So Hee Park

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.

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.

Effects of flow-induced shear stress on limbal epithelial stem cell growth and enrichment.

The roles of limbal epithelial stem cells (LESCs) are widely recognized, but for these cells to be utilized in basic research and potential clinical applications, researchers must be able to efficiently isolate them and subsequently maintain their stemness in vitro. We aimed to develop a biomimetic environment for LESCs involving cells from their in vivo niche and the principle of flow-induced shear stress, and to subsequently demonstrate the potential of this novel paradigm. LESCs, together with neighboring cells, were isolated from the minced limbal tissues of rabbits. At days 8 and 9 of culture, the cells were exposed to a steady flow or intermittent flow for 2 h per day in a custom-designed bioreactor. The responses of LESCs and epithelial cells were assessed at days 12 and 14. LESCs and epithelial cells responded to both types of flow. Proliferation of LESCs, as assessed using a BrdU assay, was increased to a greater extent under steady flow conditions. Holoclones were found under intermittent flow, indicating that differentiation into transient amplifying cells had occurred. Immunofluorescent staining of Bmi-1 suggested that steady flow has a positive effect on the maintenance of stemness. This finding was confirmed by real-time PCR. Notch-1 and p63 were more sensitive to intermittent flow, but this effect was transient. K3 and K12 expression, indicative of differentiation of LESCs into epithelial cells, was induced by flow and lasted longer under intermittent flow conditions. In summary, culture of LESCs in a bioreactor under a steady flow paradigm, rather than one of intermittent flow, is beneficial for both increasing proliferation and maintaining stemness. Conversely, intermittent flow appears to induce differentiation of LESCs. This novel experimental method introduces micro-mechanical stimuli to traditional culture techniques, and has potential for regulating the proliferation and differentiation of LESCs in vitro, thereby facilitating research in this field.

Effects of Various Patterns of Intermittent Hydrostatic Pressure on the Osteogenic Differentiation of Mesenchymal Stem Cells

This study investigated the effects of intemittent hydrostatic pressure (IHP) patterns on the responses of mesenchymal stem cells (MSCs) such as osteogenic differentiation, proliferation and senescence. For these experimental groups were set based on IHP patterns: (1) C_BM: control group with no stimulation in basal media; (2) C_OM: control group with no stimulation in osteogenic media (OM); (3) S_2H/5M: longer pressurizing (2hours) and shorter resting (5minutes) time in OM; (4) S_1H/1H: equal pressurizing and resting time (1hour) in OM; (5) S_2M/15M: shorter pressurizing (2minutes) and longer resting (15minutes) time in OM. The magnitude of IHP was 0.15 MPa. IHP was applied to corresponding groups for 4 hours a day from 3 days starting at 48 hours after seeding. Examination of DNA contents, ALP activity and its staining, quantitative real-time PCR, and β-galactosidase staining were performed. ALP amount normalized by corresponding DNA content in S_2H/5M, C_BM was significantly lower than that of the other group at day 5, which was more observable even at day 7 while S_2H/5M significantly had lower ALP count than other groups at day 5 and day 7. Other groups (S_1H/1H and S_2M/15M) showed significantly higher ALP amounts indicating the positive effect on osteogenic differentiation. Other markers indicating the degrees of differentiation showed comparable results. Based on β-galactosidase staining, it appeared that mechanical stimuli did not affect cell senescence significantly. From this study, we concluded that engagement of IHP has a potential of controlling osteogenic differentiation depending on its pattern: it can promote or suppress differentiation.

Characterization of a hybrid bone substitute composed of polylactic acid tetrapod chips and hydroxyapatite powder

This study evaluated a novel bone substitute composed of biodegradable polylactic acid (PLA) chips and hydroxyapatite (HA) powder. Using a microinjection molding technique, we manufactured tetrapod-shaped PLA chips, which are known to resist external loading. The roughness of the chip surface allowed for cell attachment. Also, no in vitro toxicity or reduced cellular proliferation was observed. The HA powder, made from porcine trabecular bones, had been characterized in previous studies. In the animal studies, pasted HA powder and PLA were implanted into the defective rabbit tibia. The control group was not treated with HA and PLA powder. Using X-ray analysis, micro-computed tomography, hematoxylin and eosin staining, and pathological examination for 12 weeks, we confirmed that the PLA/HA hybrid bone substitute has a potential of clinical application.

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.

Changes, and the Relevance Thereof, in Mitochondrial Morphology during Differentiation into Endothelial Cells.

The roles of mitochondria in various physiological functions of vascular endothelial cells have been investigated extensively. Morphological studies in relation to physiological functions have been performed. However, there have been few reports of morphological investigations related to stem cell differentiation. This was the first morphological study of mitochondria in relation to endothelial differentiation and focused on quantitative analysis of changes in mitochondrial morphology, number, area, and length during differentiation of human mesenchymal stem cells (hMSCs) into endothelial-like cells. To induce differentiation, we engaged vascular endothelial growth factors and flow-induced shear stress. Cells were classified according to the expression of von Willebrand factor as hMSCs, differentiating cells, and almost fully differentiated cells. Based on imaging analysis, we investigated changes in mitochondrial number, area, and length. In addition, mitochondrial networks were quantified on a single-mitochondrion basis by introducing a branch form factor. The data indicated that the mitochondrial number, area per cell, and length were decreased with differentiation. The mitochondrial morphology became simpler with progression of differentiation. These findings could be explained in view of energy level during differentiation; a higher level of energy is needed during differentiation, with larger numbers of mitochondria with branches. Application of this method to differentiation into other lineages will explain the energy levels required to control stem cell differentiation.

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.

Potential of Engineering Methodologies for the Application to Pharmaceutical Research

Current engineering methods and their potential for use in cell-based research are reviewed. The basis of the suggested engineering methods is that real cellular responses can be assessed when the cells are under the same conditions as in vivo. Providing various conditions for this various engineering methodologies can be adopted. Three major factors should be considered when we apply bio-mimetic conditions to cells under in vitro culture conditions. They are the surface pattern and stiffness of the substrate, physical stimuli and neighboring cells. Various outcomes affected by those factors are introduced and reviewed. In particular, those outcomes from stem cell research have been reported. Even though some limitations of adopting those factors alone or combined still exist, the potential is now widely being recognized. The readers are kindly asked to consider those methodologies in relation to pharmaceutical research.

물리적 인장 자극에 의한 줄기세포의 분화에 동반되는 미토콘드리아의 특성 변화에 관한 고찰

Recent studies have confirmed that the direction of stem cell differentiation can be modulated by physical stimuli even without biochemical growth factors. This implies stem cells attribute their differentiation to mechanical signaling directly or indirectly. However, the role or changes in mitochondria, one of the major energy sources in a cell, during differentiation have not been investigated. This study applied mechanical stretching of two different magnitudes (3% and 10%) to human mesenchymal stem cells without any relevant growth factors. We confirmed differentiation of stem cells into two different directions: osteogenic- and smooth muscle-like cells. Along with these results we found the expression of NOX4 was increased with stretching magnitude. As the expression of NOX4 is closely related to mitochondrial biogenesis and morphology various markers were also investigated. The expressions of the markers in relation to biogenesis (PGC-1alpha, TFA) and fusion (MFN1, MFN2, OPA1) were increased with stretching magnitude. However, the expression of Drp1 was observable with stretching of 3%. This study showed that the magnitude of physical stimulation affects the expression of NOX4, which has been known to be closely related to the biogenesis and morphological changes of mitochondria and stem cell differentiation. Therefore, further study of NOX4 due to physical stimulation for the control of stem cell differentiation accompanied by mitochondria functions is worth to be performed.