Ki-Taek Lim

Pulsed-Electromagnetic-Field-Assisted Reduced Graphene Oxide Substrates for Multidifferentiation of Human Mesenchymal Stem Cells

Electromagnetic fields (EMFs) can modulate cell proliferation, DNA replication, wound healing, cytokine expression, and the differentiation of mesenchymal stem cells (MSCs). Graphene, a 2D crystal of sp(2) -hybridized carbon atoms, has entered the spotlight in cell and tissue engineering research. However, a combination of graphene and EMFs has never been applied in tissue engineering. This study combines reduced graphene oxide (RGO) and pulsed EMFs (PEMFs) on the osteogenesis and neurogenesis of MSCs. First, the chemical properties of RGO are measured. After evaluation, the RGO is adsorbed onto glass, and its morphological and electrical properties are investigated. Next, an in vitro study is conducted using human alveolar bone marrow stem cells (hABMSCs). Their cell viability, cell adhesion, and extracellular matrix (ECM) formation are increased by RGO and PEMFs. The combination of RGO and PEMFs enhances osteogenic differentiation. Together, RGO and PEMFs enhance the neurogenic and adipogenic differentiation of hABMSCs. Moreover, in a DNA microarray analysis, the combination of RGO and PEMFs synergically increases ECM formation, membrane proteins, and metabolism. The combination of RGO and PEMFs is expected to be an efficient platform for stem cell and tissue engineering.

Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells.

The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.

Hierarchically Micro- and Nanopatterned Topographical Cues for Modulation of Cellular Structure and Function

Living cells receive biochemical and physical information from the surrounding microenvironment and respond to this information. Multiscale hierarchical substrates with micro- and nanogrooves have been shown to mimic the native extracellular matrix (ECM) better than conventional nanopatterned substrates; therefore, substrates with hierarchical topographical cues are considered suitable for investigating the role of physical factors in tissue functions. In this study, precisely controllable, multiscale hierarchical substrates that could mimic the micro- and nanotopography of complex ECMs were fabricated and used to culture various cell types, including fibroblasts, endothelial cells, osteoblasts, and human mesenchymal stem cells. These substrates had both microscale wrinkles and nanoscale patterns and enhanced the alignment and elongation of all the cells tested. In particular, the nanotopography on the microscale wrinkles promoted not only the adhesion, but also the functions of the cells. These findings suggest that the hierarchical multiscale substrates effectively regulated cellular structure and functions and that they can be used as a platform for tissue engineering and regenerative medicine.Living cells receive biochemical and physical information from the surrounding microenvironment and respond to this information. Multiscale hierarchical substrates with micro- and nanogrooves have been shown to mimic the native extracellular matrix (ECM) better than conventional nanopatterned substrates; therefore, substrates with hierarchical topographical cues are considered suitable for investigating the role of physical factors in tissue functions. In this study, precisely controllable, multiscale hierarchical substrates that could mimic the micro- and nanotopography of complex ECMs were fabricated and used to culture various cell types, including fibroblasts, endothelial cells, osteoblasts, and human mesenchymal stem cells. These substrates had both microscale wrinkles and nanoscale patterns and enhanced the alignment and elongation of all the cells tested. In particular, the nanotopography on the microscale wrinkles promoted not only the adhesion, but also the functions of the cells. These findings suggest that the hierarchical multiscale substrates effectively regulated cellular structure and functions and that they can be used as a platform for tissue engineering and regenerative medicine.

Measurement of Worker's Physiological and Biomechanical Responses during the Cherry Tomato Harvesting Work in a Greenhouse

Physiological signals such as body temperature, heart rate, blood pressure and heart rate variability and biomechanical workload for stress analysis were investigated during the cherry tomato harvesting work in a greenhouse. The skin temperatures raised /min, / min, and /min in standing, stooping and squatting postures, respectively. Breath rate significantly increased from 18 to 28 breaths/min during the cherry tomato harvesting work. As the heart rate during the work ranged from about 72 to 110 beats/min (bpm), the cherry tomato harvesting work appeared to be a light intensity task of less than 110 bpm. The worker`s average energy consumption rate in three positions during 43 min working time was 65.74 kcal (91 kcal/h in 70 kg). This was a light intensity of work, compared to 75 kcal/h in 70 kg of basic metabolic energy consumption rate of a worker with 70 kg weight; The maximum shear force on the disk (L5/ S1) due to static workload in the cherry tomato harvesting work was 446 N in the stooping posture, 321 N in the squatting posture and 287 N in the standing posture. Acute stress index expressed with the heart rate variability, increased parasympathetic activation up to about 70 while workers were doing most agricultural work in this study. This study provided a system to measure quantitatively workers` physiological change, kinematics and kinetic factors without any restrictions of space in the greenhouse works.

Effects of Low Intensity Ultrasound Stimulation on the Proliferation of Alveolar Bone Marrow Stem Cells

Low-intensity ultrasound stimulation produces significant multi-functional effects that are directly relevant to bone formation. It was previously found that low-intensity ultrasound stimulation enhanced bone regeneration although the exact cellular mechanism is not clear. The aim of the present study is to investigate the effect of low-intensity ultrasound stimulation on proliferation of alveolar bone marrow stem cells. Before low-intensity ultrasound stimulation, alveolar bone marrow stem cells were cultured for 24h to facilitate their attachment. The cells were cultured in medium with or without low-intensity ultrasound stimulation. The ultrasound frequency was 1 MHz. Cell cultures stimulated with ultrasound were conducted by three treatment groups - group 1: intensity (100, 200, 300, 400 and 500 mW/cm2), group 2: duty cycle (5, 10, 30 and 50%) and group 3: duration time (1, 3, 5, 10, 20 and 30 min). The effects of low-intensity ultrasound stimulation were evaluated by cell number and morphological changes. The proliferation rates of alveolar bone marrow stem cells for the particular stimulated groups were larger than those of control groups. After low-intensity ultrasound stimulation (intensity: 100 mW/cm2, duty cycle: 30% and duration time: 10 min), the alveolar bone marrow stem cell counts were significantly increased (p<0.05). This study suggested that the cell growth could be enhanced by appropriate low-intensity ultrasound stimulation.

Biomechanical Properties and Cytotoxicity of Chitosan Patch Scaffold for Artificial Eardrum

【The objectives of this study were to prepare a new artificial eardrum patch using water-insoluble chitosan for healing the tympanic membrane perforations and to investigate biomechanical properties and cyotoxicity of the chitosan patch scaffold (CPS). Tensile strength and elongation at the rupture point of CPSs were 2.49-74.05 MPa and 0.11-107.06%, respectively. As the biomechanical properties or CPSs varied with the concentration of chitosan and glycerol, the proper conditions for the CPS were found out. SEM analysis showed very smooth and uniform surface of CPSs without pores at x1000. The result of MTT test showed that CPSs had no cytotoxicity.】

Preparation and Biocompatibility of Composite Bone Scaffolds Using Gnotobiotic Pig Bones

Highly porous composite bioceramic bone scaffolds were developed using sintered gnotobiotic pig bones. These scaffolds consisted of poly-D,L-lactic acid (P(D,L)LA) and bioceramic materials of pig bone powder. The bone scaffolds were able to promote biocompatibility and possess interconnected pores that would support cell adhesion and proliferation adequately. The composite scaffolds were tested with dental pulp stem cells for cytotoxicity test. Cells seeded on the composite scaffolds were readily attached, well proliferated, as confirmed by cytotoxicity test, and cell adhesion assessment. The composite bone scaffold had no toxicity in cytotoxicity test on the extract of 0.013 g scaffold to 2 ml culture medium. The cells on the composite bone scaffold proliferated better than cells on the P(D,L)LA scaffolds.

Analysis of Physiological Bio-information, Human Physical Activities and Load of Lumbar Spine during the Repeated Lifting Work

Workers in the agricultural industry have been exposed to many work-related musculoskeletal disorders. So, our objectives in this study were to measure and analyze worker’s physiological bio-information to reduce musculoskeletal disorders in relation to agricultural works. We investigated worker’s bio-information of physiological signals during the repeated lifting work such as body temperature, heart rate, blood pressure, physical activity, and heart rate variability. Moreover, we analyzed the workloads of lumbar spine during the repeated lifting work using the 3-axis acceleration and angular velocity sensors. The changes of body temperature was not significant, but the mean heart rate increased from 90/min to 116/min significantly during 30 min of repeated lifting work (p<0.05). The average worker’s physical activity(energy consumption rate) was 206 kcal/70kg/h during the repeated lifting work. The workers’ acute stress index was more than 80, which indicated a stressful work. Also, the maximum shear force on the disk (L5/S1) of a worker’s lumbar spine in static state was 500N, and the maximum inertia moment was 139 N․m in dynamic state.

Development of a bio-electrospray system for cell and non-viral gene delivery

Bio-electrospray technology is a very attractive tool for preparing scaffolds and depositing desired solutions on various targets by electric force. In this study, we focused on the application of a bio-electrospray (BES) technique to spray cells on the target and to simultaneously deliver genetic constructs into the cells, called non-viral gene delivery-based bio-electrospray (NVG-BES). Using this method, we tried to harvest the electric charge produced during electrospray for the cellular internalization of cationic polymer/DNA nanoparticles as well as the delivery of living cells on the desired substrate. Furthermore, we optimized the voltage, culture medium and polymeric cationic charges for high transfection efficiency and cell viability during NVG-BES. As a result, the solutions used during the NVG-BES process played an important role in improving transfection efficiency. We determined that a voltage of 10 kV with PBS as the spraying solution showed high transfection efficiency, probably due to the facilitation of cationic polymer/DNA nanocomplexes in cellular internalization and their subsequent expression. In conclusion, NVG-BES, as a novel method, is expected to deliver genes to cells and simultaneously deliver transfected cells to any substrate or scaffold.

Physical Stimulation-Based Osteogenesis: Effect of Secretion In Vitro on Fluid Dynamic Shear Stress of Human Alveolar Bone-Derived Mesenchymal Stem Cells

Human alveolar bone-derived mesenchymal stem cells (hABMSCs) are promising candidates for bone therapies, which have the capacity to differentiate into osteoblasts. Recently, secretion of inducible cytokines and growth factors from mesenchymal stem cells (MSCs) has been discovered, and we also have reported the osteogenic effects of cell physical stimulation. In this study, we investigated the effects of hABMSCs-conditioned secretion media (B-CSM) on osteogenic differentiation of hABMSCs in vitro. Furthermore, we analyzed the B-CSM by proteomics array to identify inducible factors which facilitate osteogenic differentiation. To determine optimal concentration, B-CSM was firstly added at varying amounts (5, 10, 20, 40, and 60%) relative to culture medium. The viability and proliferation of hABMSCs were higher after treating with 5-20% B-CSM to the cells, compared to 40-60%. In addition, the expression of stem cells markers CD146 and STRO-1 was increased in the cells treated with 5-20% B-CSM, but decreased with 40-60%. We also found that B-CSM promoted osteogenic differentiation of hABMSCs such as mineralized nodules were strongly generated by 5-20%. B-CSM was most effective in increasing the expression of Vinculin and osteocalcin (OCN) in osteogenic differentiation of hABMSCs. Taken together, the results of our study ultimately indicate that B-CSM from hABMSCs induced by physical stimulation induce the proliferation and osteogenic differentiation of hABMSCs.Human alveolar bone-derived mesenchymal stem cells (hABMSCs) are promising candidates for bone therapies, which have the capacity to differentiate into osteoblasts. Recently, secretion of inducible cytokines and growth factors from mesenchymal stem cells (MSCs) has been discovered, and we also have reported the osteogenic effects of cell physical stimulation. In this study, we investigated the effects of hABMSCs-conditioned secretion media (B-CSM) on osteogenic differentiation of hABMSCs in vitro. Furthermore, we analyzed the B-CSM by proteomics array to identify inducible factors which facilitate osteogenic differentiation. To determine optimal concentration, B-CSM was firstly added at varying amounts (5, 10, 20, 40, and 60%) relative to culture medium. The viability and proliferation of hABMSCs were higher after treating with 5-20% B-CSM to the cells, compared to 40-60%. In addition, the expression of stem cells markers CD146 and STRO-1 was increased in the cells treated with 5-20% B-CSM, but decreased with 40-60%. We also found that B-CSM promoted osteogenic differentiation of hABMSCs such as mineralized nodules were strongly generated by 5-20%. B-CSM was most effective in increasing the expression of Vinculin and osteocalcin (OCN) in osteogenic differentiation of hABMSCs. Taken together, the results of our study ultimately indicate that B-CSM from hABMSCs induced by physical stimulation induce the proliferation and osteogenic differentiation of hABMSCs.