Functional Loss of Terminal Complement Complex Protects Rabbits from Injury-Induced Osteoarthritis on Structural and Cellular Level.

The terminal complement complex (TCC) has been described as a potential driver in the pathogenesis of posttraumatic osteoarthritis (PTOA). However, sublytic TCC deposition might also play a crucial role in bone development and regeneration. Therefore, we elucidated the effects of TCC on joint-related tissues using a rabbit PTOA model. In brief, a C6-deficient rabbit breed was characterized on genetic, protein, and functional levels. Anterior cruciate ligament transection (ACLT) was performed in C6-deficient (C6-/-) and C6-sufficient (C6+/-) rabbits. After eight weeks, the progression of PTOA was determined histologically. Moreover, the structure of the subchondral bone was evaluated by µCT analysis. C6 deficiency could be attributed to a homozygous 3.6 kb deletion within the C6 gene and subsequent loss of the C5b binding site. Serum from C6-/- animals revealed no hemolytic activity. After ACLT surgery, joints of C6-/- rabbits exhibited significantly lower OA scores, including reduced cartilage damage, hypocellularity, cluster formation, and osteophyte number, as well as lower chondrocyte apoptosis rates and synovial prostaglandin E2 levels. Moreover, ACLT surgery significantly decreased the trabecular number in the subchondral bone of C6-/- rabbits. Overall, the absence of TCC protected from injury-induced OA progression but had minor effects on the micro-structure of the subchondral bone.

Striking a new path in reducing cartilage breakdown: combination of antioxidative therapy and chondroanabolic stimulation after blunt cartilage trauma.

Cartilage injury can trigger crucial pathomechanisms, including excessive cell death and expression of matrix-destructive enzymes, which contribute to the progression of a post-traumatic osteoarthritis (PTOA). With the intent to create a novel treatment strategy for alleviating trauma-induced cartilage damage, we complemented a promising antioxidative approach based on cell and chondroprotective N-acetyl cysteine (NAC) by chondroanabolic stimulation. Overall, three potential pro-anabolic growth factors - IGF-1, BMP7 and FGF18 - were tested comparatively with and without NAC in an ex vivo human cartilage trauma-model. For that purpose, full-thickness cartilage explants were subjected to a defined impact (0.59 J) and subsequently treated with the substances. Efficacy of the therapeutic approaches was evaluated by cell viability, as well as various catabolic and anabolic biomarkers, representing the present matrix turnover. Although monotherapy with NAC, FGF18 or BMP7 significantly prevented trauma-induced cell dead and breakdown of type II collagen, combination of NAC and one of the growth factors did not yield significant benefit as compared to NAC alone. IGF-1, which possessed only moderate cell protective and no chondroprotective qualities after cartilage trauma, even reduced NAC-mediated cell and chondroprotection. Despite significant promotion of type II collagen expression by IGF-1 and BMP7, addition of NAC completely suppressed this chondroanabolic effect. All in all, NAC and BMP7 emerged as best combination. As our findings indicate limited benefits of the simultaneous multidirectional therapy, a sequential application might circumvent adverse interferences, such as suppression of type II collagen biosynthesis, which was found to be reversed 7 days after NAC withdrawal.

Hypothermia Promotes Cell-Protective and Chondroprotective Effects After Blunt Cartilage Trauma.

BACKGROUND: Cryotherapy is routinely administered after sports injuries of synovial joints. Although positive clinical effects on periarticular swelling and pain have been described, the effects on the cell biological activities of cartilage and synovial cells remain largely unknown so far. HYPOTHESIS: Local hypothermia alleviates synovial reactions and prevents chondrocyte death as well as cartilage destructive processes after blunt cartilage trauma. STUDY DESIGN: Controlled laboratory study. METHODS: Human cartilage explants were impacted by a drop-tower apparatus (0.59 J) and cultured at 24 hours or 7 days in different temperature conditions (2 hours [short term], 16 hours [medium term], or throughout [long term] at 27°C; afterwards or throughout at 37°C). Besides, isolated human fibroblast-like synoviocytes (FLS) were stimulated with traumatized cartilage conditioned medium and cultured as mentioned above up to 4 days. The effects of hypothermia were evaluated by cell viability, gene expression, type II collagen synthesis and cleavage, as well as the release of matrix metalloproteinase (MMP)-2, MMP-13, and interleukin 6 (IL-6). RESULTS: Seven days after trauma, hypothermic treatment throughout improved cell viability (short term: 10.1% [ P = .016]; medium term: 6% [ P = .0362]; long term: 12.5% [ P = .0039]). Short-term hypothermia attenuated the expression of catabolic MMP-13 (mRNA: -2.2-fold [ P = .0119]; protein: -2-fold [ P = .0238]). Whereas type II collagen synthesis (1.7-fold [ P = .0227]) was increased after medium-term hypothermia, MMP-13 expression (mRNA: -30.8-fold [ P = .0025]; protein: -10.3-fold [ P < .0001]) and subsequent cleavage of type II collagen (-1.1-fold [ P = .0489]) were inhibited. Long-term hypothermia further suppressed MMP release (pro-MMP-2: -3-fold [ P = .0222]; active MMP-2: -5.2-fold [ P = .0183]; MMP-13: -56-fold [ P < .0001]) and type II collagen breakdown (-1.6-fold [ P = .0036]). Four days after FLS stimulation, hypothermia significantly suppressed the gene expression of matrix-destructive enzymes after medium-term (MMP-3: -4.1-fold [ P = .0211]) and long-term exposure (a disintegrin and metalloproteinase with thrombospondin motifs 4 [ADAMTS4]: -4.3-fold [ P = .0045]; MMP-3: -25.8-fold [ P = .014]; MMP-13: -122-fold [ P = .0444]) and attenuated IL-6 expression by trend. CONCLUSION: After blunt cartilage trauma, initial hypothermia for only 2 hours and/or 16 hours induced significant cell-protective and chondroprotective effects and promoted the anabolic activity of chondrocytes, while the expression of matrix-destructive enzymes by stimulated FLS was attenuated by prolonged hypothermia. CLINICAL RELEVANCE: The findings of this preliminary ex vivo investigation indicate that optimized cryotherapy management after cartilage trauma might prevent matrix-degenerative processes associated with the pathogenesis of posttraumatic osteoarthritis.

Differential Interactive Effects of Cartilage Traumatization and Blood Exposure In Vitro and In Vivo.

BACKGROUND: Sport injuries of the knee often lead to posttraumatic arthritis. In addition to direct damage of the cartilage, trauma-associated intra-articular bleeding may cause hemarthrosis. Both blood exposure and trauma are known to induce cell death and inflammation and to enhance proteoglycan release in cartilage. HYPOTHESIS: Blood exposure increases chondrocyte death as well as inflammatory and degenerative processes in traumatized cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: Human macroscopically intact osteoarthritic (OA) cartilage explants were impacted by a drop-tower system (0.59 J) and cultivated with or without 10% blood. Interactive effects were studied concerning cell survival, gene expression, and the release of mediators over 24 hours and 96 hours. To evaluate the effects of trauma and hemarthrosis in vivo, a newly established blunt cartilage trauma model in the rabbit was used. Treatment of the knee joints of mature New Zealand White rabbits consisted of the following groups: control (C), arthrotomy (A), arthrotomy with cartilage trauma (AT; 1.0 J), and arthrotomy with cartilage trauma and blood injection (ATH). After 1 and 12 weeks, inflammatory mediators in the synovial fluid and histological changes of the cartilage were determined, and immunohistological staining was performed. RESULTS: The in vitro studies revealed a significant additional or synergistic effect of blood exposure on trauma-induced chondrocyte death, interleukin (IL)-1ß and prostaglandin-E2 (PGE2) release, and matrix metalloproteinase (MMP)/pro-MMP level. Singular arthrotomy in vivo induced a temporary inflammation. Histologically, cartilage trauma caused significant OA changes that were not aggravated by an additional hemarthrosis. Trauma led to a persistent deposition of terminal complement complex (TCC), being enhanced by hemarthrosis. However, trauma-induced formation of osteophytes and arthrotomy-induced elevation of tumor necrosis factor-α release were reduced by hemarthrosis. CONCLUSION: While blood exposure clearly aggravated trauma-induced OA processes in the in vitro model, a singular blood injection revealed heterogeneous effects in vivo, enhancing TCC deposition but reducing trauma-induced osteophyte formation while the histological score of traumatized cartilage was not further impaired. CLINICAL RELEVANCE: The results of this study indicate that a singular, limited bleeding event might not exacerbate early trauma-induced cartilage degeneration in joint injuries. An early removal of intra-articular blood may not prevent the final resulting cartilage damage.

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage.

INTRODUCTION: The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints. METHODS: We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1ß) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1ß and TNF-α was additionally examined by scratch assays and outgrowth experiments. RESULTS: A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1ß and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC. CONCLUSION: These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1ß and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.

Single impact cartilage trauma and TNF-α: interactive effects do not increase early cell death and indicate the need for bi-/multidirectional therapeutic approaches.

Blunt trauma of articular cartilage, often resulting from accidents or sports injuries, is associated with local inflammatory reactions and represents a major risk factor for development of post-traumatic osteoarthritis. TNF-α is increased in synovial fluid early after trauma, potentiates injury-induced proteoglycan degradation and may act proapoptotic under permissive conditions. We asked whether TNF-α also influences chondrocyte death, gene expression of catabolic and anabolic markers and the release of proinflammatory mediators in the early post-traumatic phase. Interactive effects of a defined single impact trauma (0.59 J) and TNF-α (100 ng/ml) on human early-stage osteoarthritic cartilage were investigated in vitro over 24 h. Exposure of traumatized cartilage to TNF-α did not increase chondrocyte death. IL-6-synthesis was augmented by trauma, TNF-α and combined treatment. The impact increased the release of PGE2 and PGD2 in the presence and absence of TNF-α to a similar extent while TNF-α alone showed no effect. In contrast, NOS2A-expression and nitric oxide (NO)-release were not affected by trauma but significantly increased by TNF-α. Expression of OPG and RANKL was not affected by TNF-α but modulated by trauma. TNF-α with and without trauma significantly induced MMP1 gene expression. These results indicate that TNF-α does not potentiate early cell death in early-stage osteoarthritic cartilage after blunt injury. However, trauma and TNF-α showed independent and interactive effects concerning prostaglandin and NO release. TNF-α probably contributes to cartilage degradation after trauma by an early induction of MMP1 gene expression. Our study confirms that an anti-TNF-α therapy may have inhibitory effects on catabolic and, partly, on inflammatory processes after a single impact trauma. As TNF-α does not contribute to the loss of chondrocytes in the initial post-traumatic phase, a combination with pharmaco-therapeutic strategies reducing early cell death could be reasonable.

Development of a new biomechanically defined single impact rabbit cartilage trauma model for in vivo-studies.

BACKGROUND: Clinically oriented and easy to handle animal models are urgently needed to test pharmacologic treatment of cartilage trauma to reduce the resulting tissue damage by chondrocyte apoptosis and induction of matrix-degrading enzymes. AIM: To develop a biomechanically defined cartilage trauma model. MATERIAL AND METHODS: We constructed a novel trauma device that allows biomechanically defined force application to the load-bearing region of the medial and lateral femoral condyles in adult rabbits. The fixation to the femur was specially designed to avoid uncontrolled influx of blood into the joint. The device was tested on the articular femoral surface of cadaveric rabbits. RESULTS: At a lower energy (1.0 J), the tests showed that superficial and partially deep fissuring, partial necrosis of the chondrocytes, and early proteoglycan loss occurred at the region of impact. Subchondral fractures could be excluded by micro CT. At higher energy (≥ 1.4 J), we observed more pronounced deep fissuring and in some cases complete shearing of the articular cartilage from the subchondral bone. CONCLUSION: Our model represents an easy to use method to create a biomechanically defined cartilage trauma and offers some advantages with respect to handling under aseptic surgical conditions and prevention of uncontrolled intra-articular bleeding from the bone marrow compartment for pharmacologic studies.

Single impact trauma in human early-stage osteoarthritic cartilage: implication of prostaglandin D2 but no additive effect of IL-1ß on cell survival.

Injury to articular cartilage is often associated with an inflammatory reaction and frequently results in the development of post-traumatic osteoarthritis (post-traumatic OA). Cell death, inflammation and loss of proteoglycans participate in these mechanisms with p38MAPK being one of the pivotal signaling kinases. Therefore, the interaction of trauma and of the pro-inflammatory cytokine IL-1ß was investigated in an in vitro tissue model of human osteoarthritic cartilage. Trauma was induced by impacting cartilage explants with a drop-tower system and its effect was measured in terms of cell survival, gene expression and the release of mediators. In addition, the effect of concomitant IL­1ß stimulation and p38MAPK inhibition by SB203580 was investigated. We found a significant decrease in chondrocyte viability after trauma, but no additional effect of IL-1ß stimulation. SB203580 had a tendency to improve cell survival suggesting a role for p38 signaling in cell viability after impact in an inflammatory environment. We showed that various mediators are released in response to trauma with or without IL-1ß stimulation, differing in composition and time response. Trauma resulted in an increased release of IL-6, whereas TNF-α and IL-1ß release was unaffected. Prostaglandin (PG) and NO synthesis pathways were both affected by trauma and/or IL-1ß. We demonstrate for the first time an elevated release of prostaglandin D2 (PGD2) by human articular cartilage in response to a single mechanical impact. The up-regulation of mediators was time-dependent, with a more early increase of PGD2 compared to prostaglandin E2 (PGE2) and a late induction of NO by co-stimulation with IL-1ß between 6 and 24 h.

The effect of high-energy extracorporeal shock waves on hyaline cartilage of adult rats in vivo.

The aim of this study was to determine if extracorporeal shock wave therapy (ESWT) in vivo affects the structural integrity of articular cartilage. A single bout of ESWT (1500 shock waves of 0.5 mJ/mm(2)) was applied to femoral heads of 18 adult Sprague-Dawley rats. Two sham-treated animals served as controls. Cartilage of each femoral head was harvested at 1, 4, or 10 weeks after ESWT (n = 6 per treatment group) and scored on safranin-O-stained sections. Expression of tenascin-C and chitinase 3-like protein 1 (Chi3L1) was analyzed by immunohistochemistry. Quantitative real-time polymerase chain reaction (PCR) was used to examine collagen (II)alpha(1) (COL2A1) expression and chondrocyte morphology was investigated by transmission electron microscopy no changes in Mankin scores were observed after ESWT. Positive immunostaining for tenascin-C and Chi3L1 was found up to 10 weeks after ESWT in experimental but not in control cartilage. COL2A1 mRNA was increased in samples 1 and 4 weeks after ESWT. Alterations found on the ultrastructural level showed expansion of the rough-surfaced endoplasmatic reticulum, detachment of the cell membrane and necrotic chondrocytes. Extracorporeal shock waves caused alterations of hyaline cartilage on a molecular and ultrastructural level that were distinctly different from control. Similar changes were described before in the very early phase of osteoarthritis (OA). High-energy ESWT might therefore cause degenerative changes in hyaline cartilage as they are found in initial OA.

Influence of p38MAPK inhibition on IL-1beta-stimulated human chondrocytes: a microarray approach.

Articular chondrocytes respond to extracellular influences by activating signaling pathways which change gene expression. One key signal transduction pathway of inflammatory joint disease is mediated by the p38MAPK which is known to be activated by the pro-inflammatory cytokine IL-1beta. We used the p38MAPK inhibitor SB203580 and a whole human genome microarray in an in vitro inflammation model to identify genes regulated by this pathway in human chondrocytes. We found that 1,141 genes were regulated by IL-1beta, and 646 genes were regulated by the inhibitor whereas 116 genes were co-regulated by both substances. To elucidate the overall effect of SB203580, a GoMiner pathway analysis was performed which revealed involvement of versatile biological processes. Predominantly affected terms were 'response to stimulus', 'oxygen metabolism' and 'ligase activity'. We discuss herein the relevance and function of affected fields including the involved genes and unexpected effects of p38MAPK inhibition as it relates to the context of cartilage. Our results do not predict a pro-apoptotic or cancer promoting effect and markedly extend the knowledge on p38MAPK inhibition in chondrocytes beyond primary target genes.

IL-1beta regulates FHL2 and other cytoskeleton-related genes in human chondrocytes.

In osteoarthritis (OA), cartilage destruction is associated not only with an imbalance of anabolic and catabolic processes but also with alterations of the cytoskeletal organization in chondrocytes, although their pathogenetic origin is largely unknown so far. Therefore, we have studied possible effects of the proinflammatory cytokine IL-1beta on components of the cytoskeleton in OA chondrocytes on gene expression level. Using a whole genome array, we found that IL-1beta is involved in the regulation of many cytoskeleton-related genes. Apart from well-known cytoskeletal components, the expression and regulation of four genes coding for LIM proteins were shown. These four genes were previously undescribed in the chondrocyte context. Quantitative PCR analysis confirmed significant downregulation of Fhl1, Fhl2, Lasp1, and Pdlim1 as well as Tubb and Vim by IL-1beta. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 counteracted the influence of IL-1beta on Fhl2 and Tubb expression, indicating partial involvement of this signaling pathway. Downregulation of the LIM-only protein FHL2 was confirmed additionally on the protein level. In agreement with these results, IL-1beta induced changes in the morphology of chondrocytes, the organization of the cytoskeleton, and the cellular distribution of FHL2. We conclude that L-1beta is involved in the regulation of various cytoskeletal components in human chondrocytes including the multifunctional protein FHL2. This might be relevant for the pathogenesis of OA.