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ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/β-catenin signalling, given its emerging importance in arthritis.MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types.ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/β-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hilining fibroblasts. Trajectory analyses predicted that Prg4hilining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes.ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hilining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.

Summary Objective Joint injury-induced perturbations to the endocannabinoid system (ECS), a regulator of both inflammation and nociception, remain largely uncharacterized. We employed a mouse model of ACL rupture to assess alterations to nociception, inflammation, and the ECS while using in vitro models to determine whether CB2 agonism can mitigate inflammatory signaling in macrophages and fibroblast-like synoviocytes (FLS). Design Mice underwent noninvasive ACL rupture (ACLR) via tibial compression-based loading. Nociception was measured longitudinally using mechanical allodynia and knee hyperalgesia testing. Synovitis was assessed using histological scoring and histomorphometry. Gene and protein markers of inflammation were characterized in whole joints and synovium. Immunohistochemistry assessed injury-induced alterations to CB1+, CB2+, and F4/80+ cells in synovium. To assess whether CB2 agonism can inhibit pro-inflammatory macrophage polarization, murine bone marrow-derived macrophages (mBMDM) were stimulated with IL-1β or conditioned medium from IL-1β-treated FLS and treated with vehicle (DMSO), the CB2 agonist HU308, or cannabidiol (CBD). Macrophage polarization was assessed as the ratio of M1-associated (IL1b, MMP1b, and IL6) to M2-associated (IL10, IL4, and CD206) gene expression. Human FLS (hFLS) isolated from synovial tissue of OA patients were treated with vehicle (DMSO) or HU308 following TNF-α or IL-1β stimulation to assess inhibition of catabolic/inflammatory gene expression. Results ACLR induces synovitis, progressively-worsening PTOA severity, and an immediate and sustained increase in both mechanical allodynia and knee hyperalgesia, which persist beyond the resolution of molecular inflammation. Enrichment of CB2, but not CB1, was observed in ACLR synovium at 3d, 14d, and 28d, and CB2 was found to be associated with F4/80 (+) cells, which are increased in number in ACLR synovium at all time points. The CB2 agonist HU308 strongly inhibited mBMDM M1-type polarization following stimulation with either IL-1β or conditioned medium from IL-1β-treated mFLS, which was characterized by reductions in Il1b, Mmp1b, and Il6 and increases in Cd206 gene expression. Cannabidiol similarly inhibited IL-1β-induced mBMDM M1 polarization via a reduction in Il1b and an increase in Cd206 and Il4 gene expression. Lastly, in OA hFLS, HU308 treatment inhibited IL-1β-induced CCL2, MMP1, MMP3, and IL6 expression and further inhibited TNF-α-induced CCL2, MMP1, and GMCSF expression, demonstrating human OA-relevant anti-inflammatory effects by targeting CB2. Conclusions Joint injury perturbs the intra-articular ECS, characterized by an increase in synovial F4/80(+) cells, which express CB2, but not CB1. Targeting CB2 in murine macrophages and human FLS induced potent anti-inflammatory and anti-catabolic effects, which indicates that the CB2 receptor plays a key role in regulating inflammatory signaling in the two primary effector cells in the synovium. The intraarticular ECS is therefore a potential therapeutic target for blocking pathological inflammation in future disease-modifying PTOA treatments.

Objectives: Patients with Femoroacetabular impingement (FAI) have been shown to have elevated circulating biomarkers of inflammation and cartilage catabolism, but little is known about whether corrective surgical intervention improves objective measures of hip strength and inflammation. This study measured several variables including pain, function, quality of life, symptoms, hip strength, and inflammatory and cartilage biomarkers in a cohort of subjects undergoing FAI surgery and postoperative (postop) rehabilitation. We hypothesized that surgical FAI repair and completion of a postop rehabilitation program will significantly improve patient reported outcomes (PROs), increase hip flexion and abduction strength measures, and decrease biomarkers of inflammation and cartilage degradation. Methods: This study was approved by the University of Michigan Medical School IRB (HUM00055335). Male and female patients aged 16-40 years of age, with symptomatic FAI diagnosed by concordant history, physical examination, and imaging findings were eligible for inclusion. Subjects who have undergone previous hip surgery, have a global overcoverage deformity, dysplasia, or degenerative chondral changes greater than Tonnis grade I were excluded. A single, fellowship trained, sports medicine orthopaedic surgeon performed all surgical procedures, and patients underwent a standardized postop rehabilitation program. HOOS questionnaires were assessed at each subject’s preoperative visit (preop), and at 2, 14, 26, and 52 weeks postop. Hip flexion, abduction, and extension were measured at each time point using a BioDex isokinetic dynamometer. High sensitivity C-reactive protein (HS-CRP) and cartilage oligomeric matrix protein (COMP) were measured in plasma using ELISAs. Differences between preop and postop time points were assessed using a mixed-effects model, and data are reported as mean±95% CI. Results: A total of 11 males and 6 females completed the study. Mean age of subjects was 25.2±3.8 years (range 16.0-38.0 years) and BMI of 24.2±2.0 kg/m2 (range 18.1-33.6 kg/m2). For most HOOS subscales, there was a decrease in score at 2 weeks postop, and by 14 weeks activities of daily living (ADL) improved by 18%, pain by 34%, quality of life by 69%, sport and recreation by 36%, and symptoms by 28% compared to preop values (Figure 1A-E). These improvements in HOOS subscales persisted for the duration of the study period (Figure 1A-E). No differences in hip flexion strength were observed, while extension was 20% and 31% significantly higher than preop values at 26 and 52 weeks, and for abduction patients were 34% stronger than preop values at 52 weeks. No differences in HS-CRP or COMP were observed (Figure 1F-J). Conclusion: In support of our hypothesis, we observed significant improvements in PROs of hip function, although changes in PROs preceded improvements in objective measures of hip extension and abduction strength. Despite improvements in other planes, no changes in hip flexion were observed. Plasma biomarkers of hip inflammation and cartilage turnover were not reduced by surgical intervention and rehabilitation. FAI surgery and postop rehabilitation is effective at treating pain, and improving patient reported quality of life and symptoms, with modest improvements in hip strength. However, the surgical correction of pathological hip anatomy and improvements in hip strength did not lead to significant differences in markers of inflammation or cartilage turnover.

Although numerous spinal biologics are commercially available, a cost-effective and safe bone graft substitute material for spine fusion has yet to be proven. In this study, “3D-Paints” containing varying volumetric ratios of hydroxyapatite (HA) and human demineralized bone matrix (DBM) in a poly(lactide-co-glycolide) elastomer were three-dimensional (3D) printed into scaffolds to promote osteointegration in rats, with an end goal of spine fusion without the need for recombinant growth factor. Spine fusion was evaluated by manual palpation, and osteointegration and de novo bone formation within scaffold struts were evaluated by laboratory and synchrotron microcomputed tomography and histology. The 3:1 HA:DBM composite achieved the highest mean fusion score and fusion rate (92%), which was significantly greater than the 3D printed DBM-only scaffold (42%). New bone was identified extending from the host transverse processes into the scaffold macropores, and osteointegration scores correlated with successful fusion. Strikingly, the combination of HA and DBM resulted in the growth of bone-like spicules within the DBM particles inside scaffold struts. These spicules were not observed in DBM-only scaffolds, suggesting that de novo spicule formation requires both HA and DBM. Collectively, our work suggests that this recombinant growth factor-free composite shows promise to overcome the limitations of currently used bone graft substitutes for spine fusion. IMPACT STATEMENT: Currently, there exists a no safe, yet highly effective, bone graft substitute that is well accepted for use in spine fusion procedures. With this work, we show that a three-dimensional printed scaffold containing osteoconductive hydroxyapatite and osteoinductive demineralized bone matrix that promotes new bone spicule formation, osteointegration, and successful fusion (stabilization) when implemented in a preclinical model of spine fusion. Our study suggests that this material shows promise as a recombinant growth factor-free bone graft substitute that could safely promote high rates of successful fusion and improve patient care.

Background: Owing to the debilitating nature of degenerative disc disease (DDD) and other spine pathologies, significant research has been performed with the goal of healing or regenerating the intervertebral disc (IVD). Structural complexity, coupled with low vascularity and cellularity, make IVD regeneration an extremely challenging task. Methods: Tissue engineering-based strategies utilize three components to enhance tissue regeneration; scaffold materials to guide cell growth, biomolecules to enhance cell migration and differentiation, and cells (autologous, or allogeneic) to initiate the process of tissue formation. Significant advances in IVD regeneration have been made utilizing these tissue engineering strategies. Results: The current literature demonstrates that members of the transforming growth factor beta (TGF-β) superfamily are efficacious in the regeneration of an anabolic response in the IVD and to facilitate chondrogenic differentiation. Gene therapy, though thwarted by safety concerns and the risk of ectopic transfection, has significant potential for a targeted and sustained regenerative response. Stem cells in combination with injectable, biocompatible, and biodegradable scaffolds in the form of hydrogels can differentiate into de novo IVD tissue and facilitate regeneration of the existing matrix. Therapies that address both anabolism and the inherent catabolic state of the IVD using either direct inhibitors or broad-spectrum inhibitors show extensive promise. Conclusion: This review article summarizes the genetic and molecular advances that promise to play an integral role in the development of new strategies to combat DDD and promote healing of injured discs.

Reverse total shoulder arthroplasty (RTSA) allows the deltoid to substitute for the nonfunctioning rotator cuff. To date, it is unknown whether preoperative deltoid and rotator cuff parameters correlate with clinical outcomes.We asked whether associations exist between 2-year postoperative results (ROM, strength, and outcomes scores) and preoperative (1) deltoid size; (2) fatty infiltration of the deltoid; and/or (3) fatty infiltration of the rotator cuff.A prospective RTSA registry was reviewed for patients with cuff tear arthropathy or massive rotator cuff tears, minimum 2-year followup, and preoperative shoulder MRI. Final analysis included 30 patients (average age, 71±10 years; eight males, 22 females). Only a small proportion of patients who received an RTSA at our center met inclusion and minimum followup requirements (30 of 222; 14%); however, these patients were found to be similar at baseline to the overall group of patients who underwent surgery in terms of age, gender, and preoperative outcomes scores. The cross-sectional area of the anterior, middle, and posterior deltoid was measured on axial proton density-weighted MRI. Fatty infiltration of the deltoid, supraspinatus, infraspinatus, teres minor, and subscapularis were quantitatively assessed on sagittal T1-weighted MR images. Patients were followed for Constant-Murley score, American Shoulder and Elbow Surgeons (ASES) scores, subjective shoulder value, pain, ROM, and strength. Correlations of muscle parameters with all outcomes measures were calculated.Preoperative deltoid size correlated positively with postoperative Constant-Murley score (67.27±13.07) (ρ=0.432, p=0.017), ASES (82.64±14.25) (ρ=0.377; p=0.40), subjective shoulder value (82.67±17.89) (ρ=0.427; p=0.019), and strength (3.72 pounds±2.99 pounds) (ρ=0.454; p=0.015). Quantitative deltoid fatty infiltration (7.91%±4.32%) correlated with decreased postoperative ASES scores (ρ=-0.401; p=0.047). Quantitative fatty infiltration of the infraspinatus (30.47%±15.01%) correlated with decreased postoperative external rotation (34.13°±16.80°) (ρ=-0.494; p=0.037).Larger preoperative deltoid size correlates with improved validated outcomes scores, whereas fatty infiltration of the deltoid and infraspinatus may have deleterious effects on validated outcomes scores and ROM after RTSA. The current study is a preliminary exploration of this topic; future studies should include prospective enrollment and standardized MRI with a multivariate statistical approach. Quantitative information attained from preoperative imaging not only holds diagnostic value, but, should future studies confirm our findings, also might provide prognostic value. This information may prove beneficial in preoperative patient counseling and might aid preoperative and postoperative decision-making by identifying subpopulations of patients who may benefit by therapy aimed at improving muscle properties.Level III, prognostic study.

OBJECTIVE: To determine if T1ρ magnetic resonance imaging (T1ρ MRI) could assess early articular cartilage changes in knees of asymptomatic female collegiate athletes. It was hypothesized that impact cohort would demonstrate greater changes than nonimpact cohort. DESIGN: An institutional review board–approved prospective cohort study. Blinded MRI analyses. SETTING: Participants from collegiate athletic program. Imaging at university hospital, February 2008 to July 2009. PARTICIPANTS: Inclusion criteria were female collegiate athletes in athletic season and asymptomatic. Exclusion criteria were previous/current knee injuries/surgeries. Twenty-one female NCAA Division I athletes, 11 impact (basketball players) and 10 nonimpact (swimmers) participants were consented and imaged with 3.0-T MRI (Siemens) and T1ρ sequence (University of Pennsylvania). One patient was removed (injury diagnosis). Final roster was 10 impact and 10 nonimpact participants. No difference in cohort body mass index, height, or weight. MAIN OUTCOME MEASURES: Average T1ρ relaxation times (ART) for patellar and femoral cartilage to analyze defined regions and depth and modified International Cartilage Repair Society classification. RESULTS: Statistical analyses showed that ART of radial zone of central third weight-bearing region of cartilage in basketball players was significantly greater (P = 0.041) than swimmers and ART of the superficial zone in basketball players was significantly less (P = 0.003) than that of swimmers. For both groups, the ART of super-ficial zones were significantly greater than that of radial zones (P < 0.001). Four impact athletes showed macroscopic changes (none in nonimpact cohort). CONCLUSIONS: T1ρ MRI detected early changes in articular cartilage of asymptomatic collegiate female impact athletes, with significant differences between cohorts in radial zone of central third weight-bearing region and superficial zones ART. Both cohorts showed increased ART in superficial zone. Four impact athletes showed macroscopic changes. CLINICAL RELEVANCE: This study demonstrates a quantitative MRI sequence able to detect signal differences in articular cartilage in asymptomatic athletes.