Dong Hwa Kim

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.

Comparison of physical, chemical and cellular responses to nano- and micro-sized calcium silicate/poly(ε-caprolactone) bioactive composites

In this study, we fabricated nano-sized calcium silicate/poly(ϵ-caprolactone) composite (n-CPC) and micro-sized calcium silicate/poly(ϵ-caprolactone) composite (m-CPC). The composition, mechanical properties, hydrophilicity and degradability of both n-CPC and m-CPC were determined, and in vitro bioactivity was evaluated by investigating apatite forming on their surfaces in simulated body fluid (SBF). In addition, cell responses to the two kinds of composites were comparably investigated. The results indicated that n-CPC has superior hydrophilicity, compressive strength and elastic modulus properties compared with m-CPC. Both n-CPC and m-CPC exhibited good in vitro bioactivity, with different morphologies of apatite formation on their surfaces. The apatite layer on n-CPC was more homogeneous and compact than on m-CPC, due to the elevated levels of calcium and silicon concentrations in SBF from n-CPC throughout the 14-day soaking period. Significantly higher levels of attachment and proliferation of MG63 cells were observed on n-CPC than on m-CPC, and significantly higher levels of alkaline phosphatase activity were observed in human mesenchymal stem cells (hMSCs) on n-CPC than on m-CPC after 7 days. Scanning electron microscopy observations revealed that hMSCs were in intimate contact with both n-CPC and m-CPC surfaces, and significantly cell adhesion, spread and growth were observed on n-CPC and m-CPC. These results indicated that both n-CPC and m-CPC have the ability to support cell attachment, growth, proliferation and differentiation, and also yield good bioactivity and biocompatibility.

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.

Preparation and characterization of bioactive calcium silicate and poly(ϵ-caprolactone) nanocomposite for bone tissue regeneration

A novel biocomposite of nanosized calcium silicate (n-CS) and poly(epsilon-caprolactone) (PCL) was successfully fabricated directly using n-CS slurry, not dried n-CS powder, in a solvent-casting method. The in vitro bioactivity of the composite was evaluated by investigating the apatite-forming ability in simulated body fluid. A proliferation assay with mouse L929 fibroblasts was used to test the in vitro biocompatibility. The composition, hydrophilicity, and mechanical properties were also evaluated. Results suggest that the incorporation of n-CS could significantly improve the hydrophilicity, compressive strength, and elastic modulus of n-CS/PCL composites, with the enhancements mainly dependent on n-CS content. The n-CS/PCL composites exhibit excellent in vitro bioactivity, with surface apatite formation for 40% (w/w) n-CS (C40) exceeding that of 20% (w/w) n-CS (C20) at 7 and 14 days. The Ca/P ratios of apatite formed on C20 and C40 surfaces were 1.58 and 1.61, respectively, indicating nonstoichiometric apatite with defective structure. Composites demonstrated significantly better cell attachment and proliferation than that of PCL alone, with C40 demonstrating the best bioactivity. The apatite layers that formed on the composite surfaces facilitated cell attachment (4 h) and proliferation during the early stages (1 and 4 days). Collectively, these results suggest that the incorporation of n-CS produces biocomposites with enhanced bioactivity and biocompatibility.

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.

In Vitro Evaluation of Poly ε-Caprolactone/Hydroxyapatite Composite as Scaffolds for Bone Tissue Engineering with Human Bone Marrow Stromal Cells

This study evaluated the potential of the PCL (poly -caprolactone)/HA(Hydroxyapatite) composite materials as a scaffold for bone regeneration. For this, we fabricated scaffolds utilizing salt leaching method. The PCL/HA composite scaffolds were prepared with various HA contents (20wt%, 40wt%, 60 wt %). To ensure the potential for the scaffolds, porosity tests were conducted along with SEM observations. The porosity decreased with the increase of the contents of HA particles. The porosity of the composite with the highest contents of HA was still adoptable (~85%). In addition, the PCL/HA composite scaffolds were evaluated for their ability of osteogenic differentiation with human bone marrow stromal cell (hBMSC) in vitro. Alkaline phosphatase (ALP) activity, markers for osteoblastic differentiation, and total protein contents were evaluated in hBMSCs following 14 days of cultivation. The addition of HA particles enhanced proliferation of hBMSC during the test. Also, the differentiation ability of the cells was increased as HA particles were added. In this study, we concluded that PCL/HA composite scaffolds has great potential as a scaffold for bone tissue engineering.

Hybrid nanofiber scaffolds of polyurethane and poly(ethylene oxide) using dual-electrospinning for vascular tissue engineering

The objective of this study is to investigate the potential of dual-electrospun polymer based structure for vascular tissue engineering, especially for the medium or small size blood vessels. Polyurethane(PU), which is known to be biocompatible in this area, was electrospun along with poly(ethylene oxide) (PEO). Concentration of PU was fixed at 20wt%, while that of PEO was set from 15 to 35wt%. Morphological observation (SEM and porosity) and cellular responses were tested before and after extracting PEO from the hybrid scaffolds by soaking the scaffolds into distilled water. The diameter of PEO fibers were ranged in 200≈500nm. The lower concentration of PEO tended to show beads. The porosity of the scaffolds after extracting PEO was highly increased with higher concentration of PEO as expected. Also, higher proliferation rate of smooth muscle cells was observed at higher concentration of PEO than at the lower concentration and without PEO. As conclusions, this dual electrospinning technique combined with PU and PEO is expected to overcome the current barrier of cell penetration by providing more space for cells to proliferation.

Biodegradable composite of poly ε-caprolactone/hydroxyapatite 3-D scaffolds for bone tissue engineering

Utilizing salt leaching method, a composite maaterial of PCL (Poly e-caprolactone) and HA (Hydroxyapatite) particles was suggested as a potential scaffold for bone tissue engineering. For this, composite materials were prepared with various HA contents (20wt%, 40wt%, 60wt%). To ensure the potential for the scaffolds, porosity, mechanical stiffness, proliferation tests were conducted along with SEM observations. The addition of HA particles enhanced proliferation of MG-63 during the test. Also, the mechanical stiffness was increased as HA particles were added. Even the porosity was decreased as the contents of HA particles was increased, the porosity of the composite with the highest contents of HA was still adoptable (∼85%). From the study we conducted, addition of HA particles to PCL showed promising results. However, further studies are needed such as long term tests for osteoconductivi-ties, regeneration of extracellular matrices, and differentiation utilizing BMSC (bone marrow stromal cell) with animals.