Hong-Moon Jung

Modulation of the resorption and osteoconductivity of α-calcium sulfate by histone deacetylase inhibitors

Calcium sulfate (CS) is an osteoconductive material with a long history of clinical use. However, its resorptive properties are not optimal for bone regeneration. Recently, histone deacetylase inhibitors (HDIs) have been suggested as bone regeneration tools. In this study, we investigated the effects of the HDIs sodium butyrate and trichostatin A on alpha-form CS (alphaCS) performance. MC3T3-E1 pre-osteoblasts cultured on alphaCS containing either HDI (alphaCS/HDI) showed higher levels of alkaline phosphatase activity than those cultured on alphaCS alone. The expression of genes characteristic of the osteoblast phenotype, including Runx2, osteocalcin, and bone sialoprotein, was strongly promoted by alphaCS/HDI. When cultured on alphaCS/HDIs, the osteoclastic differentiation of RAW264.7 monocytes was substantially suppressed, as measured by tartrate-resistant acid phosphatase (TRAP) activity and the expression levels of calcitonin receptor and TRAP. Neither HDI affected the CS setting time, compressive strength, or dissolution in a simulated body fluid. In a rat calvarial model of critical size bone defects, alphaCS/HDIs enhanced osteoblast differentiation, led to new bone formation, and delayed resorption, as confirmed by micro-computed tomography and histological analyses.

Comparative Evaluation of Different Crystal-Structured Calcium Sulfates as Bone-Filling Materials

The mechanical and handling properties and biological performances of two types of calcium sulfate (betaCS and alphaCS) as bone-filling materials were compared. The influence of two modifiers such as hydroxypropylmethylcellose (HPMC) and fibrin was also examined. alphaCS showed higher strength than, and similar setting time and injectability to those of betaCS. The degradation of CS in a simulated body fluid (SBF) was checked by measuring the amount of calcium released to SBF. alphaCS showed reduced calcium release than betaCS. The modifiers tended to increase the calcium release. The MC3T3-E1 preosteoblasts cultured on alphaCS showed higher levels of alkaline phosphatase (ALP) activity than those cultured on betaCS. alphaCS strongly promoted gene expression of osteoblast phenotypes including Runx2, alpha1(I) collagen, osteocalcin, and bone sialoprotein. There was no significant difference in cell adhesion and proliferation between two types of CS. The addition of modifiers to CS increased cell proliferation, ALP activity, and the gene expression. The osteoclastic differentiation of RAW264.7 monocytes was checked. The cells on both types of CS produced tartrate-resistant acid phosphatase (TRAP) activity with no significant difference. These cell response results indicated that alphaCS promoted osteoblast differentiation over betaCS but not osteoclast differentiation. Conclusively, a particular form of commercially available alphaCS possesses superior properties to betaCS in terms of mechanical properties and supports for osteoblast differentiation, suggesting that alphaCS could be an alternative to the conventionally used betaCS. The addition of HPMC and fibrin could further improve the feasibility of alphaCS as a bone-filling material.

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration.

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.

Sequential Treatment with SDF-1 and BMP-2 Potentiates Bone Formation in Calvarial Defects.

Stromal cell-derived factor-1 (SDF-1) protein and its receptor, CXCR-4, play an important role in tissue repair and regeneration in various organs, including the bone. SDF-1 is indispensable for bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. However, SDF-1 is not needed after the osteogenic induction has been activated. Since the precise condition for the additive effects of combined DF-1 and BMP-2 in bone healing had not been fully investigated, we aimed to determine the optimal conditions for SDF-1- and BMP-2-mediated bone regeneration. We examined the in vitro osteoblastic differentiation and cell migration after sequential treatments with SDF-1 and BMP-2. Based on the in vitro additive effects of SDF-1 and BMP-2, the critical size defects of mice calvaria were treated with these cytokines in various sequences. Phosphate buffered saline (PBS)-, SDF-1-, or BMP-2-soaked collagen scaffolds were implanted into the calvarial defects (n=36). Periodic percutaneous injections of PBS or the cytokine SDF-1 and BMP-2 into the implanted scaffolds were performed on days 3 and 6, postoperatively. Six experimental groups were used according to the types and sequences of the cytokine treatments. After 28 days, the mice were euthanized and bone formation was evaluated with microcomputed tomography and histology. The molecular mechanism of the additive effect of SDF-1 and BMP-2 was evaluated by analyzing intracellular signal transduction through Smad and Erk phosphorylation. The in vitro experiments revealed that, among all the treatments, the treatment with BMP-2 after SDF-1 showed the strongest osteoblastic differentiation and enhanced cell migration. Similarly, in the animal model, the treatment with SDF-1 followed by BMP-2 treatment showed the highest degree of new bone regeneration than any other groups, including the one with continuous BMP-2 treatment. This new bone formation can be partially explained by the activation of Smad and Erk pathways and enhanced cell migration. These results suggest that sequential treatment with the cytokines, SDF-1 and BMP-2, may be a promising strategy for accelerating bone regeneration in critical size defects.

The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling.

The 4–1BB is a costimulatory molecule similar to the receptor activator of NF‐κB ligand (RANKL), both of which are key factors for the differentiation of osteoclasts and are expressed mainly by activated T cells. The 4–1BB shares common signaling pathways with RANK, suggesting a potential role in osteoclastogenesis. In this study, the role of 4–1BB and 4–1BB ligand (4–1BBL) in osteoclastogenesis was investigated using 4–1BB–/– and 4–1BB+/+ mice. Osteoclast precursors normally express 4–1BB and 4–1BBL after exposure to RANKL, which was confirmed by semi‐quantitative RT‐PCR and flow cytometry. The 4–1BB–/– mice had a slightly increased bone mass accompanied by a reduced osteoclastogenic ability of 4–1BB–/– bone marrow‐derived macrophages (BMM) ex vivo. In addition, 4–1BB–/– BMM demonstrated hypophosphorylation of JNK and p38 and decreased induction of c‐Fos in response to RANKL stimulation. Retroviral transduction of wild‐type as well as partial‐length 4–1BB, which lacks TNF receptor‐associated factor 2‐binding sites for signaling, restored the osteoclastogenic ability of 4–1BB–/– BMM. Furthermore, both recombinant 4–1BB and 4–1BBL enhanced RANKL‐induced osteoclastogenesis by 4–1BB+/+ BMM and the induction of c‐Fos and NFATc1.Together, these results indicate that 4–1BBL and 4–1BB expressed on osteoclast precursors enhance RANKL‐induced osteoclastogenesis via bi–directional signaling, findings that may delineate the complex nature of the 4–1BBL and 4–1BB interaction.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpeys fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpeys fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpeys fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

Evaluation on Short Line Fault Breaking Performance of SF

In this paper, short line fault breaking performance of SF6 gas circuit breaker was evaluated by calculating the post arc current and the temperature on arc axis. For the analysis of arc-gas flow, finite volume fluid in cell method was used to solve governing equations. In arc modeling, finite element method and the method of partial characteristics were used for the calculation of Joule heating and radiation model, respectively. Also, the effect of ablated nozzle vapor was considered in the simulation. The calculated results were compared and confirmed with the experimental values.