Objectives. The objectives of this study were: 1) to examine osteophyte formation, subchondral bone advance, and bone marrow lesions (BMLs) in osteoarthritis (OA)-prone Hartley guinea pigs; and 2) to assess the disease-modifying activity of an orally administered phosphocitrate ‘analogue’, Carolinas Molecule-01 (CM-01). Methods. Young Hartley guinea pigs were divided into two groups. The first group (n = 12) had drinking water and the second group (n = 9) had drinking water containing CM-01. Three guinea pigs in each group were euthanized at age six, 12, and 18 months, respectively. Three guinea pigs in the first group were euthanized aged three months as baseline control. Radiological, histological, and immunochemical examinations were performed to assess cartilage degeneration, osteophyte formation, subchondral bone advance, BMLs, and the levels of matrix metalloproteinse-13 (MMP13) protein expression in the knee joints of hind limbs. Results. In addition to cartilage degeneration, osteophytes, subchondral bone advance, and BMLs increased with age. Subchondral bone advance was observed as early as six months, whereas BMLs and osteophytes were both observed mainly at 12 and 18 months. Fibrotic BMLs were found mostly underneath the degenerated cartilage on the medial side. In contrast, necrotic BMLs were found almost exclusively in the interspinous region. Orally administered CM-01 decreased all of these pathological changes and reduced the levels of MMP13 expression. Conclusion. Subchondral bone may play a role in cartilage degeneration. Subchondral bone changes are early events; formation of osteophytes and BMLs are later events in the OA disease process. Carolinas Molecule-01 is a promising small molecule candidate to be tested as an oral disease-modifying drug for human OA therapy. Cite this article: Y. Sun, A. J. Kiraly, A. R. Sun, M. Cox, D. R. Mauerhan, E. N. Hanley Jr. Effects of a phosphocitrate analogue on osteophyte, subchondral bone advance, and bone marrow lesions in Hartley guinea pigs. Bone Joint Res 2018;7:157–165. DOI:10.1302/2046-3758.72.BJR-2017-0253

Autologous micro-fragmented adipose tissue (MFAT) for the treatment of symptomatic knee osteoarthritis (OA) is gaining interest although there is still a lack of supportive data on safety and clinical efficacy. This study primarily aimed to identify patient- and pathology-related parameters to tighten patient selection criteria for future clinical MFAT application. Secondly, the overall (1) therapeutic response rate (TRR), (2) short-term clinical effect, (3) effect durability and (4) therapeutic safety was investigated at a minimal follow-up of 1 year. Sixty-four subjects (91 knees) with symptomatic knee OA (mild-severe on MRI) were enrolled in a prospective single-centre case series. Ethical approval was obtained from the local and academic ethical committee (#B300201733775). After liposuction, the adipose tissue was mechanically processed in a Lipogem® device which eventually produced 6–9cc MFAT. Subjects were clinically assessed by means of the KOOS, NRS, UCLA and EQ-5D at baseline and 1, 3, 6 and 12 months after injection. Adverse events were meticulously recorded. The TRR was defined according to the OMERACT-OARSI criteria. A baseline MRI was scored following the MOAKS system. Paired sample t-tests, independent t-test and Fischer's exact test were applied on appropriate variables. Multiple regression models were fit separately for patient-and pathology-specific factors. Significance level was set at α=0.05. The overall TRR was 66% at 3 months and 50% at 12 months after injection. Subgroup analysis revealed that specifically patients with no-mild bone marrow lesions (BML) had a TRR of 88% at 3 months and 75% at 12 months after MFAT injection. Therapy responders at these timepoints improved with 29.3±14.1 points and 30.8±15.3 points on KOOS pain, while non-responders deteriorated mildly. All clinical scores were significantly higher at follow-up compared to baseline (p<0.05). BMI (factor 0.17, p=0.002) and age (factor −0.48, p=0.048) were prognosticators for the TRR% at 1 month and for absolute KOOS pain improvement at 6 months, respectively. Posterior horn lesions (PHL) in the medial meniscus (p<0.001) and bone marrow lesions (p=0.003) were negative prognosticators for the TRR at respectively 6 and 12 months post-injection. An inflammatory reaction (pain, swelling or stiffness) to MFAT was reported in 79% knees and resolved spontaneously within 16.6±13.5 days after administration. The study showed a durable and satisfying TRR (up to 75% at 1 year in selected patients without BML) and clinical improvement after a single intra-articular injection with autologous MFAT. The availability of an index knee MRI is mandatory to select MFAT patients, preferably with no or mild BML and without PHL of the medial meniscus. High BMI and younger age are associated with better early outcomes. In comparison to other injection therapies such as cortisone, hyaluronic acid and PRP, MFAT appears very attractive with an effect durability of at least 1 year

Abstract. Introduction. Osteoarthritis (OA) is one of the lead causes of pain and disability in adults. Bone marrow lesions (BMLs) are one feature of subchondral bone involvement in OA. MRI images suggest changes in tissue content and properties in the affected regions however, it is not known if this alters the mechanical behavior of the bone, which could in turn affect OA progression. The aim of this study was to characterize the mechanical properties of BMLs, using a combined experimental and computational approach. Methods. Six human cadaveric patellae from donors aged 56–76 were used in this study; all exhibited BML regions under MRI. Bone plugs were taken from non-BML (n = 6) and BML (n = 7) regions within the patellae, with guidance from the MRI. The plugs were imaged at 82µm resolution using micro computed tomography (µCT) and tested under uniaxial compression. Finite element (FE) models were created for each plug from the µCT scans and morphological properties such as bone volume fraction (BV/TV) were also determined. The relationship between bone volume fraction and apparent modulus was investigated for both sample groups. Results. The BV/TV range was similar for the BML and non-BML groups (0.25–0.46 and 0.18–0.44) From the experimental tests, a moderate positive correlation was found between BV/TV and apparent modulus in the no BML group (r= 0.57) while no correlation was found in the BML group (r = −0.02). From the FE results, a different relationship between BV/TV and element elastic modulus was found for the BML and non-BML groups. Conclusions. The results of this study show that in regions of bone containing BMLs, bone volume fraction does not predict overall apparent modulus and has different relationship to local modulus, suggesting the BML associated tissue structural changes affect mechanical behavior. Funders: EPSRC

Abstract. Introduction. Osteoarthritis (OA) affects more than four million people in the UK alone. Bone marrow lesions (BMLs) are a common feature of subchondral bone pathology in OA. Both bone volume fraction and mineral density within the BML are abnormal. The aim of this study was to investigate the effect of a potential treatment (bone augmentation) for BMLs on the knee joint mechanics in cases with healthy and fully degenerated cartilage, using finite element (FE) models of the joint to study the effect of BML size. Methods. FE models of a human tibiofemoral joint were created based on models from the Open Knee project (simtk.org). Following initial mesh convergence studies, each model was manipulated in ScanIP (Synopsys-Simpleware, UK) to incorporate a BML 2mm below the surface of the tibial contact region. Models representing extreme cases (healthy cartilage, no cartilage; BML region as an empty cavity or filled with bone substitution material (200GPa)) were generated, each with different sizes of BML. Models were tested under a representative physiological load of 2kN. Results. In the absence of cartilage, the stress distribution through the bone was more localized with higher peaks in comparison to models with cartilage. In models with cartilage, BML cavity led to changes in the stress distribution through the tibia, with increasing BML size leading to higher stresses. When the BML region was represented by the substitution material very little difference was seen in comparison to models with no defect at all. Conclusions. The results of this study illustrate how the cartilage and bone behaviour in the tibiofemoral joint are linked, and that augmentation of a BML with a bone substitute has the potential to reduce adverse loading of the surrounding bone. Funders. EPSRC, NIHR. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction and Objective. Traditionally, osteoarthritis (OA) has been associated mostly with degradation of cartilage only. More recently, it has been established that other joint tissues, in particular bone, are also centrally involved. However, the link between these two tissues remains unclear. This relationship is particularly evident in post-traumatic OA (PTOA), where bone marrow lesions (BMLs), as well as fluctuating levels of inflammation, are present long before cartilage degradation begins. The process of bone-cartilage crosstalk has been challenging to study due to its multi-tissue complexity. Thus, the use of explant model systems have been crucial in advancing our knowledge. Thus, we developed a novel patellar explant model, to study bone cartilage crosstalk, in particular related to subchondral bone damage, as an alternative to traditional femoral head explants or cylindrical core specimens. The commonly used osteochondral explant models are limited, for our application, since they involve bone damage during harvest. The specifics aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response and mechanical stimulation to determine the subsequent developments of PTOA. Materials and Methods. Lewis rats (n=48) were used to obtain patellar and femoral head explants which were harvested under an institutional ethical approval license. Explants were maintained in high glucose media (containing supplements), under sterile culture conditions. Initially, we characterised undamaged patellar explants and compared them with the commonly used femoral head. First, tissue viability was assessed using an assay of metabolic activity and cell damage. Second, we created chemical and mechanical damage in the form of IL-1B treatment, and mechanical stimulation, to replicate damage. Standard biochemical assays, histological assays and microstructural assays were used to evaluate responses. For chemical damage, explants were exposed to 10ng/ml of IL-1B for 24 hours at 0, 1, 3 and 7 days after harvesting. For mechanical damage, tissues were exposed to mechanical compression at 0.5 Hz, 10 % strain for 10 cycles, for 7 days. Contralateral patellae served as controls. In both groups, sGAG, ADAMTS4, and MMP-13 were measured as an assessment of representative cartilage responses while ALP, TRAP and CTSK were assessed as a representative of bone responses. In addition to this, histomorphometric, and immunohistochemical, evaluations of each explant system were also carried out. Results. Our results confirm that the patellar explant system is an excellent ex vivo model system to study bone-cartilage crosstalk, and one which does not induce any bone damage at the time of tissue harvest. We successfully established culture conditions to maintain viability in these explants for up to 28 days. Rat IL-1B treatment resulted in increased both proteoglycan content and bone metabolism markers after 7 days when compared with the controls. To confirm this finding, qualitative immunohistochemical staining showed chondrocytes increased expression of MMP13 after treatment with IL-1B. Furthermore, we observed that the levels of ADAMTS4 decreased in 48 hours after IL-1B exposure. Contrastingly IL-1B treatment had the opposite effect on CTSK markers when compared with the control. Mechanically compressed patellae showed a decrease in compressive moduli from day 3 to day 7, suggesting that tissue remodelling may have taken place as a compensatory mechanism in response to damage. In addition, MMP13 release decreased over 48 hours after mechanical compression, while TRAP levels were increased compared with the control. Conclusions. Thus, we successfully demonstrated that IL-1B and mechanical stimulation affects both bone and cartilage tissues independently in this system, which may have relevance in the understanding of bone-cartilage crosstalk after injury and how this is involved in PTOA development

A subgroup of patients that undergo TKR surgery have evidence of neuropathic pain and central sensitization that may predispose to severe postoperative pain. This study assesses the correlation of MRI detected bone marrow lesions (BMLs) and synovitis with markers of neuropathic pain and central sensitization in patients undergoing TKR surgery and healthy volunteers. 31 patients awaiting TKR and 5 healthy volunteers were recruited. Each subject underwent a 3-T knee MRI scan that was graded for BMLs (0–45) and synovitis (0–3) using subsets of the MRI Osteoarthritis Knee Score (MOAKS). All subjects were asked to complete the PainDetect questionnaire to identify nociceptive pain (< 13), unclear pain (13–18) and neuropathic pain (>18). Correlation between BMLs and PainDetect score was the primary outcome measure. Secondary outcomes included the correlation of synovitis to PainDetect and temporal summation (TS) a measure of central sensitization to the PainDetect score. TS was determined using a monofilament to evoke pain. Pilot histological analysis of the prevalence of osteoclasts (TRAP. +. ) within BMLs versus normal subchondral bone was performed, implying a role in BML pathology. Increasing BML MOAKS score correlated with neuropathic pain (painDetect), r. s. = 0.38, p=0.013 (one-tailed). There was a positive correlation between synovitis and PainDetect score, τ =0.23, p= 0.031 (one-tailed). TS was greater in the neuropathic pain than in nociceptive pain patients, U = 18.0, p=0.003 (one-tailed). TRAP staining identified more osteoclasts within BMLs than contralateral condyle lesion free subchondral bone, z = −2.232, p = 0.026 (Wilcoxon Signed Rank Test, one-tailed). BMLs and synovitis are more prevalent in neuropathic pain and central sensitization in knee OA. Higher osteoclast prevalence was seen within BMLs which may help explain the association with BMLs and pain in OA

Painful OA is linked to CNS changes in pain processing. Temporal summation of pain (TSP) is a measure of one such CNS change, central sensitization. TSP is defined using a series (≥0.33Hz) of painful stimuli and is a predictor of postoperative pain, experienced by 20% of patients after total knee replacement (TKR) surgery. This study has developed a protocol to use functional MRI to assess CNS changes in OA pain processing. This pilot includes 3 participants with chronic knee OA pain awaiting TKR (62 ± 4.4) and 5 healthy volunteers (50 ± 13.6). 3-Tesla BOLD fMRI brain scans were recorded during short series of one second painful stimuli, applied using an automated inflatable cuff to the calf muscle of the leg with the affected knee or left side in healthy volunteers. The pain intensity at onset and during the 10 painful stimuli were recorded using a numerical rating scale. The pattern of brain activation was averaged across noxious stimuli, and the differential activation compared the 1st vs. 10th (last) stimulus. Bone marrow lesions (BMLs), synovitis and effusion size were scored from 3-Tesla knee MRI's using MOAKS scoring. TSP was raised in OA patients compared to control group (p=0.023). TSP brain activity in the chronic OA patients displayed higher signal within the subgenual anterior cingulate (sgACC) compared to healthy volunteers. Knee MRI identified OA patient's exhibited higher BML scores (p=0.038) and more knee effusion (p=0.018), but the lack of synovitis did not differ from control group (p=0.107). Enhanced TSP in chronic knee OA pain may be linked with augmented responses in emotional circuitry. BMLs and effusion size appear to contribute more with pain than synovitis. These results may help understand sensitization to improve outcomes for patients with knee OA undergoing TKR surgery

Bone marrow lesions (BMLs) have been extensively linked to the osteoarthritis (OA) disease pathway in the knee. Semi-quantitative evaluation has been unable to effectively study the spatial and temporal distribution of BMLs and consequently little is understood about their natural history. This study used a novel statistical model to precisely locate the BMLs within the subchondral bone and compare BML distribution with the distribution of denuded cartilage. MR images from individuals (n=88) with radiographic evidence of OA were selected from the Osteoarthritis Initiative. Slice-by-slice, subvoxel delineation of the lesions was performed across the paired images using the criteria laid out by Roemer (2009). A statistical bone model was fitted to each image across the cohort, creating a dense set of anatomically corresponded points which allowed BML depth, position and volume to be calculated. The association between BML and denudation was also measured semi-quantitatively by visually scoring the lesions as either overlapping or adjacent to denuded AC, or not. At baseline 75 subjects had BMLs present in at least one compartment. Of the 188 compartments with BMLs 46% demonstrated change greater than 727mm cubed, the calculated smallest detectable difference. The majority of lesions were found in medial compartments compared to lateral compartments and the patella (Figure 1A). Furthermore, in the baseline images 76.9% of all BMLs either overlapped or were adjacent to denuded bone. The closeness of this relationship in four individuals is shown in Figure 1B. The distribution of lesions follows a clear trend with the majority found in the patellofemoral joint, medial femoro-tibial joint and medial tibial compartment. Moreover the novel method of measurement and display of BMLs demonstrates that there is a striking similarity between the spatial distribution of BMLs and denuded cartilage in subjects with OA. This co-location infers the lesions have a mechanical origin much like the lesions that occur in healthy patients as a direct result of trauma. It is therefore suggested that OA associated BMLs are in fact no different from the BMLs caused by mechanical damage, but occur as a result of localised disruption to the joint mechanics, a common feature of OA

Summary Statement. OA knee with subchondral cyst formation presented differential microstructure and mechanical competence of trabecular bone. This finding sheds light on the pivot role of subchondral cyst in OA bone pathophysiology. Introduction. Subchondral bone cyst (SBC) is a major radiological finding in knee osteoarthritis (OA), together with joint space narrowing, osteophyte and sclerotic bone formation. There is mounting evidence showing that SBC originates in the same region as bone marrow lesions (BMLs). The presence of subchondral bone cyst (SBCs), in conjunction with BMLs, was associated with the severity of pain, and was able to predict tibial cartilage lolume loss and risk of joint replacement surgery in knee OA patient. It is speculated that the presence of SBCs might increase intraosseous pressure of subchondral bone, and trigger active remodeling and high turnover of surrounding trabecular bone. Yet the exact effect of SBC on the structural and mechanical properties trabecular bone, which provides the support to overlying articular cartilage, remains to be elucidated. Therefore, this study aimed to investiate the microstructure and mechanical competence of trabecular bone of knee OA in presence or absence of SBC. Patients & Methods. A total of 20 postmenopausal women (54–87 years old) with the late-stage of primary knee OA were recruited in this study. Tibial plateau specimens were collected during joint replacement surgery. The samples were grouped for comparison according to presence or absences of SBC in micro-CT images. For micro-CT examination, a cylindrical volume of region of interest (VOI) of 10mm in diameter and 1mm in height was used to cover the trabecular bone region surrounding SBC, and then a cubic VOI of 3.5×3.5×3.5mm. 3. was applied in different anatomic locations of tibial plateau, such as medial, intermediate and lateral part, for the analyses of trabecular bone microstructure. Subsequently, two cylinders of subchondral bone specimens were drilled for each sample with micro-CT guidance from lateral portion of cystic wall along the direction of physiological loading of knee joint. The specimens were processed for micro-CT and mechanical testing using MTS 858 Mini Bionix sequentially. Each specimen was compressed in a longitudinal direction at a speed of 1mm/minute; the ultimate strength and modulus of the specimens were generated. Comparisons of microstructure and mechanical properties of trabecular bone were performed between two groups using student t test. The structure-mechanics relationship was also investigated using Pearson correlation. Results. The bone volume fraction (BV/TV, %) was significantly higher in knee OA specimens in presence of SBC (32±7%) in comparison with those in absence of SBC (16±5%, p<0.001). Meanwhile there were more plate-like trabecular bone surrounding SBC (0.78±0.61) than those without SBC (1.81±0.28, p<0.001), which was indicated by structure model index (0∼3). Furthermore, the trend in conversion of rod-like (close to 3) towards plate-like trabeculae was noticed in different locations of knee OA specimens with SBC formation. Trabecular bone around SBC presented higher modulus (73±22MPa) compared with those without SBC (45±29MPa, p=0.034). The stiffer trabecular bone in presence of SBC correlated with its plate-like morphology (r=0.696, p<0.001) as well as bone volume fraction (r=0.578, p=0.004). Conclusion. Presence of SBC was associated with conversion of trabeculae towards plate-like morphology together with the increase of mechanical competence in advanced knee OA

Introduction. Anteromedial osteoarthritis of the knee (anteromedial gonarthrosis-AMG) is a common form of knee arthritis. In a clinical setting, knee arthritis has always been assessed by plain radiography in conjunction with pain and function assessments. Whilst this is useful for surgical decision making in bone on bone arthritis, plain radiography gives no insight to the earlier stages of disease. In a recent study 82% of patients with painful arthritis had only partial thickness joint space loss on plain radiography. These patients are managed with various surgical treatments; injection, arthroscopy, osteotomy and arthroplasty with varying results. We believe these varying results are in part due to these patients being at different stages of disease, which will respond differently to different treatments. However radiography cannot delineate these stages. We describe the Magnetic Resonance Imaging (MRI) findings of this partial thickness AMG as a way of understanding these earlier stages of the disease. Method. 46 subjects with symptomatic partial thickness AMG underwent MRI assessment with dedicated 3 Tesla sequences. All joint compartments were scored for both partial and full thickness cartilage lesions, osteophytes and bone marrow lesions (BML). Both menisci were assessed for extrusion and tear. Anterior cruciate ligament (ACL) integrity was also assessed. Osteophytes were graded on a four point scale in the intercondylar notch and the lateral margins of the joint compartments. Scoring was performed by a consultant radiologist and clinical research fellow using a validated MRI atlas with consensus reached for disagreements. The results were tabulated and relationships of the interval data assessed with linear by linear Chi2 test and Pearson's Correlation. Results. All cases had medial femoral cartilage loss; 22% partial and 78% full thickness. 79% showed medial tibial loss, however in no cases was there medial tibial loss without femoral loss. 10 cases had lateral compartment partial thickness cartilage loss. Again, there was no tibial loss without femoral loss present. Increasing size of intercondylar notch osteophyte is associated with increasing ACL damage (p=0.001). Independent to this, increasing ACL damage is associated with lateral femoral condyle cartilage loss (p=0.002). Throughout the knee the incidence of BMLs increased with increasing cartilage loss (p=0.025). Only 13% of medial menisci were normal. As meniscal damage increases, so does the incidence of BMLs in the same compartment (p=0.03). Discussion. We describe the MRI findings of early AMG with partial thickness joint space loss. In all cases there was medial femoral loss, either with or without tibial loss. We believe the disease begins on the medial femoral condyle and progresses through the joint in stages. Later stages are associated with damage to the other structures in the knee, such as the meniscus and the ACL. Damage to the ACL is associated with increasing osteophytosis. This description is the first step in describing the stages of early AMG. Description of these stages is important since we believe the outcome of surgical intervention may be dependant on these and they may guide future therapy