Different cartilage regions in the knee joint, furthermore, show differences in cell and ECM organisation attributed to the different mechanical loads between regions. Rolauffs et al. [93] detected four distinct superficial zone chondrocyte patterns; strings, clusters, pairs and singles in the knee joint. Different joint surfaces of the knee were typically dominated by only one of these four patterns. The predominant pattern of the femoral condyles, meniscus-covered medial tibial plateau and patellofemoral grove were strings, pairs and clusters respectively. Central regions of the tibial plateau are associated with a less organised collagen fibre orientation, which could be a consequence of high compressive loads over this region. On the other hand, peripheral regions of the tibial plateau (i.e. beneath the menisci) are associated with a more organised collagen fibre orientation more parallel to the surface. This could be a consequence of the high tensile stresses to which these regions are exposed [7, 97]. Cartilage and knee osteoarthritis One of the main features of knee OA is the loss of articular cartilage. Over the years, much of the research on OA has therefore focused on the events involved in cartilage degradation. At cartilage level, OA development is associated with cartilage fissures, swelling, chondrocyte hypertrophy and phenotypic changes in the chondrocytes. As the disease progresses, cartilage thinning and the exposure of subchondral bone occur [51]. Several important clues to the way cartilage is affected during the different stages of OA development and progression have been 29 identified during the past few decades. Proteases able to degrade cartilage constituents have been highlighted and MMP-13 and ADAMTS-5 in particular [79]. In models using genetically modified mice, deleting the catalytic domain of ADAMTS -5 protects the cartilage from degradation [98]. Furthermore, knock-in induced resistance to cleavage at the wild type ARGS↓TEGE-aggrecan site significantly protects the cartilage from degradation [99]. Induced MMP-13 deficiency also inhibits cartilage degradation [100]. Underlining the complexity of the post-traumatic OA mouse model, at least 29 different genetic modifications have been shown to be protective of cartilage erosion and 19 have been shown to increase cartilage erosion in different OA models [101]. Human OA is characterised by a whole joint disease and inducing MMP-13 deficiency did not lead to reduced osteophyte formation in a mouse OA model [100]. Many other molecular mechanisms have been implicated in OA pathogenesis and recent studies have, for example, indicated important roles for tumour growth factor (TGF)-β and complement activation in OA pathogenesis [51, 102, 103]. Cartilage and post-traumatic knee osteoarthritis Acute knee injuries are associated with radiographic OA progression and joint space narrowing indicative of cartilage erosion (see above). One major limitation of using radiographic signs to detect cartilage injury is the relatively long time from injury until these changes occur and the fact that changes indicating cartilage loss, such as joint space narrowing, may be related to other factors, such as the integrity of the menisci [104]. Recent advances in MRI have led to an increase in our understanding of early post-traumatic changes in cartilage which may precede post-traumatic OA development [105, 106]. Compositional cartilage MRI (delayed gadolinium-enhanced imaging of cartilage (dGEMRIC), T1-rho and T2 mapping) can depict information on the composition of cartilage. Whereas dGEMRIC and T1rho may distinguish changes in cartilage proteoglycan content, T2 mapping relates to the cartilage water content and indirectly to type II collagen content and orientation [105]. Before the development of these techniques, cartilage biopsies revealed important information on changes in cartilage composition after ACL injury. Suggestive of collagenase activity, biopsy samples acquired from the non weight-bearing articular cartilage of the intercondylar notch show collagen denaturation and the cleavage of type II collagen less than and more than one year post-ACL rupture [107, 108]. Interestingly, an increase in cartilage GAG content was also observed [107]. At time points after one year, lower type II collagen content in the cartilage of the intercondylar notch was observed [108]. Biopsy of the cartilage adjacent to bone marrow lesions on the lateral femur showed a decrease in GAG in median 4.5 weeks after ACL injury [49]. In line with these findings, Li et al. [109] showed elevated T1rho values (indicating GAG loss), of 30 the posterolateral tibial cartilage in ACL-injured knees at baseline. Tiderius et al. [110] showed that the estimated GAG content (assessed by dGEMRIC) was lower in both the medial and lateral femoral cartilage within mean three weeks after ACL injury. At two-year follow-up, recovery but not normalization, was observed in the lateral tibifemoral compartment whereas the estimated GAG decrease in the medial tibiofemoral compartment seemed to remain at a constant low level [111]. Potter et al. [41] demonstrated MRI-detectable cartilage lesions at the time of ACL injury in 100% of patients, primarily affecting the lateral tibial plateau and secondly the lateral femur. A progressive increase in cartilage lesion severity was observed with time lapsed after injury [41]. Synovium The non-bony cavity of the knee joint is enclosed by the synovium. It consists of the intima, a continuous layer of macrophages and specialised synovial fibroblasts (synoviocytes), and the subintima, which is the underlying tissue. The subintima is a fibrous ECM where blood and lymph vessels, nerves, stationary fibroblasts and immune cells reside. The synovial fibroblasts have a distinct phenotype. Of particular importance to the joint environment, they experience high activity from an enzyme which converts UDP-glucose to UDP-glucoronate, an essential component for hyaluronan synthesis [112]. Furthermore, synovial fibroblasts, along with superficial zone chondrocytes, produce lubricin, which is particularly essential for the boundary lubrication of cartilage [113]. The main route for nutritional access to the avascular cartilage is passive diffusion from capillaries of the synovium. They are located in the subintima, just beneath the intima. The capillary endothelial cells form a size-selective barrier allowing the flux of water, nutrients and proteins into the joint cavity. The synovial fluid-to plasma ratio of plasma proteins decreases as the molecular radius of the protein increases [114]. Importantly, the cells of the intima are loosely connected and there is no basement membrane. As a result, the interstitial fluid of the synovium and the synovial fluid form an unbroken continuum [115]. The efflux of molecules from the knee joint occurs through drainage via the lymph vessels for molecules ~2 to ~10 nm in size. Smaller molecules, for example, some cytokines, can also access the circulation via diffusion into capillaries. Larger molecules, such as hyaluronan and large aggrecan fragments, may be restricted from efflux via the lymph vessels. At high intra-joint pressures, these large molecules could create a filter cake, increasing the outflow resistance across the synovium and limiting fluid escape from the joint [115, 116]. In the event of synovitis (see below), the 31 size selectivity of the endothelial barrier is decreased, leading to an increase in the influx of large proteins and joint effusion [117]. The meniscus The meniscus is a crescent-shaped fibro-cartilaginous tissue rich in collagen type I. The molecular constituents of the meniscus are quite different from those of cartilage. For example, compared with knee-joint hyaline cartilage, the collagen fibrils are thin, the amount of aggrecan and chondroadherin is low, whereas the amount of asporin is high [118]. The tensile strength and function of the menisci are related to the circumferentially oriented collagen fibres woven together with radial fibres [119]. In principle, the menisci play an important role in reducing cartilage loads by distributing the load between the femur and tibia over a larger area. Compromising this function, such as in the case of meniscus tear or meniscectomy, leads to increased peak and average loads over the tibiofemoral cartilage [120] and a high risk of OA [19, 121, 122]. The increased loading of tibial cartilage is closely related to both the amount and type of medial meniscectomy (anterior, posterior or longitudinal) [120]. The long-term outcome of ACL injury is closely related to the integrity of the menisci (see above). However, subjects with an ACL tear (isolated or combined) present with the first radiographic signs (joint space narrowing) of cartilage degradation approximately ten years prior to subjects with an isolated meniscal tear [123]. Not only the status of the menisci at index injury but also the risk of secondary meniscus injury are important for the risk of future tibiofemoral OA after ACL injury [124]. Meniscus injury also appears to be important when it comes to developing patellofemoral OA [125, 126]. The menisci and specifically the medial meniscus may contribute to joint stability and restrain anterior translation in the ACL-deficient knee but not in knees with an intact ACL [127, 128]. This renders the medial meniscus susceptible to tears in the ACL-deficient knee and this has been advocated as an important reason to perform ACL reconstructive surgery [129]. Bone The bony parts of the knee joint, the femur, tibia and patella, have a dense and compact outer structure, the cortical bone, and a porous inner structure, the trabecular bone. In the knee, trabecular bone and cartilage are separated by the 32 osteochondral plate; a thin layer of cortical bone (the subchondral plate) and the calcified cartilage layer [130]. Bone is a metabolically active tissue. After the peak bone mass has been reached at the age of 20-30 years, bone remodelling is characterised by a higher bone resorption rate compared with the corresponding bone formation. This results in a net loss of bone mass [131]. Bone and knee osteoarthritis Bone-related alterations associated with OA include bone marrow lesions, osteophytes, subchondral bone sclerosis, cyst formation, tidemark duplication and thickening of the calcified cartilage layer [130]. Microcracks through the osteochondral plate, ingrowth of nerves, vessels and endothelial proliferation within the non-calcified cartilage are associated with OA and could increase cross talk between cartilage and bone [132, 133]. These changes, in combination with changes in bone homeostasis, may induce cartilage erosion [103, 133, 134]. An important role for increased TGF-β signalling in the subchondral bone has been suggested [103]. It is unknown whether pathological changes in bone precede or follow osteoarthritic changes at molecular level in cartilage. The parallel progression of disease in both tissues is, however, a likely scenario. This was illustrated by the increase in both serum bone sialoprotein (BSP) and COMP in subjects with chronic knee pain who went on to develop early radiographic knee OA [135]. The serum concentrations of these proteins were, furthermore, higher in subjects with bone scan abnormalities [136]. Even though these studies indicate parallel alterations in cartilage and bone homeostasis during the course of early OA development, abnormal alterations in bone typically precede those in cartilage, as visualised by different imaging techniques. For example, osteophyte formation and scintigraphic changes indicative of increased bone turnover typically occur before joint space narrowing [137]. Changes in bone shape, furthermore, predict the onset of radiographic OA [138]. These differences could, however, be related to the higher metabolic rate in bone compared with that in cartilage and the more rapid response of bone to changes in joint load. Bone and post-traumatic knee osteoarthritis In line with OA seen in subjects without previous knee joint trauma, early signs of bone disturbance are also evident after severe knee injury. Bone mineral loss in the knee of ACL-injured subjects was demonstrated after ACL reconstruction [139]. 33 In a study population of 121 subjects followed prospectively after ACL injury, it was recently shown that bone shape changes of femur, tibia and patella could be detected by advanced analyses methods applied to sequential MR images already after two years. Preliminary analyses suggest that these changes could relate to osteophyte formation. [140]. In the same study population, changes in articular bone curvature were observed within three months of the injury [141]. Buckland Wright et al. [142] demonstrated thickening of horizontal trabeculae and a high prevalence of osteophytes in the medial tibial compartment within four years of ACL injury. Taken as a whole, these findings indicate changes in bone metabolism and remodelling at an early stage after ACL injury. In subjects with an acute knee injury, cartilage and bone involvement occurs instantly, as the tibia and femur collide, resulting in cartilage injury, bone marrow lesions and osteochondral fractures [41, 44, 49, 143]. Traumatic bone marrow lesions gradually decrease in size, but fewer than half resolve within one year after the injury [144]. In most studies, their presence has not been shown to correlate to clinical outcome [34, 145]. Anterior cruciate ligament-injured subjects with a bone marrow lesion of the medial tibia, a trabecular fracture of the lateral femur or a more severe intra-articular lesion, i.e. osteochondral fracture, may, however, have poorer clinical outcome scores [39, 146, 147]. Johnson et al. [146] observed that a trabecular fracture, by the authors termed a geographical bone bruise [47], on the lateral femur was associated with the increased size and duration of effusion, a time-lag in time until the normal range of motion was regained and increased pain in the acute phase of ACL injury. Inflammation Inflammation may play an important role in the pathogenesis of OA [148]. Indicative of this, synovitis, which has been shown to be reflected by increased plasma C-reactive protein (CRP) concentrations, is a prevalent finding in OA [149]. More importantly and underlining the importance of inflammation in OA, synovitis has been shown to be prognostic of MRI detected cartilage loss in subjects without radiographic OA [150]. Increased numbers of several inflammatory cell populations have been detected in the OA joint capsule, of which macrophages, T cells and mast cells are most abundant [151]. Of particular interest, mast cells have been found at higher cell numbers in OA compared to RA joint capsule and their mediators (histamine and tryptase) at higher concentrations in the synovial fluid in subjects with OA compared to those with RA [151, 152]. Inflammatory and synovial cells may contribute to joint synovial and cartilage 34 inflammation by producing cytokines, prostaglandins and proteases, such as neutrophil elastase, aggrecanases and different MMPs [153-157]. Proteases, which are essential for the degradation of cartilage molecules, can be induced and activated by inflammatory molecules. Numerous in vitro studies have demonstrated aggrecanase and MMP activity (including collagenase activity) and injurious effects on cartilage structure and chondrocyte function (survival) in the presence of pro-inflammatory cytokines [158-161]. In fact, adding tumour necrosis factor (TNF)-α to a cartilage explant in vitro can lead to the total dissolution of the explant mediated through the upregulation of proteases in the chondrocytes [74]. In vivo, in a mouse model, Malfait et al. [162] showed that the intra-articular administration of TNF-α led to the rapid degradation of the cartilage aggrecan by the induction of aggrecanase activity. The aggrecan degradation was reversible and these findings are in line with the work of others [163]. Typically, collagen degradation, which is irreversible in in vitro cartilage explant systems, does not begin until after several days of inflammatory stimuli and not until most aggrecan molecules have been degraded [79]. Further emphasising the protective role of intact aggrecan in the in vitro cartilage explant system, aggrecanase inhibition not only prevented interleukin (IL)-1-induced aggrecan degradation but also prevented collagen degradation [160]. However, in more complex systems, increased collagen degradation may parallel that of aggrecan. In a horse model, increased synovial fluid concentrations of the C2C epitope were detected within 24 hours of lipopolysaccharide-induced joint inflammation, suggestive of increased collagenase activity [164]. Also suggesting increased protease activity, cross linked peptides of type II collagen were detected in the synovial fluid early in the acute phase of knee injury [165]. The fact that a “soup” of several pro-inflammatory cytokines or molecules leads to more rapid cartilage degradation was furthermore observed when human and bovine cartilage explants were exposed to both interleukin (IL)-1 and plasminogen compared with when explants were exposed to IL-1 alone [166]. Moreover, the combined effects of TNF-α, IL-6 and sIL-6r on human and bovine knee cartilage explants caused more GAG release than the individual cytokines alone [161]. Inflammation may also mediate pain in OA. In a recent meta-analysis, it was concluded that subjects with OA have modestly higher circulating CRP levels than controls and that there is great variation in the observed associations between different studies [167]. Circulating CRP levels appeared to be associated more with symptoms than radiographic OA changes [167]. Moreover, synovitis is associated with symptoms including pain [168]. The fact that intra-articular corticosteroids supplied to osteoarthritic joints lead to a rapid reduction in joint pain further implicates inflammation as an important mediator of pain in OA [169]. Interestingly, this effect may be related to a reduction in synovial 35 inflammation [169]. It has, however, not been demonstrated that, by inhibiting knee joint inflammation, corticosteroids are able to reduce cartilage degradation. In a randomised controlled trial, investigating intra-articular hyaluronan treatment and intra-articular hyaluronan + corticosteroid treatment, no significant MRI progression of OA was observed in either study group during the one-year course of the study [170]. Prostaglandins may contribute to sustained joint inflammation and pain. As proof of this, subjects waiting for total knee replacement displayed a dose-responsive decrease in knee synovial fluid concentrations of TNF-α, vascular endothelial growth factor and IL-6 after two weeks of non-steroidal anti-inflammatory drug treatment [171]. Pain and knee function improvements were also observed. Molecules (intact or cleavage products) of cartilage ECM or yielded from chondrocytes are able to activate toll-like receptors and complement and thereby stimulate inflammation. It has been suggested that the inflammation associated with OA is mediated primarily through this mechanism [74, 102]. In subjects with OA who undergo total knee replacement, the symptoms are reduced after the operation, which could be related to the stopped release of cartilage molecules into the synovial fluid, leading to a reduced degree of synovial inflammation subsequent to the operation [74]. However, subjects with higher synovial fluid concentrations of inflammatory markers at the time of total knee replacement experience fewer improvements in pain outcomes following surgery, underpinning the importance of pathological hemostasis in joint tissues other than cartilage for the clinical symptoms associated with OA (for example, synovitis) [172]. Inflammation and post-traumatic knee OA The trauma inflicted on joint tissues at the time of an acute knee injury initiates an immune response. This response may be exaggerated by bleeding into the joint and the degree of acute hemarthrosis may be important for both acute and chronic cartilage degradation by activating inflammatory pathways [173]. Typically, acute tissue injury leads to the release of damage-associated molecular patterns (DAMPs). These molecules, intact or proteolytically processed ECM molecules like biglycan, tenacin-C and hyaluronic acid fragments, and cell-associated molecules, such as high-mobility group box 1 (HMGB1) and uric acid are able to activate immune cells by binding to pattern recognition receptors, including toll like receptors [174-176]. In response to this activation, the immune cells release a variety of different cytokines and chemokines that notify other cells, including macrophages and neutrophils, of ongoing tissue injury [177, 178]. In the acute phase of knee injury, these events can be monitored by analysing the synovial fluid. Rapid increases in synovial fluid concentrations of pro-inflammatory 36 cytokines and proteases such as elastase and MMP-3 have been observed [153, 179, 180]. At a later stage, T-cells that have been recruited to the site of injury, together with macrophages, may induce a state of chronic inflammation, depending on the present cytokine environment [178]. These courses of events after acute knee injury have not been extensively studied. However, clinical joint effusion and investigations of synovial fluid from acutely knee-injured subjects suggest an initial hyper-inflammatory state [146, 179-181]. Importantly, these events are controlled by anti-inflammatory cytokines and protease inhibitors which may maintain homeostasis during the pro-inflammatory stage and thus reduce the proteolytic activity in cartilage [166]. The fact that both IL-1Ra and the protease inhibitor, tissue inhibitor of matrix metalloprotease (TIMP)-1 are increased in the synovial fluid after acute knee injury indicates that this could be the case [179, 180, 182]. Underpinning the importance of inflammation in post-traumatic OA development, injections of corticosteroids into the joint of ACL-transected dogs during surgery and at different time points after surgery led to reduced osteophyte size and reduced histological severity of cartilage lesions [183]. IL-1ra administered intra articularly to subjects with recent ACL injury, furthermore, led to a reduction in pain and knee functional limitations, but it is not known whether such treatment can inhibit OA progression after ACL injury [184