Role of the Sympathetic Nervous System in Mild Chronic Inflammatory Diseases: Focus on Osteoarthritis.

The sympathetic nervous system (SNS) is a major regulatory mediator connecting the brain and the immune system that influences accordingly inflammatory processes within the entire body. In the periphery, the SNS exerts its effects mainly via its neurotransmitters norepinephrine (NE) and epinephrine (E), which are released by peripheral nerve endings in lymphatic organs and other tissues. Depending on their concentration, NE and E bind to specific α- and ß-adrenergic receptor subtypes and can cause both pro- and anti-inflammatory cellular responses. The co-transmitter neuropeptide Y, adenosine triphosphate, or its metabolite adenosine are also mediators of the SNS. Local pro-inflammatory processes due to injury or pathogens lead to an activation of the SNS, which in turn induces several immunoregulatory mechanisms with either pro- or anti-inflammatory effects depending on neurotransmitter concentration or pathological context. In chronic inflammatory diseases, the activity of the SNS is persistently elevated and can trigger detrimental pathological processes. Recently, the sympathetic contribution to mild chronic inflammatory diseases like osteoarthritis (OA) has attracted growing interest. OA is a whole-joint disease and is characterized by mild chronic inflammation in the joint. In this narrative article, we summarize the underlying mechanisms behind the sympathetic influence on inflammation during OA pathogenesis. In addition, OA comorbidities also accompanied by mild chronic inflammation, such as hypertension, obesity, diabetes, and depression, will be reviewed. Finally, the potential of SNS-based therapeutic options for the treatment of OA will be discussed.

Correlation between Adrenoceptor Expression and Clinical Parameters in Degenerated Lumbar Intervertebral Discs.

Despite advanced knowledge of the cellular and biomechanical processes of intervertebral disc degeneration (IVDD), the trigger and underlying mechanisms remain unclear. Since the sympathetic nervous system (SNS) has been shown to exhibit catabolic effects in osteoarthritis pathogenesis, it is attractive to speculate that it also influences IVDD. Therefore, we explored the adrenoceptor (AR) expression profile in human IVDs and correlated it with clinical parameters of patients. IVD samples were collected from n = 43 patients undergoing lumbar spinal fusion surgery. AR gene expression was analyzed by semi-quantitative polymerase chain reaction. Clinical parameters as well as radiological Pfirrmann and Modic classification were collected and correlated with AR expression levels. In total human IVD homogenates α1A-, α1B-, α2A-, α2B-, α2C-, ß1- and ß2-AR genes were expressed. Expression of α1A- (r = 0.439), α2A- (r = 0.346) and ß2-AR (r = 0.409) showed a positive and significant correlation with Pfirrmann grade. α1A-AR expression was significantly decreased in IVD tissue of patients with adjacent segment disease (p = 0.041). The results of this study indicate that a relationship between IVDD and AR expression exists. Thus, the SNS and its neurotransmitters might play a role in IVDD pathogenesis. The knowledge of differential AR expression in different etiologies could contribute to the development of new therapeutic approaches for IVDD.

Anti-Inflammatory Effects of Endogenously Released Adenosine in Synovial Cells of Osteoarthritis and Rheumatoid Arthritis Patients.

Exogenous adenosine and its metabolite inosine exert anti-inflammatory effects in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We analyzed whether these cells are able to synthesize adenosine/inosine and which adenosine receptors (ARs) contribute to anti-inflammatory effects. The functionality of synthesizing enzymes and ARs was tested using agonists/antagonists. Both OA and RA cells expressed CD39 (converts ATP to AMP), CD73 (converts AMP to adenosine), ADA (converts adenosine to inosine), ENT1/2 (adenosine transporters), all AR subtypes (A1, A2A, A2B and A3) and synthesized predominantly adenosine. The CD73 inhibitor AMPCP significantly increased IL-6 and decreased IL-10 in both cell types, while TNF only increased in RA cells. The ADA inhibitor DAA significantly reduced IL-6 and induced IL-10 in both OA and RA cells. The A2AAR agonist CGS 21680 significantly inhibited IL-6 and induced TNF and IL-10 only in RA, while the A2BAR agonist BAY 60-6583 had the same effect in both OA and RA. Taken together, OA and RA synoviocytes express the complete enzymatic machinery to synthesize adenosine/inosine; however, mainly adenosine is responsible for the anti- (IL-6 and IL-10) or pro-inflammatory (TNF) effects mediated by A2A- and A2BAR. Stimulating CD39/CD73 with simultaneous ADA blockage in addition to TNF inhibition might represent a promising therapeutic strategy.

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.

Stimulation of TNF receptor type 2 expands regulatory T cells and ameliorates established collagen-induced arthritis in mice.

Tumor necrosis factor (TNF) and its receptors TNF receptor type 1 (TNFR1) and type 2 (TNFR2) have a central role in chronic inflammatory diseases. While TNFR1 mainly confers inflammation, activation of TNFR2 elicits not only pro-inflammatory but also anti-inflammatory effects. In this study, we wanted to investigate the anti-inflammatory therapeutic potential of selective activation of TNFR2 in mice with established collagen-induced arthritis. Mice with established arthritis induced by immunization with bovine collagen type II were treated with six injections of the TNFR2-specific agonist TNCscTNF80, given every second day. Two days after treatment cessation, the cell compositions of bone marrow, spleen and lymph nodes were analyzed. Mice were visually scored until day 30 after the start of therapy and the degree of joint inflammation was determined by histology. Treatment with TNCscTNF80 increased arthritis-induced myelopoiesis. Little effect was seen on the infiltration rate of inflammatory immature myeloid cells and on the reduction of lymphoid cells in secondary lymphoid organs. Upon treatment, frequency of regulatory T (Treg) cells in the CD4+ T-cell population was increased in both spleen and inguinal lymph nodes. In addition, the expression of TNFR2 on Treg cells was enhanced. The clinical score started to improve 1 week after cessation treatment and remained lower 30 days after initiation of therapy. The histological score also revealed amelioration of joint inflammation in TNCscTNF80-treated versus control mice. Activation of TNFR2 might provide a suitable therapeutic strategy in autoimmune arthritis by increasing the numbers of regulatory cell types, in particular Treg cells, and by attenuation of arthritis.

A Promising New Approach for the Treatment of Inflammatory Pain: Transfer of Stem Cell-Derived Tyrosine Hydroxylase-Positive Cells.

OBJECTIVES: The appearance of endogenous tyrosine hydroxylase-positive cells (TH+ cells) in collagen-induced arthritis was associated with an anti-inflammatory effect. Here we investigated putative anti-inflammatory and antinociceptive effects of the transfer of induced, bone marrow stem cell-derived TH+ cells (iTH+ cells) on murine antigen-induced arthritis (AIA). METHODS: Bone marrow-derived stem cells were differentiated into iTH+ cells. These cells were transferred to mice immunized against methylated bovine serum albumin (mBSA) 2 days before AIA was induced by injection of mBSA into one knee joint. In AIA control mice and iTH+-treated mice the severity of AIA, pain-related behavior, humoral and cellular responses, and the invasion of macrophages into the dorsal root ganglia were assessed. RESULTS: The intravenous transfer of iTH+ cells before AIA induction did not cause a sustained suppression of AIA severity but significantly reduced inflammation-evoked pain-related behavior. The iTH+ cells used for transfer exhibited enormous production of interleukin-4. A major difference between AIA control mice and iTH+-treated AIA mice was a massive invasion of the dorsal root ganglia by iNOS-negative, arginine 1-positive macrophages corresponding to an M2 phenotype. The differences in other cellular and humoral immune parameters such as release of cytokines from stimulated lymphocytes between AIA control mice and iTH+-treated mice were small. CONCLUSIONS: The transfer of iTH+ cells may cause a long-lasting reduction of arthritis-induced pain even if it does not ameliorate inflammation. The invasion of M2 macrophages into the dorsal root ganglia is likely to be an important mechanism of antinociception.

TNF inhibits catecholamine production from induced sympathetic neuron-like cells in rheumatoid arthritis and osteoarthritis in vitro.

Synovial adipose stem cells (sASC) can be differentiated into catecholamine-expressing sympathetic neuron-like cells to treat experimental arthritis. However, the pro-inflammatory tumor necrosis factor (TNF) is known to be toxic to catecholaminergic cells (see Parkinson disease), and this may prevent anti-inflammatory effects in inflamed tissue. We hypothesized that TNF exhibits inhibitory effects on human differentiated sympathetic tyrosine hydroxylase-positive (TH+) neuron-like cells. For the first time, iTH+ neuron-like sympathetic cells were generated from sACSs of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue. Compared to untreated controls in both OA and RA, TNF-treated iTH+ cells demonstrated a weaker staining of catecholaminergic markers in cell cultures of RA/OA patients, and the amount of produced noradrenaline was markedly lower. These effects were reversed by etanercept. Exposure of iTH+ cells to synovial fluid of RA patients showed similar inhibitory effects. In mixed synovial cells, significant effects of TNF on catecholamine release were observed only in OA. This study shows that TNF inhibits iTH+ synovial cells leading to the decrease of secreted noradrenaline. This might be a reason why discovered newly appearing TH+ cells in the synovium are not able to develop their possible full anti-inflammatory role in arthritis.

Marbostat-100 Defines a New Class of Potent and Selective Antiinflammatory and Antirheumatic Histone Deacetylase 6 Inhibitors.

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.

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.

Proinflammatory receptor switch from Gαs to Gαi signaling by ß-arrestin-mediated PDE4 recruitment in mixed RA synovial cells.

OBJECTIVE: In chronic inflammation, prevention of cAMP degradation by phosphodiesterase-4 (PDE4) inhibition can be anti-inflammatory therapy. However, PDE4 inhibition was uneffective in rheumatoid arthritis (RA). Recent studies demonstrated that PDE4/ß-arrestin interaction at ß-adrenoceptors resulted in switching from Gαs to Gαi signaling and ERK1/2 activation. Such a switch in signaling might elicit proinflammatory effects. We aimed to investigate this possible Gαs to Gαi signaling switch in RA and osteoarthritis (OA) mixed synoviocytes. METHODS: Synoviocytes were treated alone or with combinations of adrenergic, dopaminergic, and adenosinergic drugs, rolipram (PDE4 inhibitor), inhibitors of Gαi signaling (pertussis toxin), and blockers of protein kinase A (PKA). Under normoxic or hypoxic conditions, proinflammatory TNF was the readout-parameter. We investigated co-expression and interaction of PDE4 and ß-arrestin by imaging techniques. Expression of pERK1/2 was analyzed by western blotting. RESULTS: Mixed synoviocytes in RA and OA possessed all major Gαs-coupled neurotransmitter receptors. Under hypoxia, particularly in RA cells, Gαs-coupled receptor agonists unexpectedly increased TNF and respective antagonists decreased TNF. Under hypoxia, rolipram alone or rolipram plus Gαs agonists increased TNF, which was reversed by pertussis toxin or PKA inhibition. Co-localization and interaction of PDE4 and ß-arrestin in synovial tissue and cells was demonstrated. Gαs agonists or rolipram plus Gαs agonists increased pERK1/2 expression. CONCLUSIONS: This study in human arthritic synovial tissue presents an unexpected proinflammatory switch from Gαs to Gαi signaling, which depends on PDE4/ß-arrestin interaction. This phenomenon is most probably responsible for reduced efficacy of PDE4 inhibitors and Gαs agonists in RA.

Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis.

OBJECTIVES: Studies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH(+)) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of TH(+) cells and mode of TH induction are not known. METHODS: Synovial cells of RA/OA were isolated and cultured under normoxic/hypoxic conditions with/without stimulating enzyme cofactors of TH and inhibitors of TH. We studied TH expression and release of cytokines/catecholamines. In vivo function was tested by cell therapy with TH(+) neuronal precursor cells (TH(+) neuronal cells) in DBA/1 mice with collagen type II-induced arthritis (CIA). RESULTS: Compared with normoxic conditions, hypoxia increased TH protein expression and catecholamine synthesis and decreased release of tumour necrosis factor (TNF) in OA/RA synovial cells. This inhibitory effect on TNF was reversed by TH inhibition with α-methyl-para-tyrosine (αMPT), which was particularly evident under hypoxic conditions. Incubation with specific TH cofactors (tetrahydrobiopterin and Fe(2+)) increased hypoxia-induced inhibition of TNF, which was also reversed by αMPT. To address a possible clinical role of TH(+) cells, murine TH(+) neuronal cells were generated from mesenchymal stem cells. TH(+) neuronal cells exhibited a typical catecholaminergic phenotype. Adoptive transfer of TH(+) neuronal cells markedly reduced CIA in mice, and 6-hydroxydopamine, which depletes TH(+) cells, reversed this effect. CONCLUSIONS: The anti-inflammatory effect of TH(+) neuronal cells on experimental arthritis has been presented for the first time. In RA/OA, TH(+) synovial cells have TH-dependent anti-inflammatory capacities, which are augmented under hypoxia. Using generated TH(+) neuronal cells might open new avenues for cell-based therapy.

Norepinephrine inhibition of mesenchymal stem cell and chondrogenic progenitor cell chondrogenesis and acceleration of chondrogenic hypertrophy.

OBJECTIVE: Mesenchymal progenitor cell chondrogenesis is the biologic platform for the generation or regeneration of cartilage, but the external influence of the sympathetic nervous system on this process is not yet known. Sympathetic nerve fibers are present in articular tissue, and the sympathetic nervous system influences the musculoskeletal system by, for example, increasing osteoclastogenesis. This study was initiated to explore the role of the sympathetic neurotransmitter norepinephrine (NE) in mesenchymal stem cell (MSC)-dependent and cartilage progenitor cell (CPC)-dependent chondrogenesis. METHODS: Using human MSCs or CPCs, chondrogenic differentiation was induced in the presence of NE, the specific ß-adrenergic receptor (ß-AR) agonist isoproterenol, and the specific ß-AR antagonist nadolol. We studied sympathetic nerve fibers, tyrosine hydroxylase (TH) expression, catecholamine biosynthesis, and synovial fluid levels in human joints, as well as cartilage-specific matrix deposition during differentiation. RESULTS: TH+ sympathetic nerve fibers were present in the synovial tissue, meniscus, and subchondral bone marrow. In addition, synovial fluid from patients with knee trauma demonstrated high concentrations of NE. During MSC or CPC chondrogenesis, ß-AR were expressed. Chondrogenic aggregates treated with NE or isoproterenol synthesized lower amounts of type II collagen and glycosaminoglycans. NE and isoproterenol treatment dose-dependently increased the levels of cartilage hypertrophy markers (type X collagen and matrix metalloproteinase 13). Nadolol reversed the inhibition of chondrogenesis and the up-regulation of cartilage hypertrophy. CONCLUSION: Our findings demonstrate NE-dependent inhibition of chondrogenesis and acceleration of hypertrophic differentiation. By inhibiting cartilage repair, these sympathetic influences can be important after joint trauma. These findings may be a basis for novel neurochondrogenic therapeutic options.

Isolation, culture, and osteogenic/chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

Musculoskeletal disorders, as non-healing fractures and large bone defects, articular cartilage and subchondral bone injuries, often result in lifelong chronic pain and compromised quality of life. Although generally a natural process, failure of large bone defects to heal such as after complex fractures, resection of tumours, infections, or revisions of joint replacements remains a critical challenge that requires more appropriate solutions as those currently available. In addition, regeneration of chondral and osteochondral defects continues to be a challenge until to date. A profound understanding of the underlying mechanisms of endogenous regeneration is a prerequisite for successful bone and cartilage regeneration. Presently, one of the most promising therapeutic approaches is cell-based tissue engineering which provides a healthy population of cells to the injured site. Use of differentiated cells has severe limitations; an excellent alternative would be the application of adult marrow stromal cells/mesenchymal stem cells (MSC) which possess extensive proliferation potential and proven capability to differentiate along the osteochondral pathway. The process of osteo-/chondrogenesis can be mimicked in vitro by inducing osteo-chondroprogenitor stem cells to undergo osteogenesis and chondrogenesis through exposure of osteo-/chondrogenic favourable microenvironmental, mechanical, and nutritional conditions. This chapter provides comprehensive protocols for the isolation, expansion, and osteo-/chondrogenic differentiation of adult bone marrow-derived MSC.

Relationship between placenta growth factor 1 and vascularization, dehydroepiandrosterone sulfate to dehydroepiandrosterone conversion, or aromatase expression in patients with rheumatoid arthritis and patients with osteoarthritis.

OBJECTIVE: Proliferating pannus is in many aspects similar to placental tissue. Both fibroblast-rich tissues have high vascularity, and tissue from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA) demonstrates conversion of androgenic prehormones to downstream estrogens. We undertook this study to investigate similarities between proliferating pannus and placental tissue by focusing on angiogenic placenta growth factor 1 (PlGF-1) in patients with OA and patients with RA. METHODS: We used immunohistochemistry to study the presence of PlGF-1, its synovial distribution, and the PlGF-1-expressing synovial cell type. The relationship between PlGF-1 and conversion of the biologically inactive placental prehormone dehydroepiandrosterone sulfate (DHEAS) to the biologically active dehydroepiandrosterone (DHEA) was investigated in mixed synovial cells. The effects of DHEA on PlGF-1 expression were studied by intracellular fluorescence-activated cell sorting analysis. RESULTS: PlGF-1-positive cells were detected in the lining and sublining areas in patients with RA and patients with OA, and cellular density was similar. Double staining revealed that PlGF-1-positive cells were macrophages. In RA and OA, the density of PlGF-1-positive cells correlated positively with the density of macrophages and the density of type IV collagen-positive vessels. The supernatant concentration of (3) H-DHEA after conversion from (3) H-DHEAS and the density of aromatase-positive cells were positively correlated with the density of PlGF-1-positive cells only in OA. Low DHEA concentrations (≤10(-9) M) had stimulatory effects on PlGF-1 when compared to serum concentrations (10(-8) M to 10(-7) M) in the monocytic cell line THP-1 and in primary mixed synovial cells. CONCLUSION: PlGF-1 functions similarly in inflamed synovium and in the placenta. It is related to vessel formation and, in OA patients, to androgen/estrogen conversion. Evolutionarily conserved functions of PlGF-1 for placental phenomena are obviously also present in synovial inflammation.

Estrogen reduces cellular aging in human mesenchymal stem cells and chondrocytes.

Chondrocyte aging is associated with cartilage degeneration and senescence impairs the regenerative potential of mesenchymal stem cells (MSCs). Estrogen exerts profound effects on human physiology including articular cartilage and MSCs. The present study should analyze the effects of pre- and postmenopausal estrogen concentrations on chondrogenic cells. Physiologic premenopausal concentrations of 17ß-estradiol (E(2)) significantly decelerated telomere attrition in MSCs and chondrocytes while postmenopausal E(2) concentration had no significant effects. The estrogen agonist-antagonist tamoxifen did not affect telomere biology, but inhibited the E(2) -stimulated reduction in telomere shortening. E(2) and tamoxifen did not influence cell proliferation, cell morphology, and ß-galactosidase staining in chondrogenic cells. E(2) treatment did not affect the telomere-associated proteins TRF1 and TRF2. E(2) had no regulatory effects on the expression rates of the cell cycle regulator p21 and the DNA repair proteins SIRT1 and XRCC5. In spite of reducing telomere shortening in aging MSCs and chondrocytes, estrogen is not able to prevent somatic cells from replicative exhaustion and from finally entering senescence. The fade of telomere shortening under pre- to postmenopausal estrogen concentrations suggests, at least in part, a senescence-dependent cause for the onset of osteoarthritis in women after menopause.

Resazurin dye as a reliable tool for determination of cell number and viability in mesenchymal stem cell culture.

Human mesenchymal stem cells are a valuable cell source for tissue engineering. Determination of cell number and viability is crucial. However, this can be tested only at the end of cell culture. This study shows that Resazurin dye staining is a reliable tool for evaluation of cell number and viability in culture without cell perturbation.

Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signaling.

OBJECTIVE: We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow-derived mesenchymal stem cells (MSCs), with consideration of sex-dependent differences in cartilage repair. METHODS: Bone marrow was obtained from the iliac crest of young men. Density-gradient centrifugation-separated human MSCs proliferated as a monolayer in serum-containing medium. After confluence was achieved, aggregates were created and cultured in a serum-free differentiation medium. We added different concentrations of 17beta-estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane-impermeable E2-bovine serum albumin (E2-BSA), ICI 182.780 alone, G-1 (an agonist of G protein-coupled receptor 30 [GPR-30]), and G15 (a GPR-30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP-13) expression. RESULTS: The existence of intracellular and membrane-associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2-BSA in a dose-dependent manner. Furthermore, E2 enhanced type X collagen and MMP-13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR-30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G-1. Furthermore, the specific GPR-30 antagonist G15 reversed the GPR-30-mediated inhibition of chondrogenesis and up-regulation of hypertrophic gene expression. CONCLUSION: The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR-30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling.