TLR4 antagonism provides short-term but not long-term clinical benefit in a full-depth cartilage defect mouse model.

PURPOSE/AIM: Cartilage injury and subsequent osteoarthritis (OA) are debilitating conditions affecting millions worldwide. As there are no cures for these ailments, novel therapies are needed to suppress disease pathogenesis. Given that joint injuries are known to produce damage-associated molecular patterns (DAMPs), our central premise is that the Toll-like receptor 4 (TLR4) pathway is a principal driver in the early response to cartilage damage and subsequent pathology. We postulate that TLR4 activation is initiated/perpetuated by DAMPs released following joint damage. Thus, antagonism of the TLR4 pathway immediately after injury may suppress the development of joint surface defects. MATERIALS AND METHODS: Two groups were utilized: (1) 8-week-old, male C57BL6 mice treated systemically with a known TLR4 antagonist and (2) mice injected with vehicle control. A full-depth cartilage lesion on the midline of the patellofemoral groove was created in the right knee of each mouse. The left knee was used as a sham surgery control. Gait changes were evaluated over 4 weeks using a quantitative gait analysis system. At harvest, knee joints were processed for pathologic assessment, Nanostring® transcript expression, and immunohistochemistry (IHC). RESULTS: Short-term treatment with a TLR4 antagonist at 14-days significantly improved relevant gait parameters; improved cartilage metrics and modified Mankin scores were also seen. Additionally, mRNA expression and IHC showed reduced expression of inflammatory mediators in animals treated with the TLR4 antagonist. CONCLUSIONS: Collectively, this work demonstrates that systemic treatment with a TLR4 antagonist is protective to further cartilage damage 14-days post-injury in a murine model of induced disease.

Erythrocyte removal from bone marrow aspirate concentrate improves efficacy as intra-articular cellular therapy in a rodent osteoarthritis model.

Background: Despite the high prevalence of osteoarthritis (OA), there remains a need for additional therapeutic options. Cellular therapies with minimally manipulated cells such as bone marrow aspirate concentrates (BMAC) are increasingly popular in the U.S. but clear-cut evidence of efficacy has not been established. In theory, BMAC injections provide a source of stromal cells to stimulate healing in OA and ligamentous injuries; however, BMAC injections are also often associated with inflammation, short-term pain, and mobility impairment. Given that blood is known to trigger inflammation in joints, we hypothesized that removing erythrocytes [red blood cells (RBCs)] from BMAC preparations prior to intra-articular injection would improve efficacy for OA treatment. Methods: To test this hypothesis, BMAC was collected from the bone marrow of mice. Three treatment groups were pursued: (I) untreated; (II) BMAC; or (III) BMAC depleted of RBCs by lysis. Product was injected into the femorotibial joint of mice 7 days after OA had been induced by destabilization of the medial meniscus (DMM). To assess the impact of treatment on joint function, individual cage monitoring (ANY-mazeTM) and Digigait treadmill-based analyses were performed over 4 weeks. At study completion, joint histopathology was assessed and immune transcriptomes within joint tissues were compared using a species-specific NanoString panel. Results: Significant improvements in activity, gait parameters, and histology scores were seen in animals receiving RBC-depleted BMAC compared to untreated mice; animals treated with non-depleted BMAC did not demonstrate this same extent of consistent significant improvement. Transcriptomic analysis of joint tissues revealed significant upregulation of key anti-inflammatory genes, including interleukin-1 receptor antagonist (IRAP), in mice treated with RBC-depleted BMAC compared to animals treated with non-RBC depleted BMAC. Conclusions: These findings indicate that RBC depletion in BMAC prior to intra-articular injection improves treatment efficacy and reduces joint inflammation compared to BMAC.

Full and Partial Mid-substance ACL Rupture Using Mechanical Tibial Displacement in Male and Female Mice.

The anterior cruciate ligament (ACL) is the most commonly injured knee ligament. Surgical reconstruction is the gold standard treatment for ACL ruptures, but 20-50% of patients develop post-traumatic osteoarthritis (PTOA). ACL rupture is thus a well-recognized etiology of PTOA; however, little is known about the initial relationship between ligamentous injury and subsequent PTOA. The goals of this project were to: (1) develop both partial and full models of mid-substance ACL rupture in male and female mice using non-invasive mechanical methods by means of tibial displacement; and (2) to characterize early PTOA changes in the full ACL rupture model. A custom material testing system was utilized to induce either partial or full ACL rupture by means of tibial displacement at 1.6 or 2.0 mm, respectively. Mice were euthanized either (i) immediately post-injury to determine rupture success rates or (ii) 14 days post-injury to evaluate early PTOA progression following full ACL rupture. Our models demonstrated high efficacy in inciting either full or partial ACL rupture in male and female mice within the mid-substance of the ACL. These tools can be utilized for preclinical testing of potential therapeutics and to further our understanding of PTOA following ACL rupture.

Evaluation of electroacupuncture for symptom modification in a rodent model of spontaneous osteoarthritis.

OBJECTIVE: Faced with the frustration of chronic discomfort and restricted mobility due to osteoarthritis (OA), many individuals have turned to acupuncture for relief. However, the efficacy of acupuncture for OA is uncertain, as much of the evidence is inconclusive. The purpose of this study was to evaluate electroacupuncture (EA) in a rodent model of OA such that conclusions regarding its effectiveness for symptom or disease modification could be drawn. METHODS: Ten 12-month-old male Hartley guinea pigs-which characteristically have moderate to advanced OA at this age-were randomly assigned to receive EA for knee OA (n = 5) or anesthesia only (control group, n = 5). Treatments were performed three times weekly for 3 weeks, followed by euthanasia 2 weeks later. Gait analysis and enclosure monitoring were performed weekly to evaluate changes in movement. Serum was collected for inflammatory biomarker testing. Knee joints were collected for histology and gene expression. RESULTS: Animals receiving EA had significantly greater changes in movement parameters compared to those receiving anesthesia only. There was a tendency toward decreased serum protein concentrations of complement component 3 (C3) in the EA group compared to the control group. Structural and antioxidant gene transcripts in articular cartilage were increased by EA. There was no significant difference in total joint histology scores between groups. CONCLUSION: This study provides evidence that EA has a positive effect on symptom, but not disease, modification in a rodent model of OA. Further investigations into mechanistic pathways that may explain the efficacy of EA in this animal model are needed.