On-demand treatment with the iron chelator deferasirox is ineffective in preventing blood-induced joint damage in haemophilic mice.

INTRODUCTION: Early intervention in the devastating process of haemophilic arthropathy (HA) is highly desirable, but no disease-modifying therapy is currently available. Considering the pivotal role of iron in the development of HA, iron chelation is considered a promising therapeutic approach. A previous study in haemophilic mice demonstrated that treatment with the iron chelator deferasirox (DFX) 8 weeks before joint bleed induction, attenuated cartilage damage upon blood exposure. However, in haemophilia patients this approach is not opportune given the unpredictable occurrence of hemarthroses. AIM: To evaluate the effectiveness of on-demand DFX treatment, initiated immediately after joint bleed induction. METHODS: A joint bleed was induced in 66 factor VIII-deficient mice by infra-patellar needle puncture. Mice were randomly assigned to treatment with either placebo (drinking water) or DFX (dissolved in drinking water) throughout the study. Five weeks after joint bleed induction, inflammation and cartilage damage were assessed histologically. Joints of ten bleed naive haemophilic mice served as controls. RESULTS: A joint bleed resulted in significant inflammation and cartilage damage in the blood-exposed joint compared with those of control animals, in both the placebo and DFX group (all p = <.05). No differences in tibiofemoral or patellar inflammation (p = .305 and p = .787, respectively) nor cartilage damage (p = .265 and p = .802, respectively) were found between the blood-exposed joints of both treatment groups. CONCLUSION: On-demand treatment with DFX does not prevent joint damage following blood exposure in haemophilic mice. DFX seems unable to reach the joint in time to exert its effect before the irreversible harmful process is initiated.

Proteoglycan synthesis rate as a novel method to measure blood-induced cartilage degeneration in non-haemophilic and haemophilic rats.

INTRODUCTION: Haemophilic animal models are used to study blood-induced cartilage damage, but quantitative and sensitive outcome measures are needed. AIM: To develop a novel quantitative method for detecting early cartilage degeneration in a haemophilic rat model of blood-induced joint damage. METHODS: The 35 Sulphate incorporation (35 SO4 2- assay) was applied to tibial and patellar cartilage of wild-type rats to quantify baseline proteoglycan synthesis and to evaluate the effect of 4-day blood exposure in vitro. Next, haemarthrosis was induced in 39 FVIII-deficient rats and characterized by changes in knee joint diameter and development of bone pathology (using micro-CT). Four- and 16-day posthaemarthrosis proteoglycan synthesis rate (PSR) was assessed using the 35 SO4 2- assay, with the contralateral knee as control. RESULTS: In vitro, a decrease in PSR in tibial and patellar cartilage was demonstrated following blood exposure. In vivo, joint diameter and development of bone pathology confirmed successful induction of haemarthrosis. In the blood-exposed knee, tibial and patellar PSR was inhibited 4 and 16 days after induced haemarthrosis. Interestingly, at day 16 the proteoglycan synthesis in the contralateral knee was also inhibited to an extent correlating with that of the blood-exposed knee. CONCLUSION: For the first time, early changes in cartilage matrix synthesis upon blood exposure were quantified with the 35 SO4 2- assay in a haemophilic rat model, establishing this assay as a novel method to study blood-induced cartilage damage.

IL-1ß, in contrast to TNFα, is pivotal in blood-induced cartilage damage and is a potential target for therapy.

Joint bleeding after (sports) trauma, after major joint surgery, or as seen in hemophilia in general leads to arthropathy. Joint degeneration is considered to result from the direct effects of blood components on cartilage and indirectly from synovial inflammation. Blood-provided proinflammatory cytokines trigger chondrocytes and induce the production of cartilage-degrading proteases. In the presence of erythrocyte-derived iron, cytokines stimulate radical formation in the vicinity of chondrocytes inducing apoptosis. To unravel the role of interleukin (IL) 1ß and tumor necrosis factor (TNF) α in the pathogenesis of this blood-induced cartilage damage, the effect of antagonizing these cytokines was examined in human in vitro cultures. Addition of recombinant human IL-1ß monoclonal antibody or IL-1 receptor antagonist resulted in a dose- and time-dependent protection of cartilage from blood-induced damage. In higher concentrations, almost complete normalization of cartilage matrix proteoglycan turnover was achieved. This was accompanied by a reduction in IL-1ß and IL-6 production in whole blood cultures, whereas TNFα production remained unaffected. Interestingly, addition of a TNFα monoclonal antibody, although demonstrated to inhibit the direct (transient) effects of TNFα on cartilage, exhibited no effect on blood-induced (prolonged) cartilage damage. It is demonstrated that IL-1ß is crucial in the development of blood-induced joint damage, whereas TNFα is not. This hierarchical position of IL-1ß in blood-induced joint damage warrants studies on targeting IL-1ß to potentially prevent joint degeneration after a joint bleed.

A single intra-articular injection with IL-4 plus IL-10 ameliorates blood-induced cartilage degeneration in haemophilic mice.

The combination of interleukin (IL)-4 and IL-10 protects against blood-induced cartilage damage in vitro. It has been hypothesized that the combination of these cytokines is effective if applied early in the process of cartilage damage. The present study investigated whether a single intra-articular injection of IL-4 plus IL-10 immediately after a joint bleed limits cartilage damage in an in vivo haemophilia mouse model of blood-induced joint damage. Factor VIII knockout mice with severe haemophilia A were punctured once with a needle below the patella to induce a joint haemorrhage. Subsequently IL-4 plus IL-10 (n = 24) or vehicle (n = 24) was injected intra-articularly. After 35 days, the time needed for development of detectable joint degeneration, knee joints were examined for cartilage damage by macroscopic and microscopic evaluation. A single intra-articular injection of IL-4 plus IL-10 ameliorated progression of cartilage degeneration caused by a single joint bleed to a certain extent. No effect on inflammation was observed at this time point. A single intra-articular injection of IL-4 plus Il-10 directly after a single joint bleed limits progression of cartilage degeneration over time. Improved bioavailability (half-life) of both cytokines might improve their protective ability in the development of cartilage degeneration, and probably also inflammation.

Microencapsulated stem cells reduce cartilage damage in a material dependent manner following minimally invasive intra-articular injection in an OA rat model.

Osteoarthritis (OA) is a degenerative disease of the joints for which no curative treatment exists. Intra-articular injection of stem cells is explored as a regenerative approach, but rapid clearance of cells from the injection site limits the therapeutic outcome. Microencapsulation of mesenchymal stem cells (MSCs) can extend the retention time of MSCs, but the outcomes of the few studies currently performed are conflicting. We hypothesize that the composition of the micromaterial's shell plays a deciding factor in the treatment outcome of intra-articular MSC injection. To this end, we microencapsulate MSCs using droplet microfluidic generators in flow-focus mode using various polymers and polymer concentrations. We demonstrate that polymer composition and concentration potently alter the metabolic activity as well as the secretome of MSCs. Moreover, while microencapsulation consistently prolongs the retention time of MSC injected in rat joints, distinct biodistribution within the joint is demonstrated for the various microgel formulations. Furthermore, intra-articular injections of pristine and microencapsulated MSC in OA rat joints show a strong material-dependent effect on the reduction of cartilage degradation and matrix loss. Collectively, this study highlights that micromaterial composition and concentration are key deciding factors for the therapeutic outcome of intra-articular injections of microencapsulated stem cells to treat degenerative joint diseases.

Enhanced Extracellular Matrix Breakdown Characterizes the Early Distraction Phase of Canine Knee Joint Distraction.

OBJECTIVE: Joint distraction triggers intrinsic cartilage repair in animal models of osteoarthritis (OA), corroborating observations in human OA patients treated with joint distraction. The present study explores the still largely elusive mechanism initiating this repair process. DESIGN: Unilateral OA was induced in the knee joint of 8 dogs using the groove model; the contralateral joint served as a control. After 10 weeks, 4 animals received joint distraction, the other 4 serving as OA controls. Halfway the distraction period (after 4 weeks of a standard 8-week distraction treatment), all animals were euthanized, and joint tissues were collected. A targeted quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed of commonly involved processes including matrix catabolism/anabolism, inflammation, and known signaling pathways in OA. In addition, cartilage changes were determined on tissue sections using the canine OARSI (Osteoarthritis Research Society International) histopathology score and collagen type II (COL2A1) immunostaining. RESULTS: Midway distraction, the distracted OA joint showed an upregulation of proteolytic genes, for example, ADAMTS5, MMP9, MMP13, compared to OA alone and the healthy joints, which correlated with an increased OARSI score. Additionally, genes of the transforming growth factor (TGF)-ß and Notch pathway, and markers associated with progenitor cells were increased. CONCLUSIONS: Joint distraction initiates both catabolic and anabolic transcriptional responses. The enhanced turnover, and thereby renewal of the matrix, could be the key to the cartilage repair observed in the months after joint distraction.

Metabolic dysregulation accelerates injury-induced joint degeneration, driven by local inflammation; an in vivo rat study.

Evidence is growing for the existence of an obesity-related phenotype of osteoarthritis in which low-grade inflammation and a disturbed metabolic profile play a role. The contribution of an obesity-induced metabolic dysbalance to the progression of the features of osteoarthritis upon mechanically induced cartilage damage was studied in a rat in vivo model. Forty Wistar rats were randomly allocated 1:1 to a standard diet or a high-fat diet. After 12 weeks, in 14 out of 20 rats in each group, cartilage was mechanically damaged in the right knee joint. The remaining six animals in each group served as controls. After a subsequent 12 weeks, serum was collected for metabolic state, subchondral bone changes assessed by µCT imaging, osteoarthritis severity determined by histology, and macrophage presence assessed by CD68 staining. The high-fat diet increased statistically all relevant metabolic parameters, resulting in a dysmetabolic state and subsequent synovial inflammation, whereas cartilage degeneration was hardly influenced. The high-fat condition in combination with mechanical cartilage damage resulted in a clear statistically significant progression of the osteoarthritic features, with increased synovitis and multiple large osteophytes. Both the synovium and osteophytes contained numerous CD68 positive cells. It is concluded that a metabolic dysbalance due to a high-fat diet increases joint inflammation without cartilage degeneration. The dysmetabolic state clearly accelerates progression of osteoarthritis upon surgically induced cartilage damage supported by inflammatory responses as demonstrated by histology and increased CD68 expressing cells localized on the synovial membrane and osteophytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:881-890, 2018.

Groove model of tibia-femoral osteoarthritis in the rat.

Several experimental models of osteoarthritis in rats are used to study the pathophysiology of osteoarthritis. Many mechanically induced models have the limitation that permanent joint instability is induced by, for example, ligament transection or meniscal damage. This permanent instability will counteract the potential beneficial effects of therapy. The groove model of osteoarthritis uses a one-time trigger, surgically induced cartilage damage on the femoral condyles, and has been validated for the canine tibia-femoral compartment. The present study evaluates this model for the rat knee joint. The articular cartilage of the weight bearing surface of both femoral condyles and trochlea were damaged (grooved) without damaging the underlying subchondral bone. Severity of joint degeneration was histologically assessed, in addition to patella cartilage damage, and subchondral bone characteristics by means of (contrast-enhanced) micro-CT. Mild histological degeneration of the surgically untouched tibial plateau cartilage was observed in addition to damage of the femoral condyles, without clear synovial tissue inflammation. Contrast enhanced micro-CT demonstrated proteoglycan loss of the surgically untouched patella cartilage. Besides, a more sclerotic structure of the subchondral bone was observed. The tibia-femoral groove model in a rat results in mild knee joint degeneration, without permanent joint instability and joint inflammation. This makes the rat groove model a useful model to study the onset and progression of post-traumatic non-inflammatory osteoarthritis, creating a relatively sensitive model to study disease modifying osteoarthritic drugs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:496-505, 2017.

Hemarthrosis in hemophilic mice results in alterations in M1-M2 monocyte/macrophage polarization.

INTRODUCTION: Joint bleedings result in iron-mediated synovitis and cartilage destruction. Monocyte/macrophage polarization affects their role in iron homeostasis. This study evaluates the effects of hemarthrosis on monocyte/macrophage polarization. MATERIALS AND METHODS: Using a murine hemophilia model of acute joint bleeding and flow cytometry, we evaluated monocyte/macrophage polarization in blood, spleen, synovium, and knee lavage at day 1, 2, and 7 following the induction of hemarthrosis. RESULTS: Induction of hemarthrosis resulted in a transient shift of blood monocytes towards a M1 type (control 13 vs. 1847 counted cells at day 1; p<0.01), a temporary decrease of spleen M1 monocytes (control 2841 vs. 1086 counted cells at day 1; p=0.02), and a sustained decrease of spleen M2 red pulp macrophages (control 1853 vs. 673 counted cells at day 7; p=0.01). In addition, an increase in M1 (control 119 vs. 592 counted cells at day 1; p=0.04) and M2 (control 247 vs. 650 counted cells at day 1; p=0.02) synovial macrophages was noted. In the joint lavage, a temporary increase in M1 monocytes (control 20 vs. 125 counted cells at day 1; p=0.04) and a more sustained increase in M2 monocytes (control 73 vs. 186 counted cells at day 2; p<0.01) was observed. CONCLUSIONS: This study demonstrates alterations in monocyte/macrophage polarization following hemarthrosis resulting in a blood monocyte M1 phenotype and a combined M1-M2 monocyte/macrophage phenotype in the joint. Based on the different capabilities of M1 and M2 cells, modulating polarization of distinct monocyte/macrophage populations might represent interesting prophylactic or therapeutic approaches for joint bleedings.

Deferasirox limits cartilage damage following haemarthrosis in haemophilic mice.

Joint bleeds in haemophilia result in iron-mediated synovitis and cartilage damage. It was evaluated whether deferasirox, an iron chelator, was able to limit the development of haemophilic synovitis and cartilage damage. Haemophilic mice were randomly assigned to oral treatment with deferasirox (30 mg/kg) or its vehicle (control) (30 mg/kg). Eight weeks after start of treatment, haemarthrosis was induced. After another five weeks of treatment, blood-induced synovitis and cartilage damage were determined. Treatment with deferasirox resulted in a statistically significant (p< 0.01) decrease in plasma ferritin levels as compared to the control group (823 ng/ml ± 56 and 1220 ng/ml ±114, respectively). Signs of haemophilic synovitis, as assessed by the Valentino score [range 0 (normal) - 10 (most affected)], were not different (p=0.52) when comparing the control group with the deferasirox group. However, deferasirox treatment resulted in a statistically significant (p< 0.01) reduction in cartilage damage, as assessed by the loss in Safranin O staining [range 0 (normal) - 6 (most affected)], when comparing the deferasirox group with the control group: score 2 (65.4 % vs 4.2 %), score 3 (26.9 % vs 4.2 %), score 4 (7.7 % vs 20.8 %), score 5 (0 % vs 54.2 %), and score 6 (0 % vs 16.7 %). Treatment with deferasirox limits cartilage damage following the induction of a haemarthrosis in haemophilic mice. This study demonstrates the role of iron in blood-induced cartilage damage. Moreover, these data indicate that iron chelation may be a potential prevention option to limit the development of haemophilic arthropathy.

Haemarthrosis stimulates the synovial fibrinolytic system in haemophilic mice.

Recurrent joint bleeding is the most common manifestation of haemophilia resulting in haemophilic arthropathy (HA). The exact pathophysiology is unknown, but it is suggested that arthropathy is stimulated by liberation of fibrinolytic activators from the synovium during haemarthrosis. The aim of this study was to test the hypothesis that haemarthrosis activates the local synovial fibrinolytic system in a murine haemophilia model. The right knees of haemophilic and control mice were punctured to induce haemarthrosis. The left knees served as internal control joints. Synovial levels of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), plasmin, and alpha-2-antiplasmin (A2AP) were compared between the punctured and control knees. In haemophilic mice, an increase in synovial cells expressing urokinase-type plasminogen activator (uPA) in the right punctured knee versus the left unaffected knee was observed: (47% vs 43%) (p=0.03). Additionally, in haemophilic mice, haemarthrosis induced an increase in uPA (0.016 ng/ml vs 0.01 ng/ml) (p=0.03) and plasmin (0.53 µg/ml vs 0.46 µg/ml) (p=0.01) as promoters of fibrinolysis. Synovial levels of PAI-1 (0.32 ng/ml vs 0.17 ng/ml) (p<0.01) was also increased, whereas synovial levels of A2AP were unchanged: (0.021 µg/ml vs 0.021 µg/ml) (p=0.15). Enhanced uPA production was confirmed in human stimulated synovial fibroblast cultures and elevated levels of plasmin were confirmed harmful to human cartilage tissue explants. In this study we demonstrate that haemarthrosis in haemophilic mice induces synovial uPA expression and results in an increase in synovial plasmin levels, making the joint more vulnerable to prolonged and subsequent bleedings, and adding directly to cartilage damage.