Jui-Sheng Sun

Thermal decomposition and reconstitution of hydroxyapatite in air atmosphere

In this paper, the decomposition and reconstruction behavior of hydroxyapatite (HAP) during heating and cooling in air atmosphere were studied. The commercial HAP were chosen and gradually heated to 1500 degrees C and cooled to room temperature by a program controlled SiC heated furnace. X-ray diffraction (XRD) and Fourier-transformed infrared (FTIR) analysis were used to investigate the change of crystalline phases and functional groups of HAP at different temperatures. Weight change of samples was recorded by thermogravimetric analysis (TGA) during heating and cooling. The results revealed that HAP gradually releases its OH- ions and transforms into OHAP in the temperature of 1000-1360 degrees C. Above 1360 degrees C, the OHAP would decompose into TTCP and alpha TCP phase. The OH- stretching bands of HAP could be traced by FTIR even at the temperature of 1350 degrees C which indicates HAP decomposition. HAP does not dehydrate completely before decomposition. We speculated that some oxyapatite (OAP) might be formed during dehydration with a great amount of OHAP still left in the system even up to the temperature of decomposition. In the temperature range of 1400-1500 degrees C, there was no significant difference in XRD patterns, only TTCP and alpha TCP crystalline phases were observed. When the HAP gradually cools from 1500 degrees C, a part of TTCP and alpha TCP would directly reconstruct into OAP around 1350 degrees C. OAP existed in the temperature range of 1350-1300 degrees C during cooling. When the temperature decreased to 1290 degrees C, a part of TTCP and alpha TCP reconstructed into OHAP by rehydration reaction and OAP were rehydrated into OHAP as well. At 1100 degrees C, the rest of TTCP and alpha TCP reconstitutes into HAP. As the temperature decreases, the OHAP is gradually rehydrated and reconstituted into HAP.

Arthropod steroid hormone (20-Hydroxyecdysone) suppresses IL-1β- induced catabolic gene expression in cartilage

BackgroundIn osteoarthritis (OA), the imbalance of chondrocytes’ anabolic and catabolic factors can induce cartilage destruction. Interleukin-1 beta (IL-1β) is a potent pro-inflammatory cytokine that is capable of inducing chondrocytes and synovial cells to synthesize MMPs. The hypoxia-inducible factor-2alpha (HIF-2alpha, encoded by Epas1) is the catabolic transcription factor in the osteoarthritic process. The purpose of this study is to validate the effects of ecdysteroids (Ecd) on IL-1β- induced cartilage catabolism and the possible role of Ecd in treatment or prevention of early OA.MethodsChondrocytes and articular cartilage was harvested from newborn ICR mice. Ecd effect on chondrocytes viability was tested and the optimal concentration was determined by MTT assay. The effect of HIF-2α (EPAS1) in cartilage catabolism simulated by IL-1β (5 ng/ml) was evaluated by articular cartilage explants culture. The effects of Ecd on IL-1β-induced inflammatory conditions and their related catabolic genes expression were analyzed.ResultsInterleukin-1β (IL-1β) treatment on primary mouse articular cartilage explants enhanced their Epas1, matrix metalloproteinases (MMP-3, MMP-13) and ADAMTS-5 genes expression and down-regulated collagen type II (Col2a1) gene expression. With the pre-treatment of 10−8M Ecd, the catabolic effects of IL-1β on articular cartilage were scavenged.ConclusionIn conclusions, Ecd can reduce the IL-1β-induced inflammatory effect of the cartilage. Ecd may suppress IL-1β- induced cartilage catabolism via HIF-2α pathway.

STUDIES OF PHOTOKILLING OF BACTERIA USING TITANIUM DIOXIDE NANOPARTICLES

Metal pins used to apply skeletal traction or external fixation devices protruding through skin are susceptible to the increased incidence of pin site infection. In this work, we tried to establish the photokilling effects of titanium dioxide (TiO2) nanoparticles on an orthopedic implant with an in vitro study. In these photocatalytic experiments, aqueous TiO2 was added to the tested microorganism. The time effect of TiO2 photoactivation was evaluated, and the loss of viability of five different bacteria suspensions (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae, and Bacteroides fragilis) was examined by the viable count procedure. The bactericidal effect of TiO2 nanoparticle-coated metal plates was also tested. The ultraviolet (UV) dosage used in this experiment did not affect the viability of bacteria, and all bacteria survived well in the absence of TiO2 nanoparticles. The survival curve of microorganisms in the presence of TiO2 nanoparticles showed that nearly complete killing was achieved after 50 min of UV illumination. The formation of bacterial colonies above the TiO2 nanoparticle-coated metal plates also decreased significantly. In this study, we clearly demonstrated the bactericidal effects of titanium dioxide nanoparticles. In the presence of UV light, the titanium dioxide nanoparticles can be applicable to medical facilities where the potential for infection should be controlled.

An ultra-weak chemiluminescence study on oxidative stress in rabbits following acute thermal injury

It is not easy to detect oxygen free radicals directly because of their very short half-life. In the present study, a sensitive ultra-weak chemiluminescence detector was used to detect the generation of oxygen free radicals following thermal injury. Twelve New Zealand white rabbits were used in this study. After anesthesia, the bilateral hind-limbs were exposed to 100 degrees C water for 30 s. Six control animals were exposed to 22 degrees C water to act as a control. The chemiluminescence of whole blood and visceral organs were measured with both luminol-amplified t-butyl hydroperoxide-initiated and lucigenin-initiated methods. The results showed that chemiluminescence of blood was affected significantly by acute thermal injury. The chemiluminescence of blood increased significantly at 1 h following acute thermal injury, reached a peak at 2 h, then decreased but still remained above the control level at 4 h following thermal injury. The results for TBHP-initiated chemiluminescence from visceral organs following acute thermal injury were much higher than that of the control rabbits. The effects of lucigenin-initiated tissue chemiluminescence following acute thermal injury were not statistically significant. It is suggested that the decreased vascular antioxidant activity following local thermal injury is partially contributed by the superoxide pathway; while, the remote pathophysiologic events are mediated by the defective scavenging defenses.

The effects of calcium phosphate particles on the growth of osteoblasts.

With advances in ceramics technology, calcium phosphate bioceramics have been applied as bone substitutes for several decades. The focus of this work is to elucidate the biocompatibility of the particulates of various calcium phosphate cytotoxicities. Four different kinds of calcium phosphate powders, including beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA), beta-dicalcium pyrophosphate (beta-DCP), and sintered beta-dicalcium pyrophosphate (SDCP), were tested by osteoblast cell culture. The results were analyzed by cell count, concentration of transforming growth factor-beta 1 (TGF-beta 1), alkaline phosphatase (ALP), and prostaglandin E2 (PGE2) in culture media. The changes were most significant when osteoblasts were cultured with beta-TCP and HA bioceramics. The changes in cell population of the beta-TCP and HA were quite low in the first 3 days, then increased gradually toward the seventh day. The changes in TGF-beta 1 concentration in culture medium inversely related to the changes in cell population. The ALP titer in the culture media of the beta-TCP and HA were quite high in the first 3 days, then decreased rapidly between the third and seventh days. The concentrations of PGE2 in the culture media tested were quite high on the first day, decreased rapidly to the third day, and then gradually until the seventh day. The changes in the beta-DCP and SDCP were quite similar to those of HA and beta-TCP but much less significant. We conclude that HA and beta-TCP have an inhibitory effect on the growth of osteoblasts. The inhibitins effects of the HA and beta-TCP powders on the osteoblast cell cultures possibly are mediated by the increased synthesis of PGE2.

Icariin isolated from Epimedium pubescens regulates osteoblasts anabolism through BMP-2, SMAD4, and Cbfa1 expression

Epimedii herba is one of the most frequently used herbs in formulas prescribed for the treatment of osteoporosis in China. The main active flavonoid glucoside extracted from Epimedium pubescens is Icariin, which has been reported to enhance bone healing and reduce osteoporosis occurrence. However, the detailed molecular mechanisms remain unclear. In this present study, we examine the molecular mechanisms of icariin by using primary osteoblast cell cultures obtained from adult mice. The osteoblast cells were harvested from 8-month old female Imprinting Control Region (ICR) mice. The effects of icariin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide (NO) and caspase-3 were analyzed, along with the gene expressions of bone morphogenetic protein-2 (BMP-2), SMAD4, Cbfa1/Runx2, OPG, and RANKL. The viability of the osteoblasts reached its maximum at 10(-8)M icariin. At this concentration, icariin increased the proliferation and matrix mineralization of osteoblasts and promoted NO synthesis. With icariin treatment, the BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expressions were up-regulated; the RANKL gene expression was however down-regulated. Concurrent treatment involving the BMP antagonist (Noggin) or the NOS inhibitor (L-NAME) diminished the icariin-induced cell proliferation, ALP activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, RANKL gene expressions. In this study, we demonstrate that in vitro icariin is a bone anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions. This effect may contribute to its action on the induction of osteoblasts proliferation and differentiation, resulting in bone formation.

Simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/BMP-2 signaling pathway

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate, a rate-limiting step in cholesterol synthesis. Statins are able to reduce cardiovascular risk in hypercholesterolemic patients. In recent years, the possible effect of statins on bone tissue has received particular attention. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Our hypothesis is that simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/bone morphogenic protein (BMP)-2 signaling pathway. The viability and differentiation of osteoblasts were examined by mitochondrial activity assay, alkaline phosphatase (ALP) activity, and gene expression. The associated signaling pathways were analyzed by cytoplasmic and membrane proteins manifestation. After administration of 10(-6) M simvastatin, the ALP activity was significantly enhanced, and the expression of BMP-2, ALP, sialoprotein, and type I collagen genes were up-regulated. After simvastatin treatment, both the RasGRF1 and phospho-RasGRF1 in the cytoplasm decreased significantly, whereas those on the plasma membrane increased. A marked increase in membranous GAP-associated protein (P190) and the activated form of both phospho-extracellular signal-regulated kinase1/2 and phospho-Smad1 were also noted. In conclusion, this study shows that statins pose a positive effect on the metabolism of osteoblasts. Simvastatin can promote osteoblast viability and differentiation via membrane-bound Ras/Smad/Erk/BMP-2 pathway. Statins stimulate osteoblast differentiation in vitro and may be a promising drug for the treatment of osteoporosis in the future.

In vitro effects of low-intensity ultrasound stimulation on the bone cells

Mechanical perturbations serve as extracellular signals to a variety of cells, including bone cells. Low-intensity pulsed ultrasound produces significant multifunctional effects that are directly relevant to bone formation and resorption. Ultrasound stimulation has been shown to accelerate bone-defect healing and trabecular bone regeneration. In this study, we use an in vitro bone cell culture model to investigate the effect of low-intensity pulsed ultrasound. The rat alveolar mononuclear cell-calvaria osteoblast coculture system was used in this study. Before treatment, the bone cells were cultured for 3 days to facilitate their attachment and differentiation. Then, ultrasound exposure (frequency = 1 MHz, intensity = 0.068 W/cm(2)) or sham exposure for 20 min per day was applied until the end of the experiment. Half of the culture media were obtained on the 4th, 5th, 6th, 7th, 8th, 9th, and 10th days for the analysis of cytokines and biochemical parameters. At the end of the experiment, cells were fixed and stained for identification and quantification of the osteoblast and osteoclast cells. After low-intensity pulse ultrasound stimulation, the osteoblast cell counts were significantly increased, whereas the osteoclast cell counts were significantly decreased. The total alkaline phosphatase amount in the culture medium was increased after 7 days of ultrasound stimulation, and tumor necrosis factor-alpha in ultrasound-stimulated bone cells was significantly increased after the 7th day of culture and reached 474.77% of the control medium on the 10th day of culture. The results of this study suggest that low-intensity ultrasound treatment may have a stimulatory effect on bone-healing processes.

Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE2 synthesis

Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the detail molecular mechanisms of icariin on lipopolysaccharide (LPS)-induced osteolysis. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis. After treatment with icariin, the activity of osteoclasts differentiation maker, tatrate resistances acid phosphatease (TRAP), significantly decreased at the concentration of 10(-8)M. Icariin (10(-8)M) reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) expression. The gene expression of osteoprotegerin (OPG) was up-regulated, while receptor activator of NF-κB ligand (RANKL) was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and prostaglandin E(2) (PGE(2)). In addition, icariin had a dominant repression effect on LPS-induced hypoxia inducible factor-1α (HIF-1α) expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and I-kappa-B-alpha (IκBα), but increased the activation of p38. In conclusion, we demonstrated that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway.

Biological effects and cytotoxicity of the composite composed by tricalcium phosphate and glutaraldehyde cross-linked gelatin.

The purpose of this study was to prepare and evaluate the feasibility and cytocompatibility of a composite (GTG) as a large defect bone substitute. The composite is combined with tricalcium phosphate ceramic particles and glutaraldehyde cross-linked gelatin. Gelatin had been reported as an adhesive and biocompatible binder that could accelerate the recovery of damaged soft tissue, but the effects of gelatin when acting on the bone tissue is not clear. Thus, it is necessary to determine if the substances released from the GTG composite can facilitate the growth of bone cells. The substances released from the GTG composites after being soaked in deionized distilled water were analyzed by gas chromatography (GC), ultraviolet and visible absorption spectroscopy (UV-VIS), and inductive-coupled plasma-atomic emission spectrometry (ICP-AES). The cytotoxicity of the GTG composites was assessed by coculture of rat osteoblasts in vitro. Extracts were obtained by soaking the GTG composites in deionized distilled water for 1, 2, 4, 7, 14, 28 and 42 d. The extract mixed with complete medium in a ratio of 1:1 was added into the cell culture wells containing 1 x 10(4) cells ml(-1) osteoblasts. After culturing for 2 days, the cells attached to the surface of wells were trypsinized and the number calculated by the Neubauer counting-chamber under the optical microscope. Finally, three samples in each GTG group were examined by scanning electron microscopy (SEM) to observe the morphology of the osteoblasts attached to the surfaces of GTG composites. The examinations of osteoblasts cocultured with the developed GTG composites were used to decide the ideal concentration of glutaraldehyde as a cross-linking agent. The results of extracts cocultured with osteoblasts showed that the extracts obtained from the 2, 4 and 8% glutaraldehyde cross-linked GTG composites would inhibit the growth of osteoblasts in the first 4 soaking days. During the 4-7 days soaking, the cell numbers quickly increased with the soaking time, thereafter, the cell numbers almost reached a constant value. In the analyses of substances released from the GTG composites, it was found that the gelatin and calcium were gradually released from the GTG composites, which were supposed to be nutritious for the growth of the osteoblast. The results of osteoblasts cocultured with the GTG composites showed that the concentration of glutaraldehyde used as a cross-linking agent should be lower than 8%. Compared to the GTF (composite combined with tricalcium phosphate ceramic particles and formaldehyde cross-linked gelatin), GTG composites were much suitable for a large defect bone substitute in the near future.