Kwideok Park

Surface modification of biodegradable electrospun nanofiber scaffolds and their interaction with fibroblasts.

Biodegradable polymers, such as poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA), were dissolved individually in the proper solvents and then subjected to electrospinning process to make nanofibrous scaffolds. Their surfaces were then chemically modified using oxygen plasma treatment and in situ grafting of hydrophilic acrylic acid (AA). The fiber thickness, pore size and porosity were estimated to 200–800 nm, 2–30 μm and 94–96%, respectively, and these properties were insignificant in the PGA, PLLA and PLGA nanofibrous scaffolds. The ultimate tensile strength of PGA was about 2.5 MPa on average and that of PLGA and PLLA was less than 2 MPa. The elongation-at-break was 100–130% for the three nanofibrous scaffolds. When the surface properties of AA-grafted scaffolds were examined, higher ratios of oxygen to carbon, lower contact angles and the presence of carboxylic (–COOH) groups were identified. The properties were significantly different from those of the unmodified nanofibrous scaffolds. Fibroblasts once seeded on the scaffolds were spreading over large surface area on the AA-grafted surface as compared to the unmodified PGA, PLLA and PLGA nanofibrous scaffolds. Cultured for up to 6 days, the fibroblast proliferation was found to be much better on the surface-modified nanofibrous scaffolds. The present study showed that, with the use of plasma treatment and AA grafting, the hydrophilic functional groups could be successfully adapted on the surface of electrospun nanofibrous scaffolds. Those surface-modified scaffolds made significant improvement on cell attachment and proliferation in vitro.

Fabrication of covered porous PLGA microspheres using hydrogen peroxide for controlled drug delivery and regenerative medicine.

Poly(lactic-co-glycolic acid) (PLGA) microsphere has been a useful tool in delivering therapeutic drugs and biologically active proteins. In this study, a covered porous PLGA microsphere was manufactured using W(1)/O/W(2) double emulsion solvent evaporation method, utilizing hydrogen peroxide as a novel porogen. An enzymatic reaction between hydrogen peroxide and catalase produced oxygen bubbles and thus many internal pores within microsphere were naturally developed. When different molar ratios between lactide and glycolide in PLGA were examined, the ratio, 50:50 showed the most organized porous microstructure. Higher molecular weight of PLGA seemed to be favorable in creating a porous structure. By testing various concentrations of hydrogen peroxide, it was found that rather concentrated one was more efficient in developing a porous network in the microspheres. The source of the skin layer that covers the whole surface of the microsphere was found to be PLGA, not polyvinyl alcohol (PVA). The residual amount of hydrogen peroxide was negligible after a thorough evaporation of PLGA microsphere. When release profiles of dexamethasone (Dex) with morphologically different microspheres such as, nonporous, covered porous, and porous, were investigated for up to 28 days in vitro, their release patterns were found to be significantly different on a temporal basis. The present work demonstrated that the covered porous PLGA microspheres could be successfully fabricated using hydrogen peroxide and that the covered skin layer on the PLGA microsphere played an important role in determining the characteristic release profiles of Dex.

Controlled release of bone morphogenetic protein (BMP)-2 from nanocomplex incorporated on hydroxyapatite-formed titanium surface.

Both osteoconductivity and osteoinductivity are equally very important aspects in a new bone formation and ultimately for bone regeneration. The purpose of this study was to create an environment, not only osteoconductive but also osteoinductive on titanium (Ti) surface. To do this bone morphogenetic protein-2 (BMP-2) nanocomplex (NC) was fabricated by using an ionic interaction between BMP-2 and chondroitin sulfate (CS). Meanwhile, Ti was chemically treated, then subjected to soaking in simulated body fluid (SBF), naming the sample Ti(C)-hydroxyapatite (HA). Once the BMP-2 NC was precipitated on the Ti(C)-HA surface, along with the addition of calcium/phosphate solution, the final product was formed as Ti(C)-HA-BMP-2. The size of NC was ranged from 150 to 250nm and the amount of CS was influential in determining both NC size and zeta potential. From the SEM observation, Ti surface was found nicely covered with the crystallized apatite layer that was identified using FTIR and NMR. The immobilized BMP-2 was released in a moderate rate for 4 weeks, without an initial burst of BMP-2. When mouse osteoblast cells were seeded on different Ti substrates, cell proliferation was faster in the Ti(C)-HA-BMP-2 group, as compared to other groups. The gene expression of bone-specific markers, osteocalcin and type I collagen, was significantly upregulated with the use of BMP-2 NC. The same result was witnessed in the measurement of alkaline phosphatase activity, in which the difference was statistically significant. This study demonstrated that the delivery system of BMP-2 NC was effective in holding BMP-2 on the apatite-coated Ti surface and that the Ti surface could be modified into the environment osteoinductive as well as osteoconductive.

Fabrication of core-shell microcapsules using PLGA and alginate for dual growth factor delivery system.

To effectively harness the great potential of stem cells, we designed a dual growth factor delivery system for the application toward stem cell differentiation into specific lineages. This system carries a core-shell structure within microcapsules made of poly(L-lactide-co-glycolide) (PLGA) and alginate, which were fabricated using a coaxial electro-dropping method. Both PLGA and alginate were supplied from the inner and outer nozzles, respectively. The size and shape of microcapsules were greatly varying depending on the variables: nozzle size, applied voltage, volumetric feeding ratio (PLGA:alginate), feeding rate, and polymer concentrations. Once proper conditions were met, single or multi PLGA cores were found settled within the microcapsules. From the microscopic images, wrinkled surfaces of microcapsules were observed, along with the PLGA cores inside the alginate domain. When two different microcapsules were made, switching the position of bone morphogenetic protein (BMP)-2 and dexamethasone (Dex) for either core or shell domain, their release profiles were very unique on a temporal basis, based on their location in the microcapsules. An initial burst of biomolecules was highly suppressed when either biomolecule was loaded in the PLGA core. It was clear that the osteogenic biomolecules encapsulated in the microcapsule could be released together and their concentrations were disparate at each time point. Meanwhile as the hydrogel constructs including rat bone marrow stromal cells (BMSCs) and osteogenic factor-loaded microcapsules were cultured for up to 4 weeks, the gene expressions levels of osteopontin, type I collagen, and osteocalcin were significantly upregulated as compared to the control group. The present coaxial system was very effective in manufacturing PLGA core-alginate shell microcapsules and in encapsulating multiple biomolecules essential for stem cell differentiation.

Enhanced dermal wound neovascularization by targeted delivery of endothelial progenitor cells using an RGD-g-PLLA scaffold

Endothelial progenitor cells (EPCs), endothelial precursors that promote neovascularization in ischemic tissues, have shown the limited vascular regeneration efficacy due to their poor homing into injured sites and low survival, so that a variety of biosynthetic scaffolds have been employed as cell delivery vehicles to overcome the current cell transplantation methods. However, few paralleled studies that directly compare the efficacy of EPCs seeded within synthetic scaffolds to that of EPCs delivered by the conventional transplantation techniques used for EPC therapies have been performed. To address these issues, RGD-g-PLLA biosynthetic scaffold was developed for the targeted EPC delivery and was found to successfully support the in vitro growth and endothelial functions of EPCs. This scaffold also appeared to be good as in vivo targeted delivery carriers of EPCs as it promoted vascular regeneration in a murine dermal wound models. Furthermore, direct comparison with the intradermal EPC injection revealed that the targeted delivery of EPCs by using the RGD-g-PLLA scaffold was superior to their conventional local injection method in terms of the localization and survival/retention of the transplanted EPCs, and their vascular repairing potential. These results suggest that the development of an effective stem cell delivery system may help to maximize the tissue-repairing efficacy with a limited number of stem cells, thereby resolving the limited clinical success of current stem cell therapies that have utilized simple cell injections or infusions.

Preparation of TGF-β1-conjugated biodegradable pluronic F127 hydrogel and its application with adipose-derived stem cells

In this study, a composite hydrogel using Pluronic F127 derivatives and crosslinked hyaluronic acid (X-HA) was investigated, exploring the benefits in the induction of chondrogenic differentiation of human adipose-derived stem cells (ASCs). F127 was chemically modified through a series of reactions that produced multiple F127 derivatives. A chondrogenic growth factor, transforming growth factor-beta 1 (TGF-β1) was then coupled to the heparin-conjugated F127. X-HA was used as a physical stabilizer of the composite hydrogel (X-HA/F127). The chemical structures of F127 derivatives were analyzed using (1)H-NMR and ATR-FTIR. Sol-gel transition of the composite hydrogel was identified at body temperature. The conjugated TGF-β1 was moderately released in vitro from the composite hydrogel. Cell viability of human ASCs in the hydrogels was about 50% after in vitro culture for 3 days. As the ASCs/hydrogel were injected into nude mice subcutaneously, DAPI staining of the retrieved constructs showed that ASCs were dispersed through the hydrogel matrix. From the immunofluorescent staining of type II collagen, the TGF-conjugated group exhibited more active green signals than the others. In addition, when those constructs were loaded into the full-thickness defect of rabbit knee articular cartilage, Alcian blue staining identified the formation of cartilaginous matrix from the TGF-conjugated hydrogel. The present work indicated that X-HA/F127 composite hydrogel was thermoreversible and biodegradable, and that the TGF-conjugated hydrogel could be effective in inducing chondrogenesis of human ASCs.

N,N,N-Trimethyl chitosan nanoparticles for controlled intranasal delivery of HBV surface antigen

Hepatitis B virus surface antigen (HBsAg) loaded N,N,N-trimethyl chitosan nanoparticles (N-TMC NPs) were formulated and studied for controlled intranasal delivery. The size and surface properties of the NPs can be tuned by modifying the concentration of N-TMC and found to be 66±13, 76±9 nm for 0.25 and 0.5 wt.% respectively. Loading of 380 and 760 μl of HBsAg yielded 143±33, 259±47 nm sized spherical N-TMC NPs with highest loading efficiency and capacity of 90-93%, and 96-97% respectively. In vitro drug release analysis ensured 93% cumulative release of HBsAg antigen over prolonged period (43 days). In vivo immunological study was performed using 6-8 weeks old female BALB mice and reveals adjuvants efficiency of NPs for antigen is highly stable and better than standard. Obtained results show that N-TMC NPs can be extensively used in controlled intra nasal delivery to treat various diseases including hepatitis B and allergic rhinitis.

Biocompatible PEG Grafting on DLC-coated Nitinol Alloy for Vascular Stents:

The surfaces of Nitinol (TiNi), a popular metal alloy for arterial stents were thin-coated with diamond-like carbon (DLC) and then grafted with poly(ethylene glycol) (PEG) to increase biocompatibility. The TiNi control, DLC-coated TiNi (TiNi—DLC), and the PEG-grafted TiNi—DLC (TiNi—DLC—PEG) surface characteristics and biocompatibility were evaluated. The hydrophilicity of the TiNi—DLC—PEG significantly increased and the amount of both oxygen and nitrogen on the TiNi—DLC—PEG also increased compared to the TiNi control and TiNi—DLC due to the grafted PEG. The ratio between albumin and fibrinogen was higher on the PEG-grafted surface than the other surfaces when tested with human blood components; the platelet adhesion decreased the most on the TiNi—DLC—PEG surface, indicating improved blood compatibility. For in vivo tests using a rat model, the samples that were implanted for 6 weeks formed fibrous tissue; the tissue layer was much thinner on the PEG-grafted sample than the other two groups. The present results indicate that PEG-grafted TiNi—DLC surface may be effective in enhancing biocompatibility of blood-contacting biomaterials including vascular stents.

Self-assembled extracellular macromolecular matrices and their different osteogenic potential with preosteoblasts and rat bone marrow mesenchymal stromal cells.

Extracellular environment is a physical support that is critical to cell adhesion, migration, and differentiation. In this work, cell-derived matrices (CDMs) were obtained by separately culturing fibroblasts, preosteoblasts, and chondrocytes. The cells were grown on a coverslip and subjected to decellularization using detergents and enzymes. The resulting matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). We hypothesize that the unique compositional and structural feature of each CDM provides cells with a distinct microenvironment capable of functioning as a different signaling cue in the regulation of preosteoblast and rat bone marrow mesenchymal stromal cell (BMSC) osteogenic differentiation. SEM images show that each cell type creates its unique surface texture in a fibrillar structure. Three major macromolecules, fibronectin, type I collagen, and laminin, were clearly identified using both immunofluorescence and Western blot, in which FDM exhibited a much stronger signal of each ECM component than that of PDM or CHDM. For early cell morphology, BMSCs on the CDMs were highly elongated in a spindle-like shape. Both preosteoblasts and BMSCs proliferated well on CDMs comparable to the control. Once preosteoblasts were cultured for 2 weeks, their osteogenic activity was significantly different depending on the type of CDM. Using Alizarin red and von Kossa staining, we found that the cells on the FDM were much more osteogenic than the other groups. Furthermore, FDM was the most effective in upregulating the osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and type I collagen. In particular, we observed a 2.5-fold increase in ALP activity with FDM compared to that of control and CHDM. In stark contrast, CHDM was very poor in stimulating osteogenic differentiation of preosteoblasts. Interestingly, these results were reproducible with the use of BMSCs, which are much more heterogeneous in cell populations than preosteoblasts. CHDM was still very weak in triggering the osteogenesis of BMSCs, whereas both FDM and PDM were equally competitive. This study demonstrates that a combination of factors (surface texture and composition) shape a unique cellular microenvironment, which serves as a physical cue toward the osteogenic differentiation of preosteoblasts and BMSCs.

Dual Growth Factor Delivery Using Biocompatible Core–Shell Microcapsules for Angiogenesis

An optimized electrodropping system produces homogeneous core-shell microcapsules (C-S MCs) by using poly(L-lactic-co-glycolic acid) (PLGA) and alginate. Fluorescence imaging clearly shows the C-S domain in the MC. For release control, the use of high-molecular-weight PLGA (HMW 270 000) restrains the initial burst release of protein compared to that of low-MW PLGA (LMW 40 000). Layer-by-layer (LBL) assembly of chitosan and alginate on MCs is also useful in controlling the release profile of biomolecules. LBL (7-layer) treatment is effective in suppressing the initial burst release of protein compared to no LBL (0-layer). The difference of cumulative albumin release between HMW (7-layer LBL) and LMW (0-layer LBL) PLGA is determined to be more than 40% on day 5. When dual angiogenic growth factors (GFs), such as platelet-derived GF (PDGF) and vascular endothelial GF (VEGF), are encapsulated separately in the core and shell domains, respectively, the VEGF release rate is much greater than that of PDGF, and the difference of the cumulative release percentage between the two GFs is about 30% on day 7 with LMW core PLGA and more than 45% with HMW core PLGA. As for the angiogenic potential of MC GFs with human umbilical vein endothelial cells (HUVECs), the fluorescence signal of CD31+ suggests that the angiogenic sprout of ECs is more active in MC-mediated GF delivery than conventional GF delivery, and this difference is significant, based on the number of capillary branches in the unit area. This study demonstrates that the fabrication of biocompatible C-S MCs is possible, and that the release control of biomolecules is adjustable. Furthermore, MC-mediated GFs remain in an active form and can upregulate the angiogenic activity of ECs.