Periostin-integrin interaction regulates force-induced TGF-ß1 and α-SMA expression by hPDLSCs.

OBJECTIVE: Periostin (PN), a major matricellular periodontal ligament (PDL) protein, modulates the remodeling of the PDL and bone, especially under mechanical stress. This study investigated the requirement of PN-integrin signaling in force-induced expression of transforming growth factor-beta 1 (TGF-ß1) and alpha-smooth muscle actin (α-SMA) in human PDL stem cells (hPDLSCs). METHODS: Cells were stimulated with intermittent compressive force (ICF) using computerized controlled apparatus. Cell migration was examined using in vitro scratch assay. The mRNA expression was examined using real-time polymerase chain reaction. The protein expression was determined using immunofluorescent staining and western blot analysis. RESULTS: Stimulation with ICF for 24 h increased the expression of PN, TGF-ß1, and α-SMA, along with increased SMAD2/3 phosphorylation. Knockdown of POSTN (PN gene) decreased the protein levels of TGF-ß1 and pSMAD2/3 upon force stimulation. POSTN knockdown of hPDLSCs resulted in delayed cell migration, as determined by a scratch assay. However, migration improved after seeding these knockdown cells on pre-PN-coated surfaces. Further, the knockdown of αVß5 significantly attenuated the force-induced TGF-ß1 expression. CONCLUSION: Our findings indicate the importance of PN-αVß5 interactions in ICF-induced TGF-ß1 signaling and the expression of α-SMA. Findings support the critical role of PN in maintaining the PDL's tissue integrity and homeostasis.

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.

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.

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 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.

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

IL-1ß Damages Fibrocartilage and Upregulates MMP-13 Expression in Fibrochondrocytes in the Condyle of the Temporomandibular Joint.

The temporomandibular joint (TMJ), which differs anatomically and biochemically from hyaline cartilage-covered joints, is an under-recognized joint in arthritic disease, even though TMJ damage can have deleterious effects on physical appearance, pain and function. Here, we analyzed the effect of IL-1ß, a cytokine highly expressed in arthritic joints, on TMJ fibrocartilage-derived cells, and we investigated the modulatory effect of mechanical loading on IL-1ß-induced expression of catabolic enzymes. TMJ cartilage degradation was analyzed in 8-11-week-old mice deficient for IL-1 receptor antagonist (IL-1RA-/-) and wild-type controls. Cells were isolated from the juvenile porcine condyle, fossa, and disc, grown in agarose gels, and subjected to IL-1ß (0.1-10 ng/mL) for 6 or 24 h. Expression of catabolic enzymes (ADAMTS and MMPs) was quantified by RT-qPCR and immunohistochemistry. Porcine condylar cells were stimulated with IL-1ß for 12 h with IL-1ß, followed by 8 h of 6% dynamic mechanical (tensile) strain, and gene expression of MMPs was quantified. Early signs of condylar cartilage damage were apparent in IL-1RA-/- mice. In porcine cells, IL-1ß strongly increased expression of the aggrecanases ADAMTS4 and ADAMTS5 by fibrochondrocytes from the fossa (13-fold and 7-fold) and enhanced the number of MMP-13 protein-expressing condylar cells (8-fold). Mechanical loading significantly lowered (3-fold) IL-1ß-induced MMP-13 gene expression by condylar fibrochondrocytes. IL-1ß induces TMJ condylar cartilage damage, possibly by enhancing MMP-13 production. Mechanical loading reduces IL-1ß-induced MMP-13 gene expression, suggesting that mechanical stimuli may prevent cartilage damage of the TMJ in arthritic patients.

Integrin αMß2 is differently expressed by subsets of human osteoclast precursors and mediates adhesion of classical monocytes to bone.

Bone-degrading osteoclasts are formed through fusion of their monocytic precursors. In the population of human peripheral blood monocytes, three distinct subsets have been identified: classical, intermediate and non-classical monocytes. We have previously shown that when the monocyte subsets are cultured on bone, significantly more osteoclasts are formed from classical monocytes than from intermediate or non-classical monocytes. Considering that this difference does not exist when monocyte subsets are cultured on plastic, we hypothesized that classical monocytes adhere better to the bone surface compared to intermediate and non-classical monocytes. To investigate this, the different monocyte subsets were isolated from human peripheral blood and cultured on slices of human bone in the presence of the cytokine M-CSF. We found that classical monocytes adhere better to bone due to a higher expression of the integrin αMß2 and that their ability to attach to bone is significantly decreased when the integrin is blocked. This suggests that integrin αMß2 mediates attachment of osteoclast precursors to bone and thereby enables the formation of osteoclasts.

The immunopathogenic and immunomodulatory effects of interleukin-12 in periodontal disease.

Interleukin 12 (IL-12) is an inflammatory cytokine that promotes the response of the immune system. This cytokine has been implicated as a potent stimulator of several diseases characterized by inflammatory-induced bone destruction, such as rheumatoid arthritis and periodontitis. Yet, the exact role of IL-12 in the development and progress of periodontitis has not been clarified. Several studies have demonstrated a positive correlation between the level of IL-12 and the severity of periodontal destruction. Deletion of IL-12 in mice with periodontitis significantly suppressed the level of bone destruction. Interestingly, next to a role in modulating the pathogenesis, IL-12 also has immunological-regulatory properties. This cytokine induces expression of immunosuppressive molecules, such as indoleamine-pyrrole 2,3-dioxygenase (IDO). Thus, these findings suggest both negative and positive influences of IL-12 in periodontal disease. It is currently proposed that the diversity of action of cytokines is a molecular key which regulates biological development and homeostasis. Accordingly, the actions of IL-12 might be one of the mechanisms that regulate homeostasis of periodontal tissue during and following inflammation. Therefore, this article aims to review both destructive and protective functionalities of IL-12 with an emphasis on periodontal disease.

Effect of mechanical loading on the metabolic activity of cells in the temporomandibular joint: a systematic review.

OBJECTIVES: The purpose of this systematic review was to elucidate how different modalities and intensities of mechanical loading affect the metabolic activity of cells within the fibro-cartilage of the temporomandibular joint (TMJ). MATERIALS AND METHODS: A systematic review was conducted according to PRISMA guidelines using PubMed, Embase, and Web of Science databases. The articles were selected following a priori formulated inclusion criteria (viz., in vivo and in vitro studies, mechanical loading experiments on TMJ, and the response of the TMJ). A total of 254 records were identified. After removal of duplicates, 234 records were screened by assessing eligibility criteria for inclusion. Forty-nine articles were selected for full-text assessment. Of those, 23 were excluded because they presented high risk of bias or were reviews. Twenty-six experimental studies were included in this systematic review: 15 in vivo studies and 11 in vitro ones. CONCLUSION: The studies showed that dynamic mechanical loading is an important stimulus for mandibular growth and for the homeostasis of TMJ cartilage. When this loading is applied at a low intensity, it prevents breakdown of inflamed cartilage. Yet, frequent overloading at excessive levels induces accelerated cell death and an increased cartilage degradation. CLINICAL SIGNIFICANCE: Knowledge about the way temporomandibular joint (TMJ) fibrocartilage responds to different types and intensities of mechanical loading is important to improve existing treatment protocols of degenerative joint disease of the TMJ, and also to better understand the regenerative pathway of this particular type of cartilage.

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.

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.

Phagocytosis of Collagen Fibrils by Fibroblasts In Vivo Is Independent of the uPARAP/Endo180 Receptor.

As a crucial step in ECM remodeling, collagen degradation occurs through different processes, including both extracellular and intracellular degradation. The extracellular pathways of collagen degradation require secretion of collagenolytic proteases, whereas intracellular collagen degradation occurs in the lysosomal compartment after uptake, involving either pre-cleaved or intact fibrillar collagen. The endocytic collagen receptor uPARAP/Endo180 plays an important role in internalization of large collagen degradation products, whereas its role in the phagocytosis of fibrillar collagen has been debated. In fact, the role of this receptor in regular collagen phagocytosis in vivo has not been established. In this study, we have studied the role of uPARAP in the phagocytosis of collagen fibrils in vivo by analyzing different connective tissues of mice lacking uPARAP. Using transmission electron microscopy (TEM), we found that fibroblasts in the periosteum of tibia and calvaria, as well as in the periodontal ligament of molar and incisor, phagocytosed collagen fibrils independently of uPARAP. Quantification of phagocytosed collagen in the periodontal ligament of uPARAP-deficient mice and controls revealed no difference in the amount of fibrillar collagen taken up by uPARAP-deficient mice. The findings show that under in vivo conditions uPARAP does not play a role in the phagocytic uptake of collagen fibrils by fibroblasts. Consequently, the cellular uptake of collagen fibrils and collagen cleavage products probably occurs through fundamentally different pathways. J. Cell. Biochem. 118: 1590-1595, 2017. © 2016 Wiley Periodicals, Inc.

Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism.

Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP+ osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm2; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.

Ultrastructural aspects of foreign body giant cells generated on different substrates.

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.

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.

Inhibition of Histone Deacetylases Enhances the Osteogenic Differentiation of Human Periodontal Ligament Cells.

One of the characteristics of periodontal ligament (PDL) cells is their plasticity. Yet, the underlying mechanisms responsible for this phenomenon are unknown. One possible mechanism might be related to epigenetics, since histone deacetylases (HDACs) have been shown to play a role in osteoblast differentiation. This study was aimed to investigate the role of HDACs in osteogenic differentiation of human PDL (hPDL) cells. HDAC inhibitor trichostatin A (TSA) had no effect on cell viability as was assessed by MTT assay. Osteogenic and adipogenic differentiation was analyzed by gene expression, ALP activity and mineral deposition. Western blotting was used to investigate the effect of TSA on histone acetylation and protein expression. In the presence of the HDAC inhibitor osteogenic differentiation was induced; osteoblast-related gene expression was increased significantly. ALP activity and mineral nodule formation were also enhanced. Inhibition of HDACs did not induce differentiation into the adipocyte lineage. hPDL highly expressed HDACs of both class I (HDAC 1, 2, 3) and class II (HDAC 4, 6). During osteogenic differentiation HDAC 3 expression gradually decreased. This was apparent in the absence and presence of the inhibitor. The level of acetylated Histone H3 was increased during osteogenic differentiation. Inhibition of HDAC activity induced hyperacetylation of Histone H3, therefore, demonstrating Histone H3 as a candidate target molecule for HDAC inhibition. In conclusion, hPDL cells express a distinguished series of HDACs and these enzymes appear to be involved in osteogenic differentiation. This finding suggests a potential application of TSA for bone regeneration therapy by hPDL cells.

PLS3 mutations in X-linked osteoporosis with fractures.

Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.

Null mutation of chloride channel 7 (Clcn7) impairs dental root formation but does not affect enamel mineralization.

ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.