bmp-2 Gene-Transferred Skeletal Muscles with Needle-Type Electrodes as Efficient and Reliable Biomaterials for Bone Regeneration.

BACKGROUND: Bone morphogenetic protein-2 (bmp-2) has a high potential to induce bone tissue formation in skeletal muscles. We developed a bone induction system in skeletal muscles using the bmp-2 gene through in vivo electroporation. Natural bone tissues with skeletal muscles can be considered potential candidates for biomaterials. However, our previous system using plate-type electrodes did not achieve a 100% success rate in inducing bone tissues in skeletal muscles. In this study, we aimed to enhance the efficiency of bone tissue formation in skeletal muscles by using a non-viral bmp-2 gene expression plasmid vector (pCAGGS-bmp-2) and needle-type electrodes. METHODS: We injected the bmp-2 gene with pCAGGS-bmp-2 into the skeletal muscles of rats' legs and immediately placed needle-type electrodes there. Skeletal tissues were then observed on the 21st day after gene transfer using soft X-ray and histological analyses. RESULTS: The use of needle-type electrodes resulted in a 100% success rate in inducing bone tissues in skeletal muscles. In contrast, the plate-type electrodes only exhibited a 33% success rate. Thus, needle-type electrodes can be more efficient and reliable for transferring the bmp-2 gene to skeletal muscles, making them potential biomaterials for repairing bone defects.

Periodontal Tissue as a Biomaterial for Hard-Tissue Regeneration following bmp-2 Gene Transfer.

The application of periodontal tissue in regenerative medicine has gained increasing interest since it has a high potential to induce hard-tissue regeneration, and is easy to handle and graft to other areas of the oral cavity or tissues. Additionally, bone morphogenetic protein-2 (BMP-2) has a high potential to induce the differentiation of mesenchymal stem cells into osteogenic cells. We previously developed a system for a gene transfer to the periodontal tissues in animal models. In this study, we aimed to reveal the potential and efficiency of periodontal tissue as a biomaterial for hard-tissue regeneration following a bmp-2 gene transfer. A non-viral expression vector carrying bmp-2 was injected into the palate of the periodontal tissues of Wistar rats, followed by electroporation. The periodontal tissues were analyzed through bone morphometric analyses, including mineral apposition rate (MAR) determination and collagen micro-arrangement, which is a bone quality parameter, before and after a gene transfer. The MAR was significantly higher 3-6 d after the gene transfer than that before the gene transfer. Collagen orientation was normally maintained even after the bmp-2 gene transfer, suggesting that the bmp-2 gene transfer has no adverse effects on bone quality. Our results suggest that periodontal tissue electroporated with bmp-2 could be a novel biomaterial candidate for hard-tissue regeneration therapy.

Fgfr1 conditional-knockout in neural crest cells induces heterotopic chondrogenesis and osteogenesis in mouse frontal bones.

Most facial bones, including frontal bones, are derived from neural crest cells through intramembranous ossification. Fibroblast growth factor receptor 1 (Fgfr1) plays a pivotal role in craniofacial bone development, and loss of Fgfr1 leads to cleft palate and facial cleft defects in newborn mice. However, the potential role of the Fgfr1 gene in neural crest cell-mediated craniofacial development remains unclear. To investigate the role of Fgfr1 in neural crest cells, we analyzed Wnt1-Cre;Fgfr1flox/flox mice. Our results show that specific knockout of Fgfr1 in neural crest cells induced heterotopic chondrogenesis and osteogenesis at the interface of the anterior portions of frontal bones. We observed that heterotopic bone formation continued through postnatal day 28, whereas heterotopic chondrogenesis lasted only through the embryonic period. In summary, our results indicate that loss of Fgfr1 in neural crest cells leads to heterotopic chondrogenesis and osteogenesis.

Analysis of mineral apposition rates during alveolar bone regeneration over three weeks following transfer of BMP-2/7 gene via in vivo electroporation

Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and in vivo electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bone-staining revealed that MAR of alveolar bone was as similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining non-viral BMP-2/7 gene expression vector and in vivo electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.

Non-surgical model for alveolar bone regeneration by bone morphogenetic protein-2/7 gene therapy.

BACKGROUND: Alveolar bone is a critical tissue for tooth retention; however, once alveolar bone is lost, it may not spontaneously regenerate. Currently, bone grafts or artificial bone is commonly used for alveolar bone regeneration therapy. However, these therapies require surgical procedures, which present risks, particularly in elderly patients. Therefore, development of alveolar bone regeneration techniques that do not require surgical procedures is critical. It is well known that stem cells present in the periosteal and periodontal ligament may be induced to differentiate into osteogenic cells. This study hypothesizes that transfer of the bone morphogenetic protein-2/7 (BMP-2/7) gene into periodontal tissues via in vivo electroporation induces exogenous BMP production and causes stem cells in periodontal tissues to differentiate into osteogenic cells, enabling generation of new alveolar bone. METHOD: The BMP-2/7 gene expression vector was introduced via electroporation into the target site in periodontal tissues of the first molar of rat maxillae. RESULTS: Exogenous BMP-2 and -7 were detected in the target areas, and growth of new alveolar bone tissue was observed 5 days after gene transfer. On day 7, new alveolar bone tissues were found to connect to the original bone tissues. Moreover, mineral apposition rates of the alveolar bone after BMP-2/7 gene transfer were significantly higher than those in the control group after LacZ gene transfer. CONCLUSION: The present findings indicate that a combination of the BMP-2/7 non-viral vector and in vivo electroporation represents a promising non-surgical option for alveolar bone regeneration therapy.

Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions.

The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency-induced skeletal diseases.

Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation.

We previously developed a novel method for gene transfer, which combined a non-viral gene expression vector with transcutaneous in vivo electroporation. We applied this method to transfer the bone morphogenetic protein (BMP) gene and induce ectopic bone formation in rat skeletal muscles. At present, it remains unclear which types of cells can differentiate into osteogenic cells after BMP gene transfer by in vivo electroporation. Two types of stem cells in skeletal muscle can differentiate into osteogenic cells: muscle-derived stem cells, and bone marrow-derived stem cells in the blood. In the present study, we transferred the BMP gene into rat skeletal muscles. We then stained tissues for several muscle-derived stem cell markers (e.g., Pax7, M-cadherin), muscle regeneration-related markers (e.g., Myod1, myogenin), and an inflammatory cell marker (CD68) to follow cell differentiation over time. Our results indicate that, in the absence of BMP, the cell population undergoes muscle regeneration, whereas in its presence, it can differentiate into osteogenic cells. Commitment towards either muscle regeneration or induction of ectopic bone formation appears to occur five to seven days after BMP gene transfer.

Nicorandil inhibits osteoclast differentiation in vitro.

Nicorandil is a hybrid angina therapeutic agent that has nitric oxide (NO) action and the ability to open ATP-sensitive K+ channels (KATP channels). A transient increase in NO and intracellular Ca2+ has been demonstrated to be highly involved in the differentiation and activation of osteoclasts. The objective of this study was to verify that the pharmacological effect of nicorandil suppresses the differentiation process of osteoclasts in vitro. Although little authentic NO production was detected in the culture medium in osteoclast formation assays, NO production increased only in the presence of nicorandil. The number of osteoclasts decreased markedly at late time-points after nicorandil addition compared with the number at early time-points. Both the number of TRAP-positive multinucleated cells and the number of cells that obtained F-actin rings decreased in the presence of nicorandil in a concentration-dependent manner. The osteo assay showed that the bone resorption area was also reduced with nicorandil in a concentration-dependent manner. An inhibition recovery experiment was conducted by adding a soluble guanylyl cyclase (sGC) inhibitor (ODQ) and a KATP channel-opening inhibitor (glibenclamide) during the osteoclast formation process. In the inhibition recovery experiment, the inhibitory effect of nicorandil on osteoclastogenesis was blocked by the addition of ODQ and glibenclamide. These results suggest that both the NO and KATP channel-opening activity of nicorandil inhibit osteoclast differentiation. Further study of nicorandil may lead to the development of drugs for osteoporosis treatment.

Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts.

Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca(2+) level ([Ca(2+)]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3 increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca(2+) channels and Ca(2+) release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3 receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD3 will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.

A new method for measuring osteoclast formation by electrical impedance.

Osteoclasts are important target cells for osteoporosis treatment. Recently, a real-time cell analysis (RTCA) system was developed to observe cell morphology and adhesion; however, the use of RTCA to study osteoclastogenesis has not been reported. Here, we investigated whether osteoclast formation could be monitored in real-time using RTCA. The cell index determined via electrical impedance using RTCA, and the number of osteoclasts exhibited a significant positive correlation. RTCA was useful for determining the effect of (-)-epigallocatechin-3-gallate on the inhibition of bone resorption. We established a new method of measuring osteoclast formation in real-time using RTCA.

Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats.

Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

Zinc-Induced Effects on Osteoclastogenesis Involves Activation of Hyperpolarization-Activated Cyclic Nucleotide Modulated Channels via Changes in Membrane Potential.

Zinc is a trace element in the mammalian body, and increasing evidence shows its critical role in bone development and osteoclastogenesis. The relationships between zinc and voltage-gated ion channels have been reported; however, the effects of zinc on membrane potential and the related ion channels remain unknown. In this study, we found that zinc-induced hyperpolarization in RAW264.7 cells (RAW) was promoted by inhibition of hyperpolarization-activated cyclic nucleotide modulated channels (HCNs). In electrophysiological experiments with RAW-derived osteoclasts, HCNs were functional and generated hyperpolarization-activated inward currents (Ih) with properties similar to the Ih recorded in excitable cells such as neurons and cardiomyocytes. Quantitative PCR of HCN subunits HCN1 and HCN4 in RAW cells showed detectable levels of HCN1 mRNA and HCN4 expression was the highest of all four subunits. HCN4 knockdown decreased osteoclastic Ih and promoted osteoclastogenesis in the presence of zinc, but not in the absence of zinc. To determine the effect of membrane hyperpolarization on osteoclastogenesis, we developed a light-controllable membrane potential system in RAW cells by stably expressing the light-driven outward proton pump, Archaerhodopsin3 (Arch). Arch activation by yellow-green light hyperpolarizes the cell membrane. Light-induced hyperpolarization accelerated osteoclast differentiation in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL). Thus, HCN activation reduced the hyperpolarization-related promotion of osteoclast differentiation in the presence of zinc. This study revealed the novel role of HCN and membrane potential in non-excitable osteoclasts.

Insulinogenic sucrose+amino acid mixture ingestion immediately after resistance exercise has an anabolic effect on bone compared with non-insulinogenic fructose+amino acid mixture in growing rats.

Maximizing peak bone mass is an important factor in osteoporosis prevention. Resistance exercise increases bone mass and strength, while nutritional supplements have beneficial effects on bone loss reduction. We have previously shown that the combined intake of sucrose and amino acids (AA), which is strongly insulinogenic, efficiently increased muscle protein synthesis. To investigate the effects of sugar and an AA solution immediately after resistance exercise, we compared insulinogenic sucrose and non-insulinogenic fructose combined with an AA solution with or without resistance exercise. Sucrose intake immediately after resistance exercise increased the trabecular bone mass and compressive maximum load compared with fructose+AA intake after exercise. Additionally, combined sucrose+AA and exercise increased trabecular bone formation and decreased bone resorption more than combined fructose and exercise. Serum insulin levels were greatly increased by sucrose+AA intake with exercise. In culture experiments, neither sugar+AA affected osteoblast and osteoclast differentiation. In a gene expression study, sucrose+AA intake after resistance exercise was shown to upregulate the Runx2 expression level and decrease RANKL/OPG ratio. These results suggest that the combined intake of sucrose and an AA solution immediately after resistance exercise exerts anabolic effects on bone by altering gene expression related to bone remodeling. Although translation of our bone remodeling findings from animal to human studies has been challenging, our findings suggest that exercise with sugar+AA intake may contribute to improved bone health.

Regulation of miRNA during direct reprogramming of dental pulp cells to insulin-producing cells.

To further evaluate the multipotency of dental pulp cells, and to investigate the possible direct reprogramming of these cells, we examined their in vitro induction of direct conversion to an endocrine cell lineage. In vitro induction was carried out using similar conditions to those reported for regulating the differentiation of undifferentiated intestinal cells into endocrine progenitor cells. Specifically, the transcription factors Pdx1 and Neurog3 were transfected into rat dental pulp cells to induce their direct conversion to endocrine lineage cells. The degree of induction was evaluated by detecting insulin-producing cells. Using a miRCURY LNA microRNA Array (Exiqon), the miRNA expression profiles were comprehensively analyzed. At 10 days after induction, insulin-producing cells were detected. Based on the expression profiles, eight miRNA probes showing significant differences at 10 days after induction compared with their pre-induction baseline values were extracted after filtering. Notably, miR-183 was downregulated by less than 40% after induction. Following a target scan of miR-183, we identified 242 conserved targets, including molecules crucial for the development of pancreatic beta-cells such as Foxo1. These findings indicate that dental pulp cells have potential for direct reprogramming to insulin-producing cells. This potential ability for direct reprogramming of dental pulp cells shows promise for clinically relevant tissue engineering materials.

Parp-1 deficiency in ES cells promotes invasive and metastatic lesions accompanying induction of trophoblast giant cells during tumorigenesis in uterine environment.

Embryonic stem (ES) cells deficient in poly(ADP-ribose) polymerase-1 (Parp-1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp-1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild-type (Parp-1(+/+) ) and Parp-1(-/-) ES cells. The weights of the tumors derived from Parp-1(-/-) ES cells were lower than those of the tumors derived from Parp-1(+/+) ES cells (P < 0.05). The Parp-1(-/-) tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp-1(-/-) tumors, but not for the Parp-1(+/+) tumors (P < 0.05). Invasions were more frequently observed with the Parp-1(-/-) tumors compared with the Parp-1(+/+) tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp-1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.

Involvement of P2X receptors in the regulation of insulin secretion, proliferation and survival in mouse pancreatic ß-cells.

In order to clarify the functional role of ionotropic purinergic (P2X) receptors in pancreatic ß-cells, we examined the effect of several P2 receptor agonists and antagonists on insulin secretion by mouse pancreatic islets, mouse Beta-TC6 cell proliferation and survival of dispersed islet cells in culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the expression of mRNAs of P2X(4) receptor in mouse islets and P2X(1), P2X(2), P2X(3), P2X(4), P2X(5) and P2X(7) receptors in Beta-TC6 cells. The presence of P2X(4) receptor proteins in islets and Beta-TC6 cells was confirmed by immunofluorescent staining and Western blot analysis. We have previously found that the functional P2Y(1) receptor but not P2Y(2) and P2Y(4) receptors was present in islets. In this study we found that a nonspecific P2 receptor agonist, ATP (1 µM) stimulated insulin secretion by islets in the presence of high glucose (20 mM) in culture. The effect of ATP was partially inhibited by a P2 receptor antagonist PPADS as well as a P2Y(1) receptor antagonist MRS2179. In addition, a P2X(4) receptor potentiator ivermectin per se augmented glucose-induced insulin secretion and slightly potentiated the effect of ATP. These results suggested the involvement of P2Y(1)and P2X receptors. We also found that ATP inhibited proliferation of Beta-TC6 cells in a concentration-dependent manner during 72 h culture. The inhibitory effect of ATP was completely reversed by PPADS and partially by treating cells with small interfering RNA targeted for P2X(4) receptor mRNA. Furthermore, we found that the phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) was suppressed by treatment with ATP in Beta-TC6 cells. In addition, we found that ATP reduced the cell viability and DNA synthesis of islet cells in culture. The effect of ATP on the cell viability was blocked by PPADS or MRS2179. These results suggested that P2X receptors as well as the P2Y(1) receptor played a role in the modulation of insulin secretion, proliferation and cell viability in mouse pancreatic ß-cells.

Gene expression profile of dental pulp cells during differentiation into an adipocyte lineage.

Gene regulation during in vitro differentiation into adipocytes was examined in rat dental pulp-derived cells. Insulin, 3-isobutyl-1-methylxanthine, and dexamethasone were added to induce adipogenesis. Cells containing lipid droplets were observed after induction as in 3T3 L1 cells. Rat dental pulp-derived cells showed their potential to differentiate into adipocytes in vitro. In both types of cells, the pluripotent markers Oct-3/4 and Sox2 were downregulated during differentiation, whereas the expression of Nanog was not significantly changed during differentiation. Interestingly, in the dental pulp-derived cells, the level of Oct-3/4 was transiently induced at 1 week after induction and then significantly decreased during differentiation. Based on the expression profiles determined using GeneChip Arrays, 3418 probes across 10 clusters showed a difference in expression at 1, 2, and 3 weeks after induction versus before induction. Notably, genes in the PPAR signaling pathway including Pparγ, Fabp4, and the C/EBP family were upregulated by more than 3-fold. Upregulation of the PPAR pathways seems to be a critical signal transduction pathway in this differentiation system. These findings indicate that dental pulp-derived cells are a potential source of adipogenic cells, and their gene expression profile could be useful in future regenerative medicine applications.

Dilazep decreases lipopolysaccharide-induced nitric oxide and TNF-alpha synthesis in RAW 264 cells.

Dilazep dihydrochloride (dilazep) is used to treat ischemic dysfunction, although the mechanisms underlying the anti-inflammatory effects of the drug have not yet been elucidated. The present study evaluated the anti-inflammatory effect of dilazep. Dilazep suppressed the production of nitric oxide (NO) and the expression of TNF-alpha mRNA by lipopolysaccharide (LPS) in RAW 264 cells. However, 1400W, an inducible NO synthase inhibitor, suppressed the production of NO but did not suppress the expression of TNF-alpha mRNA following treatment with LPS. Caffeine, an adenosine antagonist, restored LPS-stimulated NO synthesis, which is suppressed by dilazep. Therefore, these observations may suggest that the suppression of NO synthesis after dilazep treatment in RAW 264 cells is caused by the inhibition of TNF-alpha expression via adenosine receptors.

A novel chemoattractant lectin, karatoxin, from the dorsal spines of the small scorpionfish Hypodytes rubripinnis.

A chemoattractant lectin from the dorsal spines of the redfin velvetfish, Hypodytes rubripinnis, was isolated using a combination of affinity chromatography techniques. The glycoprotein, with a molecular mass of 110 kDa, is named Karatoxin. Karatoxin caused agglutination of rabbit erythrocytes. This agglutination was effectively inhibited by D-mannose. In addition, Karatoxin exhibited not only mitogenic activity in the presence of murine splenocytes, but also chemotactic activity in the presence of guinea-pig neutrophils and macrophages. Thus, Karatoxin appears to be a novel chemoattractant lectin. These results suggest that the redfin velvetfish Hypodytes rubripinnis may be a novel source of biologically active substances.

5-Aza-2'-deoxycytidine treatment induces skeletal myogenic differentiation of mouse dental pulp stem cells.

OBJECTIVE: Tissue stem cells in dental pulp are assumed to possess differentiation potentials similar to mesenchymal stem cells (MSCs). The aim of this in vitro study is to examine the differentiation potentials of mouse dental pulp stem cells (DPSCs) and develop the appropriate differentiation assay systems for skeletal myogenic differentiation of these cells. METHODS: Dental pulps were extracted from mandible sections of C57/BL6 mice, and adherent dental pulp cells were isolated in culture. These cells were cultured in osteogenic or adipogenic induction medium to induce osteogenic and adipogenic differentiation. On the other hand, the skeletal myogenic differentiation potential of these cells was investigated using different conditions, such as serum-free medium, Myod1 overexpression, or 5-Aza-2'-deoxycytidine (5-Aza) treatment for DNA demethylation. Muscle-specific transcriptional factor expression was evaluated by RT-PCR, and myotube formation and myosin heavy chain expression were evaluated by phase-contrast microscopy and immunofluorescence staining, respectively. RESULTS: The adherent dental pulp cells exhibited a proliferative capacity and they showed osteogenic and adipogenic differentiation as seen in previous studies. Although the expression of Myod1 mRNA and myotube formation was not detected in serum-free conditions, the forced expression of Myod1 up-regulated the expression of Myogenin and Pax7 mRNA. However, myotube formation was not confirmed. Interestingly, myosin heavy chain expression and myotube formation were observed following 5-Aza treatment of these cells. CONCLUSIONS: These results demonstrated that mouse DPSCs possess MSC-like differentiation potential. DNA demethylation induced by 5-Aza treatment resulted in the skeletal muscle differentiation in mouse DPSCs, suggesting that DNA demethylation might trigger this differential induction of mouse DPSCs.