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.

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.

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.

The Alkyne Moiety as a Latent Electrophile in Irreversible Covalent Small Molecule Inhibitors of Cathepsin K.

Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation ( kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

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.

Osteogenic Differentiation of Human Gingival Fibroblasts Inhibits Osteoclast Formation.

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.

Gingival fibroblasts produce paracrine signals that affect osteoclastogenesis in vitro.

In periodontitis, gingival fibroblasts (GF) appear to produce a multitude of paracrine factors. However, the influence of GF-derived soluble factors on osteoclastogenesis remains unclear. In this case study, production of paracrine factors by GF was assessed under inflammatory and non-inflammatory conditions, as well as their effect on osteoclastogenesis. Human primary GF were cultured in a transwell system and primed with a cocktail of IL-1ß, IL-6 and TNF-α to mimic inflammation. GF were co-cultured directly and indirectly with human peripheral blood mononuclear cells (PBMC). Cytokines and chemokines in supernatants (flow cytometry based multiplex assay), osteoclastogenesis (TRAcP staining) and gene expression (qPCR) were quantified on days 7 and 21. Results from this case study showed that GF communicated via soluble factors with PBMC resulting in a two-fold induction of osteoclasts. Reversely, PBMC induced gene expression of IL-6, OPG and MCP-1 by GF. Remarkably, after priming of GF with cytokines, this communication was impaired and resulted in fewer osteoclasts. This could be partly explained by an increase in IL-10 expression and a decrease in MCP-1 expression. Intriguingly, the short priming of GF resulted in significantly higher expression of inflammatory cytokines that was sustained at both 7 and 21 days. GF appear to produce paracrine factors capable of stimulating osteoclastogenesis in the absence of physical cell-cell interactions. GF cultured in the presence of PBMC or osteoclasts had a remarkably inflammatory phenotype. Given profound expression of both pro- and anti-inflammatory cytokines after the inflammatory stimulus, it is probably the effector hierarchy that leads to fewer osteoclasts.

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.

Osteoclast fusion and fission.

Osteoclasts are specialized multinucleated cells with the unique capacity to resorb bone. Despite insight into the various steps of the interaction of osteoclast precursors leading to osteoclast formation, surprisingly little is known about what happens with the multinucleated cell itself after it has been formed. Is fusion limited to the short period of its formation, or do osteoclasts have the capacity to change their size and number of nuclei at a later stage? To visualize these processes we analyzed osteoclasts generated in vitro with M-CSF and RANKL from mouse bone marrow and native osteoclasts isolated from rabbit bones by live cell microscopy. We show that osteoclasts fuse not only with mononuclear cells but also with other multinucleated cells. The most intriguing finding was fission of the osteoclasts. Osteoclasts were shown to have the capacity to generate functional multinucleated compartments as well as compartments that contained apoptotic nuclei. These compartments were separated from each other, each giving rise to a novel functional osteoclast or to a compartment that contained apoptotic nuclei. Our findings suggest that osteoclasts have the capacity to regulate their own population in number and function, probably to adapt quickly to changing situations.

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.

Real-time quantification of osteoclastic resorptive activity by electric cell-substrate impedance sensing.

In several diseases, bone resorption by osteoclasts is dysregulated. Thus far, no simple technique for real-time measurement of resorption is available. Here, we introduce an impedimetric bioassay for real-time monitoring of resorption by making use of the electrical insulating properties of the resorbable substrate calcium phosphate. Different chemical stimuli were applied to (pre)osteoclasts cultured on a layer of calcium phosphate in multi-well plates containing electrodes. By this, osteoclast activity can be measured continuously over days, and the effects of stimulating or inhibiting factors can be quantified. When cells were cultured in the presence of an inflammatory factor such as IL-1ß, the resorptive activity started earlier. The measured decline in resistance was higher at culture day 5 than at cultures with M-CSF or M-CSF + RANKL (M-CSF norm. Resistance = 1, M-CSF + RANKL = 0.7, M-CSF + RANKL + IL-1ß = 0.5). However, at day 11, this difference had nearly disappeared. Likewise, bisphosphonates were shown to inhibit osteoclastic activity. Our findings illustrate the importance of real-time monitoring; wherefore, this method has high potential not only for the study of osteoclast resorptive activity in the context of osteoclast function and diseases but also could find application in high-throughput drug-testing studies.

IgA Immune Complexes Induce Osteoclast-Mediated Bone Resorption.

Objective: Autoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption. Methods: Anti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption. Results: NET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts. Conclusion: IgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.

Ae2(a,b)-deficient mice exhibit osteopetrosis of long bones but not of calvaria.

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH(i)) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na(+)-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2(a,b)(-/-) mice (deficient in the main isoforms Ae2a, Ae2b(1), and Ae2b(2)). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2(a,b)(-/-) mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2(a,b)(-/-) mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts--but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2(a,b)(-/-) mice.

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis.

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

Localization and function of the anion exchanger Ae2 in developing teeth and orofacial bone in rodents.

To explore the functions of the anion exchanger 2 (Ae2) in the development of bones and teeth we examined the distribution of Ae2 in cells involved in the formation of teeth and surrounding bone in young hamsters, mice and rats. In all three species strongest immunostaining for Ae2 was obtained in basolateral membranes of maturation ameloblasts and in osteoclasts resorbing bone. In hamsters a weaker staining was also seen in the Golgi apparatus of secretory ameloblasts, young osteoblasts and osteocytes, odontoblasts and fibroblasts of the forming periodontal ligament. In adult Ae2(a,b) (-/-) mice, in which Ae2-targeted disruption precluded the expression of Ae2a, Ae2b1 and Ae2b2 isoforms, the immunostaining for Ae2 in ameloblasts and osteoclasts was totally abolished. The enamel formation was abnormal but teeth erupted, osteoclasts in jaw bone were functional and structure of dentin and bone was normal. In another mouse model, Ae2(-/-) mice in which the expression of all five Ae2 isoforms was disrupted, teeth failed to erupt and the alveolar bone proved poorly formed with giant but apparently functional osteoclasts. Our data indicate that basolaterally located Ae2a, Ae2b1 or Ae2b2 (or a combination of these) is present in maturation ameloblasts critical for the cells' normal functioning. Although isoforms of Ae2 were also present in basolateral membranes of osteoclasts, they proved to be not critical to osteoclast resorption of orofacial bone. Poorly formed bone and the failure of teeth to erupt seen in the Ae2(-/-) mice with gene disruption affecting all isoforms may result from secondary (systemic) changes that are different from Ae2(a,b) (-/-) mice.

What Are the Peripheral Blood Determinants for Increased Osteoclast Formation in the Various Inflammatory Diseases Associated With Bone Loss?

Local priming of osteoclast precursors (OCp) has long been considered the main and obvious pathway that takes place in the human body, where local bone lining cells and RANKL-expressing osteocytes may facilitate the differentiation of OCp. However, priming of OCp away from bone, such as in inflammatory tissues, as revealed in peripheral blood, may represent a second pathway, particularly relevant in individuals who suffer from systemic bone loss such as prevalent in inflammatory diseases. In this review, we used a systematic approach to review the literature on osteoclast formation in peripheral blood in patients with inflammatory diseases associated with bone loss. Only studies that compared inflammatory (bone) disease with healthy controls in the same study were included. Using this core collection, it becomes clear that experimental osteoclastogenesis using peripheral blood from patients with bone loss diseases in prevalent diseases such as rheumatoid arthritis, osteoporosis, periodontitis, and cancer-related osteopenia unequivocally point toward an intrinsically increased osteoclast formation and activation. In particular, such increased osteoclastogenesis already takes place without the addition of the classical osteoclastogenesis cytokines M-CSF and RANKL in vitro. We show that T-cells and monocytes as OCp are the minimal demands for such unstimulated osteoclast formation. In search for common and disease-specific denominators of the diseases with inflammation-driven bone loss, we demonstrate that altered T-cell activity and a different composition-such as the CD14+CD16+ vs. CD14+CD16- monocytes-and priming of OCp with increased M-CSF, RANKL, and TNF- α levels in peripheral blood play a role in increased osteoclast formation and activity. Future research will likely uncover the barcodes of the OCp in the various inflammatory diseases associated with bone loss.

The Challenge of Teaching Essential Immunology Laboratory Skills to Undergraduates in One Month-Experience of an Osteoimmunology Course on TLR Activation.

Acquiring immunology laboratory skills during undergraduate studies is often a prerequisite for admission to Masters' programs. Many broad liberal arts and sciences honors degree colleges struggle in teaching these essentials since only limited time is usually reserved for this. Here, we describe a new 1-month-course developed to train a small group of honors students in 6 techniques that are useful for immunology research. In essence, 15 students were divided into 3 groups of 5 students where each student became involved in current osteoimmunology research. Osteoimmunology is a relatively new branch of the immunology tree, where the effects of inflammation and the immune system on bone formation and bone degradation is studied. A broad, 3 weeks experiment on the chronic effects of molecules that specifically activate toll-like receptors TLR2 and TLR4 on bone formation or osteoclast differentiation was performed just before the start of the course. Control samples and samples treated with TLR2 (group A), TLR4 (group B), or TLR2+TLR4 (group C) agonists were harvested and analyzed using quantitative PCR, ELISA, biochemistry, microscopy of enzyme-histochemically stained osteoclasts, scanning electron microscopy, and confocal microscopy. Each technique was taught for 2 days by a specialized instructor, who was present at all laboratory activities. The primary research question for each group was: how does the experimental condition affect bone formation or osteoclast formation? The secondary research question specified per technique was: how does this technique answer part of the primary research question? Pedagogically, students were encouraged to collaborate within the group to analyze the obtained data. Secondly, at the end of the course, a representative of each group collaborated to summarize the TLR activation modalities of a technique of choice. Thirdly, each group wrote a report, where introduction and discussion were graded as a group; each technique part was graded individually. The summary of the results from the 3 treatment modalities was presented orally. The student evaluation of the course was high, students remarked that the course had a curriculum overarching function, since it created an awareness and appreciation for both the joy and the blood-sweat-and-tears aspects of pipetting, and writing research articles, making interpretation of those easier.