Combined Application of Prototype Ultrasound and BSA-Loaded PLGA Particles for Protein Delivery.

PURPOSE: To develop an in vitro culture system for tissue engineering to mimic the in vivo environment and evaluate the applicability of ultrasound and PLGA particle system. METHODS: For tissue engineering, large molecules such as growth factors for cell differentiation should be supplied in a controlled manner into the culture system, and the in vivo microenvironment need to be reproduced in the system for the regulation of cellular function. In this study, portable prototype ultrasound with low intensity was devised and tested for protein release from bovine serum albumin (BSA)-loaded poly(lactic-co-glycolic acid) (PLGA) particles. RESULTS: BSA-loaded PLGA particles were prepared using various types of PLGA reagents and their physicochemical properties were characterized including particle size, shape, or aqueous wetting profiles. The BSA-loaded formulation showed nano-ranged size distribution with optimal physical stability during storage period, and protein release behaviors in a controlled manner. Notably, the application of prototype ultrasound with low intensity influenced protein release patterns in the culture system containing the BSA-loaded PLGA formulation. The results revealed that the portable ultrasound set controlled by the computer could contribute for the protein delivery in the culture medium. CONCLUSIONS: This study suggests that combined application with ultrasound and protein-loaded PLGA encapsulation system could be utilized to improve culture system for tissue engineering or cell regeneration therapy.

Molecular Regulation of Betulinic Acid on α3ß4 Nicotinic Acetylcholine Receptors.

Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3ß4 nicotinic acetylcholine receptors (α3ß4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3ß4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the ß4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor's ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative.

Differential Regulation of Human Serotonin Receptor Type 3A by Chanoclavine and Ergonovine.

Irritable bowel syndrome (IBS) is a chronic disease that causes abdominal pain and an imbalance of defecation patterns due to gastrointestinal dysfunction. The cause of IBS remains unclear, but intestinal-brain axis problems and neurotransmitters have been suggested as factors. In this study, chanoclavine, which has a ring structure similar to 5-hydroxytryptamine (5-HT), showed an interaction with the 5-HT3A receptor to regulate IBS. Although its derivatives are known to be involved in neurotransmitter receptors, the molecular physiological mechanism of the interaction between chanoclavine and the 5-HT3A receptor is unknown. Electrophysiological experiments were conducted using a two-electrode voltage-clamp analysis to observe the inhibitory effects of chanoclavine on Xenopus oocytes in which the h5-HT3A receptor was expressed. The co-application of chanoclavine and 5-HT resulted in concentration-dependent, reversible, voltage-independent, and competitive inhibition. The 5-HT3A response induced by 5-HT was blocked by chanoclavine with half-maximal inhibitory response concentration (IC50) values of 107.2 µM. Docking studies suggested that chanoclavine was positioned close F130 and N138 in the 5-HT3A receptor-binding site. The double mutation of F130A and N138A significantly attenuated the interaction of chanoclavine compared to a single mutation or the wild type. These data suggest that F130 and N138 are important sites for ligand binding and activity. Chanoclavine and ergonovine have different effects. Asparagine, the 130th amino acid sequence of the 5-HT3A receptor, and phenylalanine, the 138th, are important in the role of binding chanoclavine, but ergonovine has no interaction with any amino acid sequence of the 5-HT3A receptor. The results of the electrophysiological studies and of in silico simulation showed that chanoclavine has the potential to inhibit the hypergastric stimulation of the gut by inhibiting the stimulation of signal transduction through 5-HT3A receptor stimulation. These findings suggest chanoclavine as a potential antiemetic agent for excessive gut stimulation and offer insight into the mechanisms of 5-HT3A receptor inhibition.

Cyclic stretch increases mitochondrial biogenesis in a cardiac cell line.

Unlike stable and immobile cell line conditions, animal hearts contract and relax to pump blood throughout the body. Mitochondria play an essential role by producing biological energy molecules to maintain heart function. In this study, we assessed the effect of heart mimetic cyclic stretch on mitochondria in a cardiac cell line. To mimic the geometric and biomechanical conditions surrounding cells in vivo, cyclic stretching was performed on HL-1 murine cardiomyocytes seeded onto an elastic micropatterned substrate (10% elongation, 0.5 Hz, 4 h/day). Cell viability, semi-quantitative Q-PCR, and western blot analyses were performed in non-stimulated control and cyclic stretch stimulated HL-1 cell lines. Cyclic stretch significantly increased the expression of mitochondria biogenesis-related genes (TUFM, TFAM, ERRα, and PGC1-α) and mitochondria oxidative phosphorylation-related genes (PHB1 and CYTB). Western blot analysis confirmed that cyclic stretch increased protein levels of mitochondria biogenesis-related proteins (TFAM, and ERRα) and oxidative phosphorylation-related proteins (NDUFS1, UQCRC, and PHB1). Consequently, cyclic stretch increased mitochondrial mass and ATP production in treated cells. Our results suggest that cyclic stretch transcriptionally enhanced mitochondria biogenesis and oxidative phosphorylation without detrimental effects in a cultured cardiac cell line.

Mechanical stimuli modulate intracellular calcium oscillations: a pathological model without chemical cues.

OBJECTIVE: To control the oscillatory behavior of the intracellular calcium ([Ca2+]i) concentration in endothelial cells via mechanical factors (i.e., various hydrostatic pressures) because [Ca2+]i in these cells is affected by blood pressure. RESULTS: Quantitative analyses based on real-time imaging showed that [Ca2+]i oscillation frequency and relative concentration increased significantly when 200 mm Hg pressure, mimicking hypertension, was applied for >10 min. Peak height and peak width decreased significantly at 200 mm Hg. These trends were more marked as the duration of the 200 mm Hg pressure was increased. However, no change was observed under normal blood pressure conditions 100 mm Hg. CONCLUSION: We generated a simple in vitro model to study [Ca2+]i behavior in relation to various pathologies and diseases by eliminating possible complicating effects induced by chemical cues.

A three-dimensional hierarchical scaffold fabricated by a combined rapid prototyping technique and electrospinning process to expand hematopoietic stem/progenitor cells.

OBJECTIVE: To investigate the expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood using extracellular matrix (ECM) protein-coated three-dimensional hierarchical scaffolds. RESULTS: The expansion of HSPCs was evaluated through total nucleated cell (TNC) expansion, immuno-phenotypic analysis, and clonogenic ability. After 7 days of culture, three-dimensional cultures with fibronectin-coated scaffolds achieved the highest fold increase in TNCs (164 ± 6.9 fold) and the highest CD45(+)CD34(+) (35 %) and CD34(+)CD38(-) (32 %) ratios. CONCLUSION: Three-dimensional hierarchical scaffolds were coated with ECM protein to simulate a biomimetic environment or niche, and had a significant effect on the expansion potential of HSPCs without changing their phenotype.

Shear stress and circumferential stretch by pulsatile flow direct vascular endothelial lineage commitment of mesenchymal stem cells in engineered blood vessels.

Understanding the response of mesenchymal stem cells (MSCs) in the dynamic biomechanical vascular environment is important for vascular regeneration. Native vessel biomechanical stimulation in vitro is thought to be the most important contributor to successful endothelial differentiation of MSCs. However, the appropriate biomechanical stimulation conditions for differentiating MSCs into ECs have not been fully investigated. To accomplish an in vivo-like loading environment, a loading system was designed to apply flow induced stress and induce hMSC differentiation in vascular cells. Culturing MSCs on tubular scaffolds under flow-induced shear stress (2.5 dyne/cm(2)) for 4 days results in increased mRNA levels of EC markers (vWF, CD31, VE-cadherin and E-selectin) after one day. Furthermore, we investigated the effects of 2.5 dyne/cm(2) shear stress followed by 3% circumferential stretch for 3 days, and an additional 5% circumferential stretch for 4 days on hMSC differentiation into ECs. EC marker protein levels showed a significant increase after applying 5% stretch, while SMC markers were not present at levels sufficient for detection. Our results demonstrate that the expression of several hMSC EC markers cultured on double-layered tubular scaffolds were upregulated at the mRNA and protein levels with the application of fluid shear stress and cyclic circumferential stretch.

Texture analyses show synergetic effects of biomechanical and biochemical stimulation on mesenchymal stem cell differentiation into early phase osteoblasts.

We investigated the structural complexity and texture of the cytoskeleton and nucleus in human mesenchymal stem cells during early phase differentiation into osteoblasts according to the differentiation-induction method: mechanical and/or chemical stimuli. For this, fractal dimension and a number of parameters utilizing the gray-level co-occurrence matrix (GLCM) were calculated based on single-cell images after confirmation of differentiation by immunofluorescence staining. The F-actin and nuclear fractal dimensions were greater in both stimulus groups compared with the control group. The GLCM values for energy and homogeneity were lower in fibers of the F-actin cytoskeleton, indicating a dispersed F-actin arrangement during differentiation. In the nuclei of both stimulus groups, higher values for energy and homogeneity were calculated, indicating that the chromatin arrangement was chaotic during the early phase of differentiation. It was shown and confirmed that combined stimulation with mechanical and chemical factors accelerated differentiation, even in the early phase. Fractal dimension analysis and GLCM methods have the potential to provide a framework for further investigation of stem cell differentiation.

Mexenone protects mice from LPS-induced sepsis by EC barrier stabilization.

Blood vessels permit the selective passage of molecules and immune cells between tissues and circulation. Uncontrolled inflammatory responses from an infection can increase vascular permeability and edema, which can occasionally lead to fatal organ failure. We identified mexenone as a vascular permeability blocker by testing 2,910 compounds in the Clinically Applied Compound Library using the lipopolysaccharide (LPS)-induced vascular permeability assay. Mexenone suppressed the LPS-induced downregulation of junctional proteins and phosphorylation of VE-cadherin in Bovine Aortic Endothelial Cells (BAECs). The injection of mexenone 1 hr before LPS administration completely blocked LPS-induced lung vascular permeability and acute lung injury in mice after 18hr. Our results suggest that mexenone-induced endothelial cell (EC) barrier stabilization could be effective in treating sepsis patients.

Mechanical stimulation and the presence of neighboring cells greatly affect migration of human mesenchymal stem cells.

There are few studies regarding the effects of mechanical stimulation on cell migration although biochemical factors have been widely studied. We have investigated the effects of intermittent hydrostatic pressure (IHP) on mesenchymal stem cell migration with or without neighboring endothelial cells (EC). IHP promoted MSCs migration and the neighboring ECs helped with this. However, when IHP was applied to MSCs cultured with ECs, the opposite effect was observed. The concentration of stromal-derived factor-1 culture in medium was measured to explain the obtained results. SDF-1 concentration increased as IHP increased when MSCs were cultured alone. However, it decreased as IHP increased when MSCs and ECs were co-cultured. These results indicate that the mechanical environment should be considered when studying the migration of a cell type along with its biochemical environment.

Induction of bone ingrowth with a micropore bioabsorbable suture anchor in rotator cuff tear: an experimental study in a rabbit model.

BACKGROUND: The bioabsorbable suture anchor is probably one of the most commonly used tools in arthroscopic shoulder operations. However, there is controversy about whether the bioabsorbable anchor is replaced by bone. The object of this study is to evaluate bone ingrowth into the micropore bioabsorbable suture anchor and the differences in the biomechanical properties of a micropore anchor and a nonpore anchor. MATERIALS AND METHODS: A total of 16 microsized holes (diameter, 250 ± 50 µm; depth, 0.2 mm) were made on the bioabsorbable anchors with a microdrill. Twelve adult New Zealand White rabbits were randomly divided into two groups: group A (n = 6), the nonpore bioabsorbable suture anchor group, and group pA (n = 6), the micropore bioabsorbable suture anchor group. Microcomputed tomography was used at 4 and 8 weeks postoperatively to evaluate ingrowth by bone volume fraction (BVF), which was measured by calculating the ratio of the total volume of bone ingrowth to that of the region of interest. For pullout strength testing, 3 additional rabbits (6 limbs) were used for mechanical testing. RESULTS: The mean BVF was higher in group pA (0.288 ± 0.054) than in group A (0.097 ± 0.006). The micropore anchor had a higher pullout strength (0.520 ± 0.294 N) than the nonpore anchor (0.275 ± 0.064 N). CONCLUSION: Micropore bioabsorbable suture anchors induced bone ingrowth and showed higher pullout strength, despite processing.

Assessment of Esophageal Reconstruction via Bioreactor Cultivation of a Synthetic Scaffold in a Canine Model.

OBJECTIVES: Using tissue-engineered materials for esophageal reconstruction is a technically challenging task in animals that requires bioreactor training to enhance cellular reactivity. There have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in initial epithelialization in the special environment of peristalsis. The purpose of this study was to evaluate the potential of an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a double-layered polymeric scaffold and a custom-designed mesenchymal stem cell-based bioreactor system in a canine model. METHODS: We fabricated a novel double-layered scaffold as a tissue-engineered esophagus using an electrospinning technique. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. After 3 days of cultivation using the bioreactor system, tissue-engineered artificial esophagus was transplanted into a partial esophageal defect (5×3 cm-long resection) in a canine model. RESULTS: Scanning electron microscopy (SEM) showed that the electrospun fibers in a tubular scaffold were randomly and circumferentially located toward the inner and outer surfaces. Complete recovery of the esophageal mucosa was confirmed by endoscopic analysis and SEM. Esophagogastroduodenoscopy and computed tomography also showed that there were no signs of leakage or stricture and that there was a normal lumen with complete epithelialization. Significant regeneration of the mucosal layer was observed by keratin-5 immunostaining. Alpha-smooth muscle actin immunostaining showed significantly greater esophageal muscle regeneration at 12 months than at 6 months. CONCLUSION: Custom-designed bioreactor cultured electrospun polyurethane scaffolds can be a promising approach for esophageal tissue engineering.

Salicylideneamino-2-thiophenol enhances osteogenic differentiation through the activation of MAPK pathways in multipotent bone marrow stem cell.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Therefore, the identification of specific stimulators of bone formation is of therapeutic significance in the treatment of osteoporosis. Salicylideneamino-2-thiophenol (Sal-2) consists of two benzene rings, has been reported to possess antioxidant activity, and is an effective remedy for fever and rheumatic diseases. However, until now the effects of osteoblastic bone formation by Sal-2 were unknown. In this study, we investigated the effects of Sal-2 on osteogenic differentiation of multipotent bone marrow stromal stem cells by alizarin red S staining for osteogenic differentiation, RT-PCR and western blot for alkaline phosphatase (ALP) activity and signaling pathways, FACS analysis and immunofluorescence staining for CD44 and CD51 expression, calcium assays, and immunofluorescence staining for signaling pathways. We found that Sal-2 enhanced the osteogenic differentiation of multipotent bone marrow stromal stem cells. Sal-2 treatment induced the expression and activity of ALP, and enhanced the levels of CD44 and CD51 expression as well as Ca2+ content, in multipotent bone marrow stromal stem cells. Moreover, we found that Sal-2-induced osteogenic differentiation and expression of osteogenesis-related molecules involve the activation of the MAPK and nuclear factor-κB pathways. Our findings provide insight into both the mechanism and effects of Sal-2 on osteogenic differentiation and demonstrate that Sal-2 may be a beneficial adjuvant in stimulating bone formation in osteoporotic diseases.

Scaffolds for bone tissue engineering fabricated from two different materials by the rapid prototyping technique: PCL versus PLGA.

Three dimensional tissue engineered scaffolds for the treatment of critical defect have been usually fabricated by salt leaching or gas forming technique. However, it is not easy for cells to penetrate the scaffolds due to the poor interconnectivity of pores. To overcome these current limitations we utilized a rapid prototyping (RP) technique for fabricating tissue engineered scaffolds to treat critical defects. The RP technique resulted in the uniform distribution and systematic connection of pores, which enabled cells to penetrate the scaffold. Two kinds of materials were used. They were poly(ε-caprolactone) (PCL) and poly(D, L-lactic-glycolic acid) (PLGA), where PCL is known to have longer degradation time than PLGA. In vitro tests supported the biocompatibility of the scaffolds. A 12-week animal study involving various examinations of rabbit tibias such as micro-CT and staining showed that both PCL and PLGA resulted in successful bone regeneration. As expected, PLGA degraded faster than PCL, and consequently the tissues generated in the PLGA group were less dense than those in the PCL group. We concluded that slower degradation is preferable in bone tissue engineering, especially when treating critical defects, as mechanical support is needed until full regeneration has occurred.

Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture.

Without using biochemical agents, in this study, we sought to investigate the potential of controlling the differentiation of mesenchymal stem cells (MSCs) into a specific cell type through the use of 3D co-culturing and mechanical stimuli. MSCs and primary cultured chondrocytes were separately encapsulated into alginate beads, and the two types of beads were separated by a membrane. For the investigation a computer-controllable bioreactor was designed and used to engage intermittent hydrostatic pressure (IHP). Five different magnitudes (0.20, 0.10, 0.05, 0.02 MPa and no stimulation) of IHP were applied. The stimulation pattern was the same for all groups: 2 h/day for 7 days starting at 24 h after seeding; 2 and 15 min cycles of stimulating and resting, respectively. Biochemical (DNA and GAG contents), histological (Alcian blue), and RT-PCR (Col II, SOX9, AGC) analyses were performed on days 1, 5, 10, and 20. The results from these analyses showed that stimulation with higher magnitudes of IHP (≥0.10 MPa) were more effective on the proliferation and differentiation of co-cultured MSCs. Together, these data demonstrate the potential of using mechanical stimulation and co-culturing for the proliferation and differentiation of MSCs, even without biochemical agents.

Alternative non-oral nutrition in a rat model: a novel modified gastrostomy technique.

The gastrostomy technique is essential for esophageal reconstruction using a scaffold. To date, there are no established methods to supply nutrients through a gastrostomy tube in rats. The purpose of this study was to analyze the feasibility of a newly modified gastrostomy technique for non-oral nutrition in an adult rat model. We modified the gastrostomy technique for adult rats in a few different ways. (1) The external opening for food injection was made at the midpoint between the ears to prevent damage due to self-harm behaviour. (2) An imbedded subcutaneous tunnel was created between the internal and external openings of the gastrostomy. We compared the efficacy and safety between groups with a T-tube for biliary drainage (TT group, n=14) and a conventional silicone Foley catheter (FC group, n=7) as optimal gastrostomy tubes for in a rat model. We also evaluated the feasibility of the heparin cap connector at the end of gastrostomy tube to control food supply in the TT group (with a cap, n=7; without a cap, n=7). No mortality was observed in the TT group with a cap, whereas most rats in the FC group died within 2 weeks after the procedure. Weight loss decreased significantly in the TT group with a cap compared with all the other groups. The appearance and attitude scores were significantly better in the TT group with a cap. In addition, histologic analysis showed that the TT group a cap showed a marked decrease over time in tissue fibrosis and macrophages compared with the other experimental groups. Therefore, gastrostomy using a silicone T-tube plugged with a cap proved to be a stable and effective option for non-oral feeding in an adult rat model.

Shinjulactone A Blocks Vascular Inflammation and the Endothelial-Mesenchymal Transition.

Objective: The endothelial inflammatory response plays an important role in atherogenesis by inducing nuclear factor (NF)κB-dependent cell adhesion molecule expression and monocyte recruitment. Here, we screened for natural ligands and investigated the ability of shinjulactone A to inhibit interleukin-1ß (IL-1ß)-induced endothelial inflammatory signaling. Methods: The natural compound library included 880 single compounds isolated from medicinal plants by the Korean Medicinal Material Bank. Primary endothelial cells were pretreated with single compounds before stimulation with IL-1ß to induce endothelial inflammation. Endothelial inflammation was measured by assaying NFκB activation and monocyte adhesion. The endothelial-mesenchymal transition (EndMT) was evaluated using cell type-specific marker protein expression and morphology. Results: Shinjulactone A was identified as an efficient blocker of IL-1ß -induced NFκB activation, with a half-maximal inhibitory concentration of approximately 1 µM, and monocyte recruitment in endothelial cells. However, it did not affect lipopolysaccharide-induced NFκB activation in macrophages. Compared to Bay 11-782, a well-known NFκB inhibitor that shows considerable cytotoxicity during long-term treatment, shinjulactone A did not affect endothelial cell viability. Furthermore, it also significantly inhibited the EndMT, which is known to promote atherosclerosis and plaque instability. Conclusion: We suggest that shinjulactone A may be an effective and safe drug candidate for atherosclerosis because it targets and inhibits both endothelial inflammation and the EndMT, without impairing NFκB-dependent innate immunity in macrophages.

Combined effects of surface morphology and mechanical straining magnitudes on the differentiation of mesenchymal stem cells without using biochemical reagents.

Existing studies examining the control of mesenchymal stem cell (MSC) differentiation into desired cell types have used a variety of biochemical reagents such as growth factors despite possible side effects. Recently, the roles of biomimetic microphysical environments have drawn much attention in this field. We studied MSC differentiation and changes in gene expression in relation to osteoblast-like cell and smooth muscle-like cell type resulting from various microphysical environments, including differing magnitudes of tensile strain and substrate geometries for 8 days. In addition, we also investigated the residual effects of those selected microphysical environment factors on the differentiation by ceasing those factors for 3 days. The results of this study showed the effects of the strain magnitudes and surface geometries. However, the genes which are related to the same cell type showed different responses depending on the changes in strain magnitude and surface geometry. Also, different responses were observed three days after the straining was stopped. These data confirm that controlling microenvironments so that they mimic those in vivo contributes to the differentiation of MSCs into specific cell types. And duration of straining engagement was also found to play important roles along with surface geometry.

Shear stress magnitude is critical in regulating the differentiation of mesenchymal stem cells even with endothelial growth medium.

Human mesenchymal stem cells (MSC) were seeded onto the inner surface of a tubular silicon construct and, after 24 h, were exposed to a shearing stress of either 2.5 or 10 dyne/cm(2) for 1 day. The fluid contained endothelial growth factors in both cases. Morphological changes and cytoskeletal rearrangements were observed in the stimulated cells. Immunofluorescence staining showed that low (2.5 dyne/cm(2)) and high shear stress (10 dyne/cm(2)) resulted in the expression of von Willebrand factor (vWF) and calponin, respectively. At low shear stress, CD31 (PECAM-1) was significantly expressed whereas vWF and KDR expression was only slightly higher than those under 10 dyne/cm(2). All three markers related to smooth muscle cells (myocardin, myosin heavy chain, and SM-22α) had significantly higher expression under shear stress of 10 dyne/cm(2) compared with a 2.5 dyne/cm(2), even in endothelial growth medium. Shear stress plays a critical role in regulating MSC differentiation and must be considered for bioengineered blood vessels.