Low miR-182-5p Expressing Extracellular Vesicles Derived From Human Bone Marrow Stromal Cells of Subjects With Steroid-Induced Osteonecrosis of the Femoral Head Aggravate Disease Progression.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory, progressive disease. However, the underlying mechanisms that aggravate femoral head necrosis remain unclear. Extracellular vesicles (EVs) act as molecular carriers in intercellular communication. We hypothesize that EVs derived from human (h) bone marrow stromal cells (BMSC) resident in SONFH lesion areas promote the pathogenesis of SONFH. In the present study, we determined the modulatory effects of SONFH-hBMSCs-derived EVs on the pathogenesis of SONFH in vitro and in vivo. We found that the expression of hsa-miR-182-5p was downregulated in SONFH-hBMSCs and EVs isolated from those hBMSCs. After tail vein injection, EVs isolated from hBMSCs transfected with hsa-miR-182-5p inhibitor aggravated femoral head necrosis in the SONFH mouse model. We conclude that miR-182-5p regulates bone turnover in the SONFH mouse model via targeting MYD88 and subsequent upregulation of RUNX2 expression. We further assume that EVs derived from hBMSCs resident in SONFH lesion areas aggravate femoral head necrosis by downregulating miR-182-5p secreted from hBMSC located outside these lesions. We suggest that miR-182-5p could provide a novel target for future therapeutic approaches to treat or prevent SONFH. © 2023 American Society for Bone and Mineral Research (ASBMR).

Semaphorin 3A-Neuropilin-1 Signaling Modulates MMP13 Expression in Human Osteoarthritic Chondrocytes.

Osteoarthritis (OA) is a complex disorder of diarthrodial joints caused by multiple risk factors and is characterized by articular cartilage destruction as well as changes in other articular tissues. Semaphorin 3A (Sema3A), known to be a chemo-repellent for sensory nerve fibers, has recently been implicated in cartilage OA pathophysiology. We demonstrated that the expression of SEMA3A and its receptor neuropilin-1 (NRP1) are synchronously upregulated in chondrocytes isolated from knee cartilage of OA patients compared to non-OA control chondrocytes. In addition, we observed that during in vitro passaging of OA chondrocytes, the Nrp-1 level increases, whereas the Sema3A level decreases. In this study, we aimed to uncover how Sema3A-Nrp-1 signaling affects metabolism and viability of OA chondrocytes via siRNA-mediated inhibition of Nrp-1 expression. We observed a decreased proliferation rate and an increase in adhesion and senescence after Nrp-1 silencing. Moreover, MMP13 gene expression was reduced by approximately 75% in NRP1 knockdown OA chondrocytes, whereas MMP13 expression was induced by Sema3A treatment in control (nt siRNA) OA chondrocytes, accompanied by an impaired AKT phosphorylation. These findings suggest a potential catabolic function of Sema3A signaling in OA chondrocytes by inducing MMP13 expression and by compromising pro-survival AKT activation. We propose that targeting the Sema3A-Nrp-1 signaling axis might be an opportunity to interfere with OA pathogenesis and progression.

Involvement of complement peptides C3a and C5a in osteoarthritis pathology.

Osteoarthritis (OA) affects more than 500 million people worldwide and is among the five diseases in Germany causing the highest suffering of the patients and cost for the society. The quality of life of OA patients is severely compromised, and adequate therapy is lacking owing to a knowledge gap that acts as a major barrier to finding safe and effective solutions. Chronic, low-grade inflammation plays a central role in OA pathogenesis and is associated with both OA pain and disease progression. Innate immune pathways, such as the complement- and pattern-recognition receptor pathways, are pivotal to the inflammation in OA and key components of the innate immune system implicated in OA include DAMP-TLR signaling, the complement system, carboxypeptidase B (CPB), and mononuclear cells. Anaphylatoxins C3a and C5a are small polypeptides (77 and 74 amino acids, respectively) which are released by proteolytic cleavage of the complement components C3 and C5. The alternative complement pathway seems to play a crucial role in OA pathogenesis as these complement components, mostly C3 and its activation peptide C3a, were detected at high levels in osteoarthritic cartilage, synovial membrane, and cultured chondrocytes. Targeting the complement system by using anti-complement drugs as a therapeutic option bears the risk of major side effects such as increasing the risk of infection, interfering with cell regeneration and metabolism, and suppressing the clearance of immune complexes. Despite those adverse effects, several synthetic complement peptide antagonists show promising effects in ameliorating inflammatory cell responses also in joint tissues.

The future of basic science in orthopaedics and traumatology: Cassandra or Prometheus?

Orthopaedic and trauma research is a gateway to better health and mobility, reflecting the ever-increasing and complex burden of musculoskeletal diseases and injuries in Germany, Europe and worldwide. Basic science in orthopaedics and traumatology addresses the complete organism down to the molecule among an entire life of musculoskeletal mobility. Reflecting the complex and intertwined underlying mechanisms, cooperative research in this field has discovered important mechanisms on the molecular, cellular and organ levels, which subsequently led to innovative diagnostic and therapeutic strategies that reduced individual suffering as well as the burden on the society. However, research efforts are considerably threatened by economical pressures on clinicians and scientists, growing obstacles for urgently needed translational animal research, and insufficient funding. Although sophisticated science is feasible and realized in ever more individual research groups, a main goal of the multidisciplinary members of the Basic Science Section of the German Society for Orthopaedics and Trauma Surgery is to generate overarching structures and networks to answer to the growing clinical needs. The future of basic science in orthopaedics and traumatology can only be managed by an even more intensified exchange between basic scientists and clinicians while fuelling enthusiasm of talented junior scientists and clinicians. Prioritized future projects will master a broad range of opportunities from artificial intelligence, gene- and nano-technologies to large-scale, multi-centre clinical studies. Like Prometheus in the ancient Greek myth, transferring the elucidating knowledge from basic science to the real (clinical) world will reduce the individual suffering from orthopaedic diseases and trauma as well as their socio-economic impact.

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line.

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options.

Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts.

As events triggering early osteoarthritis onset can be related to mechanical stress and proinflammatory signaling, we investigated the effect of different mechanical strain protocols on the expression of proinflammatory genes, as well as extracellular matrix remodelling in human synovial fibroblasts. Three distinct models of tensile stretching were applied: static isotropic tensile strain at 0 Hz, 16% tension for 48 h; short-term high-frequency cyclic tension at 1 Hz, 10% tension for 4 h; and dynamic tensile stretching for 48 h, consisting of two blocks of moderate stretching at 0.2 Hz, 2%, advanced stretching at 0.5 Hz, 15%, or a combination of both. General signs of inflammation were present after static isotropic tension, whereas short-term high-frequency cyclic tension showed increased levels of IL-6 paired with diminished levels of IL-1ß. Reduced inflammatory effects of TNF-α, IL-6, and IL-1ß were observed when exposed to advanced stretching. Long-term tensile strain induced extracellular matrix remodelling at the gene and protein levels. While hyaluronan acid synthesis was increased with static tensile strain, dynamic tensile stretching had a reducing effect. Our study revealed that proinflammatory markers were activated by mechanical strain as seen in static isotropic tension and short-term high-frequency tensile strain, whereas long-term exposure induced extracellular matrix remodelling processes.

Norepinephrine Inhibits the Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells via ß2-Adrenoceptor-Mediated ERK1/2 and PKA Phosphorylation.

Bone marrow-derived mesenchymal stem cells (BMSCs) represent an alternative to chondrocytes to support cartilage regeneration in osteoarthritis (OA). The sympathetic neurotransmitter norepinephrine (NE) has been shown to inhibit their chondrogenic potential; however, their proliferation capacity under NE influence has not been studied yet. Therefore, we used BMSCs obtained from trauma and OA donors and compared the expression of adrenergic receptors (AR). Then, BMSCs from both donor groups were treated with NE, as well as with combinations of NE and α1-, α2- or ß1/2-AR antagonists (doxazosin, yohimbine or propranolol). Activation of AR-coupled signaling was investigated by analyzing ERK1/2 and protein kinase A (PKA) phosphorylation. A similar but not identical subset of ARs was expressed in trauma (α2B-, α2C- and ß2-AR) and OA BMSCs (α2A-, α2B-, and ß2-AR). NE in high concentrations inhibited the proliferation of both trauma and OA BMCSs significantly. NE in low concentrations did not influence proliferation. ERK1/2 as well as PKA were activated after NE treatment in both BMSC types. These effects were abolished only by propranolol. Our results demonstrate that NE inhibits the proliferation and accordingly lowers the regenerative capacity of human BMSCs likely via ß2-AR-mediated ERK1/2 and PKA phosphorylation. Therefore, targeting ß2-AR-signaling might provide novel OA therapeutic options.

Sensory neuropeptides are required for bone and cartilage homeostasis in a murine destabilization-induced osteoarthritis model.

Numerous studies identified a role for the sensory neuropeptides substance P (SP) and alpha calcitonin gene-related peptide (αCGRP) in osteoarthritis (OA) pain behavior. Surprisingly, little attention has been paid on how their trophic effects on cartilage and bone cells might affect structural changes of bone and cartilage in OA pathology. Here, we sought to elucidate sensory neuropeptides influence on structural alterations of bone and cartilage during murine OA pathophysiology. OA was induced by destabilization of the medial meniscus (DMM) in the right knee joint of 12 weeks old male C57Bl/6J wildtype (WT) mice and mice either deficient for SP (tachykinin 1 (Tac1)-/-) or αCGRP. By OARSI histopathological grading we observed significant cartilage matrix degradation after DMM surgery in αCGRP-deficient mice after 4 weeks whereas Tac1-/- scores were not different to sham mice before 12 weeks after surgery. Indentation-type atomic force microscopy (IT-AFM) identified a strong superficial zone (SZ) cartilage phenotype in Tac1-/- Sham mice. Opposed to WT and αCGRP-/- mice, SZ cartilage of Tac1-/- mice softened 2 weeks after OA induction. In Tac1-/- DMM mice, bone volume to total volume ratio (BV/TV) increased significantly compared to the Tac1-/- Sham group, 2 weeks after surgery. WT mice had reduced BV/TV compared to αCGRP-/- and Tac1-/- mice after 12 weeks. Increased calcified cartilage thickness and medial condyle diameter were detected in the medial tibia of all groups 8 weeks after OA induction by nanoCT analysis. Meniscal ossification occurred in all OA groups, but was significantly stronger in the absence of neuropeptides. Increased serum concentration of the respective non-deleted neuropeptide was observed in both neuropeptide-deficient mice strains. Both neuropeptides protect from age-related bone structural changes under physiological conditions and SP additionally demonstrates an anabolic effect on bone structure preservation in a pathophysiological situation. Both neuropeptide deficient mice display an intrinsic structural cartilage matrix phenotype that might alter progression of cartilage degeneration in OA.

Induction of ALP and MMP9 activity facilitates invasive behavior in heterogeneous human BMSC and HNSCC 3D spheroids.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytic, osteogenic, chondrogenic, and myogenic lineages. There is growing evidence that MSCs home into the tumor microenvironment attracted by a variety of signals such as chemokines, growth factors, and cytokines. Tumor-homing stem cells may originate from bone marrow-derived MSCs (BMSCs) or adipose tissue-derived MSCs. Recent scientific data suggest that MSCs in combination with tumor cells can either promote or inhibit tumorigenic behavior. In head and neck squamous cell carcinoma (HNSCC), BMSCs are reported to be enriched with a potential negative role. Here, we evaluated the effect of BMSCs from 4 different donors in combination with 4 HNSCC cell lines in a 3-dimensional multicellular spheroid model. Heterogeneous combinations revealed an up-regulation of gene and protein expression of osteogenic markers runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) together with a substantial secretion of matrix metalloproteinase 9. Moreover, heterogenous BMSC/tumor spheroids showed increased invasion compared with homogenous spheroids in a Boyden chamber invasion assay. Furthermore, inhibition of ALP resulted in a substantially decreased spreading of heterogeneous spheroids on laminin-rich matrix. In summary, our data suggest a prometastatic effect of BMSCs combined with HNSCC.-Wessely, A., Waltera, A., Reichert, T. E., Stöckl, S., Grässel, S., Bauer, R. J. Induction of ALP and MMP9 activity facilitates invasive behavior in heterogeneous human BMSC and HNSCC 3D-spheroids.

A humanized bone microenvironment uncovers HIF2 alpha as a latent marker for osteosarcoma.

The quest for predictive tumor markers for osteosarcoma (OS) has not well progressed over the last two decades due to a lack of preclinical models. The aim of this study was to investigate if microenvironmental modifications in an original humanized in vivo model alter the expression of OS tumor markers. Human bone micro-chips and bone marrow, harvested during hip arthroplasty, were implanted at the flanks of NOD/scid mice. We administered recombinant human bone morphogenetic protein 7 (rhBMP-7) in human bone micro-chips/bone marrow group I in order to modulate bone matrix and bone marrow humanization. Ten weeks post-implantation, human Luc-SAOS-2 OS cells were injected into the humanized tissue-engineered bone organs (hTEBOs). Tumors were harvested 5 weeks post-implantation to determine the expression of the previously described OS markers ezrin, periostin, VEGF, HIF1α and HIF2α. Representation of these proteins was analyzed in two different OS patient cohorts. Ezrin was downregulated in OS in hTEBOs with rhBMP-7, whereas HIF2α was significantly upregulated in comparison to hTEBOs without rhBMP-7. The expression of periostin, VEGF and HIF1α did not differ significantly between both groups. HIF2α was consistently present in OS patients and dependent on tumor site and clinical stage. OS patients post-chemotherapy had suppressed levels of HIF2α. In conclusion, we demonstrated the overall expression of OS-related factors in a preclinical model, which is based on a humanized bone organ. Our preclinical research results and analysis of two comprehensive patient cohorts imply that HIF2α is a potential prognostic marker and/or therapeutic target. STATEMENT OF SIGNIFICANCE: This study demonstrates the clinical relevance of the humanized organ bone microenvironment in osteosarcoma research and validates the expression of tumor markers, especially HIF2α. The convergence of clinically proven bone engineering concepts for the development of humanized mice models is a new starting point for investigations of OS-related marker expression. The validation and first data set in such a model let one conclude that further clinical studies on the role of HIF2α as a prognostic marker and its potential as therapeutic target is a condition sine qua non.

Sensory Neuropeptides and their Receptors Participate in Mechano-Regulation of Murine Macrophages.

This study aimed to analyze if the sensory neuropeptide SP (SP) and the neurokinin receptor 1 (NK1R) are involved in macrophage mechano-transduction, similar to chondrocytes, and if alpha-calcitonin gene-related peptide (αCGRP) and the CGRP receptor (CRLR/Ramp1) show comparable activity. Murine RAW264.7 macrophages were subjected to a cyclic stretch for 1⁻3 days and 4 h/day. Loading and neuropeptide effects were analyzed for gene and protein expression of neuropeptides and their receptors, adhesion, apoptosis, proliferation and ROS activity. Murine bone marrow-derived macrophages (BMM) were isolated after surgical osteoarthritis (OA) induction and proliferation, apoptosis and osteoclastogenesis were analyzed in response to loading. Loading induced NK1R and CRLR/Ramp1 gene expression and altered protein expression in RAW264.7 macrophages. SP protein and mRNA level decreased after loading whereas αCGRP mRNA expression was stabilized. SP reduced adhesion in loaded RAW264.7 macrophages and both neuropeptides initially increased the ROS activity followed by a time-dependent suppression. OA induction sensitized BMM to caspase 3/7 mediated apoptosis after loading. Both sensory neuropeptides, SP and αCGRP, and their receptors are involved in murine macrophage mechano-transduction affecting neuropeptide impact on adhesion and ROS activity. OA induction altered BMM apoptosis in response to loading indicate that OA-associated biomechanical alterations might affect the macrophage population.

Do Neuroendocrine Peptides and Their Receptors Qualify as Novel Therapeutic Targets in Osteoarthritis?

Joint tissues like synovium, articular cartilage, meniscus and subchondral bone, are targets for neuropeptides. Resident cells of these tissues express receptors for various neuroendocrine-derived peptides including proopiomelanocortin (POMC)-derived peptides, i.e., α-melanocyte-stimulating hormone (α-MSH), adrenocorticotropin (ACTH) and ß-endorphin (ß-ED), and sympathetic neuropeptides like vasoactive intestinal peptide (VIP) and neuropeptide y (NPY). Melanocortins attained particular attention due to their immunomodulatory and anti-inflammatory effects in several tissues and organs. In particular, α-MSH, ACTH and specific melanocortin-receptor (MCR) agonists appear to have promising anti-inflammatory actions demonstrated in animal models of experimentally induced arthritis and osteoarthritis (OA). Sympathetic neuropeptides have obtained increasing attention as they have crucial trophic effects that are critical for joint tissue and bone homeostasis. VIP and NPY are implicated in direct and indirect activation of several anabolic signaling pathways in bone and synovial cells. Additionally, pituitary adenylate cyclase-activating polypeptide (PACAP) proved to be chondroprotective and, thus, might be a novel target in OA. Taken together, it appears more and more likely that the anabolic effects of these neuroendocrine peptides or their respective receptor agonists/antagonists may be exploited for the treatment of patients with inflammatory and degenerative joint diseases in the future.

miR-29b regulates expression of collagens I and III in chondrogenically differentiating BMSC in an osteoarthritic environment.

Osteoarthritis (OA) is characterized by a slowly progressing, irreversible loss of articular cartilage. Tissue engineering approaches for cartilage regeneration include stem cell-based strategies but not much is known about their repair capacity in an OA microenvironment. The aim of the present study was to identify factors regulating collagen expression during chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSC) in an OA microenvironment. Coculture with OA cartilage induced miR-29b expression in BMSC which inhibited collagen I and III expression. Elevated miR-29b expression resulted in higher caspase 3/7 activity and promoted apoptosis of BMSC in part by directly inhibiting the anti-apoptotic proteins Bcl-2 and Mcl-1. Stimulation with IFN-γ induced miR-29b expression in BMSC. Our results suggest that miR-29b affects BMSC-based OA cartilage regeneration because expression of collagen III, mainly produced by undifferentiated BMSC, and collagen I, a marker for dedifferentiated chondrocytes, are inhibited by miR-29b thus influencing composition of the newly formed ECM. This might be critical to avoid formation of inferior fibrocartilage instead of hyaline cartilage. Furthermore, higher miR-29b expression promotes apoptosis either preventing excessive cell growth or reducing the number of BMSC undergoing chondrogenesis. Thus, miR-29b has both supportive but possibly also unfavourable effects on BMSC-based OA cartilage regeneration.

Peripheral Nerve Fibers and Their Neurotransmitters in Osteoarthritis Pathology.

The importance of the nociceptive nervous system for maintaining tissue homeostasis has been known for some time, and it has also been suggested that organogenesis and tissue repair are under neuronal control. Changes in peripheral joint innervation are supposed to be partly responsible for degenerative alterations in joint tissues which contribute to development of osteoarthritis. Various resident cell types of the musculoskeletal system express receptors for sensory and sympathetic neurotransmitters, allowing response to peripheral neuronal stimuli. Among them are mesenchymal stem cells, synovial fibroblasts, bone cells and chondrocytes of different origin, which express distinct subtypes of adrenoceptors (AR), receptors for vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). Some of these cell types synthesize and secrete neuropeptides such as SP, and they are positive for tyrosine-hydroxylase (TH), the rate limiting enzyme for biosynthesis of catecholamines. Sensory and sympathetic neurotransmitters are involved in the pathology of inflammatory diseases such as rheumatoid arthritis (RA) which manifests mainly in the joints. In addition, they seem to play a role in pathogenesis of priori degenerative joint disorders such as osteoarthritis (OA). Altogether it is evident that sensory and sympathetic neurotransmitters have crucial trophic effects which are critical for joint tissue and bone homeostasis. They modulate articular cartilage, subchondral bone and synovial tissue properties in physiological and pathophysiological conditions, in addition to their classical neurological features.

α-MSH modulates cell adhesion and inflammatory responses of synovial fibroblasts from osteoarthritis patients.

INTRODUCTION: The synovium is a target for neuropeptides. Melanocortins have attained particular attention as they elicit antiinflammatory effects. Although synovial fluid from patients with rheumatic diseases contains α-melanocyte-stimulating hormone (α-MSH) it is unknown whether synovial fibroblasts generate α-MSH and respond to melanocortins. METHODS: Synovial tissue was obtained from osteoarthritis (OA) patients. Cells were isolated and prepared either as primary mixed synoviocytes or propagated as synovial fibroblasts (OASFs). Melanocortin receptor (MC) and proopiomelanocortin (POMC) expression were investigated by endpoint RT-PCR, immunofluorescence and Western immunoblotting. Functional coupling of MC1 was assessed by cAMP and Ca(2+) assays. Cell adhesion was monitored by the xCELLigence system. Secretion of α-MSH, tumour necrosis factor (TNF), interleukin (IL)-6 and IL-8 was determined by ELISA. RESULTS: OASFs in vitro expressed MC1. MC1 transcripts were present in synovial tissue and appropriate immunoreactivity was detected in synovial fibroblasts in situ. OASFs contained truncated POMC transcripts but neither full-length POMC mRNA, POMC protein nor α-MSH were detectable. In accordance with this only truncated POMC transcripts were present in synovial tissue. α-MSH increased cAMP dose-dependently but did not alter calcium in OASFs. α-MSH also enhanced adhesion of OASFs to fibronectin and reduced TNF, IL-6 and IL-8 secretion in primary mixed synoviocyte cultures. In OASFs, α-MSH modulated basal and TNF/IL-1ß-mediated secretion of IL-6 and IL-8. CONCLUSION: Synovial fibroblasts express MC1in vitro and in situ. α-MSH elicits biological effects in these cells suggesting an endogenous immunomodulatory role of melanocortins within the synovium. Our results encourage in vivo studies with melanocortins in OA models.

The impact of hypoxia on mesenchymal progenitor cells of human skeletal tissue in the pathogenesis of heterotopic ossification.

PURPOSE: Mesenchymal progenitor cells (MPCs) are capable of differentiating into osteo/chondrogenic cells to contribute substantially to heterotopic ossification (HO). This study aimed to examine the impact of hypoxia on MPCs in the aetiology of HO. METHODS: MPCs from human normal and HO skeletal tissue were cultivated under normoxia and hypoxia. Gene expression of factors which have a key role in HO aetiology (BMPs, COX-1 and COX-2, etc.) were examined by real-time PCR. Tissue of both groups was analysed by immunohistochemistry. RESULTS: Under hypoxia, COX-1, -2 and SOX-9 gene expression was elevated in HO MPCs, whereas in normal muscle tissue only COX-2 was upregulated. MPCs from HO had a significantly elevated gene expression of BMP-4 and decreased expression of BMP-1 and HIF-1 under hypoxia compared to normal MPCs. Immunohistochemistry detected no significant differences between normal and HO tissue. CONCLUSIONS: Hypoxia causes an enhanced gene expression of factors, which have a key role in HO pathophysiology. A better understanding of this entity will possibly allow reducing HO rates in orthopaedic and trauma surgery.

The NC11 domain of human collagen XVI induces vasculogenic mimicry in oral squamous cell carcinoma cells.

Collagen XVI, a fibril-associated collagen with interrupted triple helix (FACIT) collagen, is involved in oral squamous cell carcinoma (OSCC) and glioblastoma progression. The NC11 domain of collagen XVI has been described previously with a strong implication in physiological processes. We detected the non-collagenous (NC) 11-domain in supernatants of OSCC cells after recombinant expression of full-length collagen XVI and in sera from OSCC patients and healthy individuals. Stable expression of NC11-green fluorescent protein (GFP) fusion protein in OSCC cells initiated proliferation control and block of anchorage-independent growth. Moreover, the NC11 domain triggered the generation of tubular-like net structures on laminin-rich matrix in contrast to mock-GFP control cells and cells expressing full-length collagen XVI. Taqman® quantitative PCR and diaminobenzidine staining in 2D- and 3D cell culture revealed a significantly increased gene and protein expression of VEGFR1, VEGFR2 and uPAR in recombinant NC11-GFP-expressing cells. Specific VEGF receptor inhibition with Axitinib or fetal calf serum heat inactivation prevented formation of tubular-like net structures. Accordantly, NC11-GFP coated culture slides led to an increase of focal adhesion contact formation and the upregulation of VEGFR1 and uPAR in three different non-transfected OSCC cell lines. In summary, we suggest that the NC11 domain of collagen XVI is a potential biomarker for OSCC and triggers vasculogenic mimicry via upregulation of endothelial receptors VEGFR1, VEGFR2 and uPAR in 2D- and 3D OSCC cell culture conditions.

Reactivity of rat bone marrow-derived macrophages to neurotransmitter stimulation in the context of collagen II-induced arthritis.

INTRODUCTION: Numerous observations indicate that rheumatoid arthritis (RA) has a bone marrow component. In parallel, local synovial changes depend on neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze whether collagen II-induced arthritis (CIA) has an impact on number, adhesion, apoptosis, and proliferation of the macrophage subset of bone marrow cells and how alterations in neurotransmitter microenvironment affect these properties. METHODS: Bone marrow-derived macrophages (BMMs) were isolated from Dark Agouti rats at different stages of CIA, and number, adhesion, caspase 3/7 activity, and proliferation were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA), and vasoactive intestinal peptide (VIP). RESULTS: Opposed to enhanced CD11b(+) (cluster of differentiation 11b-positive) and EMR1(+) (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1-positive) cells, characterizing the macrophage subset, in native bone marrow of rats with acute inflammatory arthritis, we found decreased numbers of CIA macrophages after enrichment and culture in comparison with healthy (control) animals. Adhesion studies revealed significantly reduced attachment to plastic in acute arthritis and collagen type I and fibronectin in chronic arthritis. Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA. Apoptosis remained unaffected. Neurotransmitter stimulation profoundly affected proliferation, adhesion, and apoptosis of BMMs from CIA and control rats, depending on disease time point. Cultured BMMs from CIA and control animals expressed neurotransmitter receptors for ACh, VIP and NA, but the expression profile seemed not to be affected by CIA. CONCLUSIONS: Induction of CIA distinctly inhibits proliferation of BMMs in low- and non-inflammatory phases and reduces attachment to plastic at the acute inflammatory arthritis stage and adhesion to collagen I and fibronectin at the chronic stage. Influence of neurotransmitter stimulation on adhesion, apoptosis, and proliferation is altered by CIA depending on disease stage. We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

Catecholaminergic-to-cholinergic transition of sympathetic nerve fibers is stimulated under healthy but not under inflammatory arthritic conditions.

OBJECTIVE: Density of sympathetic nerve fibers decreases in inflamed arthritic tissue tested by immunoreactivity towards tyrosine-hydroxylase (TH, catecholaminergic key enzyme). Since sympathetic nerve fibers may change phenotype from catecholaminergic to cholinergic (example: sweat glands), loss of nerve fibers may relate to undetectable TH. We aimed to investigate possible catecholaminergic-to-cholinergic transition of sympathetic nerve fibers in synovial tissue of animals with arthritis, and patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and we wanted to find a possible transition factor. METHODS: Nerve fibers were detected by immunofluorescence towards TH (catecholaminergic) and vesicular acetylcholine transporter (cholinergic). Co-culture experiments with sympathetic ganglia and lymphocytes or osteoclast progenitors were designed to find stimulators of catecholaminergic-to-cholinergic transition (including gene expression profiling). RESULTS: In mouse joints, an increased density of cholinergic relative to catecholaminergic nerve fibers appeared towards day 35 after immunization, but most nerve fibers were located in healthy joint-adjacent skin or muscle and almost none in inflamed synovial tissue. In humans, cholinergic fibers are more prevalent in OA synovial tissue than in RA. Co-culture of sympathetic ganglia with osteoclast progenitors obtained from healthy but not from arthritic animals induced catecholaminergic-to-cholinergic transition. Osteoclast mRNA microarray data indicated that leukemia inhibitory factor (LIF) is a candidate transition factor, which was confirmed in ganglia experiments, particularly, in the presence of progesterone. CONCLUSION: In humans and mice, catecholaminergic-to-cholinergic sympathetic transition happens in less inflamed tissue but not in inflamed arthritic tissue. Under healthy conditions, presence of cholinergic sympathetic nerve fibers may support the cholinergic anti-inflammatory influence recently described.

Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes.

INTRODUCTION: Osteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis. We hypothesized that signals from the subchondral bone may modulate production of matrix components, alter chondrogenic differentiation potential of cocultured bone marrow-derived mesenchymal stem cells (BMSC) and induce a phenotypic shift in differentiated OA chondrocytes. METHODS: We established a novel coculture model between BMSC, mixed cultures (BMSC and chondrocytes) and chondrocytes embedded in fibrin gel with OA and normal subchondral bone explants (OAB and NB). Tissues and cells were either derived from OA or trauma patients. In addition, we used adipose-derived stem cells (ASC) from liposuction. With gene expression analysis, biochemical assays, immunofluorescence and biomechanical tests we characterized the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiating BMSC cocultured with OAB or NB in comparison with monocultures (cultures without bone explants). RESULTS: Overall, gene expression of collagens of OAB and NB cocultured cells was reduced compared to monocultures. Concomitantly, we observed significantly lower collagen I, II and III and glycosaminoglycan (GAG) production in OAB cocultured cell lysates. In parallel, we detected increased concentrations of soluble GAGs and basic fibroblast growth factor (bFGF), interleukin (IL)-6 and IL-8 in supernatants of OAB and NB cocultures mainly at early time points. IL-1ß concentration was increased in supernatants of OAB cocultures, but not in NB cocultures. Cell-free NB or OAB explants released different amounts of IL-1ß, bFGF and soluble GAG into cell culture supernatants. In comparison to cocultures, monocultures exhibited higher Young's modulus and equilibrium modulus. Stimulation of monocultures with IL-1ß led to a downregulation of aggrecan (ACAN) gene expression and in general to induced matrix metalloprotease (MMP)2, MMP3 and MMP-13 gene expression while IL-6 and IL-8 stimulation partly reduced ACAN, MMP3 and MMP-13 gene expression. CONCLUSIONS: Our results suggest an alteration of molecular composition and mechanical properties of the newly formed ECM in subchondral bone cocultures. We suggest that soluble factors, that is interleukins and bFGF, released in cocultures exert inhibitory effects on collagen and temporary effects on proteoglycan production, which finally results in a reduction of mechanical strength of newly formed fibrillar networks.