Gotowa bibliografia na temat „Osteoclastogenesis”

Tumor necrosis factor-α(TNF-α) is a cytokine produced by monocytes, macrophages, and T cells and is induced by pathogens, endotoxins, or related substances. TNF-αmay play a key role in bone metabolism and is important in inflammatory bone diseases such as rheumatoid arthritis. Cells directly involved in osteoclastogenesis include macrophages, which are osteoclast precursor cells, osteoblasts, or stromal cells. These cells express receptor activator of NF-κB ligand (RANKL) to induce osteoclastogenesis, and T cells, which secrete RANKL, promote osteoclastogenesis during inflammation. Elucidating the detailed effects of TNF-αon bone metabolism may enable the identification of therapeutic targets that can efficiently suppress bone destruction in inflammatory bone diseases. TNF-αis considered to act by directly increasing RANK expression in macrophages and by increasing RANKL in stromal cells. Inflammatory cytokines such as interleukin- (IL-) 12, IL-18, and interferon-γ(IFN-γ) strongly inhibit osteoclast formation. IL-12, IL-18, and IFN-γinduce apoptosis in bone marrow cells treated with TNF-α in vitro, and osteoclastogenesis is inhibited by the interactions of TNF-α-induced Fas and Fas ligand induced by IL-12, IL-18, and IFN-γ. This review describes and discusses the role of cells concerned with osteoclast formation and immunological reactions in TNF-α-mediated osteoclastogenesisin vitroandin vivo.

We previously observed a novel osteoclastogenesis system that is induced by oral squamous cell carcinoma (OSCC) cells, which target osteoclast precursor cells (OPC) without upregulation of the master transcriptional factor of osteoclastogenesis, NFATc1. Here, we analyzed inflammatory cytokines that were preferentially expressed in one of the osteoclastogenic OSCC cell lines, namely NEM, compared with the subclone that had lost its osteoclastogenic properties. Based on a gene expression microarray and a protein array analyses, IL-1, IL-6, IL-8, and CXCL1 were chosen as candidates responsible for tumor-induced osteoclastogenesis. From the results of the in vitro osteoclastogenesis assay using OPCs cultured with OSCC cells or their culture supernatants, IL-1 was selected as a stimulator of both OSCC-induced and RANKL-induced osteoclastogenesis. The IL-1 receptor antagonist significantly attenuated osteoclastogenesis induced by NEM cells. The stimulatory effects of IL-1 for OSCC-induced and RANKL-induced osteoclastogenesis were effectively attenuated with cannabidiol and denosumab, respectively. These results suggest that IL-1 secreted from OSCC cells stimulates not only tumor-induced osteoclastogenesis targeting OPCs but also physiological RANKL-induced osteoclastogenesis, and this may be the biological mechanism of bone resorption induced by the infiltration of OSCC. These results also suggest that IL-1 inhibitors are candidates for therapeutic agents against bone resorption induced by OSCC.

The bone-resorbing osteoclast (OC) is essential for bone homeostasis, yet deregulation of OCs contributes to diseases such as osteoporosis, osteopetrosis, and rheumatoid arthritis. Here we show that histone deacetylase 2 (HDAC2) is a key positive regulator during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Bone marrow macrophages (BMMs) showed increased HDAC2 expression during osteoclastogenesis. HDAC2 overexpression enhanced, whereas HDAC2 deletion suppressed osteoclastogenesis and bone resorption using lentivirus infection. Mechanistically, upon RANKL activation, HDAC2 activated Akt; Akt directly phosphorylates and abrogates Forkhead box protein O1 (FoxO1), which is a negative regulator during osteoclastogenesis through reducing reactive oxygen species. HDAC2 deletion in BMMs resulted in decreased Akt activation and increased FoxO1 activity during osteoclastogenesis. In conclusion, HDAC2 activates Akt thus suppresses FoxO1 transcription results in enhanced osteoclastogenesis. Our data imply the potential value of HDAC2 as a new target in regulating osteoclast differentiation and function.

Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-β enhanced osteoclastogenesis, we confirmed the TGF-β activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-β by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-β released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.

Abstract Strontium-substituted bioactive glass (Sr-BG) has shown superior performance in bone regeneration. Sr-BG-induced osteogenesis has been extensively studied; however, Sr-BG-mediated osteoclastogenesis and the underlying molecular mechanism remain unclear. It is recognized that the balance of osteogenesis and osteoclastogenesis is closely related to bone repair, and the receptor activators of nuclear factor kappaB ligand (RANKL) signaling pathway plays a key role of in the regulation of osteoclastogenesis. Herein, we studied the potential impact and underling mechanism of strontium-substituted sub-micron bioactive glass (Sr-SBG) on RANKL-induced osteoclast activation and differentiation in vitro. As expected, Sr-SBG inhibited RANKL-mediated osteoclastogenesis significantly with the experimental performance of decreased mature osteoclasts formation and downregulation of osteoclastogenesis-related gene expression. Furthermore, it was found that Sr-SBG might suppress osteoclastogenesis by the combined effect of strontium and silicon released through inhibition of RANKL-induced activation of p38 and NF-κB pathway. These results elaborated the effect of Sr-SBG-based materials on osteoclastogenesis through RANKL-induced downstream pathway and might represent a significant guidance for designing better bone repair materials.

Lysosomes are acidic, hydrolase-rich vesicles capable of degrading most biological macromolecules. During the past several decades, much has been learned about different aspects of lysosome biogenesis. The selective phosphorylation of mannose residues on lysosomal enzymes, in conjunction with specific receptors for the mannose-6-phosphate recognition marker, has been found to be largely responsible for the targeting of newly synthesized lysosomal enzymes to lyzosomes. It is known that lysosomes receive input from both the endocytotic and biosynthetic pathways. Nevertheless the exact molecular mechanisms responsible for sorting of the biosynthetic imput involved in the lysosome biogenesis is still a matter of debate. Because osteoclast precursors do not secrete their lysosomal enzymes and osteoclasts do, the observation of modifications occuring during osteoclastogenesis is a good model to observe mechanisms responsible for lysosomal enzymes traffic. Osteoclasts are bone-degrading cells. To perform this specific task they have to reorganise the sorting of their lysosomal enzymes to be able to target them toward the bone surface in mature cells. Since few years, the differentiation of osteoclasts in vitro did help to study these cells. Osteoclast morphology has been therefore already well studied, and the nature of their specific membrane domains is now established. Sensing the proximity of a bone-like surface the cell reorganises its cytoskeleton, and creates specific membrane domains: an actin-rich ring-like zone (named actin ring) surrounded by highly ruffled membrane (named the ruffled border) where enzymes are secreted, while subsequent bone degradation products are endocytosed. Endocytosed material is then transported through the cell inside transcytotic vesicles and released at the top of the cell in an area named the functional secretory domain. Several molecular machineries are thought to control these different phenomena. The main purpose of this thesis was to identify the major regulators of lysosomal enzymes secretion and therefore to identify the molecular switches responsible for such a membrane traffic re-organisation.

Osteoclast formation is a complex process requiring the temporal activation of a yet unknown number of transcription factors. Osteoclast differentiation is dependent on two cytokines: macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). These agents induce gene expression changes during the differentiation process, presumably by inducing transcription factors. A search for genes that are regulated in the developing osteoclast was performed using both differential display and gene arrays. Differential display revealed a novel member of the krüppel-associated box (KRAB) containing transcription repressor, KROCS, which was shown to be down regulated during osteoclast formation. The potential targets for this gene remain unknown, as do the targets of the majority of the other members of the krüppel associated box (KRAB) containing family of transcription repressors. In addition to KROCS, three other genes were also identified including ADO21, PRO1859 and an endogenous retrovirus like gene and the regulation of vitamin D up-regulated protein (VDUP) was also confirmed. Array analysis identified a number of other transcription factors regulated during osteoclast formation including the up-regulated NFATc1, GABP?, FBP, EGR1 and the repressed RelB and KOX31, a KRAB containing transcription repressor. The array also identified calmodulin 1 a member of the NFAT activation pathways as up-regulated by RANKL. The expression of NFATc1 to 4 in human osteoclasts was investigated showing NFATc1 to be the most expressed NFATc in osteoclasts. The transcription variants of NFATc1 were tested for expression differences showing that the mRNAs encoding the protein isoforms B and C were most expressed. The involvement of calmodulin, calcineurin and NFATc1 involvement in osteoclast formation was further studied by the use of inhibitors. BAPTA-AM is an intracellular chelator of calcium that prevents changes in calcium concentration. Phenoxybenzamine irreversibly binds calmodulin in the presence of calcium, inhibiting the action of calmodulin. Cyclosporin A (CsA) is an inhibitor of calcineurin. Use of both BAPTA-AM and phenoxybenzamine resulted in inhibition of osteoclast formation, decreasing the percentage of multinucleated cells from 54% in control cultures to 7.9% and 7.1% respectively. Both BAPTA-AM and phenoxybenzamine treated cells showed a marked reduction in TRAP activity with only 14.5% and 16.8% respectively staining positive for TRAP. This represents an approximate 60% reduction in TRAP positive cells compared to control osteoclasts. Both BAPTA-AM treated and phenoxybenzamine treated cells were negative for bone resorption. Addition of increasing doses of cyclosporin A (CsA) to M-CSF and RANKL treated cells resulted in the inhibition of multinucleated osteoclast formation. At 1000ng/mL CsA the formation of TRAP positive cells with more than one nucleus had reduced to less than 5% from 54% without the presence of CsA. Cells treated with 1000ng/mL CsA were unable to resorb bone, however the percentage of cells that were TRAP positive remained unchanged with CsA treatment. No significant decrease in expression of cathepsin K or TRAP transcripts were observed by real-time quantitative PCR (Q-PCR) in cells treated with CsA. Although all three agents inhibited the formation of multinuclear giant cells, both BAPTA-AM and phenoxybenzamine resulted in TRAP negative cells, whereas CsA resulted in TRAP positive cells. These results implicate the intracellular calcium increase caused by RANKL and calmodulin activation as a regulator of TRAP but place the calcineurin activation of NFATc1 downstream of TRAP induction. The regulation of a series of other genes was tested to determine if some RANKL mediated regulation of osteoclast genes were 'sensitive to CsA while others were 'resistant'. Of 28 genes tested, 13 were significantly affected by CsA and were considered 'sensitive' while the RANKL mediated regulation of 15 genes was unaffected by CsA and these were considered 'resistant'. This is strong evidence for two pathways of gene activation in osteoclasts, a CsA 'sensitive' pathway involving calcineurin, NFAT and possibly other transcription factors and a CsA 'resistant' pathway of gene activation, not dependent on calcineurin. Surprisingly, the RANKL mediated induction of NFATc1 was not inhibited by CsA, suggesting that NFATc1 induction is dependent on the resistant pathway. The identity of the second pathway (or pathways) is yet to be established, however the data indicate that this pathway mediate the RANKL sensitive regulation of at least one half of genes in human osteoclasts. The corollary if that only one half of osteoclast genes are dependent on calcineurin and presumably NFATc1 activation. There was no unifying principle that separated the CsA resistant from sensitive pathways of RANKL regulation. Cell surface markers, chemokines and transcription factors were among those affected by CsA. Even classical osteoclast markers fell neatly into two categories. The RANKL mediated induction of calcitonin receptor (CalcR) was inhibited by more than 100 fold in the presence of CsA implicating NFAT/calcineurin in the regulation of CalcR expression in osteoclasts. In contrast, the RANKL mediated induction of TRAP or cathepsin K, two prominent osteoclast markers, was totally unaffected by CsA. The expression of a series of chemokines and receptors was investigated. MCP-1 and RANTES were RANKL induced, and this induction was sensitive to CsA. The CC chemokines MCP-1 and RANTES were down regulated by around 10 fold in the presence of CsA. In contrast the RANKL mediated induction of MCP-1 receptor was resistant to CsA. The existence of chemokine and receptor in the same cell provides for a RANKL inducible autocrine loop, suggesting that MCP-1 should act directly on osteoclasts. The fact that the RANKL induction of the MCP-1 receptor, CCR2B, is unaffected by CsA suggests that exogenous MCP-1 should still signal in CsA treated osteoclasts. Addition of either MCP-1 or RANTES to CsA treated cultures resulted in a recovery of 70-80% of the multinuclear TRAP positive phenotype. The MCP-1 and RANTES induced multinuclear cell could not overcome the CsA induced inhibition of bone resorption. Surprisingly, MCP-1 and RANTES induced multinucleation in the absence of RANKL (M-CSF and chemokine treated cells) resulting in 50% of the normal multinucleation present in cells treated with RANKL. The data suggest that chemokines produced by osteoclasts are involved in promoting a multinuclear phenotype. When inhibited by CsA, osteoclasts fail to produce both MCP-1 and RANTES, although their respective receptors are present. This failure to produce MCP-1 and RANTES prevents the formation of an autocrine loop. When provided with MCP-1 or RANTES the CsA inhibited osteoclasts are subsequently able to pass through to the stage of a multinucleated giant cell. Similarly, in the absence of RANKL, chemokines promote the formation of TRAP positive osteoclast-like giant cells visually indistinguishable from osteoclasts. However, the multinuclear cells formed by chemokines in the absence of RANKL were also incapable of bone resorption. In order to determine if chemokines were capable of stimulating bone resorption, after osteoclasts had formed, pre-differentiated mature osteoclasts were plated onto bone and treated with a range of cytokines. The results showed that bone resorption occurred only in cultures that were exposed continuously to RANKL. These data indicate that chemokine induction by RANKL is required for multinucleation but that RANKL is required for bone resorption. The functional testing of genes detected by array analysis proved crucial involvement of both the NFAT pathway and CC chemokines in osteoclast formation knd function. Other genes identified such as GABP and FBP, are likely to be key factors in the development of a functional osteoclast. Future works investigating human osteoclast formation should take into strong consideration the genes identified in this thesis as targets for further functional studies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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Spondyloarthropathies (or Spondyloarthritides; SpAs) are a group of heterogeneous but genetically related inflammatory disorders in which ankylosing spondylitis (AS) is considered the prototypic form. Among the genes associated with AS, HLA-B27 allele has the strongest association although the cause is still not clear. Rats transgenic for the human HLA-B27 gene (B27 rats) develop a systemic inflammation mirroring the human SpA symptoms and thus provide a useful model to study the contribution of this MHC class I molecule in the disease development. Of particular interest was the observation of absence of arthritis in B27 rats grown in germ-free conditions and a recent theory suggests that microbial dysbiosis and gut inflammation might play a key role in initiating the HLA-B27-associated diseases. Studies in our laboratory have previously demonstrated that HLA-B27 expression alters the development of the myeloid compartment within the bone marrow (BM) in B27 rat and causes loss of a specific dendritic cell (DC) population involved in self-tolerance mechanisms within the gut. The aim of this thesis was to further analyse the myeloid compartment in B27 rats with a particular focus on the osteoclast progenitors and the bone phenotype and to link this to the gut inflammation. In addition, translational studies analysed peripheral monocyte/pre-osteoclasts in AS patients and teased apart the role of cytokines in in vitro human osteoclast differentiation. To understand the dynamics of the myeloid/monocyte compartment within the B27-associated inflammation, monocytes within the bloodstream and BM of B27 rats were characterised via flow cytometry and their ability to differentiate into osteoclast was assessed in vitro. Moreover, an antibiotic regime was used to reduce the B27 ileitis and to evaluate whether this could affect the migration, the phenotype, and the osteoclastogenic potential of B27 monocytes. B27 animals display a systemic and central increase of “inflammatory” CD43low MOs, which are the main contributors to osteoclastogenesis in vitro. Antibiotic treatment reduced ileitis and also reverted the B27 monocyte phenotype. This was also associated with the reduction of the previous described TNFα-enhancement of osteoclast differentiation from B27 BM precursors. These evidences support the idea that in genetically susceptible individuals inflammation in the gut might influence the myeloid compartment within the BM; in other terms, pre-emptively educate precursor cells to acquire specific phenotype end functions after being recruited into the tissue. This might explain the enhanced differentiation of osteoclast from B27 BM progenitors and thus the HLA-B27-associated bone loss. The data shown in this thesis suggest a link between the immunity within the gut and BM haematopoiesis. This provides an attractive and novel research prospective that could help not only to increase the understanding of the HLA-B27-associated aetiopathogenesis but also to unravel the cellular crosstalk that allows the mucosal immunity to program central cell differentiation. Human translational studies on monocyte subsets, cytokines and cytokine network in AS osteoclastogenesis evidenced altered osteoclast differentiation in the presence of IL-22 although no differences in the phenotype and functions of circulating CD14+ monocytes were observed. In addition, studies on the role of TNFα and TNFRs showed a dual role of this inflammatory cytokine in the human OC differentiation. In particular, the activation of TNFR1 in monocytes in early osteoclastogenesis inhibits OC differentiation while TNFα-biasing for TNFR2 on osteoclast precursors mediates the osteoclastogenic effect. Whether similar mechanisms are involved in the TNFα-mediated joint destruction in human rheumatic diseases needs further investigations. This could contribute to the development of novel and more specific anti-TNFα agents for the treatment of bone erosion. In conclusion, taken together my studies support the idea of a crosstalk between the periphery and the central system during the inflammatory response and provide new insights to the mechanisms behind the enhancement of osteoclastogenesis in B27-associated disorders.

During osteoclast differentiation and bone resorption the redox status in the cell display a decrease in reduction and a shift to an oxidized state. Structure, metabolism and function are some of the extensive changes that cells undergo during differentiation which alters both the extra- and intracellular redox environment. Osteoclasts express enzymes such as TRAP and NADPH oxidase which generates reactive oxygen species (ROS). ROS are molecules formed by oxygen reduction which gives these radicals at least one unpaired electron and makes them very reactive and chemically unstable. These are factors which stimulates differentiation of osteoclasts and bone resorption. RAW 264.7 cells will differentiate to osteoclasts when stimulated with RANKL and to activated macrophages when stimulated with LPS.

The aim of this project was to analyze if the redox environment is affected during differentiation of RAW 264.7 cells to osteoclasts and macrophages. The reason for this was that we aimed to se if RAW 264.7 cells could be used as an in vitro system to study the effects of redox changes in osteoclasts and macrophages and their activation.

Results from Western blot showed that protein expression of the Cysteine/Glutamate transporter xCT was up regulated with LPS and downregulated with RANKL. Results from the GSH/Cys assay show that the treatments with redox modulators did not affect the levels of GSH and Cys to a measurable extent. However the levels increased for both intracellular and extracellular GSH and Cys forms at day 4 in the control and stimulated cells. Addition of the disulfide reductant DTT affected differentiation to osteoclasts, leading to smaller osteoclasts probably due to interference with fusion of mononuclear pre-osteoclasts. Thus, down regulation of the xCT transporter could be an important mechanism to maintain a low level of free thiols shown to interfere with the differentiation to osteoclasts.

Osteoporosis is a condition that results from substantially weakened bone, increasing an individual’s risk of fracture. Post-menopausal osteoporosis is the most common form of the condition, affecting 30% of post-menopausal women over the age of 50. Following the menopause, female oestrogen levels decline and this perturbs bone homeostasis by promoting an environment that is biased towards bone erosion. Osteoclasts are the cells responsible for eroding bone and are normally inhibited by oestrogen. However, the decline in oestrogen production results in increased osteoclast differentiation and activity. This rapidly decreases the bone mineral density and results in fracture-prone bone. Osteoclasts are derived from mononuclear myeloid progenitors found in the blood and bone marrow, which fuse to form large multinucleated cells that reside in the bone cavity. These progenitor cells are also responsible for replenishing monocytes, macrophages and dendritic cells. One class of receptors present on the surface of these cells, which are capable of dictating a cells function, are Fcγ receptors and modulation of Fcγ receptors has been shown to inhibit the differentiation of human monocytes to osteoclasts. This thesis investigates Fcγ receptor modulation on murine osteoclastogenesis and in order to stimulate Fcγ receptors, both IgG and IgG complexes were used. IgG complexes were generated using Staphylococcus aureus Protein A (SpA) in combination with IgG to form SpA-IgG complexes (SIC). We show that IgG and SIC are capable of engaging with Fcγ receptors resulting in the inhibition of osteoclast differentiation. Furthermore, both IgG and SIC inhibit the transcription of mRNA essential for the fusion of progenitors and enzymes for the erosion of bone matrix. Therefore, IgG and SIC are capable of inhibiting murine osteoclastogenesis. The murine model of osteoporosis was used to further investigate the ability of SIC to inhibit murine osteoclast differentiation. Previous studies have shown that when SpA is administered in vivo it is capable of binding circulating IgG to form SIC. We used this property to test the ability of SpA to bind to the surface of monocytes. SpA was found to bind with highest affinity to blood Ly6Chigh monocytes, which are known to differentiate in vitro to OCs. IgG and SIC were also able to inhibit the in vitro osteoclastogenesis of Ly6Chigh monocytes. It was hypothesised that SpA would co-opt IgG and inhibit the in vivo differentiation of progenitors to osteoclasts in the ovariectomy model of osteoporosis. To generate this animal model the ovaries were removed from the mice in order to simulate the menopause and induce bone loss. To assess the percentage of bone present after ovariectomy, we used micro-computer tomography and discovered that SpA was unable to prevent bone loss associated with ovariectomy. Therefore, SpA can bind to the surface of osteoclast progenitors but is unable to inhibit bone loss in the model of osteoporosis. In addition to studying the role of Fcγ receptor modulation of osteoclastogenesis, the role of Bcl-3 (a negative regulator of NF-κB) in osteoclast differentiation and bone remodelling was also investigated. NF-κB is an essential signalling molecule and transcription factor involved in osteoclast differentiation. Previous research has shown that in the absence of Bcl-3 (Bcl-3-/-) aberrant cytokine responses to LPS and TNF- occur. Therefore, RANKL stimulation of WT and Bcl-3-/- osteoclast precursors was done to determine whether Bcl 3 /- animals responded aberrantly to RANKL. WT and Bcl-3-/- animals were able to generate in vitro osteoclasts, which were phenotypically and transcriptionally similar. However, comparison of in vivo osteoclast progenitors revealed that Bcl-3-/- animals had reduced CD115+ osteoclast progenitors compared to WT animals. Examination of the trabecular bone present in the proximal tibia revealed that Bcl-3-/- animals had a higher percentage of bone present that WT controls. Therefore, Bcl-3 does not effect in vitro osteoclast differentiation but further work needs to be done to understand the role of Bcl 3 in bone remodelling. This thesis aimed to investigate whether SpA-IgG complexes or Bcl-3 could represent a novel avenue of therapeutic intervention in osteoporotic disease. In summation, SpA is able to form IgG complexes that can inhibit the differentiation of OCs in vitro; however, treatment of osteoporotic animals with SpA was unable to halt bone loss. This suggests that SpA-IgG complexes are able to modulate Fcγ receptors in vitro and skew progenitors from differentiation into osteoclasts but cannot overcome the prevailing pro-osteoclastogenic environment that results from ovariectomy. The presence of osteoclast progenitors was also shown to be partially dependent on Bcl-3 and as such Bcl-3 may be a novel target for therapeutic agents to target osteoclast progenitors in diseases like osteoporosis. However, the role of Bcl-3 in bone remodelling requires further investigation.

The present PhD thesis is a compendium of four publications broadening the knowledge on osteoclastogenesis under simulated bone augmentation, more especially about the effects of saliva and bone-conditioned medium on osteoclastogenesis. Resorption of bone grafts and host bone, can be a challenge especially when a bonny defect has to be regenerated or there is a lack of host bone due to a trauma, pathology, aging or tooth extraction among others. In the oral cavity, saliva is present and can reach mineralized surfaces, however, the relationship between saliva and bone resorption is yet unknown. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro, possibly affecting bone healing and bone regeneration. Bone regeneration is a common procedure in traumatology, periodontology, oral and maxillofacial surgery that involves the use of bone fillers. Bone autograft is considered to be the gold standard bone substitute due to its trinity of properties: osteoinductivity, osteoconductivity and osteogenesis. Paracrine factors released from bone autografts might contribute to the overall process of graft consolidation, however the underlying mechanisms are unknown. Here, we determined the protein spectrum released from porcine bone chips into the conditioned medium (BCM) to mimic the paracrine environment of cortical bone grafts. Some of the factors released by bone autografts could maybe influence on the autograft resorption and therefore explain why osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Here we determined whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts, still unknown. Based on the in vitro results here presented, it can be observed that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype, therefore affecting function of osteoclasts, the bone resorbing cells. Resorption of bone autografts could be attributed to some of the proteins detected on the secretions of bone autografts, termed bone conditioned medium (BCM). Proteomic analysis showed that BCM contains more than 150 proteins, among which, 43 were categorized into “secreted” and “extracellular matrix”. We discovered growth factors that are not only detectable in BCM, but potentially also target cellular processes involved in bone regeneration e.g. pleiotrophin, galectin-1, TGF-β-induced gene (TGFBI), latency-associated peptide forming a complex with TGF-β1, and TGF-β2. Results here presented on the influence of BCM on osteoclastogenesis demonstrated that activated BCM by heat is able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined regulatory mechanisms. Moreover the presented protocols on the use of BCM should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery. Taking everything together, it can be concluded that saliva affects bone resorption towards the development of a phagocytic cell line, and that not only saliva affects bone resorption but also the secretions from autologous bone grafts. There is enough evidence to conclude that bone autografts not only have three properties, but one more: a regulation property, the fourth dimension of autologous bone grafts.
La present tesis doctoral és un compendi de quatre publicacions ampliant el coneixement de l’osteoclastogènesis en les regeneracions òssies, més especialment sobre els efectes de la saliva i el medi condicionat ossi en l’osteoclastogènesis. La reabsorció dels empelts ossis i de l’os de l’hoste, pot ser un repte especialment quan un defecte ossi ha de ser regenerat en condicions desfavorables o grans atròfies com per exemple després de traumatismes, diverses patologies, edat avançada o extraccions múlti¬ples. En la cavitat oral, la saliva pot entrar en contacte amb superfícies mineralitzades, tot i això la relació entre saliva i reabsorció òssia és encara desconeguda. En la present tesis hem examinat si la saliva afecta el procés de l’osteoclastogènesis in vitro, possiblement afectant a la regeneració i cicatrització òssia. La regeneració òssia és un procés comú en traumatolo¬gia, periodòncia, cirurgia oral i maxil•lofacial que involucra l’ús de substituts ossis. Els empelts d’os autòleg són considerats l’estàndard d’or dels sub¬stituts ossis degut a la seva trinitat de propietats: osteoconductivitat, oste¬oinducció i osteogènesis. Els factors paracrins alliberats pels empelts d’os autòleg podrien contribuir en el conjunt de processos que donen com a re-sultat la consolidació del empelts, tanmateix els mecanismes que regeixen aquest processos no són coneguts. En el present treball hem pogut carac¬teritzar un conjunt de proteïnes alliberades per partícules d’os cortical porcí en el medi condicionat ossi (BCM) per imitar l’ambient paracrí dels em¬pelts d’os cortical. Alguns dels factors alliberats pels empelts d’os autòleg podrien influenciar la reabsorció òssia explicant per què els osteoclasts es formen ràpidament a la superfície de les partícules d’os autòleg en els llocs regenerats. Tot i això els mecanismes moleculars que regeixen aquest pro¬cés, encara son desconeguts. Factors solubles alliberats pels empelts d’os autòleg in vitro tenen un impacte robust a la diferenciació de cèl•lules mes¬enquimals. En la present tesis doctoral, hem determinat si aquests factors solubles son capaços de canviar la diferenciació de cèl•lules mare hemat¬opoètiques a osteoclasts, desconegut abans de realitzar els estudis aquí presentats. Basant-nos en els resultats in vitro aquí presentats, es pot observar que la saliva suprimeix l’osteoclastogènesis i promociona el desenvolupament de cèl•lules amb un fenotip fagocític, afectant a la funció dels osteoclasts, les cèl•lules encarregades de reabsorbir l’os. La reabsorció dels empelts d’os autòleg es pot atribuir a l’efecte d’algunes de les proteïnes detecta¬des en les secrecions dels auto-empelts, anomenant aquestes secrecions Medi Condicionat d’Os (BCM). Un estudi proteòmic del BCM va mostrar que aquest medi condicionat conté més de 150 proteïnes, de les quals 43 es van caracteritzar com “secretades” i presents en la matriu extracel•lular. Vàrem descobrir que alguns dels factors continguts en el BCM com per exemple pleiotropina, galectina-1 o TGF-β1 poden afectar processos cel•lulars involucrats en la regeneració òssia. El resultats presentats en aquesta tesis sobre l’influencia del BCM en l’osteoclastogènesis demostra que el BCM termo-activat és capaç d’estimular l’osteoclastogènesis in vitro. Aquests resultats in vitro suporten la noció que la reabsorció dels auto-empelts ossis pot ser que estigui estimulada per mecanismes reguladors encara no definits. En aquesta línia, els protocols presentats sobre l’ús del BCM haurien d’animar a revelar els efectes paracrins dels empelts d’os autòleg durant el procés de regeneració òssia i obrir nous camins a investi¬gacions translacionals en l’ampli camp de la cirurgia reconstructora. Resumint-ho tot, podem concloure que la saliva afecta la reabsorció òssia promocionant el desenvolupament de cèl•lules amb un fenotip fagocític, i que no només la saliva pot afectar a la reabsorció òssia, sinó que també les se¬crecions dels injerts d’os autòleg. En aquest punt, hi ha suficient evidencia per concloure que els auto-empelts d’os no només tenen tres propietats, sinó una més: la propietat reguladora, la quarta dimensió dels empelts d’os autòleg.

Glycolaldehyde (Glycol) and glyceraldehyde (Glycer) derived glycated human serum albumin (HSA) significantly reduced RANKL-induced osteoclastic TRAP activity, while glucose, fructose, glyoxal derived glycated HSA have no effect. Secreted high mobility group box protein1 (HMGB1) bind to receptor for AGE (RAGE) and play an important role for osteoclastogenesis. Next, we investigated that the effect of glycated HSA on HMGB1 secretion and RAGE expression. Both Glycol-AGE and Glycer-AGE inhibited RANKL-induced HMGB1 secretion but not altered RAGE expression levels. These result indicated that some kinds of AGEs altered osteoclastic TRAP activity through inhibition of HMGB1 secretion from the cell.
博士(理学)
Doctor of Philosophy in Science
同志社大学
Doshisha University

Bones are multifunctional passive organs of movement that supports soft tissue and directly attached muscles. They also protect internal organs and are a reserve of calcium, phosphorus and magnesium. Each bone is covered with periosteum, and the adjacent bone surfaces are covered by articular cartilage. Histologically, the bone is an organ composed of many different tissues. The main component is bone tissue (cortical and spongy) composed of a set of bone cells and intercellular substance (mineral and organic), it also contains fat, hematopoietic (bone marrow) and cartilaginous tissue. Bones are a tissue that even in adult life retains the ability to change shape and structure depending on changes in their mechanical and hormonal environment, as well as self-renewal and repair capabilities. This process is called bone turnover. The basic processes of bone turnover are: • bone modeling (incessantly changes in bone shape during individual growth) following resorption and tissue formation at various locations (e.g. bone marrow formation) to increase mass and skeletal morphology. This process occurs in the bones of growing individuals and stops after reaching puberty • bone remodeling (processes involve in maintaining bone tissue by resorbing and replacing old bone tissue with new tissue in the same place, e.g. repairing micro fractures). It is a process involving the removal and internal remodeling of existing bone and is responsible for maintaining tissue mass and architecture of mature bones. Bone turnover is regulated by two types of transformation: • osteoclastogenesis, i.e. formation of cells responsible for bone resorption • osteoblastogenesis, i.e. formation of cells responsible for bone formation (bone matrix synthesis and mineralization) Bone maturity can be defined as the completion of basic structural development and mineralization leading to maximum mass and optimal mechanical strength. The highest rate of increase in pig bone mass is observed in the first twelve weeks after birth. This period of growth is considered crucial for optimizing the growth of the skeleton of pigs, because the degree of bone mineralization in later life stages (adulthood) depends largely on the amount of bone minerals accumulated in the early stages of their growth. The development of the technique allows to determine the condition of the skeletal system (or individual bones) in living animals by methods used in human medicine, or after their slaughter. For in vivo determination of bone properties, Abstract 10 double energy X-ray absorptiometry or computed tomography scanning techniques are used. Both methods allow the quantification of mineral content and bone mineral density. The most important property from a practical point of view is the bone’s bending strength, which is directly determined by the maximum bending force. The most important factors affecting bone strength are: • age (growth period), • gender and the associated hormonal balance, • genotype and modification of genes responsible for bone growth • chemical composition of the body (protein and fat content, and the proportion between these components), • physical activity and related bone load, • nutritional factors: – protein intake influencing synthesis of organic matrix of bone, – content of minerals in the feed (CA, P, Zn, Ca/P, Mg, Mn, Na, Cl, K, Cu ratio) influencing synthesis of the inorganic matrix of bone, – mineral/protein ratio in the diet (Ca/protein, P/protein, Zn/protein) – feed energy concentration, – energy source (content of saturated fatty acids - SFA, content of polyun saturated fatty acids - PUFA, in particular ALA, EPA, DPA, DHA), – feed additives, in particular: enzymes (e.g. phytase releasing of minerals bounded in phytin complexes), probiotics and prebiotics (e.g. inulin improving the function of the digestive tract by increasing absorption of nutrients), – vitamin content that regulate metabolism and biochemical changes occurring in bone tissue (e.g. vitamin D3, B6, C and K). This study was based on the results of research experiments from available literature, and studies on growing pigs carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences. The tests were performed in total on 300 pigs of Duroc, Pietrain, Puławska breeds, line 990 and hybrids (Great White × Duroc, Great White × Landrace), PIC pigs, slaughtered at different body weight during the growth period from 15 to 130 kg. Bones for biomechanical tests were collected after slaughter from each pig. Their length, mass and volume were determined. Based on these measurements, the specific weight (density, g/cm3) was calculated. Then each bone was cut in the middle of the shaft and the outer and inner diameters were measured both horizontally and vertically. Based on these measurements, the following indicators were calculated: • cortical thickness, • cortical surface, • cortical index. Abstract 11 Bone strength was tested by a three-point bending test. The obtained data enabled the determination of: • bending force (the magnitude of the maximum force at which disintegration and disruption of bone structure occurs), • strength (the amount of maximum force needed to break/crack of bone), • stiffness (quotient of the force acting on the bone and the amount of displacement occurring under the influence of this force). Investigation of changes in physical and biomechanical features of bones during growth was performed on pigs of the synthetic 990 line growing from 15 to 130 kg body weight. The animals were slaughtered successively at a body weight of 15, 30, 40, 50, 70, 90, 110 and 130 kg. After slaughter, the following bones were separated from the right half-carcass: humerus, 3rd and 4th metatarsal bone, femur, tibia and fibula as well as 3rd and 4th metatarsal bone. The features of bones were determined using methods described in the methodology. Describing bone growth with the Gompertz equation, it was found that the earliest slowdown of bone growth curve was observed for metacarpal and metatarsal bones. This means that these bones matured the most quickly. The established data also indicate that the rib is the slowest maturing bone. The femur, humerus, tibia and fibula were between the values of these features for the metatarsal, metacarpal and rib bones. The rate of increase in bone mass and length differed significantly between the examined bones, but in all cases it was lower (coefficient b <1) than the growth rate of the whole body of the animal. The fastest growth rate was estimated for the rib mass (coefficient b = 0.93). Among the long bones, the humerus (coefficient b = 0.81) was characterized by the fastest rate of weight gain, however femur the smallest (coefficient b = 0.71). The lowest rate of bone mass increase was observed in the foot bones, with the metacarpal bones having a slightly higher value of coefficient b than the metatarsal bones (0.67 vs 0.62). The third bone had a lower growth rate than the fourth bone, regardless of whether they were metatarsal or metacarpal. The value of the bending force increased as the animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. The rate of change in the value of this indicator increased at a similar rate as the body weight changes of the animals in the case of the fibula and the fourth metacarpal bone (b value = 0.98), and more slowly in the case of the metatarsal bone, the third metacarpal bone, and the tibia bone (values of the b ratio 0.81–0.85), and the slowest femur, humerus and rib (value of b = 0.60–0.66). Bone stiffness increased as animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. Abstract 12 The rate of change in the value of this indicator changed at a faster rate than the increase in weight of pigs in the case of metacarpal and metatarsal bones (coefficient b = 1.01–1.22), slightly slower in the case of fibula (coefficient b = 0.92), definitely slower in the case of the tibia (b = 0.73), ribs (b = 0.66), femur (b = 0.59) and humerus (b = 0.50). Bone strength increased as animals grew. Regardless of the growth point tested, bone strength was as follows femur > tibia > humerus > 4 metacarpal> 3 metacarpal> 3 metatarsal > 4 metatarsal > rib> fibula. The rate of increase in strength of all examined bones was greater than the rate of weight gain of pigs (value of the coefficient b = 2.04–3.26). As the animals grew, the bone density increased. However, the growth rate of this indicator for the majority of bones was slower than the rate of weight gain (the value of the coefficient b ranged from 0.37 – humerus to 0.84 – fibula). The exception was the rib, whose density increased at a similar pace increasing the body weight of animals (value of the coefficient b = 0.97). The study on the influence of the breed and the feeding intensity on bone characteristics (physical and biomechanical) was performed on pigs of the breeds Duroc, Pietrain, and synthetic 990 during a growth period of 15 to 70 kg body weight. Animals were fed ad libitum or dosed system. After slaughter at a body weight of 70 kg, three bones were taken from the right half-carcass: femur, three metatarsal, and three metacarpal and subjected to the determinations described in the methodology. The weight of bones of animals fed aa libitum was significantly lower than in pigs fed restrictively All bones of Duroc breed were significantly heavier and longer than Pietrain and 990 pig bones. The average values of bending force for the examined bones took the following order: III metatarsal bone (63.5 kg) <III metacarpal bone (77.9 kg) <femur (271.5 kg). The feeding system and breed of pigs had no significant effect on the value of this indicator. The average values of the bones strength took the following order: III metatarsal bone (92.6 kg) <III metacarpal (107.2 kg) <femur (353.1 kg). Feeding intensity and breed of animals had no significant effect on the value of this feature of the bones tested. The average bone density took the following order: femur (1.23 g/cm3) <III metatarsal bone (1.26 g/cm3) <III metacarpal bone (1.34 g / cm3). The density of bones of animals fed aa libitum was higher (P<0.01) than in animals fed with a dosing system. The density of examined bones within the breeds took the following order: Pietrain race> line 990> Duroc race. The differences between the “extreme” breeds were: 7.2% (III metatarsal bone), 8.3% (III metacarpal bone), 8.4% (femur). Abstract 13 The average bone stiffness took the following order: III metatarsal bone (35.1 kg/mm) <III metacarpus (41.5 kg/mm) <femur (60.5 kg/mm). This indicator did not differ between the groups of pigs fed at different intensity, except for the metacarpal bone, which was more stiffer in pigs fed aa libitum (P<0.05). The femur of animals fed ad libitum showed a tendency (P<0.09) to be more stiffer and a force of 4.5 kg required for its displacement by 1 mm. Breed differences in stiffness were found for the femur (P <0.05) and III metacarpal bone (P <0.05). For femur, the highest value of this indicator was found in Pietrain pigs (64.5 kg/mm), lower in pigs of 990 line (61.6 kg/mm) and the lowest in Duroc pigs (55.3 kg/mm). In turn, the 3rd metacarpal bone of Duroc and Pietrain pigs had similar stiffness (39.0 and 40.0 kg/mm respectively) and was smaller than that of line 990 pigs (45.4 kg/mm). The thickness of the cortical bone layer took the following order: III metatarsal bone (2.25 mm) <III metacarpal bone (2.41 mm) <femur (5.12 mm). The feeding system did not affect this indicator. Breed differences (P <0.05) for this trait were found only for the femur bone: Duroc (5.42 mm)> line 990 (5.13 mm)> Pietrain (4.81 mm). The cross sectional area of the examined bones was arranged in the following order: III metatarsal bone (84 mm2) <III metacarpal bone (90 mm2) <femur (286 mm2). The feeding system had no effect on the value of this bone trait, with the exception of the femur, which in animals fed the dosing system was 4.7% higher (P<0.05) than in pigs fed ad libitum. Breed differences (P<0.01) in the coross sectional area were found only in femur and III metatarsal bone. The value of this indicator was the highest in Duroc pigs, lower in 990 animals and the lowest in Pietrain pigs. The cortical index of individual bones was in the following order: III metatarsal bone (31.86) <III metacarpal bone (33.86) <femur (44.75). However, its value did not significantly depend on the intensity of feeding or the breed of pigs.