Post-traumatic Osteoarthritis Severity Research Articles

Single Cell Profiling Reveals Unique Contributions of Senescent Macrophages across Musculoskeletal Tissues in Posttraumatic Osteoarthritis

Injury to the acute cruciate ligament (ACL) disrupts musculoskeletal tissue architecture and function, leading to atrophy and weakness associated with the development of posttraumatic osteoarthritis (PTOA). The objective of this study was to profile senescent cell burden across skeletal muscle and knee joint capsule compartments and to ascertain the effectiveness of senolytic treatment in mitigation of PTOA. Given the unique features of these tissues, we hypothesized that composition and physiological consequence of senescent cell burden would differ among muscle and joint capsule, and senolytic treatment would prevent cartilage degradation and improve muscle function. We employed a surgical ACL transection model (ACLT) and performed scRNAseq analysis on the quadriceps and joint capsule 14-days post-injury and discerned senescent status by SPiDER-βGal. We also examined the influence of senescent cells on neighboring populations in vitro. We investigated whether senolytic treatment (dasatinib and quercetin; D+Q) improves muscle and joint health 28-d post-injury. scRNAseq revealed that macrophages (MΦ) accounted for 83% of senescent cells in the quadriceps and 48% in the joint capsule. Senescent MΦ exhibited an alternatively activated phenotype (M2) . Gene ontology enrichment analysis in the quadriceps showed downregulation of positive regulation of muscle cell differentiation, and upregulation of secretory granule lumen; in the joint capsule, it revealed upregulation of glycosaminoglycan catabolic process and regulation of ossification. In vitro, we found that mesenchymal progenitors incubated with senescent MΦ conditioned media exhibited upregulation of senescence associated secretory phenotype (SASP) genes and extracellular matrix organization-related genes ( i.e.Col1a1 and Lox). We found upregulation of protein glycosylation and downregulation of chondroitin sulfate proteoglycan biosynthetic process pathways. PTOA mice exhibited significant reductions in quadriceps myofiber cross-sectional area, increased accumulation of M2-like Cd163+ MΦ, and increased fibrosis, which were blunted in D+Q-treated mice. D+Q-treated PTOA mice exhibited a trend of improved peak torque compared to vehicle-treated PTOA mice. Histochemical assessment of whole knee joints revealed compromised articular cartilage and moderate to severe PTOA in vehicle-treated mice, which was blunted in D+Q-treated mice. These findings reveal distinct transcriptional profiles of senescent macrophages isolated from unique musculoskeletal tissues, providing critical insight into the heterogeneity of senescent cell programming following traumatic injury. Further, we show that accumulation of senescent alternatively-activated MΦ likely contributes to musculoskeletal dysfunction and development of PTOA, and that senolytics may improve functional recovery. AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Primary Ankle Fusion Using an Antegrade Nail Into the Talus for Early Treatment of OTA Type C3 Distal Tibial Plafond Fractures: A Preliminary Report.

Type C3 distal tibial plafond fractures consistently show poor outcomes with high complication rates and significant risk of posttraumatic arthritis. We describe a minimally invasive technique of performing a primary ankle fusion using an anterograde tibial nail and compare our early results to traditional methods of fixation. During the acute admission, the patient undergoes an arthroscopic preparation of the ankle joint and insertion of an anterograde nail into the talus. This technique is described in detail and presented alongside a retrospective 5-year review of all adult C3 distal tibial plafond fractures from our center. Twenty-six patients (8 open fractures) had been fixed traditionally using open reduction internal fixation (24 patients) and circular frames (2 patients) with an average follow-up of 20 months. Those internally fixed had protected weightbearing for 3 months. Complications included deep infection (12%), nonunion (8%), malunion (4%), severe posttraumatic osteoarthritis (27%), and the secondary conversion to ankle replacement/fusion (12%) requiring an average of 3 reoperations.Six patients underwent primary fusion (3 open fractures) with an average follow-up of 18 months. Patients were allowed to immediately weightbear. There were no reported complications and the primary fusion group demonstrated shorter hospital stays, faster return to work, and higher mean self-reported foot and ankle score (SEFAS) compared to those treated with ORIF. C3 distal tibial plafond fractures are difficult to manage and there has not been a satisfactory method of treating them that allows early return to work, has a low risk of complications, and reduces the risk of posttraumatic tibiotalar arthritis.We present our initial results with a method that uses traditional arthroscopic techniques to prepare the tibiotalar joint together with minimally invasive anterograde tibiotalar nailing. In this initial report of a small group of patients, we found that surgery can be performed once the swelling has subsided after injury and that allowing weightbearing as tolerated did not appear to have a negative effect on initial outcomes. Level III, retrospective comparative study.

GDF8 inhibition enhances musculoskeletal recovery and mitigates posttraumatic osteoarthritis following joint injury.

Musculoskeletal disorders contribute substantially to worldwide disability. Anterior cruciate ligament (ACL) tears result in unresolved muscle weakness and posttraumatic osteoarthritis (PTOA). Growth differentiation factor 8 (GDF8) has been implicated in the pathogenesis of musculoskeletal degeneration following ACL injury. We investigated GDF8 levels in ACL-injured human skeletal muscle and serum and tested a humanized monoclonal GDF8 antibody against a placebo in a mouse model of PTOA (surgically induced ACL tear). In patients, muscle GDF8 was predictive of atrophy, weakness, and periarticular bone loss 6 months following surgical ACL reconstruction. In mice, GDF8 antibody administration substantially mitigated muscle atrophy, weakness, and fibrosis. GDF8 antibody treatment rescued the skeletal muscle and articular cartilage transcriptomic response to ACL injury and attenuated PTOA severity and deficits in periarticular bone microarchitecture. Furthermore, GDF8 genetic deletion neutralized musculoskeletal deficits in response to ACL injury. Our findings support an opportunity for rapid targeting of GDF8 to enhance functional musculoskeletal recovery and mitigate the severity of PTOA after injury.

Antibiotic Treatment Prior to Injury Abrogates the Detrimental Effects of LPS in STR/ort Mice Susceptible to Osteoarthritis Development.

Post traumatic osteoarthritis (PTOA) is a form of secondary osteoarthritis (OA) that develops in ~50% of cases of severe articular joint injuries and leads to chronic and progressive degradation of articular cartilage and other joint tissues. PTOA progression can be exacerbated by repeated injury and systemic inflammation. Few studies have examined approaches for blunting or slowing down PTOA progression with emphasis on systemic inflammation; most arthritis studies focused on the immune system have been in the context of rheumatoid arthritis. To examine how the gut microbiome affects systemic inflammation during PTOA development, we used a chronic antibiotic treatment regimen starting at weaning for 6 weeks before anterior cruciate ligament (ACL) rupture in STR/ort mice combined with lipopolysaccharide (LPS)-induced systemic inflammation. STR/ort mice develop spontaneous OA as well as a more severe PTOA phenotype than C57Bl/6J mice. By 6 weeks post injury, histological examination showed a more robust cartilage staining in the antibiotic-treated (AB) STR/ort mice than in the untreated STR/ort controls. Furthermore, we also examined the effects of AB treatment on systemic inflammation and found that the effects of LPS administration before injury are also blunted by AB treatment in STR/ort mice. The AB- or AB+LPS-treated STR/ort injured joints more closely resembled the C57Bl/6J VEH OA phenotypes than the vehicle- or LPS-treated STR/ort, suggesting that antibiotic treatment has the potential to slow disease progression and should be further explored therapeutically as prophylactic post injury. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

PRE-EXISTING KNEE OSTEOARTHRITIS AND JOINT DEPRESSION ARE ASSOCIATED WITH TOTAL KNEE ARTHROPLASTY REQUIREMENT AFTER TIBIAL PLATEAU FRACTURE IN PATIENTS ≥60 YEARS OF AGE

We aimed to determine the rate of and risk factors for post-traumatic osteoarthritis (PTOA) and total knee arthroplasty (TKA) requirement after operative management of tibial plateau fractures (TPF) in older adults.We conducted a retrospective cohort study of 182 operatively managed TPFs in 180 patients ≥60 years old over a 12-year period with minimum follow up 1 year. Data including patient demographics, clinical frailty scores, mechanism of injury, management, reoperation and mortality were recorded. Radiographs were reviewed for: Schatzker classification; pre-existing knee osteoarthritis (KOA); severe joint depression >15mm; and development of PTOA. Kaplan Meier survival analysis was performed. Regression analysis was used to identify risk factors for radiographic indication for TKA and actual TKA.Forty-seven percent were Schatzker II fractures. Radiographic KOA was present at fracture in 32.6%. Fracture fixation was performed in 95.6% cases and acute TKA in 4.4%. Thirteen patients underwent late TKA (7.5%). At five-years, 11.8% (6.0-16.7 95% CI) had required TKA and 20.9% (14.4-27.4 95% CI) had a radiographic indication for TKA. Severe joint depression and pre-existing KOA were associated with worse survival for endpoints radiographic indication for TKA and actual TKA. Severe joint depression (HR 2.49(1.35-4.61 95% CI), p=0.004), pre-existing KOA (HR 2.23(1.17-4.23), p=0.015) and inflammatory arthropathy (HR 2.4(1.04-5.53), p=0.039) were independently associated with radiographic indication for TKA.In conclusion, severe joint depression and pre-existing arthritis are independent risk factors for both severe PTOA and TKA after TPFs in older adults. These features should be considered as an indication for primary management with acute TKA.

Inhibition of CaMKK2 Decreases Progression of Post-traumatic Osteoarthritis in a Rabbit ACL Transection Model

Background and Hypothesis:Post-traumatic osteoarthritis (PTOA) is a multifactorial degenerative disease of the joint affecting 20-50% of all joint injuries with a total annual cost of $15 billion. There are no current disease-modifying therapies for PTOA. Mechanical stress due to ligament tear or impact injury triggers the release of inflammatory mediators in the joint. Resulting collagen damage, loss of proteoglycans, and cell death triggers further release of inflammatory mediators and reactive oxygen species. This cycle of inflammation leads to PTOA. We hypothesize that inhibition of Ca2+/CaM dependent protein kinase kinase 2 (CaMKK2), a kinase associated with the inflammatory effects in PTOA, will mitigate the disease-propagating mechanisms.
 Methods:We utilized a rabbit model of PTOA which involved surgical transection of the anterior cruciate ligament (ACL) to generate joint instability. Rabbits were then treated tri-weekly with either STO-609 (CaMKK2 inhibitor, 0.033 mg/kg) or saline (control) for 16 weeks. Rabbits were sacrificed at 16 weeks post-surgery. Tibiofemoral joints were harvested for staining with safranin O fast green (SO) and PTOA grading via Osteoarthritis Research Society International (OARSI) guidelines. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RNA isolation of cartilage and subchondral bone tissue was conducted for qRT-PCR. Gene expression of MMP-13, IL-6, IL-1B, ACAN, COL2A1 was quantified and normalized to GAPDH.
 Results:Histology and gross morphology showed increased PTOA severity in saline controls compared to STO-609 treated rabbits. There was no significant difference in chondrocyte apoptosis in STO-609 treated rabbits compared to saline controls based on TUNEL staining. Gene expression analyses are in progress.
 Potential Impact:This study addresses the unmet clinical need for novel disease-modifying therapeutics for PTOA. Preliminary results show that inhibition of CaMKK2 has the potential to decrease cartilage degradation after joint injuries.

Sustained inhibition of CC-chemokine receptor-2 via intraarticular deposition of polymeric microplates in post-traumatic osteoarthritis

Posttraumatic osteoarthritis (PTOA) is mostly treated via corticosteroid administration, and total joint arthroplasty continues to be the sole effective intervention in severe conditions. To assess the therapeutic potential of CCR2 targeting in PTOA, we used biodegradable microplates (µPLs) to achieve a slow and sustained intraarticular release of the CCR2 inhibitor RS504393 into injured knees and followed joint damage during disease progression. RS504393-loaded µPLs (RS-µPLs) were fabricated via a template-replica molding technique. A mixture of poly(lactic-co-glycolic acid) (PLGA) and RS504393 was deposited into 20 × 10 μm (length × height) wells in a polyvinyl alcohol (PVA) square-patterned template. After physicochemical and toxicological characterizations, the RS504393 release profile from µPL was assessed in PBS buffer. C57BL/6 J male mice were subjected to destabilization of the medial meniscus (DMM)/sham surgery, and RS-µPLs (1 mg/kg) were administered intraarticularly 1 week postsurgery. Administrations were repeated at 4 and 7 weeks post-DMM. Drug free-µPLs (DF-µPLs) and saline injections were performed as controls. Mice were euthanized at 4 and 10 weeks post-DMM, corresponding to the early and severe PTOA stages, respectively. Knees were evaluated for cartilage structure score (ACS, H&E), matrix loss (safranin O score), osteophyte formation and maturation from cartilage to bone (cartilage quantification), and subchondral plate thickness. The RS-µPL architecture ensured the sustained release of CCR2 inhibitors over several weeks, with ~ 20% of RS504393 still available at 21 days. This prolonged release improved cartilage structure and reduced bone damage and synovial hyperplasia at both PTOA stages. Extracellular matrix loss was also attenuated, although with less efficacy. The results indicate that local sustained delivery is needed to optimize CCR2-targeted therapies.Graphical abstract

Intra-articular injection of flavopiridol-loaded microparticles for treatment of post-traumatic osteoarthritis

Rapid joint clearance of small molecule drugs is the major limitation of current clinical approaches to osteoarthritis and its subtypes, including post-traumatic osteoarthritis (PTOA). Particulate systems such as nano/microtechnology could provide a potential avenue for improved joint retention of small molecule drugs. One drug of interest for PTOA treatment is flavopiridol, which inhibits cyclin-dependent kinase 9 (CDK9). Herein, polylactide-co-glycolide microparticles encapsulating flavopiridol were formulated, characterized, and evaluated as a strategy to mitigate PTOA-associated inflammation through the inhibition of CDK9. Characterization of the microparticles, including the drug loading, hydrodynamic diameter, stability, and release profile was performed. The mean hydrodynamic diameter of flavopiridol particles was ∼15 µm, indicating good syringeability and low potential for phagocytosis. The microparticles showed no cytotoxicity in-vitro, and drug activity was maintained after encapsulation, even after prolonged exposure to high temperatures (60 °C). Flavopiridol-loaded microparticles or blank (unloaded) microparticles were administered by intraarticular injection in a rat knee injury model of PTOA. We observed significant joint retention of flavopiridol microparticles compared to the soluble flavopiridol, confirming the sustained release behavior of the particles. Matrix metalloprotease (MMP) activity, an indicator of joint inflammation, was significantly reduced by flavopiridol microparticles 3 days post-injury. Histopathological analysis showed that flavopiridol microparticles reduced PTOA severity 28 days post-injury. Taken altogether, this work demonstrates a promising biomaterial platform for sustained small molecule drug delivery to the joint space as a therapeutic measure for post-traumatic osteoarthritis. Statement of significancePost-traumatic osteoarthritis (PTOA) begins with the deterioration of subchondral bone and cartilage after acute injuries. In spite of the prevalence of PTOA and its associated financial and psychological burdens, therapeutic measures remain elusive. A number of small molecule drugs are now under investigation to replace FDA-approved palliative measures, including cyclin-dependent kinase 9 (CDK9) inhibitors which work by targeting early inflammatory programming after injury. However, the short half-life of these drugs is a major hurdle to their success. Here, we show that biomaterial encapsulation of Flavopiridol (CDK9 inhibitor) in poly (lactic-co-glycolic acid) microparticles is a promising route for direct delivery and improved drug retention time in the knee joint. Moreover, administration of the flavopiridol microparticles reduced the severity of PTOA.

Poor Outcomes After Anterior Impaction Pilon Fractures.

Pilon fractures occur through high-energy axial-loading trauma and are frequently associated with complications. The goal of this study was to assess whether anterior impaction (AI) tibial pilon fractures are associated with increased rates of posttraumatic osteoarthritis (PTOA), secondary surgeries, and lower patient-reported outcomes compared with patients with non-AI pilon fractures. In this retrospective cohort study, 52 pilon fractures in 50 patients were included. The average follow-up was 25 months (range, 12 to 62 in non-AI and 12 to 66 in AI). The Kellgren and Lawrence (KL) score for PTOA, tibiotalar ratio for anterior-posterior talar subluxation, coronal tibiotalar angle, Patient-Reported Outcomes Measurement Information System score, and rates of secondary surgeries and infection were assessed. The AI group showed radiographic evidence of more advanced PTOA at the final follow-up (KL score 3.1 vs. 2.5, P = 0.021) and a higher rate of implant removal for pain (39% vs. 13%, P = 0.030). AI also had greater anterior talar subluxation on preoperative (P < 0.001) and final follow-up radiographs (P = 0.026). A higher KL score was associated with greater anterior talar displacement on preoperative (r = -0.421, P = 0.003) and final follow-up radiographs (r = -0.359, P < 0.009). No differences were seen in 1-year Patient-Reported Outcomes Measurement Information System scores. AI pilon fractures are associated with recurrent anterior talar subluxation, more severe PTOA, and a higher rate of implant removal for pain compared with non-AI fractures.

Post-Traumatic Osteoarthritis Assessment in Emerging and Advanced Pre-Clinical Meniscus Repair Strategies: A Review.

Surgical repair of meniscus injury is intended to help alleviate pain, prevent further exacerbation of the injury, restore normal knee function, and inhibit the accelerated development of post-traumatic osteoarthritis (PTOA). Meniscus injuries that are treated poorly or left untreated are reported to significantly increase the risk of PTOA in patients. Current surgical approaches for the treatment of meniscus injuries do not eliminate the risk of accelerated PTOA development. Through recent efforts by scientists to develop innovative and more effective meniscus repair strategies, the use of biologics, allografts, and scaffolds have come into the forefront in pre-clinical investigations. However, gauging the extent to which these (and other) approaches inhibit the development of PTOA in the knee joint is often overlooked, yet an important consideration for determining the overall efficacy of potential treatments. In this review, we catalog recent advancements in pre-clinical therapies for meniscus injuries and discuss the assessment methodologies that are used for gauging the success of these treatments based on their effect on PTOA severity. Methodologies include histopathological evaluation of cartilage, radiographic evaluation of the knee, analysis of knee function, and quantification of OA predictive biomarkers. Lastly, we analyze the prevalence of these methodologies using a systemic PubMed® search for original scientific journal articles published in the last 3-years. We indexed 37 meniscus repair/replacement studies conducted in live animal models. Overall, our findings show that approximately 75% of these studies have performed at least one assessment for PTOA following meniscus injury repair. Out of this, 84% studies have reported an improvement in PTOA resulting from treatment.

Effect of knee joint loading on chondrocyte mechano-vulnerability and severity of post-traumatic osteoarthritis induced by ACL-injury in mice

ObjectiveThe objective of this study is to understand the role of altered in vivo mechanical environments in knee joints post anterior cruciate ligament (ACL)-injury in chondrocyte vulnerability against mechanical stimuli and in the progression of post-traumatic osteoarthritis (PT-OA). MethodsDifferential in vivo mechanical environments were induced by unilateral ACL-injury (uni-ACL-I) and bilateral ACL-injury (bi-ACL-I) in 8-week-old female C57BL/6 mice. The gait parameters, the mechano-vulnerability of in situ chondrocytes, Young's moduli of cartilage extracellular matrix (ECM), and the histological assessment of OA severity (OARSI score) were compared between control and experimental groups at 0∼8-weeks post-ACL-injury. ResultsWe found that bi-ACL-I mice experience higher joint-loading on their both injured limbs, but uni-ACL-I mice balance their joint-loading between injured and uninjured hind limbs resulting in a reduced joint-loading during gait. We also found that at 4- and 8-week post-injury the higher weight-bearing hind limbs (i.e., bi-ACL-I) had the increased area of chondrocyte death induced by impact loading and higher OARSI score than the lower weight-bearing limbs (uni-ACL-I). Additionally, we found that at 8-weeks post-injury the ECM became stiffer in bi-ACL-I joints and softer in uni-ACL-I joints. ConclusionsOur results show that ACL-injured limbs with lower in vivo joint-loading develops PT-OA significantly slower than injured limbs with higher joint-loading during gait. Our data also indicate that articular chondrocytes in severe PT-OA are more fragile from mechanical impacts than chondrocytes in healthy or mild PT-OA. Thus, preserving physiologic joint-loads on injured joints will reduce chondrocyte death post-injury and may delay PT-OA progression.

A prospective randomized double-blind clinical trial to assess the effects of leukocyte-reduced platelet-rich plasma on pro-inflammatory, degradative, and anabolic biomarkers after closed pilon fractures.

Posttraumatic osteoarthritis (PTOA) significantly affects patients with pilon fractures even after adequate anatomical reduction, and treatment strategies targeting the biologic mediators of PTOA are needed. This study was designed to determine the effects of intra-articular injection of platelet-rich plasma (PRP) on synovial fluid (SF) biomarkers for patients undergoing open reduction and internal fixation (ORIF) of pilon fractures. Patients undergoing staged management of pilon fractures were enrolled in a prospective, double-blinded, randomized, and placebo-controlled clinical trial to determine the effects of a single intra-articular injection of leukocyte-reduced PRP on SF biomarkers. Arthrocentesis of the injured and uninjured ankles was performed at the time of external skeletal fixation (ESF) and ORIF. Patients were randomized to receive either autogenous leukocyte-reduced PRP or saline (control) via intra-articular injection into the injured ankle at the time of ESF. SF biomarker concentrations were compared-uninjured, injured pretreatment, and saline-injected or PRP-injected. Eleven patients (PRP, n = 5; saline, n = 6) completed the study. Twenty-one uninjured, and 11 injured pretreatment, five PRP-treated, and six saline-treated SF samples were analyzed. PRP-treated SF contained significantly higher levels of PDGF-AA (p = 0.046) and significantly lower levels of MMP-3 (p = 0.042), MMP-9 (p = 0.009), IL-1β (p = 0.049), IL-6 (p < 0.01), IL-8 (p = 0.048), and PGE2 (p < 0.04). This study provided mechanistic data to suggest that a single intraarticular injection of leukocyte-reduced PRP is associated with anti-inflammatory, anti-degradative, and anabolic responses compared with saline control. These findings provide the impetus for investigating long-term clinical outcomes after PRP injection as an orthobiologic adjunct to ORIF for mitigating the incidence and severity of PTOA after pilon fractures.

Bromodomain-containing-protein-4 and cyclin-dependent-kinase-9 inhibitors interact synergistically in vitro and combined treatment reduces post-traumatic osteoarthritis severity in mice

Joint injury rapidly induces expression of primary response genes (PRGs), which activate a cascade of secondary genes that destroy joint tissues and initiate post-traumatic osteoarthritis (PTOA). Bromodomain-containing-protein-4 (Brd4) and cyclin-dependent-kinase-9 (CDK9) cooperatively control the rate-limiting step of PRG transactivation, including pro-inflammatory genes. This study investigated whether Brd4 and CDK9 inhibitors suppress inflammation and prevent PTOA development in vitro and in a mouse PTOA model. The effects of Brd4 and CDK9 inhibitors (JQ1 and Flavopiridol) on PRG and associated secondary damage were rigorously tested in different settings. Short-term effects of inflammatory stimuli (IL-1β, IL-6, TNF) on human chondrocyte PRG expression were assessed by RT-PCR and microarray after 5-h. We quantified glycosaminoglycan release from IL-1β-treated bovine cartilage explants after 3-6 days, and osteoarthritic changes in mice after ACL-rupture using RT-PCR (2-24hrs), in vivo imaging of MMP activity (24hrs), AFM-nanoindentation (3-7days), and histology (3days-4wks). Flavopiridol and JQ1 inhibitors act synergistically, and a combination of both almost completely prevented the activation of most IL-1β-induced PRGs in vitro by microarray analysis, and prevented IL-1β-induced glycosaminoglycan release from cartilage explants. Mice given the drug combination showed reduced IL-1β and IL-6 expression, less in vivo MMP activity, and lower synovitis (1.5 vs 4.9) and OARSI scores (2.8 vs 6.0) than untreated mice with ACL-rupture. JQ1 and Flavopiridol work synergistically to reduce injury response after joint trauma, suggesting that targeting Brd4 and/or CDK9 could be a viable strategy for PTOA prevention and treatment of early OA.

Prediction and Potential Preventions for the Development of Posttraumatic Osteoarthritis after the Terrible Triad Injury: A Multicenter Risk Factors Study

Objective A multicenter study to evaluate risk factors for the development of moderate or severe posttraumatic osteoarthritis (PTOA) and to find potential preventions. Methods We conducted a retrospective multicenter study including the terrible triad injury (TTI) patients with surgical treatment from January 2007 to November 2014. Demographics, injury information, and treatment history were obtained retrospectively. According to the Broberg and Morrey criterion, 198 included patients were sorted into two groups: the mild or no PTOA and moderate or severe PTOA. Uni- and multivariate logistic regression analyses were used to identify risk factors for moderate or severe PTOA. Results Moderate or severe PTOA was present in 64 patients (32.3%). Significant risk factors were Mason III radial head fracture (OR 4.049, 95% CI 1.877-8.736, p < 0.001), medial collateral ligament injury (OR 5.120, 95% CI 1.261-20.790, p = 0.022), and heavy use of elbow (OR 2.333, 95% CI 1.060-5.136, p = 0.035). Besides, patients suffered subluxation (p = 0.007) and those with more risk factors had a higher risk to develop moderate or severe PTOA. Conclusions Moderate or severe PTOA was common after the TTI. Patients need to be counseled about avoiding heavy use of the elbow, especially for those with Mason III radial head fractures. Surgeons should be aware of the recurrent instability of the elbow.

LPS-Induced Inflammation Prior to Injury Exacerbates the Development of Post-Traumatic Osteoarthritis in Mice.

ABSTRACTOsteoarthritis (OA) is a debilitating and painful disease characterized by the progressive loss of articular cartilage. Post‐traumatic osteoarthritis (PTOA) is an injury‐induced type of OA that persists in an asymptomatic phase for years before it becomes diagnosed in ~50% of injured individuals. Although PTOA is not classified as an inflammatory disease, it has been suggested that inflammation could be a major driver of PTOA development. Here we examined whether a state of systemic inflammation induced by lipopolysaccharide (LPS) administration 5‐days before injury would modulate PTOA outcomes. RNA‐seq analysis at 1‐day post‐injury followed by micro‐computed tomography (μCT) and histology characterization at 6 weeks post‐injury revealed that LPS administration causes more severe PTOA phenotypes. These phenotypes included significantly higher loss of cartilage and subchondral bone volume. Gene expression analysis showed that LPS alone induced a large cohort of inflammatory genes previously shown to be elevated in synovial M1 macrophages of rheumatoid arthritis (RA) patients, suggesting that systemic LPS produces synovitis. This synovitis was sufficient to promote PTOA in MRL/MpJ mice, a strain previously shown to be resistant to PTOA. The synovium of LPS‐treated injured joints displayed an increase in cellularity, and immunohistological examination confirmed that this increase was in part attributable to an elevation in type 1 macrophages. LPS induced the expression of Tlr7 and Tlr8 in both injured and uninjured joints, genes known to be elevated in RA. We conclude that inflammation before injury is an important risk factor for the development of PTOA and that correlating patient serum endotoxin levels or their state of systemic inflammation with PTOA progression may help develop new, effective treatments to lower the rate of PTOA in injured individuals. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Transgenic conversion of \u03c9-6 to \u03c9-3 polyunsaturated fatty acids via fat-1 reduces the severity of post-traumatic osteoarthritis

BackgroundDietary fatty acid (FA) content has been shown to influence the development of post-traumatic osteoarthritis (PTOA) in obesity. We used the fat-1 transgenic mouse to examine the hypothesis that endogenous reduction of ω-6 to ω-3 FA ratio, under the same dietary conditions, would mitigate metabolic inflammation and the pathogenesis of PTOA in obese male and female mice.MethodsMale and female fat-1 and wild-type littermates were fed either a control diet or an ω-6 FA-rich high-fat diet and underwent destabilization of the medial meniscus (DMM) surgery to induce PTOA. OA severity, synovitis, and osteophyte formation were determined histologically, while biomarker and lipidomic analyses were performed to evaluate levels of adipokines, insulin, pro-/anti-inflammatory cytokines, and FAs in serum and joint synovial fluid. Multivariable models were performed to elucidate the associations of dietary, metabolic, and mechanical factors with PTOA.ResultsWe found that elevated serum levels of ω-3 FAs in fat-1 mice as compared to wild-type controls fed the same diet resulted in reduced OA and synovitis in a sex- and diet-dependent manner, despite comparable body weights. The fat-1 mice showed trends toward decreased serum pro-inflammatory cytokines and increased anti-inflammatory cytokines. Multivariable analysis for variables predicting OA severity in mice resulted in correlations with serum FA levels, but not with body weight.ConclusionsThis study provides further evidence that circulating FA composition and systemic metabolic inflammation, rather than body weight, may be the major risk factor for obesity-associated OA. We also demonstrate the potential genetic use of ω-3 FA desaturase in mitigating PTOA in obese patients following injury.

Joint injury-induced angiogenesis and thrombospondin 2

Purpose: It is controversial whether inhibition of angiogenesis slows the progression of post-traumatic osteoarthritis (PTOA). The matricellular protein thrombospondin-2 (TSP2) is a candidate for inhibiting intraarticular angiogenesis after joint injury as it is a well-characterized endogenous anti-angiogenic protein. In addition, prior literature demonstrates the inhibition of matrix metalloproteinase (MMP) bioavailability by TSP2. However, little is known of the involvement of TSP2 in joint trauma, injury-induced angiogenesis and MMP activity, and PTOA progression. The purpose of this study was to characterize the endogenous TSP2 response following traumatic joint injury in mice. Methods: With IACUC approval, noninvasive ACLR was induced in adult TSP2-GFP reporter mice and C57Bl/6 mice via tibial compression. Limbs were processed for histology at 3d, 14d, and 28d post-ACLR (n=3-4/timepoint). The proportion of TSP2+ cells in synovium was quantified in injured and contralateral limbs of TSP2-GFP mice using k-means clustering of reporter intensity. ACLR-induced angiogenesis was assessed in C57Bl/6 mice via CD31 immunostaining and in vivo near-infrared imaging (NIR) utilizing the AngioSense-agent (Perkin Elmer), which enables in vivo assessment of vascular changes. Synovia from C57Bl/6 ACLR and contralateral knees were dissected, homogenized in Trizol, and analyzed for TSP2, MMP2, MMP3, MMP13 gene expression via rtPCR at 3d and 14d post-ACLR (n=5-8/timepoint), representing timepoints of early and late inflammation. To determine which cytokines induce TSP2 in fibroblast-like synoviocytes (FLS) and whether ACLR-associated in vivo inflammation affects the in vitro FLS cytokine response, primary mouse FLS were isolated from knee synovium of 7d post-ACLR and contralateral knees (n=3/group). FLS were induced with IL-1β and TNFα (10 ng/ml) for 48 hours and analyzed for TSP2 and MMP13 gene expression. Gene expression was normalized to ATP5b and paired t-tests performed. Results: ACLR induced intraarticular angiogenesis at 14d and 28d, assessed by influx of CD31+ cells. Abundant CD31+ staining was observed in synovium, menisci, and osteophytes of injured knees up to 28d post-ACLR (Figure. 1 A-C). An observed increase in in vivo AngioSense signal of the injured knee 7d post-ACLR (P=0.021) corroborates this finding and indicates acute vascular changes (Figure. 1D-E). ACLR also induced significant upregulation of MMP-2, -3, and -13 mRNA by synovium at 3d and 14d post-ACLR (Figure. 2K). In TSP2-GFP reporter mice, injured knees exhibited abundant synovial TSP2 expression, as early as 3d and up to 28d (at which point mice exhibit moderate PTOA severity; data not shown). At 3d and 14d, TSP2 expression is confined to the synovium, and by 28d, developing osteophytes are also TSP2(+). At all timepoints, synovial TSP2 expression is localized to the subintima. The intima of injured joints, although hyperplastic, remains TSP2(-). The cruciate ligaments, menisci, articular cartilage, and subchondral bone remain TSP2(-) (Figure. 2A-H). Quantitative image analysis indicates ACLR-induced TSP2-GFP expression in ∼27% of synovial cells at 14d (P=0.017) and ∼24% at 28d (P=0.018) post-ACLR, compared to near-zero expression in contralateral synovia (Figure. 2J). rtPCR confirms strong TSP2 upregulation in injured C57Bl/6 synovium at 3d (P=.006) and 14d (P=.007) (Figure. 2I). Surprisingly, both IL-1β and TNFα downregulated TSP2 in FLS from healthy (IL-1β - P=.009; TNFα - P=.001) and injured (IL-1β - P=.001; TNFα - P=.10) joints. TNFα-treated FLS from injured joints exhibited significantly less downregulation compared to healthy FLS (P=0.025) (Figure. 3B). Both IL-1β and TNFα induced a strong MMP13 response in healthy (P=0.002; P=0.004) and injured (P=0.001; P=0.006) FLS (Figure 3A), and there was no difference in the magnitude of MMP13 expression between healthy and injured FLS. Conclusions: TSP2 is strongly upregulated by the synovium following ACLR. This coincides with a synovial MMP-2, -3, and -13 response, increased intraarticular angiogenesis, and the time course of post-injury inflammation. Interestingly, TSP2 expression is confined to the subintimal region of the synovium. Our in vitro data shows that primary FLS do not upregulate TSP2 in response to IL-1β or TNFα, and interestingly, FLS from injured joints do not exhibit differentially-elevated TSP2 compared to FLS from contralateral joints, despite abundant in vivo synovial TSP2 expression following ACLR. Thus, ongoing work is identifying local TSP2+ cell populations and ligands that induce TSP2 expression in FLS. Given evidence on the anti-angiogenic capacity of TSP2, ongoing work is assessing angiogenesis, inflammation, catabolism, and PTOA severity in global TSP2-KO mice. Our ultimate objective is to mitigate injury-induced angiogenesis with exogenous TSP2 treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Combinatorial Prg4 and Il-1ra Gene Therapy Protects Against Hyperalgesia and Cartilage Degeneration in Post-Traumatic Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA.

Increased susceptibility to develop spontaneous and severe post-traumatic osteoarthritis in DOT1L-deficient mice

Increased susceptibility to develop spontaneous and severe post-traumatic osteoarthritis in DOT1L-deficient mice

ZCCHC6 (TUT7) deletion inhibit the expression of IL-6 and MMP-13 and reduces the severity of post-traumatic osteoarthritis in a murine model

Purpose: Approximately 20-50% of patients diagnosed with osteoarthritis (OA) develop the disease in joints that previously sustained trauma. RNA terminal uridyltransferases (TUTs), including Zcchc6 (zinc-finger CCHC-domain containing protein 6/TUT7), have been implicated in the post-transcriptional regulation of inflammatory mediators. However, role of Zcchc6 (TUT7) in the pathogenesis of OA or post-traumatic OA has not been explored. Methods: All murine studies were approved by the IACUC and the studies with deidentified, discarded human cartilage were approved by the IRB. Zcchc6 knockout mice were generated by gene targeting (KOMP, UC-Davis). Human or murine chondrocytes were prepared by the enzymatic digestion of the OA cartilage or knee cartilage respective and treated with recombinant human or mouse IL-1β (5ng/ml, R&D Systems, #: 201-LB-025 and #:401-ML-005 respectively) for in vitro studies. IL-6 and MMP13 gene and protein expression in chondrocytes was analyzed by qPCR and immunoblotting respectively. IL-6 mRNA stability was determined by Actinomycin-D chase experiments. For in vivo studies, post-traumatic OA in the knee joints of Zcchc6 deficient male mice and wild type littermates was induced by the surgical destabilization of medial meniscus (DMM) and mice were sacrificed after eight weeks of DMM and the severity of OA was determined per OARSI guidelines for murine OA. Contra lateral knee joints with sham surgery served as controls. Expression of MMP13 (Santa Cruz Biotechnology, #sc-30071), Type-II Collagen cleavage (IBEX Pharmaceuticals, #C1,2C) and Zcchc6 (Santa Cruz Biotechnology, #sc-137947) in vivo were analyzed by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed using the RNAScope technology. RNA immunoprecipitation (RIP) was performed using validated antibodies. siRNAs were used to deplete the expression of ZCCHC6. Results: Knee cartilage chondrocytes prepared from Zcchc6 KO mice showed increased levels of IL-6 and MMP13 mRNA expression compared to WT chondrocytes in response to IL-1β stimulation. Of interest was the observation that the protein levels of IL-6 and MMP13 were significantly downregulated in IL-1β stimulated Zcchc6 KO chondrocytes lysate and culture supernatants in comparison to WT chondrocytes. FISH and RIP analysis revealed that ZCCHC6 protein binds to IL-6 mRNAs in primary human OA chondrocytes under pathological conditions. Furthermore, IL-6 mRNA half-life was reduced by 50% in Zcchc6 KO murine chondrocytes and ZCCHC6 depleted human primary chondrocytes. The expression of Zcchc6 in the knee joints of wild type mice with DMM surgery was highly upregulated in comparison to control joints. Zcchc6 KO mice expressed low levels of MMP13 and showed lesser matrix degradation in the joints with DMM surgery compared to joints of the WT mice and the control contralateral joints and developed less severe OA as determined by Safranin-O-Fast green staining followed by OARSI scoring. Synovitis was also found to be decreased in the KO mice DMM joints in comparison to WT DMM joints. Conclusions: Our data demonstrate that Zcchc6 is upregulated in DMM joints and functions as a positive regulator of IL-6 and MMP13 expression in murine chondrocytes and cartilage in vivo. Severity of the posttraumatic OA and cartilage degradation was significantly low in Zcchc6 KO mice and correlated with reduced expression of MMP-13. These results identify ZCCHC6 as a potential therapeutic target for the treatment of OA.