Ji Won Shin

Fabrication and characterization of novel nano- and micro-HA/PCL composite scaffolds using a modified rapid prototyping process

Novel three-dimensional scaffolds consisting of nano- and microsized hydroxyapatite (HA)/poly(epsilon-caprolactone) (PCL) composite were fabricated using a modified rapid-prototyping (RP) technique for bone tissue engineering applications. The size of the nano-HA ranged from 20 to 90 nm, whereas that of the micro-HA ranged from 20 to 80 microm. The scaffold macropores were well interconnected, with a porosity of 72-73% and a pore size of 500 microm. The compressive modulus of the nano-HA/PCL and micro-HA/PCL scaffolds was 3.187 +/- 0.06 and 1.345 +/- 0.05 MPa, respectively. The higher modulus of the nano-HA/PCL composite (n-HPC) was to be likely caused by a dispersion strengthening effect. The attachment and proliferation of MG-63 cells on n-HPC were better than that on the micro-HA/PCL composite (m-HPC) scaffold. The n-HPC was more hydrophilic than the m-HPC because of the greater surface area of HA exposed to the scaffold surface. This may give rise to better cell attachment and proliferation. Bioactive n-HA/PCL composite scaffold prepared using a modified RP technique has a potential application in bone tissue engineering.

In Vitro and Animal Study of Novel Nano-Hydroxyapatite/Poly(ɛ-Caprolactone) Composite Scaffolds Fabricated by Layer Manufacturing Process

The purpose of this study was to propose a computer-controllable scaffold structure made by a layer manufacturing process (LMP) with addition of nano- or micro-sized particles and to investigate the effects of particle size in vitro. In addition, the superiority of this LMP method over the conventional scaffolds made by salt leaching and gas forming process was investigated through animal study. Using the LMP, we have created a new nano-sized hydroxyapatite/poly(epsilon-caprolactone) composite (n-HPC) scaffold and a micro-sized hydroxyapatite/poly(epsilon-caprolactone) composite (m-HPC) scaffold for bone tissue engineering applications. The scaffold macropores were well interconnected, with a porosity of 73% and a pore size of 500 microm. The compressive modulus of the n-HPC and m-HPC scaffolds was 6.76 and 3.18 MPa, respectively. We compared the cellular responses to the two kinds of scaffolds. Both n-HPC and m-HPC exhibited good in vitro biocompatibility. Attachment and proliferation of mesenchymal stem cells were better on the n-HPC than on the m-HPC scaffold. Moreover, significantly higher alkaline phosphatase activity and calcium content were observed on the n-HPC than on the m-HPC scaffold. In an animal study, the LMP scaffolds enhanced bone formation, owing to their well-interconnected pores. Radiological and histological examinations confirmed that the new bony tissue had grown easily into the entire n-HPC scaffold fabricated by LMP. We suggest that the well-interconnected pores in the LMP scaffolds might encourage cell attachment, proliferation, and migration to stimulate cell functions, thus enhancing bone formation in the LMP scaffolds. This study shows that bioactive and biocompatible n-HPC composite scaffolds prepared using an LMP have potential applications in bone tissue engineering.

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.

Enhanced differentiation of mesenchymal stem cells into NP-like cells via 3D co-culturing with mechanical stimulation.

This study proposes a three-dimensional co-culturing system of mesenchymal stem cells (MSCs) and nucleus pulposus (NP) cells from New Zealand white male rabbits to differentiate MSCs into NP-like cells. The preferable ratio of MSCs to NP cells and the effects of mechanical stimulation were investigated without biochemical reagents. The preferable ratio was investigated without mechanical stimulation using five groups: Group I (MSC control); Group II (NP cell control); and Groups III, IV, and V, for which the ratios of NP cells to MSCs were 1:1, 1:2, and 2:1, respectively. During culture for 10 days without stimulation, the proliferation of MSCs did not increase after day 4. NP cells proliferated more when co-cultured as in Group V. However, the degree of differentiation of MSCs increased significantly in Group V. The differentiation of NP cells decreased gradually over time. When mechanical stimulation was applied to Groups I, II, and V, it contributed to the differentiation of MSCs into NP-like cells, as well as to that of NP cells, but did not contribute to the proliferation of either cell type. The contribution of mechanical stimulation to differentiation was also confirmed by RT-PCR.

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.

Manufacturing of Multi-Layered Nanofibrous Structures Composed of Polyurethane and Poly(ethylene oxide) as Potential Blood Vessel Scaffolds

One of the current limitations in using electrospun nanofibrous materials for tissue engineering is that cells have difficulty penetrating into the materials. For this, multi-layered electrospun structures composed of polyurethane (PU) and poly(ethylene oxide) (PEO) were fabricated and tested in vitro. A 20% (w/v) PU solution was electrospun for 30 min, while a 20% (w/v) PEO solution was electrospun for 5, 15 or 30 min, alternatively. Then, the PEO was extracted by immersing the structure in distilled water to make multi-layered structure. The characteristics of fabricated structures were examined by SEM, FT-IR spectroscopy, mechanical tests and cell penetration test. The bioactivities of smooth muscle cells (SMCs) on these scaffolds were assessed by quantifying DNA, collagen and glycosaminoglycan (GAG) levels. Although hybrid PEO-extracted scaffolds had a little of residual PEO, they were more penetrable than PU alone scaffolds. Also, they showed higher bioactivity than PU-alone scaffolds. The results of this study provided potential of this structure in the application not only to the development of artificial blood vessels but also to other types for tissue engineering.

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.