Young Ki Min

Preparation and characterization of PLGA microspheres by the electrospraying method for delivering simvastatin for bone regeneration

Microparticles formulated from poly (D,L-lactic-co-glycolide) (PLGA), a biodegradable polymer, have been investigated extensively as a drug delivery system. In this study, solid tiny PLGA microspheres were fabricated using the electrospraying method. PLGA polymer was dissolved in dichloromethane (DCM), and the solution was electrosprayed. The electrospraying conditions were adjusted so that the stream ejected from the needle was divided into spheres instead of continuous fibers or irregular-shaped particles. Several experiments were carried out using the PLGA-DCM source solution with different amounts of simvastatin (SIM), a drug that enhances bone regeneration, to understand this drug delivery system. The surface morphology and microstructure of the microspheres formed were characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and differential scanning calorimetry. The in vitro experiments on drug loading and drug release behavior of the microspheres suggested a drug encapsulation efficacy >90%. The drug was continuously released from the microspheres for >3 weeks. Other experiments, such as MTT, cell attachment and proliferation and reverse transcription-polymerase chain reaction showed good biocompatibility of the electrosprayed PLGA microspheres, which increased in the presence of SIM. Thus, electrosprayed PLGA microspheres have potential as a drug delivery system and application in bone tissue engineering.

Fabrication of polyvinyl alcohol/gelatin nanofiber composites and evaluation of their material properties

Electrospinning of polyvinyl alcohol (PVA), gelatin (GE), and a PVA/GE blend was conducted with the aim of fabricating biodegradable scaffolds for tissue engineering. The process parameters including the concentration of GE in PVA/GE blends, electrical field, and tip-to-collector distance (TCD) were investigated. Electrospinning processes were conducted at three different GE concentrations (PVA/GE = 2/8, 6/4, and 8/2), and the voltage and TCD were varied from 18 to 24 kV and 7 to 20 cm, respectively. The average diameter of the electrospun PVA, GE, and PVA/GE blend fibers ranged from 50 to 150 nm. The TCD had significant effects on the average diameter of the PVA/GE nanofiber, while changes in the voltage did not significantly affect the diameter of the PVA/GE nanofiber. The miscibility of the PVA/GE blend fibers was examined by differential scanning calorimetry, and X-ray diffraction was used to determine the crystallinity of the membrane. Tensile strength was measured to evaluate the physical properties of the membrane. Based on the combined results of this study, the PVA/GE membrane holds great promise for use in tissue engineering applications, especially in bone or drug delivery systems.

Bioactive glass incorporation in calcium phosphate cement-based injectable bone substitute for improved in vitro biocompatibility and in vivo bone regeneration.

In this work, we fabricated injectable bone substitutes modified with the addition of bioactive glass powders synthesized via ultrasonic energy-assisted hydrothermal method to the calcium phosphate-based bone cement to improve its biocompatibility. The injectable bone substitutes was initially composed of a powder component (tetracalcium phosphate, dicalcium phosphate dihydrate and calcium sulfate dehydrate) and a liquid component (citric acid, chitosan and hydroxyl-propyl-methyl-cellulose) upon which various concentrations of bioactive glass were added: 0%, 10%, 20% and 30%. Setting time and compressive strength of the injectable bone substitutes were evaluated and observed to improve with the increase of bioactive glass content. Surface morphologies were observed via scanning electron microscope before and after submersion of the samples to simulated body fluid and increase in apatite formation was detected using x-ray diffraction machine. In vitro biocompatibility of the injectable bone substitutes was observed to improve with the addition of bioactive glass as the proliferation/adhesion behavior of cells on the material increased. Human gene markers were successfully expressed using real time-polymerase chain reaction and the samples were found to promote cell viability and be more biocompatible as the concentration of bioactive glass increases. In vivo biocompatibility of the samples containing 0% and 30% bioactive glass were evaluated using Micro-CT and histological staining after 3 months of implantation in male rabbits’ femurs. No inflammatory reaction was observed and significant bone formation was promoted by the addition of bioactive glass to the injectable bone substitute system.

Bone formation of a porous Gelatin-Pectin-biphasic calcium phosphate composite in presence of BMP-2 and VEGF.

A composite scaffold of gelatin (Gel)-pectin (Pec)-biphasic calcium phosphate (BCP) was fabricated for the successful delivery of growth factors. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were coated on the Gel-Pec-BCP surface to investigate of effect of them on bone healing. Surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive X-ray spectroscopy. The results obtained from Fourier transform infrared spectroscopy showed that BMP-2 and VEGF were successfully coated on Gel-Pec-BCP hydrogel scaffolds. MC3T3-E1 preosteoblasts were cultivated on the scaffolds to investigate the effect of BMP-2 and VEGF on cell viability and proliferation. VEGF and BMP-2 loaded on Gel-Pec-BCP scaffold facilitated increased cell spreading and proliferation compared to Gel-Pec-BCP scaffolds. In vivo, bone formation was examined using rat models. Bone formation was observed in Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds within 4 weeks, and was greatest with Gel-Pec-BCP/BMP-2 scaffolds. In vitro and in vivo results suggest that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds could enhance bone regeneration.

Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering

In order to augment bone formation, a new biodegradable scaffold system was fabricated using different ratios of hydroxyapatite (HAp) blended with synthetic polymer polycaprolactone (PCL) and natural polymer gelatin (GE) followed by electrospinning method. Three different concentrations of HAp were used in PCL/GE to obtain a blend of 10, 30, and 50% (w/v) HAp–PCL/GE. These HAp-loaded PCL/GE blends were then compared with PCL/GE blends by different mechanical and biological in vitro and in vivo studies to understand the applicability of the system. Scanning electron microscopy, X-ray diffraction analysis, and tensile strength measurement were done to obtain physical properties. Fifty Percent HAp–PCL/GE blends possessed the highest mechanical strength. In vitro cytotoxicity and proliferation of osteoblast cells on the PCL/GE and HAp–PCL/GE scaffolds were examined and shown that addition of HAp in PCL/GE was beneficial by increasing cell viability (>85%) proliferation and cell-surface attachment. Expression of collagen and osteopontin was also found higher in 50% HAp–PCL/GE blends than the others. On the other hand, in vivo bone formation was examined using rat models and increased bone formation was observed in 50% HAp–PCL/GE blends within 6 weeks. Based on the combined results of this study, HAp–PCL/GE membranes were found to hold great promise for use in tissue engineering applications, especially in bone tissue engineering.

Effect of the Heat-exposure on Peripheral Sudomotor Activity Including the Density of Active Sweat Glands and Single Sweat Gland Output.

Tropical inhabitants are able to tolerate heat through permanent residence in hot and often humid tropical climates. The goal of this study was to clarify the peripheral mechanisms involved in thermal sweating pre and post exposure (heat-acclimatization over 10 days) by studying the sweating responses to acetylcholine (ACh), a primary neurotransmitter of sudomotor activity, in healthy subjects (n=12). Ten percent ACh was administered on the inner forearm skin for iontophoresis. Quantitative sudomotor axon reflex testing, after iontophoresis (2 mA for 5 min) with ACH, was performed to determine directly activated (DIR) and axon reflex-mediated (AXR) sweating during ACh iontophoresis. The sweat rate, activated sweat gland density, sweat gland output per single gland activated, as well as oral and skin temperature changes were measured. The post exposure activity had a short onset time (p<0.01), higher active sweat rate [(AXR (p<0.001) and DIR (p<0.001)], higher sweat output per gland (p<0.001) and higher transepidermal water loss (p<0.001) compared to the pre-exposure measurements. The activated sweat rate in the sudomotor activity increased the output for post-exposure compared to the pre-exposure measurements. The results suggested that post-exposure activity showed a higher active sweat gland output due to the combination of a higher AXR (DIR) sweat rate and a shorter onset time. Therefore, higher sudomotor responses to ACh receptors indicate accelerated sympathetic nerve responsiveness to ACh sensitivity by exposure to environmental conditions.

HAp granules encapsulated oxidized alginate-gelatin-biphasic calcium phosphate hydrogel for bone regeneration.

Bone repair in the critical size defect zone using 3D hydrogel scaffold is still a challenge in tissue engineering field. A novel type of hydrogel scaffold combining ceramic and polymer materials, therefore, was fabricated to meet this challenge. In this study, oxidized alginate-gelatin-biphasic calcium phosphate (OxAlg-Gel-BCP) and spherical hydroxyapatite (HAp) granules encapsulated OxAlg-Gel-BCP hydrogel complex were fabricated using freeze-drying method. Detailed morphological and material characterizations of OxAlg-Gel-BCP hydrogel (OGB00), 25wt% and 35wt% granules encapsulated hydrogel (OGB25 and OGB35) were carried out for micro-structure, porosity, chemical constituents, and compressive stress analysis. Cell viability, cell attachment, proliferation and differentiation behavior of rat bone marrow-derived stem cell (BMSC) on OGB00, OGB25 and OGB35 scaffolds were confirmed by MTT assay, Live-Dead assay, and confocal imaging in vitro experiments. Finally, OGB00 and OGB25 hydrogel scaffolds were implanted in the critical size defect of rabbit femoral chondyle for 4 and 8 weeks. The micro-CT analysis and histological studies conducted by H&E and Massons trichrome demonstrated that a significantly higher (***p<0.001) and earlier bone formation happened in case of 25% HAp granules encapsulated OxAlg-Gel-BCP hydrogel than in OxAlg-Gel-BCP complex alone. All results taken together, HAp granules encapsulated OxAlg-Gel-BCP system can be a promising 3D hydrogel scaffold for the healing of a critical bone defect.

Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends

In this study, the optimized formulations of polycaprolactone (PCL) combined with poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL), and biphasic calcium phosphate (BCP) were analyzed in terms of cytocompatibility with bone-related cells, hemocompatibility, and in vivo bone-regenerating capacity to determine their potentials for bone tissue regeneration. Fiber morphology of PCL/GEL and PCL/BCP electrospun mats considerably differs from that of the PCL membrane. Based on the contact angle analyses, the addition of GEL and PLGA was shown to reduce the hydrophobicity of these membranes. The assessment of in vitro cytocompatibility using MC3T3-E1 cells indicated that all of the membranes were suitable for pre-osteoblast proliferation and adhesion, with PCL/BCP having a significantly higher reading after seven days of incubation. The results of the in vitro hemocompatibility of the different fibrous scaffolds suggest that coagulation and platelet adhesion were higher for hydrophobic membranes (PCL and PCL/PLGA), while hemolysis can be associated with fiber morphology. The potential of the membranes for bone regeneration was determined by analyzing the microCT data and tissue sections of samples implanted in 5 mm sized defects (one and two months). Although all of the membranes were suitable for pre-osteoblast proliferation, in vivo bone regeneration after two months was found to be significantly higher in PCL/BCP (p < 0.001).

Heat acclimation affects circulating levels of prostaglandin E2, COX-2 and orexin in humans.

We examined serum levels of prostaglandin E2 (PGE2), cyclooxygenase (COX)-2 and orexin before and after heat acclimation (HA) to test the hypothesis that decreased basal body temperature due to HA correlate with circulating levels of these key thermoregulatory molecules. Nine healthy human male volunteers were recruited (age, 21.9±2.7 years). The subjects were exposed to half-body immersion in hot water (42±0.5°C) at the same time of day (2-5p.m.) on alternate days for 3 weeks. The HA protocol included 10 bouts of 30min immersion. All experiments were performed in an automated climate chamber (temperature, 26.0±0.5°C; relative humidity, 60±3.0%; air velocity, <1m/s). Tympanic and skin temperatures were measured, and mean body temperature was calculated. The difference in body weight was used to estimate total sweat loss. Serum levels of PGE2, COX-2 and orexin were analyzed before and after HA. Body temperature decreased significantly (P<0.05) after HA, whereas sweat volume increased significantly (P<0.01). Serum PGE2, COX-2 and orexin concentrations decreased significantly compared to those at pre-acclimation (P<0.001, P<0.01, P<0.01, respectively). Our data suggest that decreased basal body temperature after HA is associated with decreases in thermoregulatory molecules, such as PGE2, COX-2 and orexin.

Resistance to reinfection in rats induced by irradiated metacercariae of Clonorchis sinensis

A study was made to observe the association between the resistance to reinfection induced by irradiated metacercariae (MC) of Clonorchis sinensis and antigen specific Th1- and Th2-type cytokine productions in rats. Rats were infected with 20 MC of C. sinensis, previously exposed to a single dose of gamma irradiation, which varied from 0 to 100 Gy. All of them, single dose of 12 Gy showed higher IgG antibody titer with lowest worm recovery. Thus, 50 MC were used to challenge infection in rats previously infected with 20 MC irradiated at 12 Gy and the highest resistance to challenge infection was observed. The results of lymphocyte proliferation with specific antigen, ES Ag were shown no difference of proliferative responses as compared with primary and challenge infection at 12 Gy irradiation dose. In the case of cytokines production were observed that interferon (IFN-gamma) and interlukin (IL-2) were significantly enhanced, while IL-4 and IL-10 was almost unchanged to make comparison between primary and secondary infection at 12 Gy irradiation dose. In conclusion, the single dose of 12 Gy could be adopted for induction of the highest resistance to challenge infection. Up-regulation of Th1 type cytokines, IFN-gamma and IL-2 may be affected to develop vaccine by irradiated MC.