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.

TGF-Beta Induces Activin A Production in Dermal Fibroblasts Derived from Patients with Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic, ultra-rare disease of heterotopic ossification caused by genetic defects in the ACVR1 gene. The mutant ACVR1 receptor, when triggered by an inflammatory process, leads to heterotopic ossification of the muscles and ligaments. Activin A has been discovered as the main osteogenic ligand of the FOP ACVR1 receptor. However, the source of Activin A itself and the trigger of its production in FOP individuals have remained elusive. We used primary dermal fibroblasts from five FOP patients to investigate Activin A production and how this is influenced by inflammatory cytokines in FOP. FOP fibroblasts showed elevated Activin A production compared to healthy controls, both in standard culture and osteogenic transdifferentiation conditions. We discovered TGFß1 to be an FOP-specific stimulant of Activin A, shown by the upregulation of the INHBA gene and protein expression. Activin A and TGFß1 were both induced by BMP4 in FOP and control fibroblasts. Treatment with TNFα and IL6 produced negligible levels of Activin A and TGFß1 in both cell groups. We present for the first time TGFß1 as a triggering factor of Activin A production in FOP. As TGFß1 can promote the induction of the main driver of FOP, TGFß1 could also be considered a possible therapeutic target in FOP treatment.

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.

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.

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.

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.

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.

M-CSF priming of osteoclast precursors can cause osteoclastogenesis-insensitivity, which can be prevented and overcome on bone.

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.

A challenge with Porphyromonas gingivalis differentially affects the osteoclastogenesis potential of periodontal ligament fibroblasts from periodontitis patients and non-periodontitis donors.

AIM: Porphyromonas gingivalis (Pg) may cause an immune-inflammatory response in host cells leading to bone degradation by osteoclasts. We investigated the osteoclast-inducing capacity of periodontal ligament fibroblasts from periodontitis patients and non-periodontitis donors after a challenge with viable Pg. MATERIALS AND METHODS: PDLFs from periodontitis patients (n = 8) and non-periodontitis donors (n = 7) were incubated for 6 h with or without viable Pg and subsequently co-cultured with osteoclast precursors from peripheral blood mononuclear cells (PBMCs). The number of multinucleated tartrate-resistant acid phosphatase-positive cells was determined at 21 days. Expression of osteoclastogenesis-associated genes was assessed after infection of PDLFs mono-cultures and in PDLFs-PBMCs co-cultures. Resorption activity was analysed on bone slices. RESULTS: Pg induced the expression of osteoclastogenesis-associated genes by PDLFs. After bacterial challenge the formation of osteoclast-like cell was decreased in co-cultures of PBMCs with non-periodontitis PDLFs, but not with PDLFs from periodontitis patients. CONCLUSION: PDLFs from sites free of periodontitis respond to an infection with Pg by tempering formation of osteoclast-like cells, probably promoting clearance of the infection. PDLFs from periodontitis sites are desensitized to a Pg challenge in terms of their osteoclast-inducing capacity.

Passaging of gingival fibroblasts from periodontally healthy and diseased sites upregulates osteogenesis-related genes.

To investigate biological processes of the periodontium, in vitro primary cell models have been established. To study the biology of the gingiva, primary gingival fibroblast cell models are widely used. For such experiments, cells need to be expanded and passaged. A key assumption is that primary cells maintain most of their original characteristics they have in situ. The aim of this research is to explore the impact of early passaging on selected gene expression of human gingival fibroblast cells. For this purpose, gene expression from the outgrowth of the resected tissues until the fourth passage was followed for nine tissue samples, from both healthy and diseased sites. Micrographs were taken from the cultures, RNA was extracted from the samples of each passage and quantitative PCR was performed for selected genes representing various biological processes. Epithelial cells were present during the first outgrowth, but were no longer present in the second passage. Our results indicate that the morphology of the gingival fibroblast cells does not change with passaging and that passages 2-4 contain only gingival fibroblasts. Gene expression of M-CSF, TNF-α, TLR4, POSTN and FAPα was unchanged by passaging, the expression of IL-6, IL-1ß and TLR2 decreased due to passaging and the expression of in particular the selected osteogenesis genes (ALP, RUNX2, Osteonectin, COL1A), OPG and MKI67 increased with passaging. Worldwide, use of the same passage in laboratory experiments using primary cell cultures is the standard. Our results support this, since for certain genes, in particular osteogenesis genes, expression may alter solely due to passaging.

Design and evaluation of an immunology and pathology course that is tailored to today's dentistry students.

Curricular reform provides new opportunities to renovate important pillars of the dentistry curriculum, such as immunology and pathology, with novel approaches that appeal to new generations of students. When redesigning a course that integrates both immunology and pathology at the level that provides dentistry students with sustainable knowledge that is useful for their entire career, several challenges must be met. The objective of the present study was to describe the considerations involved in the design phase of such a new course. First, the course should be compatible with the new view on the incorporation of more active learning and teaching methods. Practically, this means that the course design should contain fewer lectures and more seminars and tutorials, where the students have fewer contact hours and actively engage in using recently acquired knowledge within a contextual background. A mandatory session of team-based learning provides opportunities to apply knowledge in combination with academic reasoning skills, teamwork, and communication. Second, for a 4-week course, choices must be made: students will not become immunologists nor pathologists in such a short period. A governing principle for this course's design is that it should be based on understanding the basic principles of immunology and pathology. The ultimate goal for the students is to make the course immuno-logical and patho-logical, challenging them to reach a next level but clearly without oversimplification. Part of the course design should allow room for students to further study an immunological topic of their own choice, thereby contributing to their immunological curiosity and to their academic development. Third, to make it tailored to a new generation of dentists, examples from the field of dentistry are actively integrated in all aspects of the course. Finally, the era of ChatGPT provides novel opportunities to use generative artificial intelligence (AI) tools in the learning process, but it demands critical thinking of how to use it in a newly designed course. A mid-course evaluation revealed that students acknowledged that immunology and pathology were presented as an integrated course. The final course evaluation endorsed the use of these various educational methods. These methods proved to be appropriate and logical choices for reaching the learning goals of the course.

Osteogenic differentiation of periodontal ligament fibroblasts inhibits osteoclast formation.

One of the deficits of knowledge on bone remodelling, is to what extent cells that are driven towards osteogenic differentiation can contribute to osteoclast formation. The periodontal ligament fibroblast (PdLFs) is an ideal model to study this, since they play a role in osteogenesis, and can also orchestrate osteoclastogenesis.when co-cultured with a source of osteoclast-precursor such as peripheral blood mononuclear cells (PBMCs). Here, the osteogenic differentiation of PdLFs and the effects of this process on the formation of osteoclasts were investigated. PdLFs were obtained from extracted teeth and exposed to osteogenic medium for 0, 7, 14, or 21 out of 21 days. After this 21-day culturing period, the cells were co-cultured with peripheral blood mononuclear cells (PBMCs) for an additional 21 days to study osteoclast formation. Alkaline phosphatase (ALP) activity, calcium concentration, and gene expression of osteogenic markers were assessed at day 21 to evaluate the different stages of osteogenic differentiation. Alizarin red staining and scanning electron microscopy were used to visualise mineralisation. Tartrate-resistant acid phosphatase (TRAcP) activity, TRAcP staining, multinuclearity, the expression of osteoclastogenesis-related genes, and TNF-α and IL-1ß protein levels were assessed to evaluate osteoclastogenesis. The osteogenesis assays revealed that PdLFs became more differentiated as they were exposed to osteogenic medium for a longer period of time. Mineralisation by these osteogenic cells increased with the progression of differentiation. Culturing PdLFs in osteogenic medium before co-culturing them with PMBCs led to a significant decrease in osteoclast formation. qPCR revealed significantly lower DCSTAMP expression in cultures that had been supplemented with osteogenic medium. Protein levels of osteoclastogenesis stimulator TNF-α were also lower in these cultures. The present study shows that the osteogenic differentiation of PdLFs reduces the osteoclastogenic potential of these cells. Immature cells of the osteoblastic lineage may facilitate osteoclastogenesis, whereas mature mineralising cells may suppress the formation of osteoclasts. Therefore, mature and immature osteogenic cells may have different roles in maintaining bone homeostasis.

Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB.

Disequilibrium between bone-forming osteoblasts and bone-resorbing osteoclasts is central to many bone diseases. Here, we show that dysregulated expression of translationally controlled isoforms of CCAAT/enhancer-binding protein beta (C/EBPbeta) differentially affect bone mass. Alternative translation initiation that is controlled by the mammalian target of rapamycin (mTOR) pathway generates long transactivating (LAP(*), LAP) and a short repressive (LIP) isoforms from a single C/EBPbeta transcript. Rapamycin, an inhibitor of mTOR signalling increases the ratio of LAP over LIP and inhibits osteoclastogenesis in wild type (WT) but not in C/EBPbeta null (c/ebpbeta(-/-)) or in LIP knock-in (L/L) osteoclast precursors. C/EBPbeta mutant mouse strains exhibit increased bone resorption and attenuated expression of MafB, a negative regulator of osteoclastogenesis. Ectopic expression of LAP and LIP in monocytes differentially affect the MafB promoter activity, MafB gene expression and dramatically affect osteoclastogenesis. These data show that mTOR regulates osteoclast formation by modulating the C/EBPbeta isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression.

T cell-mediated increased osteoclast formation from peripheral blood as a mechanism for Crohn's disease-associated bone loss.

The pathophysiology of osteoporosis in patients with Crohn's disease (CD) is still not completely elucidated. In this study, we evaluated osteoclastogenesis from peripheral blood cells of CD patients and studied the role of lymphocytes and inflammatory cytokines in this process. Peripheral blood mononuclear cells from seven patients with quiescent CD and matched healthy controls were isolated, and separated into T cells, B cells, and a T- and B-cell depleted fraction. In various culture combinations, osteoclast formation in the absence of the osteoclastogenic factors RANKL and M-CSF was assessed by scoring the number of tartrate-resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs). Cytokine levels in culture supernatants were measured. Formation of heterogeneous cell clusters in culture was noticed; a process that was inhibited by anti-LFA-1. In CD cultures, mean cluster area was up to threefold higher than in control cultures, and shown to be induced by T cells. Over tenfold higher numbers of TRACP(+) MNCs were found in CD cultures, but exclusively in cultures containing T cells. Formation of cell clusters correlated strongly with formation of TRACP(+) MNCs. Both cell cluster formation and osteoclast formation were related to IL-17 levels in vitro. In conclusion, osteoclastogenesis, preceded by cell cluster formation, is T cell-mediated and increased in patients with quiescent CD. Our findings suggest heterotypic interactions between osteoclast precursors and T cells to be a triggering step in osteoclast formation in CD. Furthermore, our results propose a possible role for IL-17 in osteoclastogenesis in CD patients, and as such in CD-associated bone loss.

Vitamin C in plasma and leucocytes in relation to periodontitis.

AIM: To test the hypothesis that vitamin C concentrations in plasma, polymorphonuclear neutrophilic leucocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) are lower in periodontitis patients compared with healthy controls. METHODS: Twenty-one untreated periodontal patients and 21 healthy controls matched for age, gender, race and smoking habits were selected. Dietary vitamin C intake was assessed by a self-administered dietary record. Fasting blood samples were obtained and analysed for vitamin C concentrations in plasma, PMNs and PBMCs by means of high-pressure liquid chromatography (HPLC). RESULTS: Plasma vitamin C was lower in periodontitis patients compared with controls (8.3 and 11.3 mg/l, respectively, p = 0.03). Only in the control group a positive correlation was present between vitamin C intake and plasma values. No differences could be assessed between patients and controls regarding vitamin C dietary intake and levels in PMNs and PBMCs. In the patient group, pocket depth appeared to be negatively associated with the vitamin C concentration in PMNs. CONCLUSION: Although the relationship between low plasma vitamin C levels and periodontitis is clear, the disease cannot be explained by insufficient vitamin C storage capacity of leucocytes; the question remains through which mechanism low plasma vitamin C levels are related to periodontitis.

Limitations of Jaw Movement in Fibrodysplasia Ossificans Progressiva: A Review.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by heterotopic ossification (HO) of the skeletal muscles, fascia, tendons and ligaments. Patients often experience limitations in jaw function due to HO formation in the maxillofacial region. However, no studies have yet analyzed the age of onset and location of HO and the type of restrictions it may yield in the maxillofacial region. The aim of this study was to evaluate all existing literature on the site of onset of HO and associated functional restrictions of the jaw. To this end, a scoping review was performed focusing on limitations of jaw movement in FOP patients. The literature search resulted in 725 articles, of which 30 articles were included for full study after applying the exclusion criteria. From these articles 94 FOP patients were evaluated for gender, age, presence and age at which HO started in the maxillofacial region, location of HO, whether HO was caused spontaneous or traumatic and maximum mouth opening. Formation of HO is slightly more common in female patients compared to male patients, but the age of HO onset or the maximum mouth opening does not differ between genders. Trauma-induced HO occurred at a significantly younger age than spontaneous HO. Interestingly, a difference in maximum mouth opening was observed between the different ossified locations in the maxillofacial region, with ossification of the masseter muscle resulting in the smallest and ossification of the zygomatic arch resulting in the largest maximum mouth opening. This review revealed that the location of the maxillofacial region affected by HO determines the degree of limitations of the maximum mouth opening. This finding may be important for establishing clinical guidelines for the dental management of FOP patients.