Anti-inflammatory effects of the prostacyclin analogue iloprost in an in vitro model of inflamed human dental pulp cells.

Iloprost's anti-inflammatory effects on human dental pulp stem cells (HDPCs) are currently unknown. We hypothesized that iloprost could downregulate the expression of inflammatory-related genes and protein in an inflamed HDPC in vitro model. To induce inflammation, the HDPCs were treated with a cocktail of interleukin-1 beta, interferon-gamma, and tumour necrosis alpha, at a ratio of 1:10:100. Iloprost (10-6  M) was then added or not to the cultures. Interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA expression were assessed by real-time polymerase chain reaction. IL-6 protein expression was assessed by enzyme-linked immunosorbent assay. The results were analysed using one-way ANOVA or the Kruskal-Wallis test. The cytokine cocktail induced more robust IL-6 expression than LPS treatment. Iloprost slightly, yet significantly, downregulated IL-6 and IL-12 mRNA expression. These findings suggest that iloprost might be used as a beneficial component in vital pulp therapy.

Mutation in the CCAL1 locus accounts for bidirectional process of human subchondral bone turnover and cartilage mineralization.

OBJECTIVES: To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients. METHODS: OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation. RESULTS: Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively. CONCLUSIONS: The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humans via DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies.

Osteopontin induces osteogenic differentiation by human periodontal ligament cells via calcium binding domain-ALK-1 interaction.

BACKGROUND: Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN-induced osteogenic differentiation of human PDL cells. METHODS: Full length rhOPN (FL-OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium-binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL-OPN, N142, C122, or C122δ-coated surfaces. Real-time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein-protein interaction. In vivo analysis was performed using a rat calvaria defect model. RESULTS: Human PDL cells seeded on FL-OPN and C122-coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL-OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor-like kinase (ALK-1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK-1 receptor. C122, but not C122δ coated surfaces, induced the expression of p-Smad-1 and this induction was inhibited by an ALK-1 inhibitor and RNAi against ALK-1. In vivo data showed that 3D porous scaffold containing C-122 enhanced new bone formation as compared to scaffold alone. CONCLUSION: The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK-1 receptor.

LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa ß (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA.

Recently, it was shown that interleukin-1ß (IL-1ß) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C-), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31- Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

Cyclic tensile force-upregulated IL6 increases MMP3 expression by human periodontal ligament cells.

OBJECTIVE: Cyclic tensile force (CTF) modulates physiological responses of periodontal ligament (PDL) cells. PDL cells are mechanosensitive and are able to maintain tissue homeostasis; a process mediated by the expression of particular cytokines including interleukin 6 (IL6). It is unknown whether CTF-induced IL6 regulates the expression of MMPs, enzymes needed for tissue remodeling. DESIGN: Human PDL cells were subjected to 10% elongation strain of CTF at a frequency of 60 rpm continuously for 6 h. RNA and proteins were extracted and analyzed for IL6 and MMP expression by quantitative real-time PCR and ELISA, respectively. Using a neutralizing anti-IL6 antibody and addition of recombinant human IL6 at concentrations of 0.1, 1, 10 ng.mL-1 were performed to clarify whether CTF-upregulated IL6 increased MMP expression. Inhibitors of intracellular signaling molecules were employed to reveal possible pathway(s) of IL6-induced MMP expression. RESULTS: CTF-induced IL6 expression coincided with an increased MMP3 expression. A neutralizing anti-IL6 antibody attenuated the CTF-increased MMP3 expression, whereas stimulating the cells with recombinant human IL6 increased MMP3 expression. Both PI3K and MAPK pathways were essential in the IL6 induced expression of MMP3. CONCLUSION: Our findings suggest a role of CTF in the modulation of expression of IL6 and MMP3 and thus in the regulation of homeostasis and remodeling of the periodontal ligament.

Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells.

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y 1 receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y 1 receptor antagonist), MRS 2365 (a specific P2Y 1 agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y 1 in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca 2+ through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.

Conditional mouse models support the role of SLC39A14 (ZIP14) in Hyperostosis Cranialis Interna and in bone homeostasis.

Hyperostosis Cranialis Interna (HCI) is a rare bone disorder characterized by progressive intracranial bone overgrowth at the skull. Here we identified by whole-exome sequencing a dominant mutation (L441R) in SLC39A14 (ZIP14). We show that L441R ZIP14 is no longer trafficked towards the plasma membrane and excessively accumulates intracellular zinc, resulting in hyper-activation of cAMP-CREB and NFAT signaling. Conditional knock-in mice overexpressing L438R Zip14 in osteoblasts have a severe skeletal phenotype marked by a drastic increase in cortical thickness due to an enhanced endosteal bone formation, resembling the underlying pathology in HCI patients. Remarkably, L438R Zip14 also generates an osteoporotic trabecular bone phenotype. The effects of osteoblastic overexpression of L438R Zip14 therefore mimic the disparate actions of estrogen on cortical and trabecular bone through osteoblasts. Collectively, we reveal ZIP14 as a novel regulator of bone homeostasis, and that manipulating ZIP14 might be a therapeutic strategy for bone diseases.

Interleukin-1ß induces human cementoblasts to support osteoclastogenesis.

Injury of the periodontium followed by inflammatory response often leads to root resorption. Resorption is accomplished by osteoclasts and their generation may depend on an interaction with the cells in direct contact with the root, the cementoblasts. Our study aimed to investigate the role of human cementoblasts in the formation of osteoclasts and the effect of interleukin (IL)-1ß hereupon. Extracted teeth from healthy volunteers were subjected to sequential digestion by type I collagenase and trypsin. The effect of enzymatic digestion on the presence of cells on the root surface was analyzed by histology. Gene expression of primary human cementoblasts (pHCB) was compared with a human cementoblast cell line (HCEM). The pHCBs were analyzed for their expression of IL-1 receptors as well as of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). In a co-culture system consisting of osteoclast precursors (blood monocytes) and pHCBs, the formation of osteoclasts and their resorptive activity was assessed by osteo-assay and ivory slices. The cells obtained after a 120 min enzyme digestion expressed the highest level of bone sialoprotein, similar to that of HCEM. This fraction of isolated cells also shared a similar expression pattern of IL-1 receptors (IL1-R1 and IL1-R2). Treatment with IL-1ß potently upregulated RANKL expression but not of OPG. pHCBs were shown to induce the formation of functional osteoclasts. This capacity was significantly stimulated by pretreating the pHCBs with IL-1ß prior to their co-culture with human blood monocytes. Our study demonstrated that cementoblasts have the capacity to induce osteoclastogenesis, a capacity strongly promoted by IL-1ß. These results may explain why osteoclasts can be formed next to the root of teeth.

TNF-α has both stimulatory and inhibitory effects on mouse monocyte-derived osteoclastogenesis.

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF-α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31hi Ly-6C- ), myeloid blasts (CD31+ Ly-6C+ ), and monocytes (CD31- Ly-6Chi ) were sorted from mouse bone marrow using flow cytometry and cultured with M-CSF and RANKL, with or without TNF-α. Surprisingly, TNF-α prevented the differentiation of TRAcP+ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL-1ß or IL-6. When monocytes were pre-cultured with M-CSF and RANKL followed by exposure to TNF-α, a stimulatory effect was found. TNF-α also stimulated monocytes' osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF-α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down-regulated while TNF-αR1 and TNF-αR2 were up-regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF-α, indicating an altered ratio of bound-RANK/unbound-RANK. Our findings suggest a diverse role of TNF-α on monocytes' osteoclastogenesis: it affects the RANK-signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre-cultured with M-CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte-derived osteoclast formation away from the bone.

Interleukin-12 Induces Receptor Activator of Nuclear Factor-Kappa B Ligand Expression by Human Periodontal Ligament Cells.

BACKGROUND: Increased level of proinflammatory cytokine interleukin (IL)-12 correlates with the severity of periodontitis. Yet, a possible role of IL-12 in periodontal disease has not been clarified. The aim of this study is to investigate whether IL-12 affects expression of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), a potent osteoclast-stimulating factor, by human periodontal ligament (hPDL) cells. METHODS: To determine the effect of IL-12, hPDL cells were incubated with recombinant human IL-12 (p70) in a dose- (0 to 10 ng/mL) and time-dependent manner. Expression of RANKL was evaluated at mRNA and protein levels. Underlying signaling pathways of IL-12 were determined by using specific inhibitors. RESULTS: Under the influence of IL-12, hPDL cells expressed significantly higher levels of RANKL. Expression was mediated by signal transducer and activator of transcription 4 and NF-κB signaling pathways. Conditioned medium of IL-12-incubated cells proved to contain molecule(s) that induced RANKL expression. Addition of suramin (G protein-coupled receptor inhibitor) and ethylene glycol tetraacetic acid (calcium chelator) suggested existence of intermediate molecule(s) that could activate heterotrimeric G protein signaling in a calcium-dependent pathway. CONCLUSIONS: Expression of RANKL by hPDL cells significantly increased after IL-12 treatment. Therefore, this study supports a close interrelationship between immune and skeletal systems and suggests an osteolytic role of IL-12 in pathogenesis of periodontal disease.

Cyclic Tensile Strain Reduces TNF-α Induced Expression of MMP-13 by Condylar Temporomandibular Joint Cells.

To investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ-derived cells to tumor necrosis factor-α (TNF-α), and whether mechanical loading affects this response. The effect of TNF-α (0.1-10 ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT-qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12 h with TNF-α (10 ng/ml), followed by 8 h of 6% cyclic tensile strain, and gene expression of MMPs was quantified. TNF-α (10 ng/ml) reduced the expression of the matrix proteins collagen types I and II after 6 h of incubation. Aggrecan gene expression was increased in the presence of 0.1 ng/ml TNF-α. The fossa and disc cells responded to TNF-α with an increased expression of the aggrecanase ADAMTS4. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect. Cells isolated from the different cartilaginous structures reacted differently to TNF-α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF-α induced MMP-13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF-α. J. Cell. Physiol. 232: 1287-1294, 2017. © 2016 Wiley Periodicals, Inc.

Histone deacetylase inhibition enhances in-vivo bone regeneration induced by human periodontal ligament cells.

Periodontal ligament cells have the potential to differentiate into bone forming osteoblasts and thus represent a good cellular candidate for bone regeneration. This study aimed to investigate the effect of inhibition of histone deacetylases, using the inhibitor Trichostatin A (TSA), on bone regeneration by human periodontal ligament cells (hPDLCs) in a mouse calvaria bone defect. METHODS: RUNX2 protein and its acetylation was analyzed by immunoprecipitation and western blotting. The effect of TSA on osteogenic differentiation of hPDLCs was investigated using in vitro 3D cultures. hPDLCs were pre-incubated with and without TSA and implanted in mouse calvaria defects with polycaprolactone/polyethylene glycol (PCL/PEG) co-polymer scaffold. Micro-CT scanning and bone histomorphometric analysis were used to quantify the amount of bone. Survival of hPDLCs as xenogenic grafts was verified by immunohistochemistry with anti-human ß1-integrin. The immunological response of mice against hPDLCs xenografts was evaluated by measuring total IgG and hPDLCs-specific IgG. RESULTS: Beside affecting histone protein, TSA also induced hyper-acetylation of RUNX2 which might be a crucial mechanism for enhancing osteogenesis by hPDLCs. TSA enhanced mineral deposition by hPDLCs in in vitro 3D cultures and had no effect on cell viability. In vivo bone regeneration of mouse calvaria defects was significantly enhanced by TSA pre-treated hPDLCs. By using anti-human ß1 integrin hPDLCs were shown to differentiate into osteocyte-like cells that were present in newly formed bone. hPDLCs, as a xenograft, slightly but not significantly induced an immunological response in recipient mice as demonstrated by the level of total IgG and hPDLCs-specific IgG. CONCLUSION: Inhibition of histone deacetylases by TSA enhanced in vivo bone regeneration by hPDLCs. The data strongly suggest a novel approach to regenerate bone tissue.

Monocyte Heterogeneity: Consequences for Monocyte-Derived Immune Cells.

Blood monocytes are precursors of dendritic cells, macrophages, and osteoclasts. They are a heterogeneous cell population with differences in size, phenotype, and function. Although monocytes maintain several tissue-specific populations of immune cells in homeostasis, their contribution to populations of dendritic cells, macrophages, and osteoclasts is significantly increased in inflammation. Identification of a growing number of functionally different subsets of cells within populations of monocyte-derived immune cells has recently put monocyte heterogeneity into sharp focus. Here, we summarize recent findings in monocyte heterogeneity and their differentiation into dendritic cells, macrophages, and osteoclasts. We also discuss these advances in the context of the formation of functionally different monocyte-derived subsets of dendritic cells, macrophages, and osteoclasts.

Osteonecrosis of the Jaw-a Bone Site-Specific Effect of Bisphosphonates.

A known complication that can occur in patients using bisphosphonates (BPs) is osteonecrosis of the jaw (ONJ). ONJ features bone exposure that may be associated with severe pain, swelling, local infection, and pathological fracture of the jaw. Current literature indicates that a complex combination of factors is necessary to induce ONJ. Several hypotheses about the pathophysiology of ONJ were previously reported. Here, we review these hypotheses and introduce new ideas and suggestions on this topic, focusing on bone site-specific cells, and the effect that BPs and other anti-resorptive drugs have on those cells. Gaining more insight into bone site-specific effects may help to better understand the pathogenesis ONJ, and contribute to the development of new bone site-specific anti-resorptive drugs.

New Insights into the Effects of Chronic Kidney Failure and Dialysate Exposure on the Peritoneum.

♦ INTRODUCTION: Chronic uremia and the exposure to dialysis solutions during peritoneal dialysis (PD) induce peritoneal alterations. Using a long-term peritoneal exposure model, we compared the effects of chronic kidney failure (CKD) itself and exposure to either a 'conventional' or a 'biocompatible' dialysis solution on peritoneal morphology and function. ♦ METHODS: Wistar rats (Harlan, Zeist, the Netherlands) were grouped into: normal kidney function (NKF), CKD induced by 70% nephrectomy, CKD receiving daily peritoneal infusions with 3.86% glucose Dianeal (CKDD), or Physioneal (both solutions from Baxter Healthcare, Castlebar, Ireland) (CKDP). At 16 weeks, a peritoneal function test was performed, and histology, ultrastructure, and hydroxyproline content of peritoneal tissue were assessed. ♦ RESULTS: Comparing CKD with NKF, peritoneal transport rates were higher, mesothelial cells (MC) displayed increased number of microvilli, blood and lymph vasculature expanded, vascular basal lamina appeared thicker, with limited areas of duplication, and fibrosis had developed. All alterations, except lymphangiogenesis, were enhanced by exposure to both dialysis fluids. Distinct MC alterations were observed in CKDD and CKDP, the latter displaying prominent basolateral protrusions. In addition, CKDP was associated with a trend towards less fibrosis compared to CKDD. ♦ CONCLUSIONS: Chronic kidney failure itself induced peritoneal alterations, which were in part augmented by exposure to glucose-based dialysis solutions. Overall, the conventional and biocompatible solutions had similar long-term effects on the peritoneum. Importantly, the latter may attenuate the development of fibrosis.

IL-1ß differently stimulates proliferation and multinucleation of distinct mouse bone marrow osteoclast precursor subsets.

Osteoclasts are bone-resorbing cells and targets for treating bone diseases. Previously, we reported that distinct murine osteoclast precursor subsets, such as early blasts (CD31(hi) Ly-6C(-)), myeloid blasts (CD31(+) Ly-6C(+)), and monocytes (CD31(-) Ly-6C(hi)), respond differently to the osteoclastogenesis-inducing cytokines, macrophage colony-stimulating factor, and receptor activator for nuclear factor κB ligand. It is unknown, however, how these cell types respond to the osteoclast-stimulating inflammatory cytokine interleukin 1ß. This study aims to investigate the effect of interleukin 1ß on osteoclastogenesis derived from different mouse bone marrow precursors. Early blasts, myeloid blasts, and monocytes were sorted from mouse bone marrow cells using flow cytometry. Cells were cultured on plastic or on bone slices in the presence of macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand, without or with interleukin 1ß (0.1-10 ng/ml). We found that interleukin 1ß stimulated multinucleation and bone resorption of osteoclasts derived from the 3 precursors at different rates. The most large osteoclasts (>20 nuclei) and highest level of bone resorption (16.3%) was by myeloid blast-derived osteoclasts. Interleukin 1ß particularly accelerated proliferation of early blasts and the most small osteoclasts (3-5 nuclei) formed on plastic. Life span varied among osteoclasts derived from different precursors: large osteoclasts (>2400 µm(2)) formed most rapidly (75 h) from myeloid blasts but had a short life span (30 h). Monocytes needed the longest time (95 h) for the generation of such large osteoclasts, but these cells had a longer life span (50 h). Our results indicate that the different bone marrow osteoclast precursors are differently stimulated by interleukin 1ß with respect to proliferation, multinucleation, life span, and bone resorption.

Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone.

Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of osteoclasts. The data may suggest that the heterogeneity of osteoclasts is partially due to the way the osteoblasts induce their formation.

Different Blood-Borne Human Osteoclast Precursors Respond in Distinct Ways to IL-17A.

Osteoclasts are bone-degrading cells that are formed through fusion of their monocytic precursors. Three distinct subsets of monocytes have been identified in human peripheral blood: classical, intermediate, and non-classical monocytes. They are known to play different roles in physiology and pathology, but their capacity to differentiate into osteoclasts and whether inflammatory cytokines influence this differentiation is unknown. We hypothesized that classical, intermediate, and non-classical monocytes generate functionally different osteoclasts and that they respond in different ways to the inflammatory cytokine interleukin-17A (IL-17A). To investigate this, the different monocyte subsets were isolated from human peripheral blood and osteoclastogenesis was induced with the cytokines M-CSF and RANKL, with or without IL-17A. We found that all subsets are able to differentiate into osteoclasts in vitro, and that both osteoclastogenesis and subsequent bone resorption was distinctly affected by IL-17A. Osteoclastogenesis and bone resorption by osteoclasts derived from classical monocytes remained unaffected by IL-17A, while osteoclast formation from intermediate monocytes was inhibited by the cytokine. Surprisingly, bone resorption by osteoclasts derived from intermediate monocytes remained at similar levels as control cultures, indicating an increased bone resorbing activity by these osteoclasts. Limited numbers of osteoclasts were formed from non-classical monocytes on bone and no bone resorption was detected, which suggest that these cells belong to a cell lineage different from the osteoclast. By providing more insight into osteoclast formation from human blood monocytes, this study contributes to the possible targeting of specific osteoclast precursors as a therapeutic approach for diseases associated with inflammatory bone loss.

The number of immune cells is lower in healthy oral mucosa compared to skin and does not increase after scarring.

OBJECTIVE: Depending on the location of injury, wounds can heal with different outcomes. In addition foetal wounds heal fast without scar formation, while scars are a common feature of regular skin repair. Since inflammation is very limited in these wounds reduced numbers or even absence of immune cells might be responsible for scarless foetal wound healing. It is thought that various immune cells, such as macrophages, neutrophils and T-cells, play a role in aberrant wound healing and the fibrotic process seen in scar formation in the adult skin. Similar to the foetus, oral wounds show comparable healing properties by means of accelerated reepithelialization and negligible scar formation. It is possible that reduced inflammatory reaction as a result of lower numbers of immune cells are present in oral wounds compared to skin wounds. DESIGN: Here we investigated the presence of various immune cells in human skin and oral mucosa, with or without scars. The presence or absence of these cells may play a role in the different modes of healing observed between the two types of tissue. Mast cells, neutrophils, M1/M2 macrophages, T-cells and blood vessels were localized in healthy and scarred skin and oral mucosa (scars>1 year old). RESULTS: Oral mucosa had significantly fewer neutrophils, macrophages, mannose receptor-positive M2 macrophages, but more blood vessels. Scars contained similar numbers of immune cells compared to healthy tissues. CONCLUSIONS: Less immune cells in the healthy oral mucosa may induce a diminished immune reaction when wounding occurs, and could explain the better healing capacity of the oral mucosa.