Heungsoo Shin - Academia.edu (original) (raw)
Papers by Heungsoo Shin
Macromolecular Bioscience, 2010
Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepare... more Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepared. The contact angles and mechanical properties of crosslinked PCL-gelatin nanofibers decrease as the gelatin content increases. The proliferation of myoblasts is higher in the crosslinked PCL-gelatin nanofibers than in the PCL nanofibers, and the formation of myotubes is only observed on the crosslinked PCL-gelatin nanofibers. The expression level of myogenin, myosin heavy chain, and troponin T genes is increased as the gelatin content is increased. The results suggest that PCL-gelatin nanofibers crosslinked with genipin can be used as a substrate to modulate proliferation and differentiation of myoblasts, presenting potential applications in muscle tissue engineering.
Biomacromolecules, 2010
Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical ... more Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical applications like implants, drugs and cell delivery carriers because of their high biocompatibility, high water contents and excellent permeability for nutrients and metabolites. Especially, in situ forming hydrogel systems have received much attention because of their easy application based on minimal invasive techniques. Chemical cross-linking systems fabricated using enzymatic reactions have various advantages, such as high biocompatibility and easy control of reaction rates under mild condition. In this study, we report enzyme-triggered injectable and biodegradable hydrogels composed of Tetronic-tyramine conjugates. The Tetronic-tyramine conjugates were synthesized by first reacting Tetronic with succinic anhydride and subsequent conjugation with tyramine using DCC/NHS as coupling reagents. The chemical structure of Tetronic-succinic anhydride-tyramine (Tet-SA-TA) copolymer was characterized by (1)H NMR and FTIR. The hydrogels were prepared from a Tet-SA-TA solution above 3 wt % in the presence of horseradish peroxidase (HRP) and H(2)O(2) under physiological conditions. Their mechanical property, gelation time, swelling ratio and degradation time were evaluated at different polymer, HRP, and H(2)O(2) concentrations. In addition, a cyto-compatibility study was performed using the MC3T3-E1 cell line. In the cytotoxicity test, it was clear that the Tet-SA-TA hydrogel had no apparent cytotoxicity except for the hydrogel formed with 0.25 wt % H(2)O(2) due to the cytotoxicity of residual H(2)O(2). In conclusion, the obtained results demonstrated that the Tet-SA-TA hydrogel has great potential for use as an injectable scaffold for tissue engineering and as a drug carrier for controlled drug delivery systems.
Biomacromolecules, 2008
Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential f... more Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.
Biomaterials, 2007
Success in tissue engineering requires an understanding of how cells integrate the signals presen... more Success in tissue engineering requires an understanding of how cells integrate the signals presented from the microenvironment created by biomaterial scaffolds to alter their responses. Besides the presence of chemical stimuli, there is growing evidence that the spatial organization of cells and tissue within a 3-dimensional (3-D) extracellular matrix (ECM) context is a critical element in controlling cellular function. Therefore, in order to direct cells toward a desirable tissue structure, it is necessary to engineer biomaterials to have spatiotemporal control of the presentation of regulatory signals. Given that, micro-patterning techniques have profited by combining micro-fabrication technology with the chemical conjugation of biologically active molecules to provide new culture systems where cells can be cultured within a specific geometry. The micro-engineered environments have been developed as 2-and 3-D structures, which have proven greatly useful as versatile platforms to study cell, biomaterial, and ECM interactions on both macroscopic and microscopic levels. The main focus of this review is a brief summary of the use of micro-engineered substrates in the analysis of cell-biomaterial interactions with the aim to provide an introductory overview of practical applications available in the literature. In particular, topics regarding (1) the soft-lithography technique to prepare micro-patterned substrates for the spatial control of cell adhesion, (2) biomaterials stiffnessdependent cellular responses, and (3) the microarray techniques for analysis of cell/biomaterials interactions are discussed. r
Biomaterials, 2009
Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Ther... more Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Therefore, myotube formation plays an important role in restoring muscular functions, and substrates to promote the differentiation of myoblasts to myotubes need to be developed for muscle tissue engineering. In this study, we developed electrically conductive composite fibers of poly(L-lactide-co-3-caprolactone) (PLCL) blended with polyaniline (PANi) using an electrospinning method, and then investigated the effect of these composite fibers on the differentiation of myoblasts. The prepared PLCL/PANi fibers showed no significant difference in fiber diameter or contact angle, regardless of the incorporation of PANi. The fibers containing 30% PANi (PLCL/PANi-30) maintained elastic properties of maximum elongation at break (160 AE 14.4%). The composite fibers were cytocompatible, as the DNA content on each fiber was similar for up to 8 days of C2C12 myoblast culture. After 4 days of culture, the number of cells positive for sarcomeric myosin was 3.6-times greater on the electrically conductive fibers (21 AE 1 and 19 AE 2 for PLCL/ PANi-15 and -30 fibers, respectively) than on the PLCL/PANi-0 fibers (6 AE 2). Furthermore, the level of myogenin expression detected on day 8 of culture on PLCL/PANi-15 was approximately 1.6-fold greater than the PLCL/PANi-0 fibers. Similar results were observed for the expression of other genes including troponin T (2-fold greater) and the myosin heavy chain gene (3-fold greater). These results indicate that electrically conductive substrates can modulate the induction of myoblasts into myotube formation without additional electrical stimulation, suggesting that these fibers may have potential as a temporary substrate for skeletal tissue engineering.
Macromolecular Bioscience, 2008
In this work, electrically conductive polyaniline (PAni) doped with camphorsulfonic acid (CPSA) i... more In this work, electrically conductive polyaniline (PAni) doped with camphorsulfonic acid (CPSA) is blended with poly(L-lactide-co-e-caprolactone) (PLCL), and then electrospun to prepare uniform nanofibers. The CPSA-PAni/PLCL nanofibers show a smooth fiber structure without coarse lumps or beads and consistent fiber diameters (which range from 100 to 700 nm) even with an increase in the amount of CPSA-PAni (from 0 to 30 wt.-%). However, the elongation at break decreases from 391.54 AE 9.20% to 207.85 AE 6.74% when 30% of CPSA-PAni is incorporated. Analysis of the surface of the nanofibers demonstrates the presence of homogeneously blended CPSA-PAni. Most importantly, a four-point probe analysis reveals that electrical properties are maintained in the nanofibers where the conductivity is significantly increased from 0.0015 to 0.0138 S Á cm À1 when the nanofibers are prepared with 30% CPSA-PAni. The cell adhesion tests using human dermal fibroblasts, NIH-3T3 fibroblasts, and C2C12 myoblasts demonstrate significantly higher adhesion on the CPSA-PAni/PLCL nanofibers than pure PLCL nanofibers. In addition, the growth of NIH-3T3 fibroblasts is enhanced under the stimulation of various direct current flows. The CPSA-PAni/PLCL nanofibers with electrically conductive properties may potentially be used as a platform substrate to study the effect of electrical signals on cell activities and to direct desirable cell function for tissue engineering applications.
Tissue Engineering Part A, 2008
Tissue engineering has become an alternative method to traditional surgical treatments for the re... more Tissue engineering has become an alternative method to traditional surgical treatments for the repair of bone defects, and an appropriate scaffold supporting bone formation is a key element in this approach. In the present study, nanofibrous organic and inorganic composite scaffolds containing nano-sized demineralized bone powders (DBPs) with biodegradable poly(L-lactide) (PLA) were developed using an electrospinning process for engineering bone. To assess their biocompatibility, in vitro osteogenic differentiation of human mandible-derived mesenchymal stem cells (hMSCs) cultured on PLA or PLA/DBP composite nanofiber scaffolds were examined. The mineralization of hMSCs cultured with osteogenic supplements on the PLA/DBP nanofiber scaffolds was remarkably greater than on the PLA nanofiber scaffold during the first 14 days of culture but reached the same level after 21 days. The in vivo osteoconductive effect of PLA/DBP nanofibrous scaffolds was further investigated using rats with critical-sized skull defects. Micro-computerized tomography revealed that a greater amount of newly formed bone extended across the defect area in PLA/DBP scaffolds than in the nonimplant and PLA scaffolds 12 weeks after implantation and that the defect size was almost 90% smaller. Therefore, PLA/DBP composite nanofiber scaffolds may serve as a favorable matrix for the regeneration of bone tissue.
Macromolecular Research, 2007
Mechanical stimulation is known to activate several cellular signal transduction pathways, leadin... more Mechanical stimulation is known to activate several cellular signal transduction pathways, leading to the induction of signaling molecules and extracellular matrix (ECM) proteins, thereby modulating cellular activities, such as proliferation and survival. In this study, primary human dermal fibroblasts (HDFs) were seeded onto chitosan-based scaffolds, and then cultured for 3 weeks in a bioreactor under a cyclic strain of 1 Hz frequency. Compared to control samples cultured under static conditions, the application of a cyclic strain stimulated the proliferation of HDFs in 1 week, and by week 3 the thickness of the cell/scaffold composites increased 1.56 fold. Moreover, immunohistochemical staining of the culture media obtained from the cell/scaffold samples subjected to the cyclic strain, revealed increases in the expression and secretion of ECM proteins, such as fibronectin and collagen. These results suggest that the preconditioning of cell/scaffold composites with a cyclic strain may enhance the proliferation of HDFs, and even facilitate integration of the engineered artificial dermal tissue into the host graft site.
Journal of Biomaterials Science-polymer Edition, 2008
Recently, much attention has been given to the fabrication of tissue-engineering scaffolds with n... more Recently, much attention has been given to the fabrication of tissue-engineering scaffolds with nano-scaled structure to stimulate cell adhesion and proliferation in a microenvironment similar to the natural extracellular matrix milieu. In the present study, blends of gelatin and poly(L-lactide-co-epsilon-caprolactone) (PLCL) (blending ratio: 0, 30, 70 and 100 wt% gelatin to PLCL) were electrospun to prepare nano-structured non-woven fibers for the development of mechanically functional engineered skin grafts. The resulting nanofibers demonstrated the uniform and smooth fibers with mean diameters ranging from approx. 50 to 500 nm with interconnected pores, regardless of the composition. The contact angle decreased with increasing amount of gelatin in the blend and the water content of the nanofibers increased concurrently. PLCL nanofibers retained significant levels of recovery following application of uniaxial stress; GP-3 with 70% PLCL blend returned to the original length within less than 10% of deformation following 200% of uniaxial elongation. The overall tensile strength was inversely affected by increase in the gelatin content and degradation rates of the nanofibers were accelerated as the gelatin concentration increased. When seeded with human primary dermal fibroblasts and keratinocytes on the nanofibers, both initial cell adhesion and proliferation rate increased as a function of the gelatin content in the blend. Additionally, the total cell number was significantly greater on the nanofiber scaffolds than on polymer-coated glasses, indicating that nanofibrous structure facilitates cell proliferation. Taken together, gelatin/PLCL blend nanofiber scaffolds may serve as a promising artificial extracellular matrix for regeneration of mechanically functional skin tissue.
Langmuir, 2010
The noble synthesis method for hydroxyapatite (HAp) nanoparticles was exploited using a fairly si... more The noble synthesis method for hydroxyapatite (HAp) nanoparticles was exploited using a fairly simple reaction of Ca(OH)(2) and H(3)PO(4), which does not generate residual harmful anions and consequently does not need an additional washing process. HAp nanoparticles were found to yield from dicalcium phosphate dehydrate (DCPD) as the only intermediate phase, which was monitored by in situ observation study using X-ray diffraction (XRD), Fourier transform infrared (FT-IR), (1)H and (31)P magic-angle spinning (MAS) NMR. Furthermore, we found that the phase evolution of HAp was preceded by heteronucleation of HAp onto the DCPD surface. The combination of scanning electron microscopy (SEM) and inductively coupled plasma atomic emission spectroscopy (ICP-ES) analysis gave more information on the HAp crystallization process, which was found to be retarded by the residual Ca(OH)(2) and slow diffusion process of Ca ions into the interface between HAp and DCPD. These results demonstrate that the synthesis of pure HAp nanoparticles with high throughput can be achieved by controlling the residual Ca(OH)(2) and diffusion process of Ca ions.
![Research paper thumbnail of Modulation of Osteogenic Differentiation of Human Mesenchymal Stem Cells by Poly[(L-lactide)- co -( ε -caprolactone)]/Gelatin Nanofibers](https://attachments.academia-assets.com/47531511/thumbnails/1.jpg)
](Macromolecular Bioscience, 2009
Developing biomaterial scaffolds to elicit specific cell responses is important in many tissue en... more Developing biomaterial scaffolds to elicit specific cell responses is important in many tissue engineering applications. We hypothesized that the chemical composition of the scaffold may be a key determinant for the effective induction of differentiation in human mesenchymal stem cells (hMSCs). In this study, electrospun nanofibers with different chemical compositions were fabricated using poly[(L-lactide)-co-(e-caprolactone)] (PLCL) and gelatin. Scanning electron microscopy (SEM) images showed a randomly arranged structure of nanofibers with diameters ranging from 400 nm to 600 nm. The incorporation of gelatin in the nanofibers stimulated the adhesion and osteogenic differentiation of hMSCs. For example, the well-stretched and polygonal morphology of hMSCs was observed on the gelatin-containing nanofibers, while the cells cultured on the PLCL nanofibers were contracted. The DNA content and alkaline phosphatase activity were significantly increased on the PLCL/gelatin blended nanofibers. Expression of osteogenic genes including alkaline phosphatase (ALP), osteocalcin (OCN), and collagen type I-a2 (Col I-a2) were also upregulated in cells cultured on nanofibers with gelatin. Mineralization of hMSCs was analyzed by von Kossa staining and the amount of calcium was significantly enhanced on the gelatin-incorporated nanofibers. These results suggest that the chemical composition of the underlying scaffolds play a key role in regulating the osteogenic differentiation of hMSCs.
![Research paper thumbnail of Control of Osteogenic Differentiation and Mineralization of Human Mesenchymal Stem Cells on Composite Nanofibers Containing Poly[lactic- co -(glycolic acid)] and Hydroxyapatite](https://attachments.academia-assets.com/47531515/thumbnails/1.jpg)
](Macromolecular Bioscience, 2010
We fabricated composite fibrous scaffolds from blends of poly(lactide-co-glycolide) (PLGA) and na... more We fabricated composite fibrous scaffolds from blends of poly(lactide-co-glycolide) (PLGA) and nano-sized hydroxyapatite (HA) via electrospinning. SEM-EDX and AFM analysis demonstrated that HA was homogeneously dispersed in the nanofibers, and the roughness increased along with the amount of incorporated HA. When hMSCs were cultured on these PLGA/HA composite nanofibers, we found that incorporation of HA on the nanofibers did not affect cell viability whereas increased ALP activity and expression of osteogenic genes as well as the calcium mineralization of hMSCs. Our results indicate that the composite nanofibers can be offered as a potential bone regenerative biomaterial for stem cell based therapies.
Macromolecular Bioscience, 2008
The effect of NBM incorporation in PLA nanofibers on their mechanical properties and the differen... more The effect of NBM incorporation in PLA nanofibers on their mechanical properties and the differentiation and mineralization of osteoblasts was studied. At 20% NBM, the Young's modulus of the nanofibers was 37.78 AE 4.23, significantly larger than that of pure PLA nanofibers. MC3T3-E1 pre-osteoblasts attached to both types of nanofibers and developed full osteogenic phenotypes. A profound effect of NBM on the mineralization of MC3T3-E1 pre-osteoblasts was confirmed, suggesting that NBM/PLA composite nanofibers exhibit properties similar to those of the native collagen-rich mineralized bone matrix, and could therefore serve as a temporary substrate for facilitating the differentiation and mineralization of bone-forming cells.
Macromolecular Bioscience, 2008
Biomacromolecules, 2003
Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an inj... more Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.
Biomacromolecules, 2001
A novel macromer, oligo(poly(ethylene glycol) fumarate) (OPF), was synthesized by the reaction be... more A novel macromer, oligo(poly(ethylene glycol) fumarate) (OPF), was synthesized by the reaction between poly(ethylene glycol) (PEG) of molecular weight 1000 (PEG 1.0K) and fumaryl chloride. The oligo(PEG fumarate) (OPF 1.0K) was modified with a peptide known to modulate cellular functions, Gly-Arg-Gly-Asp (GRGD), after being activated with 4-nitrophenyl chloroformate (NPC). The determined yield of the GRGD modification in 0.1 M sodium bicarbonate buffer of pH 8.3 was 83% as determined by NMR measurements. The OPF 1.0K and the OPF 1.0K modified with GRGD were cross-linked with an unsaturated biodegradable polyester, poly(propylene fumarate) (PPF), by photopolymerization. The cross-linked PPF was characterized by Fourier transform infrared (FT-IR) spectroscopy and contact angle measurements. The equilibrium contact angle of water on the cross-linked PPF surface decreased with the incorporation of OPF 1.0K and the OPF 1.0K modified with GRGD. The results suggest that the OPF macromer can be used for the preparation of functionalized networks incorporating cell adhesion specific sequences.
Biomacromolecules, 2003
This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric n... more This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric network based on poly(propylene fumarate) (PPF) and the cross-linking agent PPF-diacrylate (PPF-DA). Using a methyl tetrazolium (MTT) assay, the effect of the concentrations of PPF and PPF-DA on the cytotoxicity of its unreacted macromers, cross-linked networks, and degradation products was examined. The influence of network structure properties on cell viability and attachment to the cross-linked material was also investigated. The unreacted macromers exhibited a time- and dose-dependent cytotoxic response that increased with more PPF-DA in the mixture. Conversely, the cross-linked networks formed with more PPF-DA did not demonstrate an adverse response because increases in conversion and cross-linking density prevented the extraction of toxic products. Fibroblast attachment was observed on the PPF/PPF-DA networks with the highest double bond conversions. The degradation products, obtained from the complete breakdown of the networks in basic conditions, displayed a dose-dependent cytotoxic response. These results show that there are concerns regarding the biocompatibility of injectable, biodegradable PPF/PPF-DA networks but also sheds light onto potential mechanisms to reduce the cytotoxic effects.
Biomaterials, 2003
Tissue engineering and regenerative medicine is an exciting research area that aims at regenerati... more Tissue engineering and regenerative medicine is an exciting research area that aims at regenerative alternatives to harvested tissues for transplantation. Biomaterials play a pivotal role as scaffolds to provide three-dimensional templates and synthetic extracellular-matrix environments for tissue regeneration. It is often beneficial for the scaffolds to mimic certain advantageous characteristics of the natural extracellular matrix, or developmental or would healing programs. This article reviews current biomimetic materials approaches in tissue engineering. These include synthesis to achieve certain compositions or properties similar to those of the extracellular matrix, novel processing technologies to achieve structural features mimicking the extracellular matrix on various levels, approaches to emulate cell-extracellular matrix interactions, and biologic delivery strategies to recapitulate a signaling cascade or developmental/would-healing program. The article also provides examples of enhanced cellular/tissue functions and regenerative outcomes, demonstrating the excitement and significance of the biomimetic materials for tissue engineering and regeneration.
Biomacromolecules, 2003
A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being i... more A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate-2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/ AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications.
Macromolecular Bioscience, 2010
Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepare... more Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepared. The contact angles and mechanical properties of crosslinked PCL-gelatin nanofibers decrease as the gelatin content increases. The proliferation of myoblasts is higher in the crosslinked PCL-gelatin nanofibers than in the PCL nanofibers, and the formation of myotubes is only observed on the crosslinked PCL-gelatin nanofibers. The expression level of myogenin, myosin heavy chain, and troponin T genes is increased as the gelatin content is increased. The results suggest that PCL-gelatin nanofibers crosslinked with genipin can be used as a substrate to modulate proliferation and differentiation of myoblasts, presenting potential applications in muscle tissue engineering.
Biomacromolecules, 2010
Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical ... more Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical applications like implants, drugs and cell delivery carriers because of their high biocompatibility, high water contents and excellent permeability for nutrients and metabolites. Especially, in situ forming hydrogel systems have received much attention because of their easy application based on minimal invasive techniques. Chemical cross-linking systems fabricated using enzymatic reactions have various advantages, such as high biocompatibility and easy control of reaction rates under mild condition. In this study, we report enzyme-triggered injectable and biodegradable hydrogels composed of Tetronic-tyramine conjugates. The Tetronic-tyramine conjugates were synthesized by first reacting Tetronic with succinic anhydride and subsequent conjugation with tyramine using DCC/NHS as coupling reagents. The chemical structure of Tetronic-succinic anhydride-tyramine (Tet-SA-TA) copolymer was characterized by (1)H NMR and FTIR. The hydrogels were prepared from a Tet-SA-TA solution above 3 wt % in the presence of horseradish peroxidase (HRP) and H(2)O(2) under physiological conditions. Their mechanical property, gelation time, swelling ratio and degradation time were evaluated at different polymer, HRP, and H(2)O(2) concentrations. In addition, a cyto-compatibility study was performed using the MC3T3-E1 cell line. In the cytotoxicity test, it was clear that the Tet-SA-TA hydrogel had no apparent cytotoxicity except for the hydrogel formed with 0.25 wt % H(2)O(2) due to the cytotoxicity of residual H(2)O(2). In conclusion, the obtained results demonstrated that the Tet-SA-TA hydrogel has great potential for use as an injectable scaffold for tissue engineering and as a drug carrier for controlled drug delivery systems.
Biomacromolecules, 2008
Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential f... more Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.
Biomaterials, 2007
Success in tissue engineering requires an understanding of how cells integrate the signals presen... more Success in tissue engineering requires an understanding of how cells integrate the signals presented from the microenvironment created by biomaterial scaffolds to alter their responses. Besides the presence of chemical stimuli, there is growing evidence that the spatial organization of cells and tissue within a 3-dimensional (3-D) extracellular matrix (ECM) context is a critical element in controlling cellular function. Therefore, in order to direct cells toward a desirable tissue structure, it is necessary to engineer biomaterials to have spatiotemporal control of the presentation of regulatory signals. Given that, micro-patterning techniques have profited by combining micro-fabrication technology with the chemical conjugation of biologically active molecules to provide new culture systems where cells can be cultured within a specific geometry. The micro-engineered environments have been developed as 2-and 3-D structures, which have proven greatly useful as versatile platforms to study cell, biomaterial, and ECM interactions on both macroscopic and microscopic levels. The main focus of this review is a brief summary of the use of micro-engineered substrates in the analysis of cell-biomaterial interactions with the aim to provide an introductory overview of practical applications available in the literature. In particular, topics regarding (1) the soft-lithography technique to prepare micro-patterned substrates for the spatial control of cell adhesion, (2) biomaterials stiffnessdependent cellular responses, and (3) the microarray techniques for analysis of cell/biomaterials interactions are discussed. r
Biomaterials, 2009
Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Ther... more Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Therefore, myotube formation plays an important role in restoring muscular functions, and substrates to promote the differentiation of myoblasts to myotubes need to be developed for muscle tissue engineering. In this study, we developed electrically conductive composite fibers of poly(L-lactide-co-3-caprolactone) (PLCL) blended with polyaniline (PANi) using an electrospinning method, and then investigated the effect of these composite fibers on the differentiation of myoblasts. The prepared PLCL/PANi fibers showed no significant difference in fiber diameter or contact angle, regardless of the incorporation of PANi. The fibers containing 30% PANi (PLCL/PANi-30) maintained elastic properties of maximum elongation at break (160 AE 14.4%). The composite fibers were cytocompatible, as the DNA content on each fiber was similar for up to 8 days of C2C12 myoblast culture. After 4 days of culture, the number of cells positive for sarcomeric myosin was 3.6-times greater on the electrically conductive fibers (21 AE 1 and 19 AE 2 for PLCL/ PANi-15 and -30 fibers, respectively) than on the PLCL/PANi-0 fibers (6 AE 2). Furthermore, the level of myogenin expression detected on day 8 of culture on PLCL/PANi-15 was approximately 1.6-fold greater than the PLCL/PANi-0 fibers. Similar results were observed for the expression of other genes including troponin T (2-fold greater) and the myosin heavy chain gene (3-fold greater). These results indicate that electrically conductive substrates can modulate the induction of myoblasts into myotube formation without additional electrical stimulation, suggesting that these fibers may have potential as a temporary substrate for skeletal tissue engineering.
Macromolecular Bioscience, 2008
In this work, electrically conductive polyaniline (PAni) doped with camphorsulfonic acid (CPSA) i... more In this work, electrically conductive polyaniline (PAni) doped with camphorsulfonic acid (CPSA) is blended with poly(L-lactide-co-e-caprolactone) (PLCL), and then electrospun to prepare uniform nanofibers. The CPSA-PAni/PLCL nanofibers show a smooth fiber structure without coarse lumps or beads and consistent fiber diameters (which range from 100 to 700 nm) even with an increase in the amount of CPSA-PAni (from 0 to 30 wt.-%). However, the elongation at break decreases from 391.54 AE 9.20% to 207.85 AE 6.74% when 30% of CPSA-PAni is incorporated. Analysis of the surface of the nanofibers demonstrates the presence of homogeneously blended CPSA-PAni. Most importantly, a four-point probe analysis reveals that electrical properties are maintained in the nanofibers where the conductivity is significantly increased from 0.0015 to 0.0138 S Á cm À1 when the nanofibers are prepared with 30% CPSA-PAni. The cell adhesion tests using human dermal fibroblasts, NIH-3T3 fibroblasts, and C2C12 myoblasts demonstrate significantly higher adhesion on the CPSA-PAni/PLCL nanofibers than pure PLCL nanofibers. In addition, the growth of NIH-3T3 fibroblasts is enhanced under the stimulation of various direct current flows. The CPSA-PAni/PLCL nanofibers with electrically conductive properties may potentially be used as a platform substrate to study the effect of electrical signals on cell activities and to direct desirable cell function for tissue engineering applications.
Tissue Engineering Part A, 2008
Tissue engineering has become an alternative method to traditional surgical treatments for the re... more Tissue engineering has become an alternative method to traditional surgical treatments for the repair of bone defects, and an appropriate scaffold supporting bone formation is a key element in this approach. In the present study, nanofibrous organic and inorganic composite scaffolds containing nano-sized demineralized bone powders (DBPs) with biodegradable poly(L-lactide) (PLA) were developed using an electrospinning process for engineering bone. To assess their biocompatibility, in vitro osteogenic differentiation of human mandible-derived mesenchymal stem cells (hMSCs) cultured on PLA or PLA/DBP composite nanofiber scaffolds were examined. The mineralization of hMSCs cultured with osteogenic supplements on the PLA/DBP nanofiber scaffolds was remarkably greater than on the PLA nanofiber scaffold during the first 14 days of culture but reached the same level after 21 days. The in vivo osteoconductive effect of PLA/DBP nanofibrous scaffolds was further investigated using rats with critical-sized skull defects. Micro-computerized tomography revealed that a greater amount of newly formed bone extended across the defect area in PLA/DBP scaffolds than in the nonimplant and PLA scaffolds 12 weeks after implantation and that the defect size was almost 90% smaller. Therefore, PLA/DBP composite nanofiber scaffolds may serve as a favorable matrix for the regeneration of bone tissue.
Macromolecular Research, 2007
Mechanical stimulation is known to activate several cellular signal transduction pathways, leadin... more Mechanical stimulation is known to activate several cellular signal transduction pathways, leading to the induction of signaling molecules and extracellular matrix (ECM) proteins, thereby modulating cellular activities, such as proliferation and survival. In this study, primary human dermal fibroblasts (HDFs) were seeded onto chitosan-based scaffolds, and then cultured for 3 weeks in a bioreactor under a cyclic strain of 1 Hz frequency. Compared to control samples cultured under static conditions, the application of a cyclic strain stimulated the proliferation of HDFs in 1 week, and by week 3 the thickness of the cell/scaffold composites increased 1.56 fold. Moreover, immunohistochemical staining of the culture media obtained from the cell/scaffold samples subjected to the cyclic strain, revealed increases in the expression and secretion of ECM proteins, such as fibronectin and collagen. These results suggest that the preconditioning of cell/scaffold composites with a cyclic strain may enhance the proliferation of HDFs, and even facilitate integration of the engineered artificial dermal tissue into the host graft site.
Journal of Biomaterials Science-polymer Edition, 2008
Recently, much attention has been given to the fabrication of tissue-engineering scaffolds with n... more Recently, much attention has been given to the fabrication of tissue-engineering scaffolds with nano-scaled structure to stimulate cell adhesion and proliferation in a microenvironment similar to the natural extracellular matrix milieu. In the present study, blends of gelatin and poly(L-lactide-co-epsilon-caprolactone) (PLCL) (blending ratio: 0, 30, 70 and 100 wt% gelatin to PLCL) were electrospun to prepare nano-structured non-woven fibers for the development of mechanically functional engineered skin grafts. The resulting nanofibers demonstrated the uniform and smooth fibers with mean diameters ranging from approx. 50 to 500 nm with interconnected pores, regardless of the composition. The contact angle decreased with increasing amount of gelatin in the blend and the water content of the nanofibers increased concurrently. PLCL nanofibers retained significant levels of recovery following application of uniaxial stress; GP-3 with 70% PLCL blend returned to the original length within less than 10% of deformation following 200% of uniaxial elongation. The overall tensile strength was inversely affected by increase in the gelatin content and degradation rates of the nanofibers were accelerated as the gelatin concentration increased. When seeded with human primary dermal fibroblasts and keratinocytes on the nanofibers, both initial cell adhesion and proliferation rate increased as a function of the gelatin content in the blend. Additionally, the total cell number was significantly greater on the nanofiber scaffolds than on polymer-coated glasses, indicating that nanofibrous structure facilitates cell proliferation. Taken together, gelatin/PLCL blend nanofiber scaffolds may serve as a promising artificial extracellular matrix for regeneration of mechanically functional skin tissue.
Langmuir, 2010
The noble synthesis method for hydroxyapatite (HAp) nanoparticles was exploited using a fairly si... more The noble synthesis method for hydroxyapatite (HAp) nanoparticles was exploited using a fairly simple reaction of Ca(OH)(2) and H(3)PO(4), which does not generate residual harmful anions and consequently does not need an additional washing process. HAp nanoparticles were found to yield from dicalcium phosphate dehydrate (DCPD) as the only intermediate phase, which was monitored by in situ observation study using X-ray diffraction (XRD), Fourier transform infrared (FT-IR), (1)H and (31)P magic-angle spinning (MAS) NMR. Furthermore, we found that the phase evolution of HAp was preceded by heteronucleation of HAp onto the DCPD surface. The combination of scanning electron microscopy (SEM) and inductively coupled plasma atomic emission spectroscopy (ICP-ES) analysis gave more information on the HAp crystallization process, which was found to be retarded by the residual Ca(OH)(2) and slow diffusion process of Ca ions into the interface between HAp and DCPD. These results demonstrate that the synthesis of pure HAp nanoparticles with high throughput can be achieved by controlling the residual Ca(OH)(2) and diffusion process of Ca ions.
Macromolecular Bioscience, 2009
Developing biomaterial scaffolds to elicit specific cell responses is important in many tissue en... more Developing biomaterial scaffolds to elicit specific cell responses is important in many tissue engineering applications. We hypothesized that the chemical composition of the scaffold may be a key determinant for the effective induction of differentiation in human mesenchymal stem cells (hMSCs). In this study, electrospun nanofibers with different chemical compositions were fabricated using poly[(L-lactide)-co-(e-caprolactone)] (PLCL) and gelatin. Scanning electron microscopy (SEM) images showed a randomly arranged structure of nanofibers with diameters ranging from 400 nm to 600 nm. The incorporation of gelatin in the nanofibers stimulated the adhesion and osteogenic differentiation of hMSCs. For example, the well-stretched and polygonal morphology of hMSCs was observed on the gelatin-containing nanofibers, while the cells cultured on the PLCL nanofibers were contracted. The DNA content and alkaline phosphatase activity were significantly increased on the PLCL/gelatin blended nanofibers. Expression of osteogenic genes including alkaline phosphatase (ALP), osteocalcin (OCN), and collagen type I-a2 (Col I-a2) were also upregulated in cells cultured on nanofibers with gelatin. Mineralization of hMSCs was analyzed by von Kossa staining and the amount of calcium was significantly enhanced on the gelatin-incorporated nanofibers. These results suggest that the chemical composition of the underlying scaffolds play a key role in regulating the osteogenic differentiation of hMSCs.
Macromolecular Bioscience, 2010
We fabricated composite fibrous scaffolds from blends of poly(lactide-co-glycolide) (PLGA) and na... more We fabricated composite fibrous scaffolds from blends of poly(lactide-co-glycolide) (PLGA) and nano-sized hydroxyapatite (HA) via electrospinning. SEM-EDX and AFM analysis demonstrated that HA was homogeneously dispersed in the nanofibers, and the roughness increased along with the amount of incorporated HA. When hMSCs were cultured on these PLGA/HA composite nanofibers, we found that incorporation of HA on the nanofibers did not affect cell viability whereas increased ALP activity and expression of osteogenic genes as well as the calcium mineralization of hMSCs. Our results indicate that the composite nanofibers can be offered as a potential bone regenerative biomaterial for stem cell based therapies.
Macromolecular Bioscience, 2008
The effect of NBM incorporation in PLA nanofibers on their mechanical properties and the differen... more The effect of NBM incorporation in PLA nanofibers on their mechanical properties and the differentiation and mineralization of osteoblasts was studied. At 20% NBM, the Young's modulus of the nanofibers was 37.78 AE 4.23, significantly larger than that of pure PLA nanofibers. MC3T3-E1 pre-osteoblasts attached to both types of nanofibers and developed full osteogenic phenotypes. A profound effect of NBM on the mineralization of MC3T3-E1 pre-osteoblasts was confirmed, suggesting that NBM/PLA composite nanofibers exhibit properties similar to those of the native collagen-rich mineralized bone matrix, and could therefore serve as a temporary substrate for facilitating the differentiation and mineralization of bone-forming cells.
Macromolecular Bioscience, 2008
Biomacromolecules, 2003
Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an inj... more Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.
Biomacromolecules, 2001
A novel macromer, oligo(poly(ethylene glycol) fumarate) (OPF), was synthesized by the reaction be... more A novel macromer, oligo(poly(ethylene glycol) fumarate) (OPF), was synthesized by the reaction between poly(ethylene glycol) (PEG) of molecular weight 1000 (PEG 1.0K) and fumaryl chloride. The oligo(PEG fumarate) (OPF 1.0K) was modified with a peptide known to modulate cellular functions, Gly-Arg-Gly-Asp (GRGD), after being activated with 4-nitrophenyl chloroformate (NPC). The determined yield of the GRGD modification in 0.1 M sodium bicarbonate buffer of pH 8.3 was 83% as determined by NMR measurements. The OPF 1.0K and the OPF 1.0K modified with GRGD were cross-linked with an unsaturated biodegradable polyester, poly(propylene fumarate) (PPF), by photopolymerization. The cross-linked PPF was characterized by Fourier transform infrared (FT-IR) spectroscopy and contact angle measurements. The equilibrium contact angle of water on the cross-linked PPF surface decreased with the incorporation of OPF 1.0K and the OPF 1.0K modified with GRGD. The results suggest that the OPF macromer can be used for the preparation of functionalized networks incorporating cell adhesion specific sequences.
Biomacromolecules, 2003
This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric n... more This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric network based on poly(propylene fumarate) (PPF) and the cross-linking agent PPF-diacrylate (PPF-DA). Using a methyl tetrazolium (MTT) assay, the effect of the concentrations of PPF and PPF-DA on the cytotoxicity of its unreacted macromers, cross-linked networks, and degradation products was examined. The influence of network structure properties on cell viability and attachment to the cross-linked material was also investigated. The unreacted macromers exhibited a time- and dose-dependent cytotoxic response that increased with more PPF-DA in the mixture. Conversely, the cross-linked networks formed with more PPF-DA did not demonstrate an adverse response because increases in conversion and cross-linking density prevented the extraction of toxic products. Fibroblast attachment was observed on the PPF/PPF-DA networks with the highest double bond conversions. The degradation products, obtained from the complete breakdown of the networks in basic conditions, displayed a dose-dependent cytotoxic response. These results show that there are concerns regarding the biocompatibility of injectable, biodegradable PPF/PPF-DA networks but also sheds light onto potential mechanisms to reduce the cytotoxic effects.
Biomaterials, 2003
Tissue engineering and regenerative medicine is an exciting research area that aims at regenerati... more Tissue engineering and regenerative medicine is an exciting research area that aims at regenerative alternatives to harvested tissues for transplantation. Biomaterials play a pivotal role as scaffolds to provide three-dimensional templates and synthetic extracellular-matrix environments for tissue regeneration. It is often beneficial for the scaffolds to mimic certain advantageous characteristics of the natural extracellular matrix, or developmental or would healing programs. This article reviews current biomimetic materials approaches in tissue engineering. These include synthesis to achieve certain compositions or properties similar to those of the extracellular matrix, novel processing technologies to achieve structural features mimicking the extracellular matrix on various levels, approaches to emulate cell-extracellular matrix interactions, and biologic delivery strategies to recapitulate a signaling cascade or developmental/would-healing program. The article also provides examples of enhanced cellular/tissue functions and regenerative outcomes, demonstrating the excitement and significance of the biomimetic materials for tissue engineering and regeneration.
Biomacromolecules, 2003
A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being i... more A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate-2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/ AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications.