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1.
Osteoarthritis Cartilage ; 32(5): 514-525, 2024 May.
Article En | MEDLINE | ID: mdl-38242312

OBJECTIVE: The complement cascade as major fluid phase innate immune system is activated during progression of osteoarthritis (OA). Generated anaphylatoxins and the corresponding receptors C3aR and C5aR1 are associated with the calcification of blood vessels and involved in osteogenic differentiation. This study aims on elucidating whether complement activation products contribute to cartilage calcification of OA cartilage. METHOD: Human articular chondrocytes were osteogenically differentiated in vitro in the presence or absence of C3a, C5a, and bone morphogenetic protein (BMP) 2. Furthermore, macroscopically intact (OARSI grade ≤ 1) and highly degenerated human cartilage (OARSI grade ≥ 3) was used for C3aR and C5aR1 histochemistry. Calcification of the cartilage was assessed by Alizarin Red S and von Kossa staining. RESULTS: C3a and C5a amplified matrix mineralization during in vitro osteogenesis, while inhibition of the corresponding receptors impaired calcium deposition. Moreover, C3aR and C5aR1 expression was upregulated during osteogenic differentiation and also in degenerated cartilage. Additionally, anaphylatoxin receptor expression was positively associated with calcification of native cartilage tissue and calcium deposition during osteogenic differentiation. Finally, the pro-hypertrophic growth factor BMP2 induced the expression of C5aR1. CONCLUSIONS: Our findings indicate that anaphylatoxins and their receptors play a decisive role in cartilage calcification processes during OA progression.


Calcinosis , Osteoarthritis , Humans , Anaphylatoxins/metabolism , Osteogenesis , Calcium/metabolism , Cartilage/metabolism , Complement C5a/metabolism , Complement C5a/pharmacology
2.
Elife ; 122023 10 19.
Article En | MEDLINE | ID: mdl-37855367

Accumulation of dysfunctional chondrocytes has detrimental consequences on the cartilagehomeostasis and is thus thought to play a crucial role during the pathogenesis of osteoarthritis(OA). However, the underlying mechanisms of phenotypical alteration in chondrocytes areincompletely understood. Here, we provide evidence that disruption of the intracellularvimentin network and consequent phenotypical alteration in human chondrocytes results in anexternalization of the intermediate filament. The presence of the so-called cell surfacevimentin (CSV) on chondrocytes was associated with the severity of tissue degeneration inclinical OA samples and was enhanced after mechanical injury of cartilage tissue. By meansof a doxorubicine-based in vitro model of stress-induced premature senescence (SIPS), wecould confirm the connection between cellular senescence and amount of CSV. AlthoughsiRNA-mediated silencing of CDKN2A clearly reduced the senescent phenotype as well asCSV levels of human chondrocytes, cellular senescence could not be completely reversed.Interestingly, knockdown of vimentin resulted in a SIPS-like phenotype and consequentlyincreased CSV. Therefore, we concluded that the integrity of the intracellular vimentinnetwork is crucial to maintain cellular function in chondrocytes. This assumption could beconfirmed by chemically- induced collapse of the vimentin network, which resulted in cellularstress and enhanced CSV expression. Regarding its biological function, CSV was found to beassociated with enhanced chondrocyte adhesion and plasticity. While osteogenic capacitiesseemed to be enhanced in chondrocytes expressing high levels of CSV, the chondrogenicpotential was clearly compromised. Overall, our study reinforces the importance of thevimentin network in maintenance of the chondrogenic phenotype and introduces CSV as anovel membrane-bound marker of dysfunctional chondrocytes.


Intermediate Filaments , Osteoarthritis , Humans , Intermediate Filaments/metabolism , Chondrocytes/metabolism , Vimentin/metabolism , Cellular Senescence/genetics , Osteoarthritis/metabolism
3.
Cell Mol Biol Lett ; 28(1): 76, 2023 Sep 30.
Article En | MEDLINE | ID: mdl-37777764

During aging and after traumatic injuries, cartilage and bone cells are exposed to various pathophysiologic mediators, including reactive oxygen species (ROS), damage-associated molecular patterns, and proinflammatory cytokines. This detrimental environment triggers cellular stress and subsequent dysfunction, which not only contributes to the development of associated diseases, that is, osteoporosis and osteoarthritis, but also impairs regenerative processes. To counter ROS-mediated stress and reduce the overall tissue damage, cells possess diverse defense mechanisms. However, cellular antioxidative capacities are limited and thus ROS accumulation can lead to aberrant cell fate decisions, which have adverse effects on cartilage and bone homeostasis. In this narrative review, we address oxidative stress as a major driver of pathophysiologic processes in cartilage and bone, including senescence, misdirected differentiation, cell death, mitochondrial dysfunction, and impaired mitophagy by illustrating the consequences on tissue homeostasis and regeneration. Moreover, we elaborate cellular defense mechanisms, with a particular focus on oxidative stress response and mitophagy, and briefly discuss respective therapeutic strategies to improve cell and tissue protection.


Osteoarthritis , Osteoporosis , Humans , Reactive Oxygen Species/metabolism , Oxidative Stress/physiology , Osteoarthritis/metabolism , Cell Differentiation , Cellular Senescence
5.
Cells ; 12(6)2023 03 13.
Article En | MEDLINE | ID: mdl-36980228

Terminal complement complex (TCC) deposition was identified in human degenerated discs. To clarify the role of terminal complement activation in disc degeneration (DD), we investigated respective activating mechanisms and cellular effects in annulus fibrosus (AF) cells. Isolated cells from human AF, nucleus pulposus (NP), and endplate (EP) were stimulated with human serum alone or with zymosan and treated with either the C3 inhibitor Cp40 or the C5 antibody eculizumab. Complement activation was determined via anaphylatoxin generation and TCC deposition detection. Thereby, induced catabolic effects were evaluated in cultured AF cells. Moreover, C5 cleavage under degenerative conditions in the presence of AF cells was assessed. Zymosan-induced anaphylatoxin generation and TCC deposition was significantly suppressed by both complement inhibitors. Zymosan induced gene expression of ADAMTS4, MMP1, and COX2. Whereas the C3 blockade attenuated the expression of ADAMTS4, the C5 blockade reduced the expression of ADAMTS4, MMP1, and COX2. Direct C5 cleavage was significantly enhanced by EP conditioned medium from DD patients and CTSD. These results indicate that terminal complement activation might be functionally involved in the progression of DD. Moreover, we found evidence that soluble factors secreted by degenerated EP tissue can mediate direct C5 cleavage, thereby contributing to complement activation in degenerated discs.


Annulus Fibrosus , Intervertebral Disc Degeneration , Intervertebral Disc , Humans , Intervertebral Disc Degeneration/metabolism , Intervertebral Disc/metabolism , Matrix Metalloproteinase 1/metabolism , Cyclooxygenase 2/metabolism , Zymosan/metabolism , Complement Activation
6.
Biomolecules ; 13(2)2023 01 22.
Article En | MEDLINE | ID: mdl-36830586

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.


Cartilage, Articular , Osteoarthritis , Animals , Rabbits , Complement Membrane Attack Complex/pharmacology , Cartilage, Articular/pathology , Osteoarthritis/pathology , Anterior Cruciate Ligament/pathology , Anterior Cruciate Ligament/surgery , Chondrocytes/pathology
7.
Front Bioeng Biotechnol ; 10: 965302, 2022.
Article En | MEDLINE | ID: mdl-36159664

Joint injuries are known to induce pathomechanisms that might lead to posttraumatic osteoarthritis (PTOA). In this regard, statins with their pleiotropic effects could represent potential therapeutic agents in preventing the development of PTOA. Therefore, we investigated the effects of simvastatin and fluvastatin in a drop-tower-based human ex vivo cartilage trauma model. After 7 days, a mechanical impact (0.59 J) resulted in a decrease of the cell viability and increased expression of catabolic enzymes in cartilage explants. Simvastatin and fluvastatin treatment of impacted cartilage demonstrated cell protective effects in a concentration dependent manner. Moreover, statin therapy exhibited chondroprotective effects as demonstrated by attenuated expression of MMP-2 and MMP-13 as well as subsequent breakdown of collagen type II (after impact). Further analysis indicated antioxidative properties of the statins by upregulating the gene expression of SOD2 and suppression that of NOX2 and NOX4. Despite its protective effects, simvastatin impaired the biosynthesis of collagen type II, which was confirmed during chondrogenic redifferentiation of high passage chondrocytes. However, while long-term administration of statins for 4 weeks impaired chondrogenic redifferentiation, addition of simvastatin at low concentrations for 1 week exhibited a slightly promoting effect. In conclusion, our data imply that simvastatin and fluvastatin are suitable in terms of initial harm reduction after cartilage trauma.

9.
Dis Model Mech ; 15(5)2022 05 01.
Article En | MEDLINE | ID: mdl-35394023

Osteoporosis is a systemic metabolic skeletal disease characterized by low bone mass and strength associated with fragility fractures. Oxidative stress, which results from elevated intracellular reactive oxygen species (ROS) and arises in the aging organism, is considered one of the critical factors contributing to osteoporosis. Mitochondrial (mt)ROS, as the superoxide anion (O2-) generated during mitochondrial respiration, are eliminated in the young organism by antioxidant defense mechanisms, including superoxide dismutase 2 (SOD2), the expression and activity of which are decreased in aging mesenchymal progenitor cells, accompanied by increased mtROS production. Using a mouse model of osteoblast lineage cells with Sod2 deficiency, we observed significant bone loss in trabecular and cortical bones accompanied by decreased osteoblast activity, increased adipocyte accumulation in the bone marrow and augmented osteoclast activity, suggestive of altered mesenchymal progenitor cell differentiation and osteoclastogenesis. Furthermore, osteoblast senescence was increased. To date, there are only a few studies suggesting a causal association between mtROS and cellular senescence in tissue in vivo. Targeting SOD2 to improve redox homeostasis could represent a potential therapeutic strategy for maintaining bone health during aging.


Osteoblasts , Osteoporosis , Superoxide Dismutase , Animals , Mice , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteoporosis/metabolism , Phenotype , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism
10.
Cells ; 11(7)2022 03 25.
Article En | MEDLINE | ID: mdl-35406671

Accumulation of senescent chondrocytes is thought to drive inflammatory processes and subsequent cartilage degeneration in age-related as well as posttraumatic osteoarthritis (OA). However, the underlying mechanisms of senescence and consequences on cartilage homeostasis are not completely understood so far. Therefore, suitable in vitro models are needed to study chondrocyte senescence. In this study, we established and evaluated a doxorubicin (Doxo)-based model of stress-induced premature senescence (SIPS) in human articular chondrocytes (hAC). Cellular senescence was determined by the investigation of various senescence associated (SA) hallmarks including ß-galactosidase activity, expression of p16, p21, and SA secretory phenotype (SASP) markers (IL-6, IL-8, MMP-13), the presence of urokinase-type plasminogen activator receptor (uPAR), and cell cycle arrest. After seven days, Doxo-treated hAC displayed a SIPS-like phenotype, characterized by excessive secretion of SASP factors, enhanced uPAR-positivity, decreased proliferation rate, and increased ß-galactosidase activity. This phenotype was proven to be stable seven days after the removal of Doxo. Moreover, Doxo-treated hAC exhibited increased granularity and flattened or fibroblast-like morphology. Further analysis implies that Doxo-mediated SIPS was driven by oxidative stress as demonstrated by increased ROS levels and NO release. Overall, we provide novel insights into chondrocyte senescence and present a suitable in vitro model for further studies.


Chondrocytes , Osteoarthritis , Cellular Senescence/genetics , Chondrocytes/metabolism , Doxorubicin/pharmacology , Humans , Osteoarthritis/metabolism , beta-Galactosidase/metabolism
11.
Int J Mol Sci ; 22(14)2021 Jul 06.
Article En | MEDLINE | ID: mdl-34298867

The hexosamine biosynthetic pathway (HBP) is essential for the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the building block of glycosaminoglycans, thus playing a crucial role in cartilage anabolism. Although O-GlcNAcylation represents a protective regulatory mechanism in cellular processes, it has been associated with degenerative diseases, including osteoarthritis (OA). The present study focuses on HBP-related processes as potential therapeutic targets after cartilage trauma. Human cartilage explants were traumatized and treated with GlcNAc or glucosamine sulfate (GS); PUGNAc, an inhibitor of O-GlcNAcase; or azaserine (AZA), an inhibitor of GFAT-1. After 7 days, cell viability and gene expression analysis of anabolic and catabolic markers, as well as HBP-related enzymes, were performed. Moreover, expression of catabolic enzymes and type II collagen (COL2) biosynthesis were determined. Proteoglycan content was assessed after 14 days. Cartilage trauma led to a dysbalanced expression of different HBP-related enzymes, comparable to the situation in highly degenerated tissue. While GlcNAc and PUGNAc resulted in significant cell protection after trauma, only PUGNAc increased COL2 biosynthesis. Moreover, PUGNAc and both glucosamine derivatives had anti-catabolic effects. In contrast, AZA increased catabolic processes. Overall, "fueling" the HBP by means of glucosamine derivatives or inhibition of deglycosylation turned out as cells and chondroprotectives after cartilage trauma.


Biosynthetic Pathways/drug effects , Cartilage Diseases/drug therapy , Chondrocytes/drug effects , Chondrocytes/metabolism , Glucosamine/pharmacology , Hexosamines/metabolism , Uridine Diphosphate N-Acetylglucosamine/pharmacology , Biomarkers/metabolism , Cartilage/drug effects , Cartilage/metabolism , Cartilage Diseases/metabolism , Cell Survival/drug effects , Collagen Type II/metabolism , Female , Gene Expression/drug effects , Glycosylation/drug effects , Humans , Male , Middle Aged , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Phosphorylation/drug effects
12.
Eur Spine J ; 30(8): 2247-2256, 2021 08.
Article En | MEDLINE | ID: mdl-34169354

PURPOSE: Formation of terminal complement complex (TCC), a downstream complement system activation product inducing inflammatory processes and cell lysis, has been identified in degenerated discs. However, it remains unclear which molecular factors regulate complement activation during disc degeneration (DD). This study investigated a possible involvement of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) and the lysosomal protease cathepsin D (CTSD). METHODS: Disc biopsies were collected from patients suffering from DD (n = 43) and adolescent idiopathic scoliosis (AIS, n = 13). Standardized tissue punches and isolated cells from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were stimulated with 5% human serum (HS) alone or in combination with IL-1ß, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed. RESULTS: In DD tissue cultures, IL-1ß stimulation decreased the percentage of TCC + cells in AF and EP (P < 0.05), whereas CTSD stimulation significantly increased TCC deposition in NP (P < 0.01) and zymosan in EP (P < 0.05). Overall, the expression of CD46, CD55 and CD59 significantly increased in all isolated cells during culture (P < 0.05). Moreover, cellular TCC deposition was HS concentration dependent but unaffected by IL-1ß, CTSD or zymosan. CONCLUSION: These results suggest a functional relevance of IL-1ß and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1ß may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology.


Intervertebral Disc Degeneration , Intervertebral Disc , Adolescent , Cathepsin D , Cells, Cultured , Complement Membrane Attack Complex , Humans , Interleukin-1beta
13.
Eur Spine J ; 30(1): 217-226, 2021 01.
Article En | MEDLINE | ID: mdl-32936402

PURPOSE: The complement system is a crucial part of innate immunity. Recent work demonstrated an unexpected contribution to tissue homeostasis and degeneration. This study investigated for the first time, in human disc tissues, the deposition profile of the complement activation product terminal complement complex (TCC), an inflammatory trigger and inducer of cell lysis, and its inhibitor CD59, and their correlation with the degree of disc degeneration (DD). METHODS: Disc biopsies were collected from patients diagnosed with DD (n = 39, age 63 ± 12) and adolescent idiopathic scoliosis (AIS, n = 10, age 17 ± 4) and compared with discs from healthy Young (n = 11, age 7 ± 7) and Elder (n = 10, age 65 ± 15) donors. Immunohistochemical detection of TCC and CD59 in nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) was correlated with age, Pfirrmann grade and Modic changes. RESULTS: Higher percentage of TCC+ cells was detected in the NP and EP of DD compared to Elder (P < 0.05), and in the EP of Young versus Elder (P < 0.001). In DD, TCC deposition was positively correlated with Pfirrmann grade, but not with Modic changes, whereas for Young donors, a negative correlation was found with age, indicating TCC's involvement not only in DD, but also in early stages of skeletal development. Higher CD59 positivity was found in AIS and DD groups compared to Young (P < 0.05), and it was negatively correlated with the age of the patients. CONCLUSION: TCC deposition positively correlated with the degree of disc degeneration. A functional relevance of TCC may exist in DD, representing a potential target for new therapeutics.


Annulus Fibrosus , Intervertebral Disc Degeneration , Intervertebral Disc , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Complement Activation , Complement Membrane Attack Complex , Humans , Infant , Infant, Newborn , Magnetic Resonance Imaging , Middle Aged , Young Adult
14.
Int J Mol Sci ; 21(5)2020 Feb 25.
Article En | MEDLINE | ID: mdl-32106481

Traumatic injuries of the knee joint result in a wide variety of pathomechanisms, which contribute to the development of so-called posttraumatic osteoarthritis (PTOA). These pathogenetic processes include oxidative stress, excessive expression of catabolic enzymes, release of damage-associated molecular patterns (DAMPs), and synovial inflammation. The present review focuses on the underlying pathomechanisms of PTOA and in particular the behavior and fate of the surviving chondrocytes, comprising chondrocyte metabolism, regulated cell death, and phenotypical changes comprising hypertrophy and senescence. Moreover, possible therapeutic strategies, such as chondroanabolic stimulation, anti-oxidative and anti-inflammatory treatment, as well as novel therapeutic targets are discussed.


Chondrocytes/metabolism , Knee Injuries/complications , Osteoarthritis, Knee/metabolism , Animals , Cell Death , Chondrocytes/pathology , Humans , Osteoarthritis, Knee/etiology , Osteoarthritis, Knee/pathology , Oxidative Stress
15.
J Clin Med ; 9(1)2020 Jan 18.
Article En | MEDLINE | ID: mdl-31963737

This study aimed to assess associations between serum cartilage oligomeric matrix protein (sCOMP) and phenotypic characteristics in late-stage hip and knee Osteoarthritis (OA) as well as its correlation with further serum markers of possible comorbidities in the Ulm Osteoarthritis Study. Moreover, the prognostic relevance of preoperative sCOMP concentrations for short-term functionality and pain outcomes after hip or knee joint replacement was explored. Preoperative serum samples and detailed information about the health status (i.e., WOMAC scores, Hannover Functionality Status (FFbH)) of 754 OA patients undergoing total joint replacement were included. Spearman rank-correlation coefficients and multiple linear regression models were used to evaluate the relationships between sCOMP, other serum markers, and health outcomes. There was a significant positive association between sCOMP and markers of renal (cystatin C, creatinine, and eGFR) and cardiac (e.g., NT-proBNP) impairment. Since renal failure might cause accumulation of sCOMP, additional adjustment with eGFR was performed. Preoperative sCOMP levels in knee OA but not hip OA patients were positively associated with FFbH, WOMAC function sub-scale and total WOMAC scale as well as the post-operative WOMAC stiffness sub-scale six months after surgery. Our data clearly demonstrate an association between sCOMP and renal function as well as other confounding factors, which should be considered in future biomarker studies.

16.
Front Bioeng Biotechnol ; 8: 624096, 2020.
Article En | MEDLINE | ID: mdl-33553127

The incidence of musculoskeletal diseases is steadily increasing with aging of the population. In the past years, extracellular vesicles (EVs) have gained attention in musculoskeletal research. EVs have been associated with various musculoskeletal pathologies as well as suggested as treatment option. EVs play a pivotal role in communication between cells and their environment. Thereby, the EV cargo is highly dependent on their cellular origin. In this review, we summarize putative mechanisms by which EVs can contribute to musculoskeletal tissue homeostasis, regeneration and disease, in particular matrix remodeling and mineralization, pro-angiogenic effects and immunomodulatory activities. Mesenchymal stromal cells (MSCs) present the most frequently used cell source for EV generation for musculoskeletal applications, and herein we discuss how the MSC phenotype can influence the cargo and thus the regenerative potential of EVs. Induced pluripotent stem cell-derived mesenchymal progenitor cells (iMPs) may overcome current limitations of MSCs, and iMP-derived EVs are discussed as an alternative strategy. In the last part of the article, we focus on therapeutic applications of EVs and discuss both practical considerations for EV production and the current state of EV-based therapies.

17.
Cell Death Dis ; 10(10): 683, 2019 09 17.
Article En | MEDLINE | ID: mdl-31527653

Joint injuries are highly associated with cell death and development of posttraumatic osteoarthritis (PTOA). The present study focused on necroptosis as a possible modality of chondrocyte death after cartilage trauma and its relevance in OA disease in general. For this purpose, apoptosis- and necroptosis-associated markers were determined in highly degenerated (ICRS ≥ 3) as well as macroscopically intact cartilage tissue (ICRS ≤ 1) by means of real-time PCR and immunohistochemistry (IHC). Moreover, influence of blunt trauma and/or stimulation with cycloheximide (CHX), TNF-a, and caspase-inhibitor zVAD were investigated in cartilage explants (ICRS ≤ 1). Further characterization of necroptosis was performed in isolated chondrocytes. We found that gene expression levels of RIPK3 (4.2-fold, P < 0.0001) and MLKL (2.7-fold, P < 0.0001) were elevated in highly degenerated cartilage tissue, which was confirmed by IHC staining. After ex vivo trauma and/or CHX/TNF stimulation, addition of zVAD further enhanced expression of necroptosis-related markers as well as release of PGE2 and nitric oxide, which was in line with increased cell death and subsequent release of intracellular HMGB1 and dsDNA in CHX/TNF stimulated chondrocytes. However, trauma and/or chemically induced cell death and subsequent release of pro-inflammatory mediators could be largely attenuated by RIPK1-inhibitor necrostatin 1 or antioxidant N-acetylcysteine. Overall, the study provided clear evidence of necroptotic cell death in OA disease. Moreover, a possible link between cartilage injury and necroptotic processes was found, depending on oxidative stress and cytokine release. These results contribute to further understanding of cell death in PTOA and development of novel therapeutic approaches.


Necroptosis/physiology , Necrosis/metabolism , Osteoarthritis/complications , Gene Expression , Humans , Osteoarthritis/pathology
18.
Int J Mol Sci ; 20(12)2019 Jun 14.
Article En | MEDLINE | ID: mdl-31207966

Joint injuries are highly associated with the development of post-traumatic osteoarthritis. Previous studies revealed cell- and matrix-protective effects of N-acetylcysteine (NAC) after ex vivo cartilage trauma, while chondroanabolic stimulation with bone morphogenetic protein 7 (BMP7) enhanced type II collagen (COL2) expression. Here, as a next step, we investigated the combined and individual efficacy of intra-articular antioxidative and chondroanabolic treatment in a rabbit in vivo cartilage trauma model. Animals were randomly divided into group A (right joint: trauma (T); left joint: T+BMP7) and group B (right joint: T+NAC; left joint: T+BMP7+NAC). Condyles were impacted with the use of a spring-loaded impact device to ensure defined, single trauma administration. After 12 weeks, histopathological analysis was performed and the presence of matrix metalloproteinase 13 (MMP-13) and COL2 was assessed. Trauma-induced hypocellularity, MMP-13 expression, and cell cluster formation were reduced in NAC-treated animals. In contrast, BMP7 further increased cluster formation. Moreover, synovial concentrations of COL2 carboxy propeptide (CPII) and proteoglycan staining intensities were enhanced in NAC- and NAC+BMP7-treated joints. For the first time, the efficacy of NAC regarding early harm reduction after blunt cartilage trauma was demonstrated in vivo. However, parallel administration of BMP7 was not significantly superior compared to NAC alone.


Acetylcysteine/therapeutic use , Cartilage/metabolism , Osteoarthritis/drug therapy , Regeneration , Wounds, Nonpenetrating/complications , Acetylcysteine/pharmacology , Animals , Bone Morphogenetic Protein 7/pharmacology , Bone Morphogenetic Protein 7/therapeutic use , Cartilage/drug effects , Cartilage/injuries , Cartilage/physiology , Collagen Type II/metabolism , Female , Matrix Metalloproteinase 13/metabolism , Osteoarthritis/etiology , Rabbits , Wounds, Nonpenetrating/drug therapy
19.
Int J Mol Sci ; 20(2)2019 Jan 21.
Article En | MEDLINE | ID: mdl-30669608

Osteoarthritis (OA) is a progressive joint disease characterized by a continuous degradation of the cartilage extracellular matrix (ECM). The expression of the extracellular glycoprotein thrombospondin-4 (TSP-4) is known to be increased in injured tissues and involved in matrix remodeling, but its role in articular cartilage and, in particular, in OA remains elusive. In the present study, we analyzed the expression and localization of TSP-4 in healthy and OA knee cartilage by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot. We found that TSP-4 protein expression is increased in OA and that expression levels correlate with OA severity. TSP-4 was not regulated at the transcriptional level but we detected changes in the anchorage of TSP-4 in the altered ECM using sequential protein extraction. We were also able to detect pentameric and fragmented TSP-4 in the serum of both healthy controls and OA patients. Here, the total protein amount was not significantly different but we identified specific degradation products that were more abundant in sera of OA patients. Future studies will reveal if these fragments have the potential to serve as OA-specific biomarkers.


Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Gene Expression , Osteoarthritis, Knee/diagnosis , Osteoarthritis, Knee/genetics , Thrombospondins/genetics , Aged , Aged, 80 and over , Biomarkers , Extracellular Matrix/metabolism , Female , Humans , Male , Middle Aged , Osteoarthritis, Knee/metabolism , Protein Transport , Severity of Illness Index , Thrombospondins/blood , Thrombospondins/metabolism
20.
J Cell Mol Med ; 22(1): 77-88, 2018 01.
Article En | MEDLINE | ID: mdl-28834244

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.


Anabolic Agents/therapeutic use , Antioxidants/therapeutic use , Cartilage, Articular/pathology , Chondrocytes/pathology , Wounds, Nonpenetrating/drug therapy , Acetylcysteine/pharmacology , Acetylcysteine/therapeutic use , Aged , Aged, 80 and over , Anabolic Agents/pharmacology , Antioxidants/pharmacology , Biomarkers/metabolism , Bone Morphogenetic Protein 7/pharmacology , Bone Morphogenetic Protein 7/therapeutic use , Cell Survival/drug effects , Chondrocytes/drug effects , Chondrocytes/metabolism , Collagen Type II/metabolism , Cytoprotection/drug effects , Extracellular Matrix/metabolism , Fibroblast Growth Factors/pharmacology , Fibroblast Growth Factors/therapeutic use , Gene Expression Regulation/drug effects , Humans , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor I/therapeutic use , Middle Aged , Wounds, Nonpenetrating/pathology
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