The alarmins high mobility group box protein 1 and S100A8/A9 display different inflammatory profiles after acute knee injury.

OBJECTIVE: To compare the concentrations of high mobility group box 1 protein (HMGB1) and S100A8/A9 in synovial fluid between patients with knee injuries and osteoarthritis (OA), and knee healthy subjects. To investigate associations of alarmin levels with different joint injuries and with biomarkers of inflammation, Wnt signaling, complement system, bone and cartilage degradation. METHODS: HMGB1 and S100A8/A9 were measured in synovial fluid by immunoassays in patients with knee injuries, with OA and from knee healthy subjects, and were related to time from injury and with biomarkers obtained from previous studies. Hierarchical cluster and enrichment analyses of biomarkers associated to HMGB1 and S100A8/A9 were performed. RESULTS: The synovial fluid HMGB1 and S100A8/A9 concentrations were increased early after knee injury; S100A8/A9 levels were negatively associated to time after injury and was lower in the old compared to recent injury group, while HMGB1 was not associated to time after injury. The S100A8/A9 levels were also increased in OA. The initial inflammatory response was similar between the alarmins, and HMGB1 and S100A8/A9 shared 9 out of 20 enriched pathways. The alarmins displayed distinct response profiles, HMGB1 being associated to cartilage biomarkers while S100A8/A9 was associated to proinflammatory cytokines. CONCLUSIONS: HMGB1 and S100A8/A9 are increased as an immediate response to knee trauma. While they share many features in inflammatory and immunoregulatory mechanisms, S100A8/A9 and HMGB1 are associated to different downstream responses, which may have impact on the OA progression after acute knee injuries.

Early stage blockade of the alarmin HMGB1 reduces cartilage destruction in experimental OA.

OBJECTIVE: The alarmin HMGB1 is an endogenous molecule that is released into the extracellular space upon trauma or cell activation. Extracellular HMGB1 initiates innate immune responses and besides mediating inflammation, has osteoclast-activating features and mediates pain, all important features in OA. The aim of this study was to examine the involvement of HMGB1 in experimental OA and to explore the effect of local anti-HMGB1-therapy on disease progression. METHOD: OA was induced in mice by surgical destabilization of knee joints and HMGB1 expression and localization was assessed by immunohistochemistry. For therapy evaluation, HMGB1-neutralizing antibodies were injected intraarticularly, alone or encapsulated in an injectable hyaluronan-based delivery vehicle. Human primary chondrocytes were stimulated with rHMGB1 and analyzed by qPCR and cytometric bead-array. RESULTS: HMGB1 immunostaining of mouse OA joints demonstrated intra- and pericellular expression in chondrocytes, overlapping with proteoglycan depleted areas. Intra-articular injection of anti-HMGB1 antibodies had cartilage-protective effects, comparable to treatment with a TNF inhibitor. Direct and vehicle-based delivery had similar ameliorating effects and the effect of a single, early injection could not be enhanced by repeated injections. In vitro stimulation of chondrocytes with rHMGB1 affected chondrocyte function by inducing protein expression of IL6 and IL8 and downregulating mRNA of COL2A1. CONCLUSIONS: Our results suggest that the alarmin HMGB1 might be a new target for OA therapy development as we could observe an aberrant HMGB1 expression in mouse OA joints, stimulation of chondrocytes with rHMGB1 induced cytokine production and decreased matrix production and finally that HMGB1 blockade suppressed disease progression.

An in vivo cross-linkable hyaluronan gel with inherent anti-inflammatory properties reduces OA cartilage destruction in female mice subjected to cruciate ligament transection.

OBJECTIVE: To explore the possibility of cartilage protection in osteoarthritis (OA) by intraarticular injection of a chemically modified hyaluronan (HA) gel and investigate whether the chemical modifications provide intrinsic anti-inflammatory activity. METHOD: OA was induced in C57BL/6 mice by anterior cruciate ligament transection (ACLT) and HA gel or carbazate-modified component was injected intra-articularly. Assessment of cartilage rescue was performed by histology, immunohistochemistry and TUNEL analysis. Serum levels of proinflammatory cytokines were evaluated with cytometric bead array, measuring IL-1ß, TNF, IFN-γ, KC/CXCL1 and MCP-1. RESULTS: Intraarticular injection of the HA gel showed significantly reduced cartilage destruction and decreased osteophyte formation. Besides the biological and biomechanical effects of HA, we investigated lipid peroxidation products as an alternative inflammatory and potential mechanism contributing to OA. To address this, injection of the carbazate-modified component alone was performed, which also demonstrated a cartilage-saving effect. Besides the cartilage amelioration effects, decreased apoptosis, 4-hydroxynonenal (4-HNE) and MHC class II staining was recorded. No changes in serum levels of proinflammatory cytokines were detected. CONCLUSION: We have shown that the HA gel has an anti-destructive effect on articular cartilage (AC). Our results demonstrated that the carbazate-modified component could suppress apoptotic events, potentially by quenching of ROS/LPO products such as 4-HNE in OA joints. Modification of the HA molecule offers opportunities to introduce (covalent) coupling of additional molecules to the gel, with controlled retention and subsequent release in the joint.

Cartilage repair of experimentally 11 induced osteochondral defects in New Zealand White rabbits.

Articular cartilage has a limited capacity for self-repair in adult humans, and methods used to stimulate regeneration often result in re-growth of fibrous cartilage, which has lower durability. No current treatment option can provide complete repair. The possibility of growth factor delivery into the joint for cartilage regeneration after injury would be an attractive treatment option. A full thickness osteochondral defect of 4 mm in diameter and 2 mm deep was created by mechanical drilling in the medial femoral condyle in 20 female adult New Zealand White rabbits. In an attempt to improve regeneration a hyaluronic hydrogel system, with or without bone morphogenetic protein-2 (BMP-2) was delivered intraarticularly. The contralateral joint defect was treated with saline as control. Throughout the study, rabbits were clinically examined and after 12 (n = 6) or 24 (n = 9) weeks, the rabbits were euthanized and the joints evaluated by histology. The defects healed with fibrocartilage like tissue, and the filling of the defects ranged from less than 25% to complete. The healing of the defects varied both inter- and intra-group wise. Treatment with hyaluronan gel with or without BMP-2 had no effect on cartilage regeneration compared with controls. Instead, severe ectopic bone formation was found in seven joints treated with BMP-2. In conclusion, the present study shows that neither treatment with hyaluronic gel alone, nor in combination with BMP-2, improves the healing of an induced cartilage defect in rabbits. It further shows that BMP-2 can induce ectopic bone formation, which severely affects the functionality of the joint.