Biomaterials are widely used in medical practice to help maintain, improve or restore diseased tissues or organs. The successful integration of biomaterials with host tissue depends on substratum surface properties, as well as host tissue quality and its regulatory environment. The overall goal of this dissertation is to incorporate these three factors to achieve better biomaterial-host tissue interactions. Important surface properties include surface topography, surface energy, chemical composition and surface charge. We designed a new titanium (Ti) substratum with modified surface chemical composition by preventing the contamination when in contact with the atmosphere. The new Ti surface has lower carbon contamination and promotes osteoblast differentiation phenotype. The osteogenic effect is synergistic with micrometer and sub-micrometer scale surface structures. To further investigate the effects of bone quality on peri-implant bone formation, we developed a novel mouse femoral medullary bone formation model. This new model will facilitate research evaluating the effects of biomaterial surface treatments in host animals with deficient bone development, including genetically engineered mice. Finally, we studied sexual dimorphism in the response of osteoblasts to systemic regulatory hormones 1¦Á,25-dihydroxyvitamin D3 and 17¦Â-estradiol. The results showed intrinsic differences in male and female osteoblasts with respect to their differentiation and their responses to hormones, suggesting that host chromosomal sex should be considered in biomaterial research. Taken together, this research provides fundamental information on biomaterial surface properties and the regulation of host tissue response, which are important in guiding biomaterial design and evaluation.

The need for anabolic therapies to increase bone formation in difficult orthopaedic circumstances and to treat osteoporosis is an area of intense research focus. There is a current interest in the Wnt signalling pathway as a target for such treatment, with accumulating evidence for a role of this pathway in bone formation. Ectoderm Neural Cortex 1 (ENC1) is a Wnt target gene, not previously studied in bone, which was observed in our laboratory to be up-regulated in an anabolic surgical model of bone formation. The involvement of ENC1 in the differentiation of neuronal and adipocytic cells has previously been reported; therefore, this thesis investigates the expression of ENC1 in cells of the bone and the role of ENC1 during osteoblast differentiation. ENC1 transcript expression was localised to osteoblastic, chondrocytic and osteocytic cells in sections of healing fracture callus and normal mouse bone by in situ hybridisation. The expression of ENC1 was confirmed in differentiating primary osteoblasts and in osteoblastic and osteosarcoma cell lines by quantitative real time PCR and western blotting. ENC1 exists as two protein isoforms of 67 and 57kD in size, which are translated from alternatively spliced ENC1 transcripts. Both isoforms of the protein were detected in differentiating cultures of the pre-osteoblast cell line MC3T3-E1. To address the function of ENC1 in osteoblast differentiation, shRNA knockdown of the endogenous transcript was undertaken in MG63 osteosarcoma cells and in the MC3T3-E1 pre-osteoblastic differentiation model. Stable expression of shRNA targeted to both ENC1 spliceforms resulted in reduced accumulation of alkaline phosphatase positive nodules and alkaline phosphatase transcripts in MG63 cell culture. This reduction was not seen with targeted knockdown of 67kD ENC1 alone. Stable tetracycline-inducible shRNA knockdown targeted to both 57 and 67kD ENC1 isoforms in MC3T3-E1 cells resulted in a significant reduction of Alizarin Red S stained mineralised nodules. When expression of 67kD ENC1 alone was reduced, however, a significant increase in MC3T3-E1 nodule formation was observed. This knockdown had no effect on the expression of early genes involved in osteoblast differentiation Runx2 and osterix, but changes in expression of alkaline phosphatase and osteocalcin mRNA mirrored nodule formation. ENC1 is a member of the BTB-Kelch family of proteins. Some members of this family have recently been found to act as substrate adaptors for the E3 ubiquitin ligase, binding to the cullin 3 component of the complex. These adaptor proteins function to bring a substrate protein within the vicinity of the E2 ubiquitin-conjugating enzyme, thus targeting it for ubiquitination and subsequent proteasomal degradation. The ability of ENC1 to interact with cullin 3 was investigated as a possible mechanism by which it may affect a role in osteoblast differentiation. Full length ENC1 showed robust binding to cullin 3 and weak binding was seen between the N-terminally truncated 57kD isoform and cullin 3. ENC1, therefore, may act as a substrate adaptor protein for the cullin 3 based E3 ubiquitin ligase. These data present ENC1 as a novel candidate protein involved in osteoblast differentiation, and suggest the possible involvement of this protein in proteasomal degradation of a substrate involved in osteoblast differentiation. The ENC1 isoforms and the associated functional pathways thus are possible future therapeutic targets to treat bone loss and enhance or accelerate fracture healing.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21004号 / 医博第4350号 / 新制||医||1028(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 戸口田 淳也, 教授 妻木 範行, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bone is currently the second most transplanted tissue, second only to blood. However, significant hurdles including graft supply and implant failure continue to plague researchers and clinicians. Currently, standard clinical procedures include autologous and allogeneic grafting. Autologous grafts may achieve functional repair; yet, they are available in limited supply and are associated with donor site morbidity. Allogeneic grafts are available in greater supply, but have a higher risk of infection. To overcome the disadvantages of current grafts, tissue engineering has become a major focus for the regeneration of bone. The goal of tissue engineering is to use a multidisciplinary approach to create biomimetic constructs that stimulate osteogenic regeneration to heal bone defects and restore tissue function. Biodegradable scaffolds are used in tissue engineering strategies as an interim template for tissue regeneration. The scaffold architecture provides mechanical support for cell attachment and tissue regeneration. Biocompatible poly(lactic-co-glycolic acid) (PLGA) has been processed through a number of techniques to create porous 3D architectures. Hydroxyapatite (HAP) and tricalcium phosphate have been used in conjunction with polymer scaffolds due to their osteoconductivity and biocompatibility, but they often lack osteoinductivity and are resistant to biodegradation. Conversely, amorphous calcium phosphate (ACP) is a mineral that solubilizes under aqueous conditions, releasing calcium and phosphate ions, which have been postulated to enhance osteoblast differentiation and mineralization. Controlled dissolution can be achieved by stabilizing ACP with divalent cations such as zinc or copper. Furthermore, incorporation of such osteogenic ACPs within a biodegradable PLGA scaffold could enhance the osteoconductivity of the scaffold while providing calcium and phosphate ions to differentiating osteoprogenitor cells, thereby stimulating osteogenesis when implanted in vivo. In this research, the effect of zinc on the differentiation of osteoprogenitor cells was investigated. Zinc supplementation of the culture media had no stimulatory effect on cell proliferation or differentiation. ACPs were synthesized using zirconium (ZrACP) and zinc (ZnACP) as stabilizers to achieve sustained ion release. Elevated concentrations suggested sustained ion release over the course of 96 hours and enhanced solubility of ZrACP and ZnACP. X-ray diffraction analysis showed a conversion of ZrACP to a semi-crystalline material after 96 hours, but ZnACP showed no conversion after 96 hours. Composite scaffolds were fabricated by incorporating HAP, zirconium-stabilized ACP (ZrACP), or zinc-stabilized ACP (ZnACP) into a sintered PLGA microsphere matrix and then characterized to determine the effect of the minerals on the in vitro differentiation of MC3T3-E1 cells. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate powders distributed on the surface of the microspheres. Measurements of mechanical properties indicated that incorporation of 0.5 wt% calcium phosphates resulted in a 30% decrease in compressive modulus. When cells were cultured in the scaffolds, composite ACP scaffolds stimulated proliferation and ALP activity, while HAP scaffolds stimulated osteoblast gene expression. Overall, the results of this work indicate the addition of calcium phosphate minerals to PLGA scaffolds supported cell growth and stimulated osteogenic differentiation, making the scaffolds a promising alternative for bone tissue regeneration. / Ph. D.

No description available.

Dental and orthopaedic implants are currently the solutions of choice for teeth and joint replacements with success rates continually improving, but they still have undesirable failure rates in patients who are compromised by disease or age, and who in many cases are the ones most in need. The success of titanium (Ti) implants depends on their ability to osseointegrate with the surrounding bone and this, in turn, is greatly dependent on the surface characteristics of the device. Advancements in surface analysis and surface modification techniques have improved the biological performance of metallic implants by mimicking the hierarchical structure of bone associated with regular bone remodeling. In this process, damaged bone is resorbed by osteoclasts, which produce resorption lacunae containing high microroughness generated after mineral dissolution under the ruffled border, as well as superimposed nanoscale features created by the collagen fibers left at the surface. Indeed, increasing Ti surface roughness at the micro and sub-microscale level has been shown to increase osteoblast differentiation in vitro, increase bone-to-implant contact in vivo, and accelerate healing times clinically. Recently, the clinical application of surface nanomodification of implants has been evaluated. Still, most clinically-available devices remain smooth at the nanoscale and fundamental questions remain to be elucidated about the effect of nanoroughness on the initial response of osteoblast lineage cells. Another property that could be used to control osteoblast development and the process of osseointegration is the electrical surface charge of implants. The presence of endogenous electrical signals in bone has been implicated in the processes of bone remodeling and repair. The existence of these native signals has prompted the use of external electrical stimulation to enhance bone growth in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports confirming its beneficial effects on bone formation. However, the use of electrical stimulation on Ti implants to enhance osseointegration is less understood, in part because of the lack of in vitro models that truly represent the in vivo environment. In addition, an aspect that has not been thoroughly examined is the electrical implication of implant corrosion and its effect on the surrounding tissue. Implants are exposed to extreme conditions in the body such as high pH during inflammation, and cyclic loads. These circumstances may lead to corrosion events that generate large electrochemical currents and potentials, and may cause abnormal cell and tissue responses that could be partly responsible for complications such as aseptic loosening of implants. Consequently, Ti implants with tailored surface characteristics such as nanotopography and electrical polarization, could promote bone healing and osseointegration to ensure successful outcomes for patients by mimicking the biological environment of bone without the use of systemic drugs. The objective of this thesis is to understand how surface nanostructural and electrical characteristics of Ti and Ti alloy surfaces may affect osteoblast lineage cell response in vitro for normal tissue regeneration and repair. Our central hypothesis is that combined micro/nanostructured surfaces, as well as direct stimulation of Ti surfaces with fixed direct current (DC) potentials, can enhance osteoblast differentiation.

In vivo, les cellules osseuses évoluent dans un microenvironnement complexe, tridimensionnel et interagissent avec celui-ci à de nombreuses échelles, depuis le nanomètre (tropocollagène) jusqu’à des structures de plusieurs centaines de micromètres (trabécules). Paradoxalement, la majeure partie de nos connaissances sur la physiologie cellulaire est issue d’expériences réalisées sur des cellules cultivées sur du plastique et en deux dimensions. Ces différences ne peuvent qu’avoir une influence sur le comportement des cellules, qui n’entretiennent plus les mêmes relations spatiales entre elles, ainsi qu’avec leur environnement. De plus, si ces dernières années, nombre d’études ont été réalisées sur l’influence de la topographie à des échelles nano et micrométriques, peu d’études ont montré le rôle de la géométrie du substrat à une échelle tissulaire, soit au sein de structures supérieures à 100 µm. Afin d’étudier l’influence de la macroarchitecture du substrat sur le comportement cellulaire, des céramiques en hydroxyapatite à architecture contrôlée ont été ensemencées avec des cellules primaires de calvaria de souris. Une première étude a été entreprise sur des substrats macroarchitecturés, présentant des sillons de différentes géométries : sillons semi-circulaires (Wave), sillons triangulaires à angle de 90° ou à angle de 45°. Plus la géométrie du substrat était refermée (45°>90°>Wave), plus la différenciation ostéoblastique était rapide. Cela s’est traduit par une augmentation des niveaux d’expression génique et protéique d’ostéocalcine et de sclérostine, indiquant la présence d’ostéocytes au sein de l’important tissu déposé par les cellules. De plus, au sein de la géométrie à l’angle le plus fermé (i.e. « 45° »), des structures fibreuses minéralisées, orientées parallèlement au fond du substrat ont été observées. Cette orientation s’est confirmée au niveau cellulaire, avec une orientation similaire des fibres de stress et un étirement des noyaux cellulaires. La géométrie du substrat influence donc le comportement des cellules en modifiant très probablement leur signalisation intracellulaire. Ces investigations ont été poursuivi par le développement d’un modèle d’ostéogénèse 3D sous perfusion au sein du bioréacteur BOSE ElectroForce® 5270 BioDynamic®de la plateforme Equipex IVTV, afin d’explorer les interactions cellulaires-substrat en réponse à des contraintes mécaniques (forces de cisaillement). Le dépôt tissulaire était particulièrement abondant au sein des pores triangulaires à angle de 45°, confirmant les données obtenues sur les substrats macroarchitecturés et laissant penser que ce type de pores est le plus à même de permettre une différenciation ostéoblastique optimale. Les résultats de ces travaux pourront permettre des avancées dans la compréhension de la biologie de l’os, mais également dans la conception d’implants innovants destinés à la réparation de défaux osseux, avec une ostéointégration stimulée via la présence de structures à géométrie fermée, tel que des sillons triangulaires à angles de 45°. / In vivo, cells reside in a complex and three-dimensional microenvironment, with which they interact at multiple scales, from the nanometer (tropocollagen) to structures of several hundred of micrometers (trabeculae). However, most of our knowledge on cell physiology has been obtained from cells grown in Petri dishes, on plastic and in two dimensions. In those conditions, the spatial relationships between cells and their environment can only be deeply modified. Moreover, if the impact of substrate closure at a cellular level is particularly well documented, very few studies have shown its role at a tissue level (i.e. greater than 100 µm), and thus focused mostly on the matrix deposition rather than on the osteoblastic differentiation. In order to study the effects of substrate macroarchitecture on cells, primary mouse calvarial cells were seeded on hydroxyapatite-based bioceramics, made from wax molds by 3D printing. A first study was conducted on macroarchitectured substrates. These bioceramics have three patterns of different degrees of closure: semi-circular grooves (Wave), triangular grooves with 90° angle and triangular grooves with 45° angle. The tighter was the substrate geometry (45°> 90°> Wave), the faster was osteoblastic differentiation. This resulted in increased levels of gene and protein expression of osteocalcin and sclerostin, indicating the presence of osteocytes inside the tissue layed by cells. Moreover, in the tightest geometry (i.e. 45°), mineralized fibrous structures, oriented parallel to the bottom substrate were observed. This orientation was confirmed at the cellular level, with a similar orientation of stress fibers and a stretch of cell nuclei. Thus, the substrate macroarchitecture influences the cellular behavior by, most likely, modifying the intracellular signaling. These investigations were pursued with the development of a 3D model of osteogenesis under perfusion, in the BOSE 5270 ElectroForce® BioDynamic® bioreactor of the IVTV Equipex platform, to explore cell-substrate interactions in response to mechanical stress (shear forces). Tissue deposition was particularly abundant in the triangular pores with 45° angles, confirming our previous observations and suggesting that this geometry was able to promote osteoblast differentiation.Our results could lead to breakthroughs in the understanding of the bone biology but also in the design of innovative implants for the repair of bone defects, with a stimulated osseointegration throught the presence of structures with closed geometries, such as triangular grooves with 45° angles.

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.

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.

Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression. However, the transcriptional regulation of OA gene expression by BMP-2 is still unknown. The aim of this study was to characterize BMP-2-induced transcription factors that regulate OA gene expression during osteoblast differentiation. The stimulatory effects of BMP-2 on OA transcription were established by cloning the proximal 0.96kb of rat OA promoter region in a luciferase reporter vector in various osteogenic cell types. Further, by deletion and mutagenesis analyses of the cloned OA promoter, key binding sites for osteogenic transcription factors namely, Runx2, Smad1, Smad4 and homeodomain proteins (Dlx3, Dlx5 and Msx2) were identified and characterized. Utilizing specific siRNAs to knock down Runx2, Smad1, Smad4, Dlx3, Dlx5 or Msx2 proteins in osteoblasts, we found that Runx2, Smad1, Smad4, Dlx3 and Dlx5 proteins up-regulate OA transcription, whereas, Msx2 down-regulated OA gene expression. These specific effects of transcription factors on OA promoter regulation were confirmed by forced expression of transcription factors. Most notably, BMP-2-stimulated cooperative and synergistic interactions between Runx2-Smad1 proteins and Dlx3-Dlx5 proteins that up-regulate OA promoter activity. Electrophoretic mobility shift and supershift assays demonstrated that BMP-2 stimulates interactions between Runx2, Smad1 and Smad4 and homeodomain transcription factors with the OA promoter regions flanking the -585 Runx2 binding site, the -248 Smad binding site and the region between the -852 and the -843 homeodomain binding sites relative to transcription start site. The OA promoter region was occupied by Runx2 and also Dlx3 transcription factors during proliferation stages of osteoblast differentiation. As the osteoblasts progress from proliferation to matrix maturation stages of differentiation, the OA promoter was predominantly occupied by Runx2 and to a lesser extent Dlx5 in response to BMP-2. Finally, during matrix mineralization stages of osteoblast differentiation, BMP-2-induced a robust recruitment of Dlx5, Smad1, Dlx3 and Msx2 proteins with simultaneous dissociation of Runx2 from the OA promoter region. In conclusion, the BMP-2-induced osteogenic transcription factors Runx2, Smad1, Smad4, Dlx3, Dlx5 and Msx2 provide key molecular switches that regulate OA transcription during osteoblast differentiation. / Cell Biology