Les mécanismes par lesquels les charges mécaniques et électriques agissent sur le tissu osseux dans son ensemble, et sur les ostéoblastes, en particulier, sont encore mal compris. La réponse des ostéoblastes soumis à un seul type de stimulus physique a été comparée à celle obtenue par des combinaisons de plusieurs signaux mécaniques et/ou électriques. Dans la perspective d’améliorer l’ostéointégration des biomatériaux, nos études ont porté principalement sur les deux composants essentiels pour le succès de la greffe d’un biomatériau : la matrice extracellulaire (MEC) qui sert d’interface entre le biomatériau et l’hôte ainsi que les facteurs angiogéniques. Nous avons étudié les réponses des ostéoblastes à des contraintes mécaniques complexes basées sur des signaux de « basse amplitude haute fréquence » (BAHF) appliquées à un modèle de culture 3D (hydroxyapatite macroporeux). Nous montrons donc qu’une stimulation mécanique simple (3Hz) peut être potentialisée par des BAHF appropriées (25 Hz). Un dispositif a été développé pour appliquer des contraintes mécaniques très BAHF sur des modèles de culture 2D. La synthèse de la MEC est favorisée et ses propriétés ostéogéniques sont augmentées sous BAHF. Les BAHF n’ont pas d’effet sur le VEGF. Un autre dispositif a permis d’appliquer un champ électrique aux cultures cellulaires. Quelques paramètres nous indiquent que les cellules perçoivent le champ électrique, mais nous retenons que le VEGF n’est pas affecté. En revanche, la combinaison de ces stimulations physiques (contrainte mécanique très BAHF et champ électrique) augmente l’expression de plusieurs facteurs impliqués dans l’angiogénèse (VEGF, TGFβ1, FGF2…). Les sollicitations complexes définies dans cette thèse pourraient être un outil pour fonctionnaliser un substitut osseux cellularisé / Over the course of a day, weight bearing bones experience numerous stimulations : mechanical loadings varying in magnitude and frequency, but also electric fields. However, the biological effects of mechanical strain or electrical field on bone cells are poorly understood. In the present in vitro study, osteoblasts were submitted to only one kind of physical stimulus or a combination of stimuli, and the responses were compared. In the perspective of improving the qualities of bone substitute, we analysed parameters essential for a successfull osteointegration : the extracellular matrice (ECM) as host-biomaterial interface, and angiogenic factors which induce implant vascularization. We investigated the effects of complex mechanical strains based on signals of "low magnitude / high frequency" (LMHF) applied to 3D cultures (macroporous hydroxyapatite). Our study shows that an appropriate combined strain regimen (3 Hz+25Hz) has the potential to functionalise cellularized bone-like constructs. ECM synthesis was promoted by LMHF and the osteogenic properties of this ECM were enhanced while VEGF was not affected. Another system was developed to apply an electric field to cell cultures. Some parameters indicated that cells are sensitive to electric fields ; however VEGF expression was not affected. In contrast, when the physical stimulations were combined (LMHF strain + electric field) gene expression of factors implicated in angiogenesis (VEGF, TGFß1, FGF2...) was increased. The complex stimuli whose effects were analysed in this work could be used as a tool for the functionalization of a cellularized bone substitutes

Hydroxyapatite (HA) has received wide attention in orthopedics, due to its biocompatibility and osseointegration ability. Despite these advantages, the brittle nature and low fracture toughness of HA often results in rapid wear and premature fracture of implant. Hence, there is a need to improve the fracture toughness and wear resistance of HA without compromising its biocompatibility. The aim of the current research is to explore the potential of nanotubes as reinforcement to HA for orthopedic implants. HA- 4 wt.% carbon nanotube (CNT) composites and coatings are synthesized by spark plasma sintering and plasma spraying respectively, and investigated for their mechanical, tribological and biological behavior. CNT reinforcement improves the fracture toughness (>90%) and wear resistance (>66%) of HA for coating and free standing composites. CNTs have demonstrated a positive influence on the proliferation, differentiation and matrix mineralization activities of osteoblasts, during in-vitro biocompatibility studies. In-vivo exposure of HA-CNT coated titanium implant in animal model (rat) shows excellent histocompatibility and neobone integration on the implant surface. The improved osseointegration due to presence of CNTs in HA is quantified by the adhesion strength measurement of single osteoblast using nano-scratch technique. Considering the ongoing debate about cytotoxicity of CNTs in the literature, the present study also suggests boron nitride nanotube (BNNT) as an alternative reinforcement. BNNT with the similar elastic modulus and strength as CNT, were added to HA. The resulting composite having 4 wt.% BNNTs improved the fracture toughness (~85%) and wear resistance (~75%) of HA in the similar range as HA-CNT composites. BNNTs were found to be non-cytotoxic for osteoblasts and macrophages. In-vitro evaluation shows positive role of BNNT in osteoblast proliferation and viability. Apatite formability of BNNT surface in ~4 days establishes its osseointegration ability.

Na ossificação endocondral, osteoclastos (Oc) reabsorvem os remanescentes de cartilagem, e osteoblastos (Ob) depositam matriz óssea. Bisfosfonatos (Bps) inibem a ação dos Oc. Foi avaliado o efeito dos Bps alendronato (Aln) e etidronato (Etn) em joelhos de ratos jovens (in vivo) e na cultura primária de Oc (in vitro). O material in vivo foi analisado por MEV, MET e ML (morfologia e histoquímica para TRAP). RNA foi extraído para análise por RT-PRC e proteínas para análise por WB, que também foram extraídos após o tratamento da cultura com Bps. O tratamento com Etn revelou lâmina epifiseal desorganizada com extensa área de cartilagem; a MEV mostrou pouco osso trabecular com lacunas de reabsorção, que não foram observadas com Aln. O Aln revelou numerosos Oc TRAP-positivos latentes, confirmados por MET. In vivo os Bps diminuem a expressão dos genes analisados; In vitro o Aln diminui somente a expressão de Runx2, menos expresso com Etn, assim como Spp1. A expressão proteica variou entre os grupos. Aln é o mais potente em inibir os Oc enquanto o Etn atua sobre os Ob. / In endochondral ossification, clastic cells (Oc) resorb the calcified cartilage, while osteoblasts (Ob) form new bone. Bisphosphonates (Bps) inhibit the action of Oc. The effect of the Bps alendronate (Aln) and etidronate (Etn) on the knees of young rats (in vivo) and in primary cultures of Oc (in vitro) was evaluated. The specimens were analyzed by SEM, TEM, and LM or TRAP histochemistry. RNA was extracted to analysis by RT-PRC and protein to analysis by WB. RNA and protein were also extracted after the treatment of cultures with Bps. Rats treated with Etn exhibited a disorganized epiphyseal plate containing large area of cartilage; SEM showed few bone trabeculae with resorption lacunae, which were not observed in Aln specimens. Aln showed numerous latent Oc by TRAP histochemistry and TEM. In vivo, the Bps decreased the expression of all analyzed genes; in vitro, Aln decreased only the expression of Runx2 as well as SPP1, which expression was less with Etn. Protein expression varied among the groups. Aln is more potent for inhibiting the Oc, while Etn acts on Ob.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22646号 / 医博第4629号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 松田 秀一, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bone is an important organ influenced by mechanical load, hormones, nutrition and disease. During bone remodelling, osteoclasts resorb bone and osteoblasts form new bone. Osteoblasts are derived from mesenchymal stem cells (MSCs) such as adipose-derived stromal cells (ASCs). The mitogen-activated protein kinase (MAPK) pathway has been shown to interfere with osteoblast differentiation from an early stage. Runt related transcription factor 2 (RUNX2) exerts an effect downstream from p38 MAPK. RUNX2 phosphorylation by p38 MAPK may increase osteoblast differentiation markers such as alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor kB ligand (RANKL). Palmitoleic acid (PLA), an omega-7 monounsaturated fatty acid (MUFA), promotes anti-osteoclastogenic effects, however, the effects of PLA on osteoblasts has not been reported. Osteoporosis is a condition which has debilitating effects in the elderly. Unsaturated fatty acids (UFA) have been studied for their beneficial effects on human health for a number of years. Polyunsaturated fatty acids (PUFA) have been studied as a potential therapeutic agent to prevent and assist in managing the condition. Few studies have been conducted on the effects of MUFA on bone therefore this study aimed to investigate the effects of PLA on osteoblast differentiation using ASCs and MG-63 osteosarcoma cells as an osteoblast model. ASCs and MG-63 osteosarcoma cell lines were exposed to PLA (20-100 μM) in osteogenic media (OM). The effects of PLA on cell viability was evaluated on undifferentiated cells. Thereafter, cells were exposed to PLA for 7, 14 or 21 days. Subsequently ALP activity, calcium mineralisation, gene expression, protein expression and adipogenesis were assessed. In this study, PLA had no significant effects on cell viability in undifferentiated cells. Furthermore, PLA had no significant effects on ALP activity, calcium mineralisation or phosphorylated extracellular signal-regulated kinase (pERK)/extracellular signal-regulated kinase (ERK) expression in differentiating cells, however, ALP activity increased at 7 day in ASCs and 21 days in MG-63 cells. Alizarin Red S staining increased at 21 days in both cell lines with a significant increase in the ASCs, however, calcium nodules were not visible. In the ASCs, PLA significantly increased the gene expression of ALP at 7 and 14 days compared to control (p<0.01 and p<0.05) while RANKL was significantly decreased at 7 days compared to the control (p<0.05). In the MG-63 cells, RUNX2 and OCN were significantly reduced at 7 days compared to control (p<0.05) and ALP, RUNX2, Osx and RANKL were significantly reduced at 14 days compared to control (p<0.001 and p<0.05). In the ASCs, lipid accumulation was not present after 21 days while in MG-63 cells, there was a significant increase in lipid accumulation at a high concentration of PLA after 21 days compared to control (p<0.05). This is the first study to explore the effects of PLA on osteoblast formation using ASCs and MG-63 osteosarcoma cells. Results suggest that PLA exerted changes in the ASCs and MG-63 cells during osteoblast differentiation, however, these changes were not significant. To conclude, PLA showed some significant effects on osteoblast-specific gene expression, however, most of the osteoblast-specific gene expression was downregulated, particularly in the MG-63 cells, after PLA treatment. / Dissertation (MSc)--University of Pretoria, 2020. / Physiology / MSc / Unrestricted

Les progrès dans la compréhension de la biologie osseuse ont permis d’identifier le rôle du système nerveux sensoriel dans la formation osseuse, le remodelage et la régénération tissulaire. Cependant, le rôle précis du système nerveux sensoriel sur la l’ostéogénèse reste encore méconnu. La première partie de ce travail a été d’analyser le rôle des neurones du ganglion de la racine dorsale (DRG) sur la différenciation ostéoblastique des cellules souches mésenchymateuse (MSCs). Pour répondre à cette question, nous avons utilisé une plate-forme microfluidique, qui tente de mimer l’innervation sensorielle du tissu osseux. Dans la seconde partie de cette étude, nous avons cherché à mieux caractériser la sous-population de neurones DRG impliqués dans la régulation directe de la différenciation des MSCs vers le lignage ostéoblastique. En conclusion, l’ensemble des résultats permettent de montrer que: i) les neurones sensoriels ont un effet positif et direct sur la différenciation ostéoblastique des cellules ostéoprogénitrices, ii) la voie de signalisation Wnt/β-caténine est impliquée dans cette transduction du signal; iii) cet effet est principalement régulé par des neurones sensorimoteur, iv) qui peuvent induire la libération locale de facteurs neuroactifs. / Advances in the understanding of bone biology have identified the sensory nervous system as a critical regulator in the orchestration of bone formation, remodeling, and repair. However, the precise role of the sensory nervous system on bone tissue, particularly on osteoprogenitor cells, remains unknown. Firstly, we were interested in clarifying whether dorsal root ganglion (DRG) neurons would be able to induce the osteoblast differentiation by acting directly on mesenchymal stem cells (MSCs). Afterwards, we attempted to understand whether the canonical Wnt signaling pathway could be implicated in the DRG neurons-induced osteoblastogenesis. In the second part of this study, we aimed at better characterizing the subset of DRG neurons involved in the direct regulation of osteoblast differentiation from MSCs. In this work we provide several novel insights: i) we show that sensory neurons have a positive and direct effect on osteoblast differentiation of osteoprogenitor cells, ii) by activating the Wnt/β-catenin signaling pathway; and iii) we suggest that this effect is mainly regulated by sensorimotor neurons, iv) which possibly mediate the local release of neuroactive factors.

Les surcharges en fer, qu’elles soient d’origine génétique ou secondaire, favorisent la baisse de densité minérale osseuse et par conséquent l’apparition d’une ostéoporose. Des liens entre surcharge en fer et perte osseuse ont pu être établis in vivo et in vitro, néanmoins les mécanismes mis en jeu, notamment sur la cellule ostéoblastique, restent incomplètement caractérisés. Notre objectif a donc été de préciser les mécanismes cellulaires conduisant à l’altération du phénotype et de l’activité ostéoblastique observée en présence d’un excès de fer. La réalisation préalable d’une étude transcriptomique sur la lignée ostéoblastique humaine MG-63 nous a permis d’identifier plusieurs gènes susceptibles de voir leur niveau d’ARNm régulé par le fer. Il a été fait l’hypothèse que ces différents gènes pouvaient être impliqués dans la survenue des pertes osseuses observées au cours des surcharges en fer. Ainsi l’expression du gène SPNS2, dont la protéine permet l’export de la Sphingosine-1-Phosphate (S1P), a été identifiée comme potentiellement induite par un excès de fer. Les relations entre l’expression du gène SPNS2 et un excès de fer ont ainsi été investiguées et les résultats obtenus ont mis en évidence une augmentation fer-dépendante de l’ARNm du gène SPNS2 dans la lignée MG-63, non retrouvée dans d’autres types cellulaires. Cette caractérisation nous a ainsi conduits à déterminer, dans la lignée MG-63, l’impact fonctionnel d’une exposition au fer sur l’export cellulaire de la S1P. Nous avons donc pour cela mis au point une méthode d’étude basée sur une stratégie « fluxomique » nous permettant d’évaluer l’efflux de la S1P au moyen d’un outil de spectrométrie de masse. Nos résultats objectivent une diminution des capacités de synthèse et d’export de la S1P en présence de fer et ceci malgré la surexpression du gène SPNS2. La diminution concomitante de l’expression du récepteur S1PR1 et du gène COL1A1 codant pour la chaîne α du collagène de type I suggère un impact fonctionnel de la baisse de concentration en S1P extracellulaire sur la cellule MG-63. La mise en évidence, dans un modèle ostéoblastique, d’une altération fer-dépendante de l’axe de signalisation S1P/S1PR ouvre de nouvelles perspectives quant à la compréhension des mécanismes mis en jeu lors des pertes osseuses associées aux surcharges en fer. / Osteoporosis may complicate genetic or secondary iron overload as reported in clinical and animal studies. However, the mechanisms leading to disrupted bone homeostasis are still to be fully elucidated. In vitro, iron exposure of both osteoblast and osteoclast cell models induces phenotypic and functional impairment, but the molecular mechanisms of iron excess on bone cell physiology are not well characterized, particularly in osteoblast. Our objective was to study the impact of iron overload on osteoblast biology and characterize the molecular mechanisms involved. Transcriptomic analysis previously performed by our group on MG-63 osteoblast-like cell-line to identify iron-modulated genes revealed that expression of SPNS2 gene, which encodes a transporter for the signaling lipid sphingosine 1-phosphate (S1P), is potentially induced by iron. The purpose of this work was to characterize the SPNS2 iron-related regulation and analyze its potential impact on S1P efflux and the S1P/S1PR signaling pathway in MG-63 cells. Our findings showed that iron exposure induces a dose-dependent increase of SPNS2 mRNA levels in MG-63 osteoblast-like cells that was not observed in hepatocyte and enterocyte cell models. We then performed a fluxomic assay on MG-63 cells to investigate iron potential impact on S1P efflux. Unexpectedly, our data showed that extracellular S1P levels were decreased in presence of iron excess and its associated SPNS2 upregulation. Furthermore, based on the observed iron associated S1PR1 and COL1A1 decrease, the defect in S1P export system seems to have functional consequence on MG-63 cells. These results suggest that iron may affect osteoblast S1P/SPR signaling and potentially alter a wide range of bone processes, thus participating in bone impairment in situations of chronic iron overload. These data open a new door for the understanding of mechanisms involved in iron-induced osteoporosis.

A thesis submitted to the College of Dental Medicine of Nova Southeastern University of the degree of Master of Science in Dentistry. Introduction: One of the challenges in orthodontics is obtaining desired tooth movement. A recent development in orthodontics offers patients more comfort by minimizing pain, and shortening the treatment time. Low level laser therapy (LLLT) emerged as a technology that may accelerate the velocity of tooth movement and shorten the orthodontic treatment period. LLLT (630-1000nm) has been shown to modulate various biological processes including wound healing and bone remodeling. Bone remodeling is one of the biological processes that ensue during tooth movement. Bone remodeling is a continuous process characterized by bone deposition at sites of tension and bone resorption on the pressure sites. At cellular level bone remodeling is regulated by receptor activator of NF-κB (RANK) and receptor activator of NF-κB ligand (RANKL) and Osteoprotegrin (OPG). RANK and RANKL promotes osteoclastic differentiation and promotes bone resorption. OPG is a soluble decoy receptor that competes with RANK for binding to RANKL and inhibits the osteoclastic activity. The goal of the research is to understand the regulatory effects of LLLT on bone metabolism at the cellular level. Furthermore, the purpose of this study is to evaluate the critical parameters of low level lasers on the early stage of ostegenic regulation of human osteoblast cells. In this study, CRL-1427 cells derived from human osteosarcoma which have an osteoblast phenotype were used as cell model. Methods: Human osteoblast cells CRL1427(ATCC, Manassas, VA) were cultured in minimum essential medium supplemented with 10% fetal bovine serum and 1% antibiotics and incubated in at 37°C with 5% CO2. The monolayer of cells after reaching 70-80% confluency were irradiated with a single dose of Galium Aluminum Arsenide (Ga-Al-As) laser, with a wave length of 830nm and output power of 85mW with 0.6, 1.5, 1.8 Joules/cm2 energy exposure regimes. The mRNA expressions of Alkaline phosphatase (ALP), Osteoprotegerin (OPG), RANKL and RANK were compared after seven days by quantitative real time PCR. Results: We observed that treatment of CRL 1427 cells with LLLT (1.5, 1.8 Joules/cm2) irradiation significantly increased the expression of ALP, OPG, RANKL and RANK mRNAs compared to the control group (P≤0.05). There was no significant difference between the control group and mRNA expression of ALP, OPG, RANK, or RANKL at 0.6J/cm2of irradiation. Conclusion: LLLT irradiation can directly influence the expression of genes associated with bone metabolism and potentially represents a mechanism that facilitates rapid bone remodeling.

Indiana University-Purdue University Indianapolis (IUPUI) / The hormonal and cellular mechanisms controlling bone formation are not completely understood. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity and bone formation. However, female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone volume/total volume. Previously, our laboratory found ovariectomized Pyk2-KO mice supplemented with 17β-estradiol (E2) exhibited a greater increase in bone volume than WT mice treated with E2. The overall hypotheses of our studies are that Pyk2 regulates OB activity by modulating the E2-signaling cascade and that a Pyk2-inhibitor will promote OB activity and be suitable for bone regeneration applications. In Aim1, we determined the mechanism of action of Pyk2 and E2 in OBs. Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In the presence of E2 or raloxifene, a selective estrogen receptor (ER) modulator, both matrix formation and mineralization were further increased in Pyk2-KO OBs, but not WT OBs. Consistent with a role of Pyk2 in E2 signaling, Pyk2-depletion led to the proteasome-mediated degradation of ERα, but not ERβ. Finally, we found Pyk2-depletion and E2 have an additive effect on ERK phosphorylation, known to increase cell differentiation and survival. In Aim2, we developed a Pyk2-inhibitor loaded hydrogel and evaluated its viscosity, gelation time, swelling, degradation, and release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin exhibited viscosity and shear-thinning behavior suitable for use as an injectable-carrier. Importantly, the Pyk2-inhibitor-hydrogel was cytocompatible, retained its inhibitory activity against Pyk2 leading to an increase in OB activity. In conclusion, therapeutic strategies targeting Pyk2 may improve systemic bone formation, while Pyk2-inhibitor loaded hydrogels may be suitable for targeted bone regeneration in craniofacial and/or the other skeletal defects.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is the most frequent cancer to involve the skeleton, with over 80% of myeloma patients developing lytic bone disease (MMBD). Importantly, MM-associated bone lesions rarely heal even when patients are in complete remission. Bone marrow stromal cells (BMSCs) isolated from MM patients have a distinct genetic profile and an impaired osteoblast (OB) differentiation capacity when compared to BMSCs from healthy donors. Utilizing an in vivo model of MMBD and patient samples, we showed that BMSCs from tumor-bearing bones failed to differentiate into OBs weeks after removal of MM cells. Both Runx2 and Osterix, the master transcription factors for OB differentiation, remained suppressed in these BMSCs. However, the molecular mechanisms for MM-induced long-term OB suppression are poorly understood. We characterized both Runx2 and Osterix promoters in murine pre-osteoblast MC4 cells by chromatin immunoprecipitation (ChIP). The transcriptional start sites (TSSs) of Runx2 and Osterix in untreated MC4 cells were co-occupied by transcriptionally active histone 3 lysine 4 tri-methylation (H3K4me3) and transcriptionally repressive histone 3 lysine 27 tri-methylation (H3K27me3), termed the “bivalent domain”. These bivalent domains became transcriptionally silent with increasing H3K27me3 levels when MC4 cells were co-cultured with MM cells or treated with TNF-α, an inflammatory cytokine increased in MM bone marrow microenvironment. The increasing H3K27me3 levels induced by MM cells or TNF-α were associated with the downregulation of the H3K27 demethylase JMJD3 in MC4 cells and murine BMSCs. Knockdown of JMJD3 in MC4 cells was sufficient to inhibit OB differentiation. Further, ectopic overexpression of JMJD3 in MC4 cells partially rescued the suppression of osteoblast differentiation induced by TNFa. We also found that pre-incubation of MC4 cells with the NF-kB inhibitor quinazoline (QNZ) before TNF-a treatment prevented the downregulation of JMJD3. In agreement with our in vitro findings, BMSCs from MM patients had persistently decreased JMJD3 expression compared to healthy BMSCs. Our findings together demonstrate that decreased JMJD3 expression in BMSCs contributes to the long-term OB suppression in MMBD by remodeling histone landscapes at the Runx2 and Osterix TSSs. Thus, developing strategies to restore JMJD3 expression in BMSCs should increase bone formation and possibly decrease tumor burden in MM.