The Differential Effect of Metformin on Osteocytes, Osteoblasts, and Osteoclasts.

PURPOSE OF REVIEW: Metformin is an anti-glycemic agent, which is widely prescribed to diabetes patients. Although its alleged role on bone strength has been reported for some time, this review focuses primarily on the recent mechanistical insights of metformin on osteocytes, osteoblasts, and osteoclasts. RECENT FINDINGS: Overall, metformin contributed to steering anabolic activity in osteocytes. It caused lower expression in osteocytes of the negative regulators of bone formation sclerostin and DKK1. Likewise, the osteoclastogenesis function of osteoblasts was also skewed towards lower RANKL and higher OPG expressions. Osteoblast lineage cells generally responded to metformin by activating bone formation parameters, such as alkaline phosphatase activity, higher expression of anabolic members of the Wnt pathway, transcription factor Runx2, bone matrix protein proteins, and subsequent mineralization. Metformin affected osteoclast formation and activity in a negative way, reducing the number of multinucleated cells in association with lower expression of typical osteoclast markers and with inhibited resorption. A common denominator studied in all three cell types is its beneficial effect on activating phosphorylated AMP kinase (AMPK) which is associated with the coordination of energy metabolism. Metformin differentially affects bone cells, shifting the balance to more bone formation. Although metformin is a drug prescribed for diabetic patients, the overall bone anabolic effects on osteocytes and osteoblasts and the anti-catabolic effect on osteoclast suggest that metformin could be seen as a promising drug in the bone field.

Transcriptomic Differences Underlying the Activin-A Induced Large Osteoclast Formation in Both Healthy Control and Fibrodysplasia Ossificans Progressiva Osteoclasts.

Fibrodysplasia Ossificans Progressiva (FOP) is a very rare genetic disease characterized by progressive heterotopic ossification (HO) of soft tissues, leading to immobility and premature death. FOP is caused by a mutation in the Activin receptor Type 1 (ACVR1) gene, resulting in altered responsiveness to Activin-A. We recently revealed that Activin-A induces fewer, but larger and more active, osteoclasts regardless of the presence of the mutated ACVR1 receptor. The underlying mechanism of Activin-A-induced changes in osteoclastogenesis at the gene expression level remains unknown. Transcriptomic changes induced by Activin-A during osteoclast formation from healthy controls and patient-derived CD14-positive monocytes were studied using RNA sequencing. CD14-positive monocytes from six FOP patients and six age- and sex-matched healthy controls were differentiated into osteoclasts in the absence or presence of Activin-A. RNA samples were isolated after 14 days of culturing and analyzed by RNA sequencing. Non-supervised principal component analysis (PCA) showed that samples from the same culture conditions (e.g., without or with Activin-A) tended to cluster, indicating that the variability induced by Activin-A treatment was larger than the variability between the control and FOP samples. RNA sequencing analysis revealed 1480 differentially expressed genes induced by Activin-A in healthy control and FOP osteoclasts with p(adj) < 0.01 and a Log2 fold change of ≥±2. Pathway and gene ontology enrichment analysis revealed several significantly enriched pathways for genes upregulated by Activin-A that could be linked to the differentiation or function of osteoclasts, cell fusion or inflammation. Our data showed that Activin-A has a substantial effect on gene expression during osteoclast formation and that this effect occurred regardless of the presence of the mutated ACVR1 receptor causing FOP.

The Effect of Sclerostin and Monoclonal Sclerostin Antibody Romosozumab on Osteogenesis and Osteoclastogenesis Mediated by Periodontal Ligament Fibroblasts.

Sclerostin is a bone formation inhibitor produced by osteocytes. Although sclerostin is mainly expressed in osteocytes, it was also reported in periodontal ligament (PDL) fibroblasts, which are cells that play a role in both osteogenesis and osteoclastogenesis. Here, we assess the role of sclerostin and its clinically used inhibitor, romosozumab, in both processes. For osteogenesis assays, human PDL fibroblasts were cultured under control or mineralizing conditions with increasing concentrations of sclerostin or romosozumab. For analyzing osteogenic capacity and alkaline phosphatase (ALP) activity, alizarin red staining for mineral deposition and qPCR of osteogenic markers were performed. Osteoclast formation was investigated in the presence of sclerostin or romosozumab and, in PDLs, in the presence of fibroblasts co-cultured with peripheral blood mononuclear cells (PBMCs). PDL-PBMC co-cultures stimulated with sclerostin did not affect osteoclast formation. In contrast, the addition of romosozumab slightly reduced the osteoclast formation in PDL-PBMC co-cultures at high concentrations. Neither sclerostin nor romosozumab affected the osteogenic capacity of PDL fibroblasts. qPCR analysis showed that the mineralization medium upregulated the relative expression of osteogenic markers, but this expression was barely affected when romosozumab was added to the cultures. In order to account for the limited effects of sclerostin or romosozumab, we finally compared the expression of SOST and its receptors LRP-4, -5, and -6 to the expression in osteocyte rich-bone. The expression of SOST, LRP-4, and LRP-5 was higher in osteocytes compared to in PDL cells. The limited interaction of sclerostin or romosozumab with PDL fibroblasts may relate to the primary biological function of the periodontal ligament: to primarily resist bone formation and bone degradation to the benefit of an intact ligament that is indented by every chew movement.

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.

Intra-pulpal connective tissue formation and the advanced carious lesion: Is chondrogenesis and heterotopic ossification a response to pulpal inflammation?

AIMS: (a) The aim of this study was to investigate both the formation of dense connective tissue within the dental pulp, and its association with pulpal inflammation in teeth with advanced carious lesions; and (b) to investigate in vitro whether inflammation affects the expression of markers related to chondrogenesis/osteogenesis in pulp cells. MATERIALS AND METHODS: Radiology and Histology: Forty-six teeth with advanced carious lesions were radiographically investigated for intra-pulpal radiodense structures. Specimens were processed for histology and stained with haematoxylin/eosin and proteoglycan-specific stains. The intra-pulpal connective tissue was scored as pulp stones or ectopic connective tissue. Cell culture: pulpal cells from human third molars (n = 5) were cultured in chondrogenic medium +/- TLR2/4 agonists. Expression of the genes IL6, TLR2/4, SOX9, COL1A1, COL2A1, TGFB1, RUNX2 and ALPL was assessed by qPCR. Proteoglycan content within cultures was assessed spectrophotometrically. RESULTS: Radiodense structures were discovered in about half of all pulps. They were associated with ectopic connective tissue (χ2  = 8.932, p = .004, OR = 6.80, 95% CI: [1.84, 25.19]) and with pulp stones (χ2  = 12.274, df = 1, p < .001, OR = 22.167, 95% CI: [2.57, 200.00]). The morphology of the ectopic tissue resembled cartilage and was associated with inflammatory infiltration of the pulp (χ2  = 10.148, p = .002, OR = 17.77, 95% CI: [2.05, 154.21]). After continuous stimulation of cultured cells with TLR2/4 agonists, the expression of two inflammatory markers increased: IL6 at Days 7 (p = .020) and 14 (p = .008); TLR2 at Days 7 (p = .023) and 14 (p = .009). Similarly, expression of chondrogenic markers decreased: SOX9 at Day 14 (p = .035) and TGFB1 at Day 7 (p = .004), and the osteogenic marker COL1A1 at Day 7 (p = .007). Proteoglycan content did not differ between unstimulated and stimulated cells. CONCLUSIONS: Ectopic connective tissue resembling cartilage can form in teeth affected by advanced carious lesions. This tissue type is radiographically visible and is associated with inflammatory infiltration of the pulp. Although TLR2/4 agonists led to an inflammatory response in cell culture of pulp cells, the effect on the expression of osteogenic/chondrogenic markers was limited, suggesting that immune cells are needed for connective tissue formation in vivo.

Increased Bone Resorption during Lactation in Pycnodysostosis.

Pycnodysostosis, a rare autosomal recessive skeletal dysplasia, is caused by a deficiency of cathepsin K. Patients have impaired bone resorption in the presence of normal or increased numbers of multinucleated, but dysfunctional, osteoclasts. Cathepsin K degrades collagen type I and generates N-telopeptide (NTX) and the C-telopeptide (CTX) that can be quantified. Levels of these telopeptides are increased in lactating women and are associated with increased bone resorption. Nothing is known about the consequences of cathepsin K deficiency in lactating women. Here we present for the first time normalized blood and CTX measurements in a patient with pycnodysostosis, exclusively related to the lactation period. In vitro studies using osteoclasts derived from blood monocytes during lactation and after weaning further show consistent bone resorption before and after lactation. Increased expression of cathepsins L and S in osteoclasts derived from the lactating patient suggests that other proteinases could compensate for the lack of cathepsin K during the lactation period of pycnodysostosis patients.

Effects of L-PRF and A-PRF+ on periodontal fibroblasts in in vitro wound healing experiments.

OBJECTIVE: To determine whether leukocyte-platelet-rich fibrin (L-PRF) and advanced platelet-rich fibrin (A-PRF+) differ in their in vitro capacity to induce proliferation and migration of periodontal fibroblasts. BACKGROUND: L-PRF and A-PRF + are autologous materials used in periodontal regenerative surgery. They derive from blood from patients, but have different characteristics. The literature is controversial regarding the effects of the two PRF preparations on periodontal tissue fibroblasts. MATERIALS AND METHODS: L-PRF and A-PRF + membranes were prepared from eight patients and incubated in 3 mL of culture medium for 2 days. Gingival fibroblasts (G-F) and periodontal ligament fibroblast (PDL-F) primary cells were retrieved from 7 donors. These cells were pre-cultured for 1 day in wound healing experiment plates leaving a gap of 500 ± 50 µm in a concentration of 3.3 x 105 cells/mL. 70 µL of the cell suspension was placed in each half of the well. Thereafter, the pre-cultured L-PRF and A-PRF + supernatants were added to the experimental plates, and the fibroblasts were incubated for another 24 h. Medium alone (NEG) and fibroblast growth factor II (FGF) were used as controls. Subsequently, cell migration was registered for 24 h with live cell imaging in a time frame microscope at 5% CO2 in air at 37°C. Images were analyzed using ImageJ. Cell proliferation and cell viability were measured. RESULTS: L-PRF and A-PRF + induced higher cell proliferation than FGF and NEG. Both A-PRF + and L-PRF induced significant faster artificial wound closure than controls. Both PRF conditioned media induced faster cell migration in the initial phase (P < .01), but in the stoppage phase, the induced migration was higher for the A-PRF+, compared with L-PRF (P < .01). CONCLUSION: L-PRF and A-PRF + have a stimulatory effect on migration and proliferation of periodontal fibroblasts, and artificial wound closure was longer sustained by A-PRF + than L-PRF.

Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells.

Osteoblasts derived from mouse skulls have increased osteoclastogenic potential compared to long bone osteoblasts when stimulated with 1,25(OH)2 vitamin D3 (vitD3). This indicates that bone cells from specific sites can react differently to biochemical signals, e.g., during inflammation or as emitted by bioactive bone tissue-engineering constructs. Given the high turn-over of alveolar bone, we hypothesized that human alveolar bone-derived osteoblasts have an increased osteogenic and osteoclastogenic potential compared to the osteoblasts derived from long bone. The osteogenic and osteoclastogenic capacity of alveolar bone cells and long bone cells were assessed in the presence and absence of osteotropic agent vitD3. Both cell types were studied in osteogenesis experiments, using an osteogenic medium, and in osteoclastogenesis experiments by co-culturing osteoblasts with peripheral blood mononuclear cells (PBMCs). Both osteogenic and osteoclastic markers were measured. At day 0, long bones seem to have a more late-osteoblastic/preosteocyte-like phenotype compared to the alveolar bone cells as shown by slower proliferation, the higher expression of the matrix molecule Osteopontin (OPN) and the osteocyte-enriched cytoskeletal component Actin alpha 1 (ACTA1). This phenotype was maintained during the osteogenesis assays, where long bone-derived cells still expressed more OPN and ACTA1. Under co-culture conditions with PBMCs, long bone cells also had a higher Tumor necrose factor-alfa (TNF-α) expression and induced the formation of osteoclasts more than alveolar bone cells. Correspondingly, the expression of osteoclast genes dendritic cell specific transmembrane protein (DC-STAMP) and Receptor activator of nuclear factor kappa-Β ligand (RankL) was higher in long bone co-cultures. Together, our results indicate that long bone-derived osteoblasts are more active in bone-remodeling processes, especially in osteoclastogenesis, than alveolar bone-derived cells. This indicates that tissue-engineering solutions need to be specifically designed for the site of application, such as defects in long bones vs. the regeneration of alveolar bone after severe periodontitis.

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.

Burst, Short, and Sustained Vitamin D3 Applications Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells.

Incorporation of 1,25(OH)2 vitamin D3 (vitD3) into tissue-engineered scaffolds could aid the healing of critical-sized bone defects. We hypothesize that shorter applications of vitD3 lead to more osteogenic differentiation of mesenchymal stem cells (MSCs) than a sustained application. To test this, release from a scaffold was mimicked by exposing MSCs to exactly controlled vitD3 regimens. Human adipose stem cells (hASCs) were seeded onto calcium phosphate particles, cultured for 20 days, and treated with 124 ng vitD3, either provided during 30 min before seeding ([200 nM]), during the first two days ([100 nM]), or during 20 days ([10 nM]). Alternatively, hASCs were treated for two days with 6.2 ng vitD3 ([10 nM]). hASCs attached to the calcium phosphate particles and were viable (~75%). Cell number was not affected by the various vitD3 applications. VitD3 (124 ng) applied over 20 days increased cellular alkaline phosphatase activity at Days 7 and 20, reduced expression of the early osteogenic marker RUNX2 at Day 20, and strongly upregulated expression of the vitD3 inactivating enzyme CYP24. VitD3 (124 ng) also reduced RUNX2 and increased CYP24 applied at [100 nM] for two days, but not at [200 nM] for 30 min. These results show that 20-day application of vitD3 has more effect on hASCs than the same total amount applied in a shorter time span.

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.

The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.

The Osteocyte as a Novel Key Player in Understanding Periodontitis Through its Expression of RANKL and Sclerostin: a Review.

PURPOSE OF REVIEW: Periodontitis is the inflammation-associated bone loss disease of the alveolar bone that surrounds teeth. Classically, the emphasis on the etiology of periodontitis has been on the products of periodontal pathogens that lead to an inflammatory response of the soft tissues of the periodontium, eventually leading to activation of osteoclasts that degrade the alveolar bone. Until recently, the response of osteocytes that populate the alveolar bone, and that are known for their regulatory role in bone anabolism and catabolism, has not been addressed. RECENT FINDINGS: This review demonstrates that osteocytes play a key contributing role in periodontitis progression in various experimental mouse and rat periodontitis models. Osteocytes are the key expressing cells of both osteoclast differentiation factor RANKL as well as osteoblast activity regulator sclerostin. Targeted deletion of RANKL in osteocytes prevents osteoclast formation, thereby impairing periodontitis, despite the pressure of periodontitis-associated bacteria. Antibodies against the osteocyte-derived protein sclerostin inhibit and partially revert periodontitis by stimulating bone formation. Experimental mouse and rat periodontitis models strongly indicate a key role for the bone-encapsulated osteocyte in understanding periodontitis etiology.

T Cell Proliferation Is Induced by Chronically TLR2-Stimulated Gingival Fibroblasts or Monocytes.

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF-PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC-GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3-), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, showed a significantly (p < 0.0001) increased proinflammatory cytokine production of TNF-α and IL-1ß up to 21 days only in TLR2-activated PBMC with concomitant T cell proliferation, suggesting a role for monocytes. In conclusion, chronic TLR activation mediates the shift in cell populations during infection. Particularly, TLR2 activators play an important role in T cell proliferation and proinflammatory cytokine production by monocytes, suggesting that TLR2 activation represents a bridge between innate and adaptive immunity.

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.

Hypoxia negatively affects senescence in osteoclasts and delays osteoclastogenesis.

Cellular senescence, that is, the withdrawal from the cell cycle, combined with the acquirement of the senescence associated secretory phenotype has important roles during health and disease and is essential for tissue remodeling during embryonic development. Osteoclasts are multinucleated cells, responsible for bone resorption, and cell cycle arrest during osteoclastogenesis is well recognized. Therefore, the aim of this study was to investigate whether these cells should be considered senescent and to assess the influence of hypoxia on their potential senescence status. Osteoclastogenesis and bone resorption capacity of osteoclasts, cultured from CD14+ monocytes, were evaluated in two oxygen concentrations, normoxia (21% O2 ) and hypoxia (5% O2 ). Osteoclasts were profiled by using specific staining for proliferation and senescence markers, qPCR of a number of osteoclast and senescence-related genes and a bone resorption assay. Results show that during in vitro osteoclastogenesis, osteoclasts heterogeneously obtain a senescent phenotype. Furthermore, osteoclastogenesis was delayed at hypoxic compared to normoxic conditions, without negatively affecting the bone resorption capacity. It is concluded that osteoclasts can be considered senescent, although senescence is not uniformly present in the osteoclast population. Hypoxia negatively affects the expression of some senescence markers. Based on the direct relationship between senescence and osteoclastogenesis, it is tempting to hypothesize that contents of the so-called senescence associated secretory phenotype (SASP) not only play a functional role in matrix resorption, but also may regulate osteoclastogenesis.

Osteogenic and osteoclastogenic potential of jaw bone-derived cells-A case study.

Though the stem cell properties of tooth-derived periodontal ligament and gingival cells have been widely documented, surprisingly little is known about both the osteogenic and osteoclastogenic differentiation capacities of the more clinically relevant jaw bone-derived cells. These cells could be considered being recruited during bone healing such as after tooth extraction, after placing an implant, or after surgical or traumatic injury. Here, we compared the osteoblast and osteoclastogenesis features of four consecutive bone outgrowths with periodontal ligament and gingiva cells. For osteogenesis assay, cells were cultured in osteogenic medium, whereas in osteoclastogenesis assays, cells were cultured in the presence of human peripheral blood mononuclear cells (PBMCs) as a source of osteoclast precursors. After osteogenic stimulus, all six cell types responded by an increased expression of osteoblast markers RUNX2 and DMP1. Periodontal ligament cells expressed significantly higher levels of RUNX2 compared to all bone outgrowths. Alkaline phosphatase enzyme levels in periodontal ligament cells reached earlier and higher peak expression. Mineral deposits were highest in periodontal ligament, gingiva and the first bone outgrowth. Osteoclastogenesis revealed a stepwise increase of secreted pro-osteoclastogenesis proteins M-CSF, IL-1ß, and TNF-α in the last three consecutive bone cultures. OPG mRNA showed the opposite: high expression in periodontal and gingiva cells and the first outgrowth. Osteoclast numbers were similar between the six cultures, both on bone and on plastic. This first study reveals that jaw bone outgrowths contain bone remodelling features that are slightly different from tooth-associated cells.

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.

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.