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

OBJECTIVE: To determine the effects of vitamin C and insulin on osteoblasts harvested from neonatal mouse calvaria. To determine the effects of experimental media (vitamin C and insulin and a combination) on the ex-vivo live bone organ culture model and explore the capacity of addition of osteoblasts to allow for bone formation within a critical defect. To use the chick chorioallantoic membrane (CAM) model to explore bone formation within critical bone defect. METHODS: Osteoblasts were harvested from neonatal mice were tested using four types of experimental media: control DMEM, media prepared with 150 μg/ml vitamin C, 10 nM media, or a combination of both vitamin C, insulin, and a combination of vitamin C and insulin media. Cell were cultured for 18 days at 37°C. Neutral red was done to identify cellular activity and silver nitrate to detect calcium deposits. Two types of scaffolds were inserted into the defect: collagen membrane scaffold and NuOss (xenograft) with collagen scaffold. After 30 days the samples were collected for histological analysis. Neonatal mouse calvaria were harvested and a 2mm critical defect made on each calvaria. Each calvaria received a scaffold of collagen or NuOss with or without osteoblasts with one of three experimental media within the CAM model. After 7 days, the amnion membrane of the egg was dropped and a window was made. The calvaria with the scaffold samples were placed on the amnion membrane. The eggs were incubated for 10 days then the experiment was terminated. Calvaria were collected and processed for histological evaluation. RESULTS: Neutral red and silver nitrate of 2D in-vitro cells revealed calcium deposits in culture well using vitamin C media, cell cultured with insulin media showed calcium deposits and cell morphological change, and cell cultured with a combination of vitamin C and insulin media showed the most calcium deposits and morphological changed. Ex-Vivo samples with collagen scaffold had bone thickening but not enough nutrients for bone regeneration, despite the addition of cells. The collagen scaffold is a more suitable material than xenograft due to particle size. The CAM model showed new bone formation and new vessels were most abundant in areas closest to lining cells in collagen samples. Samples with additional osteoblasts added showed greater results. NuOss scaffold samples did not show the same bone formation or vessel growth. CONCLUSIONS: The results indicate and confirm the basic principles of tissue engineering. In order to have bone regeneration more cells allow for better results. The quality of the scaffold is important and should have stability as well as enough space for cellular migration and recruitment for new blood vessel to support regeneration of bone to its original state.

Indiana University-Purdue University Indianapolis (IUPUI) / The proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry.

Zirconia has been established as a promising material for dental implants. Various surface treatment methods have been utilized to promote better osseointegration and improve the success rate of dental implants. However, a better understanding of the influences of topographic characteristics on cell attachment, proliferation, and differentiation is needed. Different surface topographic zirconia specimens, As sintered, Mild rough, Moderate rough, and Rough zirconia groups were fabricated with sandblasting method in various distances and stages. The surface texture, microstructure, and wettability were inspected with the optical profiler, SEM, and contact angle measurement respectfully. Human primary osteoblast cells were cultured on the four groups of zirconia with different surface modifications in 24 well plates and on plates without test material as control. Crystal violet and triton X-100 solution were used to evaluate cell attachment efficiency at 9 hours and proliferation rate at 7, 14, and 21 days after seeding. ALP activity was measured with fluorometric assay. The expression of osteocalcin was measured with an ELISA kit. Alizarin red staining was conducted to evaluate the mineralization. The cell morphologies were inspected under SEM after cell fixation and critical point drying process. The data were analyzed with one-way ANOVA for experiment on each time interval and two-way ANOVA for all time points. Tukey post hoc test was used for pairwise comparison. P value < 0.05 was considered statistically significant. Topographic parameters and contact angle measured in As sintered, Mild rough, Moderate rough, and Rough surface groups were as follow: Sa = 0.23, 0.50, 2.13, 5 µm, Sal = 49.88, 21.20, 30.42, 49.87 µm, Sdq = 64.64, 248.60, 511, 734.66 µm/mm, Sk = 0.7, 1.54, 4.19,16 µm, Spk = 0.31, 0.64, 1.47, 5.13 µm, Svk = 0.35, 0.71, 5.96, 6.18 µm respectively, and contact angle = 64.6°, 55.2°, 43.5°, 38.6° respectively. The result showed that Rough zirconia group induced the highest cell attachment efficiency at 9 hours (p<.001). As sintered zirconia group promoted significant cell proliferation rate at 21 days (p<.001) while Rough zirconia group drastically down-regulated cell proliferation (p<0.001). The most elevated levels of ALP, osteocalcin, and mineralization expressions at 21 days were found in the Moderate rough group with significant differences (p<.001, p<.001, p<.001 accordingly) Within the limitations of this in vitro study, it can be concluded 1) the modified microroughened zirconia surfaces of Sa 5 µm would promote human osteoblast attachment but down-regulate cell proliferation, 2) the As sintered zirconia surface would stimulate cell proliferation, and 3) the microroughened surfaces of Sa 2 µm would up-regulate osteoblast differentiation. These findings could be incorporated into designing and fabricating the dental implant surfaces for optimal osseointegration.

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.

The interaction of osteoclasts and osteoblasts regulates bone density and the maintenance of osseointegration of implants. To investigate osteoblastic response to strain, rat osteosarcoma cells were cultured on titanium plates and subjected to one of three treatments: a neutral control (0 microstrain), tension (1000 microstrain), and compression (1000 microstrain). Treatments were applied via 4 point bending for fifteen minutes each day to a strain of 1mm/m at a sinusoidal frequency of 1Hz. Cells were measured for DNA as indicator of proliferation, alkaline phosphatase as indicator of phenotype and total protein and calcium deposition as indicators of matrix formation. Results indicated that DNA and total protein differed significantly between treatments. The alkaline phosphatase levels and calcification of matrix did not. The differences in the levels of DNA indicate different growth patterns between treatments. The total protein levels imply distinctions in matrix deposition. The alkaline phosphatase and calcium levels did not vary significantly, implying no difference in bone-specificity or calcification.

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