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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. [ABSTRACT FROM AUTHOR]

Abstract: The human osteosarcoma cell line Saos-2 is widely used as a model system for human osteoblastic cells, though its phenotypic stability has not been ascertained. We therefore propagated these cells over 100 passages and compared relevant phenotypic properties. In general, higher passage cells exhibited higher proliferation rates and lower specific alkaline phosphatase activities, though mineralization was significantly more pronounced in cultures of late passage cells. Whereas expression of most genes investigated did not vary profoundly, some genes exhibited remarkable differences. Decorin, for instance, that has been discussed as a regulator of proliferation and mineralization, is strongly expressed only in early passage cells, and two receptors for pleiotrophin and midkine exhibited an almost mutually exclusive expression pattern in early and late passage cells, respectively. Our observations indicate that special care is required when results obtained with Saos-2 cells with different culture history are to be compared. [Copyright &y& Elsevier]

Cellular senescence is associated with various age‐related disorders and is assumed to play a major role in the pathogenesis of osteoarthritis (OA). Based on this, we tested a senolytic combination therapy using Dasatinib (D) and Quercetin (Q) on aged isolated human articular chondrocytes (hACs), as well as in OA‐affected cartilage tissue (OARSI grade 1–2). Stimulation with D + Q selectively eliminated senescent cells in both, cartilage explants and isolated hAC. Furthermore, the therapy significantly promoted chondroanabolism, as demonstrated by increased gene expression levels of COL2A1, ACAN, and SOX9, as well as elevated collagen type II and glycosaminoglycan biosynthesis. Additionally, D + Q treatment significantly reduced the release of SASP factors (IL6, CXCL1). RNA sequencing analysis revealed an upregulation of the anabolic factors, inter alia, FGF18, IGF1, and TGFB2, as well as inhibitory effects on cytokines and the YAP‐1 signaling pathway, explaining the underlying mechanism of the chondroanabolic promotion upon senolytic treatment. Accordingly, stimulation of untreated hAC with conditioned medium of D + Q‐treated cells similarly induced the expression of chondrogenic markers. Detailed analyses demonstrated that chondroanabolic effects could be mainly attributed to Dasatinib, while monotherapeutical application of Quercetin or Navitoclax did not promote the chondroanabolism. Overall, D + Q therapy restored the chondrogenic phenotype in OA hAC most likely by creating a pro‐chondroanabolic environment through the reduction of SASP factors and upregulation of growth factors. This senolytic approach could therefore be a promising candidate for further testing as a disease‐modifying osteoarthritis drug. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article offers information related to the treatment of articular cartilage injuries. Topics discussed include major risk factor for development of osteoarthritis, first-in-human trial on the potential use of autologous nasal septum chondrocytes for articular cartilage repair, lateral integration into adjacent cartilage as an important factor for long-term clinical success and tissue-engineered cartilage based on nasal chondrocytes.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nasal septum defects can currently only be reconstructed using autologous cartilage grafts. In this study, we examine the reconstruction of septal cartilage defects in a rabbit model using porcine decellularized nasal septal cartilage (DNSC) functionalized with recombinant platelet-derived growth factor-BB (PDFG-BB). The supportive function of the transplanted DNSC was estimated by the degree of septum deviation and shrinkage using magnetic resonance imaging (MRI). The biocompatibility of the transplanted scaffolds was evaluated by histology according to international standards. A study group with an autologous septal transplant was used as a reference. In situ regeneration of cartilage defects was assessed by histological evaluation 4 and 16 weeks following DNSC transplantation. A study group with non-functionalized DNSC was introduced for estimation of the effects of PDFG-BB functionalization. DNSC scaffolds provided sufficient structural support to the nasal septum, with no significant shrinkage or septal deviations as evaluated by the MRI. Biocompatibility analysis after 4 weeks revealed an increased inflammatory reaction of the surrounding tissue in response to DNSC as compared to the autologous transplants. The inflammatory reaction was, however, significantly attenuated after 16 weeks in the PDGF-BB group whereas only a slight improvement of the biocompatibility score was observed in the untreated group. In situ regeneration of septal cartilage, as evidenced by the degradation of the DNSC matrix and production of neocartilage, was observed in both experimental groups after 16 weeks but was more pronounced in the PDFG-BB group. Overall, DNSC provided structural support to the nasal septum and stimulated in situ regeneration of the cartilage tissue. Furthermore, PDFG-BB augmented the regenerative potential of DNSC and enhanced the healing process, as demonstrated by reduced inflammation after 16 weeks. [ABSTRACT FROM AUTHOR]

Spondyloepiphyseal dysplasia tarda (SEDT) is a genetically heterogeneous disorder often associated with the early onset of osteoarthrosis. The X-linked recessive form (SEDL) affects men and is characterized by reduced height, arm span exceeding total height, and barrel chest deformity. The radiographic phenotype comprises a hump-shaped deformity of vertebral bodies and mild epiphyseal dysplasia of the femoral head associated with early signs of hip arthrosis. The disorder is caused by mutations in the SEDL (or sedlin) gene on Xp22.12-p22.31. In 4 male patients from a German family, we identified a new nonsense mutation in SEDL exon 4 (C74A). The carrier status for the mutation could be confirmed in 2 women of the family, both of whom show no obvious signs of bone and joint diseases. SEDT should be kept in mind as a differential diagnosis in men with early "primary" bilateral osteoarthrosis [ABSTRACT FROM AUTHOR]

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-1β (IL-1β) 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-1β, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed. Results: In DD tissue cultures, IL-1β 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-1β, CTSD or zymosan. Conclusion: These results suggest a functional relevance of IL-1β and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1β may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are multipotent cells that are considered indispensable in regeneration processes after tissue trauma. MSCs are recruited to damaged areas via several chemoattractant pathways where they function as “actors” in the healing process by the secretion of manifold pro- and anti-inflammatory, antimicrobial, pro- and anticoagulatory, and trophic/angiogenic factors, but also by proliferation and differentiation into the required cells. On the other hand, MSCs represent “targets” during the pathophysiological conditions after severe trauma, when excessively generated inflammatory mediators, complement activation factors, and damage- and pathogen-associated molecular patterns challenge MSCs and alter their functionality. This in turn leads to complement opsonization, lysis, clearance by macrophages, and reduced migratory and regenerative abilities which culminate in impaired tissue repair. We summarize relevant cellular and signaling mechanisms and provide an up-to-date overview about promising future therapeutic MSC strategies in the context of severe tissue trauma. [ABSTRACT FROM AUTHOR]

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)–1β 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. [ABSTRACT FROM AUTHOR]

Multipotent mesenchymal stromal cells (MSC) exert immune-modulatory effects and support tissue regeneration in various local trauma models. In case of a polytrauma, high amounts of danger-associated molecular patterns are released, leading to a systemic increase of inflammatory mediators. The influence of such a complex inflammatory microenvironment on human MSC is mainly unknown so far. Therefore, we investigated the effects of a defined serum-free polytrauma “cocktail” containing ILͳbeta, IL6, IL8 and the anaphylatoxins C3a and C5a, in concentrations corresponding to those measured in the blood of polytrauma patients, on human MSC in vitro. The polytrauma cocktail induced directed migration of MSC with C3a representing its major soluble chemoattractive agent. Furthermore, the polytrauma cocktail and IL1beta upregulated the expression of MMP1 indicating a potential role of IL1beta to enhance MSC migration in the tissue context. COX2, PTGES and TSG6 were also found to be upregulated upon stimulation with the polytrauma cocktail or IL1beta, but not through other single factors of the polytrauma cocktail in pathophysiologically relevant concentrations. An RNA expression array of 84 inflammation-related genes revealed that both the polytrauma cocktail and IL1beta induced C3, CSF1, TLR3 and various chemokines without major qualitative or quantitative differences. These results indicate that IL1beta is a crucial mediator of the polytrauma cocktail in terms of immune-modulation and MMP1 expression. Thus, upon encountering the primary sterile, inflammatory milieu of a polytrauma, endogenous or systemically transfused MSC might be able to migrate to sites of injury, secrete TSG6 and PGE2 and to influence macrophage biology as observed in local trauma models. [ABSTRACT FROM AUTHOR]

Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration. [ABSTRACT FROM AUTHOR]

Highlights: [•] Films of controlled thickness based on mesoporous silica nanoparticles of different size and shape are made by spin-coating. [•] The films are shown to be biocompatible in vitro. [•] Particles are taken up by cells cultivated on these films. [•] Intracellular delivery of cargo present inside the pores of the particles is demonstrated. [Copyright &y& Elsevier]

The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Hexagonally arranged Gold nanoparticles with controllable diameters and inter-particle distances were deposited on thick SiO2 layers on top of Si wafers and used as masks during subsequent reactive ion etching. In this way, arrays of nanopillars are obtained with well-defined diameters (10/30 nm), inter-pillar distances (50–120 nm) and heights (20–35 nm), all on the nanoscale. Such nanotopographies served as substrate for multipotent mesenchymal stromal cells (MSC) and human osteoblasts (OB) allowing to study cellular responses to purely topographically patterned interfaces. Focus was put on adhesion, proliferation and differentiation of the cells. It turned out experimentally that adhesion is comparable for both cell types practically independent of topographical details at the substrate surface. Topography induced proliferation enhancement, however, is again independent of geometrical details in case of MSC, but significantly sensitive to pillar height in case of OB with a clear preference towards short nanopillars (20 nm). A high sensitivity to topographic details is also observed for osteogenic differentiation of MSC, in that case with a preference towards higher nanopillars (50 nm). The present experimental data also allow the important conclusion that cell proliferation and differentiation can be optimized simultaneously by fine-tuning nanoscaled topographical parameters. [Copyright &y& Elsevier]

Abstract: The skeletal and the immune system are not two independent systems, rather, there are multifaceted and complex interactions between the different cell types of both systems and there are several shared cytokines. As a part of the innate immunity, the complement system was found to be an important link between bone and immunity. Complement proteins appear to be involved in bone development and homeostasis, and specifically influence osteoblast and osteoclast activity. This review describes the complex mutual regulation of the two systems, and indicates some of the negative side effects as a result of inappropriate or excessive complement activation. [Copyright &y& Elsevier]

Bone has a high capacity for self-renewal and repair. Prolonged local secretion of interleukin 1β (IL-1β), however, is known to be associated with severe bone loss and delayed fracture healing. Since induction of bone resorption by IL-1β may not sufficiently explain these pathologic processes, we investigated, in vitro, if and how IL-1β affects migration of multipotent mesenchymal stromal cells (MSC) or osteoblasts. We found that homogenous exposure to IL-1β significantly diminished both nondirectional migration and site-directed migration toward the chemotactic factors platelet-derived growth factor (PDGF)-BB and insulinlike growth factor 1 (IGF-1) in osteoblasts. Exposure to a concentration gradient of IL-1β induced an even stronger inhibition of migration and completely abolished the migratory response of osteoblasts toward PDGF-BB, IGF-1, vascular endothelial growth factor A (VEGF-A) and the complement factor C5a. IL-1β induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK) activation and inhibition of these signaling pathways suggested an involvement in the IL-1β effects on osteoblast migration. In contrast, basal migration of MSC and their migratory activity toward PDGF-BB was found to be unaffected by IL-1β. These results indicate that the presence of IL-1β leads to impaired recruitment of osteoblasts which might influence early stages of fracture healing and could have pathological relevance for bone remodeling in inflammatory bone disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Multiple epiphyseal dysplasia (MED) is one of the more common generalised skeletal dysplasias. Due to its clinical heterogeneity diagnosis may be difficult. Mutations of at least six separate genes can cause MED. Joint deformities, joint pain and gait disorders are common symptoms. Case Presentation: We report on a 27-year-old male patient suffering from clinical symptoms of autosomal recessive MED with habitual dislocation of a multilayered patella on both sides, on the surgical treatment and on short-term clinical outcome. Clinical findings were: bilateral hip and knee pain, instability of femorotibial and patellofemoral joints with habitual patella dislocation on both sides, contractures of hip, elbow and second metacarpophalangeal joints. Main radiographic findings were: bilateral dislocated multilayered patella, dysplastic medial tibial plateaus, deformity of both femoral heads and osteoarthritis of the hip joints, and deformity of both radial heads. In the molecular genetic analysis, the DTDST mutation g.1984T > A (p.C653S) was found at the homozygote state. Carrier status was confirmed in the DNA of the patient's parents. The mutation could be considered to be the reason for the patient's disease. Surgical treatment of habitual patella dislocation with medialisation of the tibial tuberosity led to an excellent clinical outcome. Conclusions: The knowledge of different phenotypes of skeletal dysplasias helps to select genes for genetic analysis. Compared to other DTDST mutations, this is a rather mild phenotype. Molecular diagnosis is important for genetic counselling and for an accurate prognosis. Even in case of a multilayered patella in MED, habitual patella dislocation could be managed successfully by medialisation of the tibial tuberosity. [ABSTRACT FROM AUTHOR]

Dysplasia epiphysealis hemimelica (DEH) is a rare developmental disorder. The underlying pathophysiology is largely unclear. Its diagnosis is based on clinical findings and may be difficult due to its low incidence and close relationship to other disorders such as osteochondroma. We describe a 13-year-old boy who presented with a unilateral lesion of the left medial femoral condyle and left ankle. In addition to standard diagnostic tools such as radiographs and MRI, arthroscopy-guided biopsy was performed; histologic/immunohistochemical findings from cartilage-bone specimens confirmed the diagnosis and provided novel information toward a disease mechanism. The cellular phenotype of clustered chondrocytes exhibited characteristics of chondroprogenitor cells and terminally differentiated cells, suggesting dysregulation of resident progenitor cells. No other surgery was performed and during a 2 year period, we observed spontaneous ossification of the lesion associated with decreased joint impairment. Immunohistochemical analysis of the lesion provided a more accurate diagnosis and may contribute to unraveling potential novel mechanisms involved in its pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Mesenchymal stem cells are multipotent cells able to differentiate into different mesenchymal lineages. Studies in the past had suggested that two of these mesenchymal differentiation directions, the chondrogenic and the myogenic differentiation, are negatively regulated by the transcription factor NF-κB. Although osteogenic differentiation has been extensively studied, the influence of NF-κB on this differentiation lineage was not subject of detailed analyses in the past. We have analyzed the consequences of TNF-α treatment and genetic manipulation of the NF-κB pathway for osteogenic differentiation of hMSCs. Treatment of hMSCs during differentiation with TNF-α activates NF-κB and this results in enhanced expression of osteogenetic proteins like bone morphogenetic protein2 (BMP-2) and alkaline phosphatase (ALP). In addition, enhanced matrix mineralization was observed. The direct contribution of the NF-κB pathway was confirmed in cells that express a constitutively active version of the NF-κB-inducing kinase IKK2 (CA-IKK2). The IKK2/NF-κB-induced BMP-2 up-regulation results in the enhancement of RUNX2 and Osterix expression, two critical regulators of the osteogenic differentiation program. Interestingly, a genetic block of the NF-κB pathway did not interfere with osteogenic differentiation. We conclude that TNFα mediated NF-κB activation, although not absolutely required for BMP-2 expression and matrix mineralization nevertheless supports osteogenic differentiation and matrix mineralization by increasing BMP-2 expression. Our results therefore suggest that NF-κB activation may function in lineage selection during differentiation of hMSCs by fostering osteogenic differentiation at the expense of other differentiation lineages. [Copyright &y& Elsevier]

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 L-1β on components of the cytoskeleton in OA chondrocytes on gene expression level. Using a whole genome array, we found that IL-1β 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 FhI1, Fhl2, Lasp1, and Pdlim1 as well as Tubb and Vim by IL-1β. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 counteracted the influence of IL-1β 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-1β induced changes in the morphology of chondrocytes, the organization of the cytoskeleton, and the cellular distribution of FHL2. We conclude that L-1β 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. [ABSTRACT FROM AUTHOR]

Background: CCN-proteins are known to be involved in development, homeostasis and repair of mesenchymal tissues. Since these processes implicate recruitment of cells with the potential to be committed to various phenotypes, we studied the effect of CYR61/CCN1 and WISP3/CCN6 on migration of human bone marrow derived mesenchymal stroma cells (MSCs) in comparison to in vitro osteogenic differentiated MSCs using a modified Boyden chamber assay. Results: CYR61 and WISP3 were purified as fusion proteins with a C-terminal Fc-tag from baculovirus infected SF21 cells using protein G sepharose columns. CYR61 and WISP3 stimulated cell migration of undifferentiated MSCs in a dose-dependent manner. CYR61 and WISP3 had similar effects on committed osteogenic precursor cells. Checkerboard analysis revealed that CYR61 and WISP3 stimulated true directed cell migration (chemotaxis) of MSCs and committed osteogenic precursors. In MSCs the chemotactic activity of WISP3 but not CYR61 was mediated through integrin α΍7#x03B2;5. Conclusion: Our results indicate that CYR61 and WISP3 can function as soluble ligands transmitting chemotactic signals to human MSCs but differ in the involvement of integrin α΍7#x03B2;5. This may be relevant for their possible role in connective tissue repair. [ABSTRACT FROM AUTHOR]

Abstract: Insulin-like growth factors (IGFs) are known to be key regulators of bone growth, remodeling, and repair. Since all these processes depend on the recruitment of cells with the potential to be committed to the osteoblastic lineage, we studied possible effects of IGF-I and -II on migration of human mesenchymal progenitor cells (MPC) using a modified Boyden chamber assay. The results were compared to those of primary osteoblasts and in vitro-osteogenic-differentiated MPC. IGF-I and -II stimulated cell migration of all these cell populations in a dose-dependent manner from 1 to 100ng/mL. The maximal chemotactic index (CI) was 4–5 for MPC and primary osteoblasts and about 3 for in vitro-differentiated MPC. Checkerboard analysis revealed that IGFs stimulated true directed cell migration (chemotaxis) and not simply chemokinesis. Addition of an antibody against the type I IGF receptor (αIR3) completely abolished (MPC) or markedly reduced (primary osteoblasts) the chemotactic effects of each of the IGFs. IGFBP-3 itself had no direct effect, while IGFBP-5 stimulated MPC migration at concentrations of 80 and 160ng/mL. Parallel application of IGFBP-3 had borderline inhibitory effects while the addition of 40ng/mL of IGFBP-5 enhanced the chemotactic effect of IGF-I on MPC. In conclusion, our results show that IGF-I and -II are chemotactic factors for MPC and indicate that IGFBP-5 both modulates the IGF-I effect and directly stimulates migration of human mesenchymal progenitor cells. [Copyright &y& Elsevier]

Background/Aims: The aim of our study was to investigate interactions of mesenchymal progenitor cells (MPCs) with collagen matrices. Methods: Human bone-marrow-derived MPCs were cultivated in collagen type I gels with and without inhibition of β1-integrin by a specific antibody. Collagen gel contraction, cell morphology, expression of integrin subunits and several genes related to matrix synthesis and turnover as well as MPC differentiation were analyzed over 14 days. Results: Human MPCs markedly contracted free-floating collagen gels. Contraction was nearly completely inhibited by blocking β1-integrin. Cellular morphology was elongated in the absence and mostly round in the presence of the antibody. Expression of integrin α1, α2 and β1 subunits showed several changes partly dependent on β1-integrin blocking. Expression of matrix metalloproteinase-1 was elevated irrespective of β1-integrin blocking and tenascin-C was subsequently induced during gel contraction. Spontaneous induction of chondrogenic, osteogenic or adipogenic differentiation was observed neither in the presence nor in the absence of the β1-integrin antibody. Conclusion: Our results indicate that the interaction of human MPCs with fibrillar collagen type I involves β1- and α-integrin subunits and induces changes in gene expression related to extracellular matrix synthesis and turnover but not differentiation to the chondrogenic, osteogenic or adipogenic phenotype. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Vascular endothelial growth factor (VEGF) has been indicated to play a role during endochondral ossification by stimulation of blood vessel invasion into hypertrophic cartilage resulting in its replacement by trabecular bone. We could demonstrate a dose-dependent chemoattractive effect of VEGF-A and PlGF-1, but not VEGF-E or VEGF-C, on human mesenchymal progenitor cells. Quantitative realtime PCR revealed the expression of VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4), which markedly declined during osteogenic differentiation. In addition, expression of neuropilin-1 and -2 was detected by RT-PCR. In an in vitro kinase assay, we could demonstrate activation of VEGFR-1 and VEGFR-2 upon stimulation with specific ligands. These findings are consistent with the idea that the chemotactic effect of VEGF-A on MPC is mediated via VEGFR-1, and that VEGF-A and PlGF-1, have a functional role for recruitment of osteoprogenitor cells in the course of endochondral bone formation or remodeling. [Copyright &y& Elsevier]

Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome, an autosomal dominant disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular system. There is a remarkable degree of clinical variability both within and between families with Marfan syndrome as well as in individuals with related disorders of connective tissue caused by FBN1 mutations and collectively termed type-1 fibrillinopathies. The so-called neonatal region in FBN1 exons 24-32 comprises one of the few generally accepted genotype-phenotype correlations described to date. In this work, we report 12 FBN1 mutations identified by temperature-gradient gel electrophoresis screening of exons 24-40 in 127 individuals with Marfan syndrome or related disorders. The data reported here, together with other published reports, document a significant clustering of mutations in exons 24-32. Although all reported mutations associated with neonatal Marfan syndrome and the majority of point mutations associated with atypically severe presentations have been found in exons 24-32, mutations associated with classic Marfan syndrome occur in this region as well. It is not possible to predict whether a given mutation in exons 24-32 will be associated with classic, atypically severe, or neonatal Marfan syndrome. [ABSTRACT FROM AUTHOR]

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To describe the 5 years' trajectories in functionality and pain of patients with hip or knee osteoarthritis and arthroplasty and analyze the association of these with long-term patients survival. Patients with OA receiving total hip or knee arthroplasty were recruited and completed two sets of standardized questionnaires for functionality and pain 6, 12, and 60 months postoperatively. Multivariate mixed models were conducted to assess trajectories over time and the resulting improvement per month during the last time period was included in a landmark-model to estimate adjusted hazard ratios for mortality. In total 809 patients with joint replacement were included (mean age 65.0 years, 62.2% female), 407 patients died (median follow-up 18.4 years). Both instruments of functionality and pain showed extensive improvement during the first 6 months. Baseline and change in functionality (both p < 0.001) and pain (p = 0.02) during the first 6 months were associated with mortality. Better values in functionality corresponded with improved survival whereas the association with the pain scores was inverse. In patients with hip and knee OA, an explicit improvement in function is seen within the first 6 months after arthroplasty. In addition, especially the functionality scores at baseline as well as their improvement showed an association with long-term patient survival. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]