Se Heang Oh

Fabrication and characterization of hydrophilic poly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-molding particulate-leaching method

Porous PLGA/PVA scaffolds were fabricated by blending poly(lactic-co-glycolic acid) (PLGA) with polyvinyl alcohol (PVA) to improve the hydrophilicity and cell compatibility of the scaffolds for tissue engineering applications. PLGA/PVA blend scaffolds with different PVA compositions up to 20wt% were fabricated by a melt-molding particulate-leaching method (non-solvent method). The prepared scaffolds were investigated by scanning electron microscopy (SEM), mercury intrusion porosimetry, the measurements of water contact angles and bi-axial tensile strengths, etc. for their surface and bulk characterizations. The scaffolds exhibited highly porous and open-cellular pore structures with almost same surface and interior porosities (pore size, 200-300 microm; porosity, about 90%). The PLGA/PVA blend scaffolds with PVA compositions more than 5% were easily wetted in cell culture medium without any prewetting treatments, which is highly desirable for tissue engineering applications. In vitro cell compatibility of the control hydrophobic PLGA and hydrophilized PLGA/PVA (5wt%) blend scaffolds was compared by the culture of human chondrocytes in the scaffolds and the following analyses by MTT assay and SEM observation. It was observed that the PLGA/PVA blend scaffold had better cell adhesion and growth than the control PLGA scaffold. For in vivo evaluation of tissue compatibility, the scaffolds were implanted into the skull defects of rabbits. The results were evaluated by histology examinations. The PLGA/PVA (5wt%) blend scaffold showed better bone ingrowth into the scaffold and new bone formation inside the scaffold than the PLGA scaffold. It seems that 5% addition of PVA to PLGA to fabricate PLGA/PVA blend scaffolds is enough for improving the hydrophilicity and cell compatibility of the scaffolds.

Development of a composite vascular scaffolding system that withstands physiological vascular conditions.

Numerous scaffolds that possess ideal characteristics for vascular grafts have been fabricated for clinical use. However, many of these scaffolds may not show consistent properties when they are exposed to physiologic vascular environments that include high pressure and flow, and they may eventually fail due to unexpected rapid degradation and low resistance to shear stress. There is a demand to develop a more durable scaffold that could withstand these conditions until vascular tissue matures in vivo. In this study, vascular scaffolds composed of poly(epsilon-caprolactone) (PCL) and collagen were fabricated by electrospinning. Morphological, biomechanical, and biological properties of these composite scaffolds were examined. The PCL/collagen composite scaffolds, with fiber diameters of approximately 520 nm, possessed appropriate tensile strength (4.0+/-0.4 MPa) and adequate elasticity (2.7+/-1.2 MPa). The burst pressure of the composite scaffolds was 4912+/-155 mmHg, which is much greater than that of the PCL-only scaffolds (914+/-130 mmHg) and native vessels. The composite scaffolds seeded with bovine endothelial cells (bECs) and smooth muscle cells (bSMCs) showed the formation of a confluent layer of bECs on the lumen and bSMCs on the outer surface of the scaffold. The PCL/collagen composite scaffolds are biocompatible, possess biomechanical properties that resist high degrees of pressurized flow over long term, and provide a favorable environment that supports the growth of vascular cells.

Oxygen generating scaffolds for enhancing engineered tissue survival.

One of the continued challenges in engineering clinically applicable tissues is the establishment of vascularization upon implantation in vivo. Although the effectiveness of an enhanced angiogenic response using various growth factors has been demonstrated in many tissue systems, the rate of angiogenesis could not be accelerated. In this study we investigated whether incorporating oxygen generating biomaterials into tissue engineered constructs would provide a sustained oxygen release over an extended period of time. We examined whether oxygen generating biomaterials are able to maintain cell viability while also maintaining structural integrity of a 3-D construct. Calcium peroxide-based oxygen generating particles were incorporated into 3-D scaffolds of Poly(d,l-lactide-co-glycolide) (PLGA). The scaffolds were designed to generate oxygen over the course of 10 days and simultaneously maintain sufficient mechanical integrity. Scaffolds containing oxygen generating materials maintained elevated levels of oxygen when incubated under hypoxic conditions. Further, these biomaterials were able to extend cell viability growth under hypoxic conditions. These findings indicate that the use of oxygen generating biomaterials may allow for increased cell survivability while neovascularization is being established after implantation. Such scaffolds may play an important role in tissue engineering where currently oxygen diffusion limits the engineering of large tissue implants.

In vitro and in vivo degradation behavior of acetylated chitosan porous beads

Chitosans with different degree of acetylation (DA, 10–50%) were synthesized by the acetylation reaction of deacetylated chitosan and acetic anhydride with different ratios. The porous beads (approx. 500 μm) fabricated from the acetylated chitosans were used to investigate the degradation behaviors of chitosans with different DA in vitro and in vivo. The in vitro degradation behavior of the acetylated chitosan beads was investigated in solutions of lysozyme and/or N-acetyl-β-D-glucosaminidase (NAGase), which are enzymes for chitosan present in the human body. It was observed that the degradation rate of acetylated chitosans can be controlled by adjusting the DA value: the degradation increased with increasing DA value of the acetylated chitosans. It seemed that NAGase plays an important role for the full degradation of chitosans in the body, even though NAGase itself can not initiate the degradation of chitosans. The in vitro degradation behavior of the chitosans in the mixture solution of lysozyme and NAGase was more similar to the in vivo degradation behavior than in the single lysozyme or NAGase solution. It may be owing to the sequential degradation reaction of chitosans in the mixture solution of lysozyme and NAGase (initial degradation by lysozyme to low-molecular-weight species or oligomers and the following degradation by NAGase to monomer forms). The in vivo degradation rate of acetylated chitosan beads was faster than the in vitro degradation rate. The acetylated chitosan porous beads with different DA value (and thus different degradation time) can be widely applicable as cell carriers for tissue-engineering applications.

Fabrication and characterization of porous alginate/polyvinyl alcohol hybrid scaffolds for 3D cell culture

Porous alginate/polyvinyl alcohol (PVA) hybrid scaffolds as bioartificial cell scaffolds were fabricated to improve cell compatibility as well as flexibility of the scaffolds. The alginate/PVA hybrid scaffolds with different PVA compositions up to 50 wt% were fabricated by a modified freeze-drying method including the physical cross-linking of PVA and the following chemical cross-linking of alginate. The prepared alginate/PVA hybrid scaffolds were characterized by morphology observations using scanning electron microscopy (SEM), the measurements of porosity and average pore sizes and the measurements of compressive strength and modulus. The scaffolds exhibited highly porous, open-cellular pore structures with almost the same surface and cross-sectional porosities (total porosities about 85%, regardless of PVA composition) and the pore sizes from about 290 μm to about 190 μm with increasing PVA composition. The alginate/PVA hybrid scaffolds were more soft and elastic than the control alginate scaffold without significant changes of mechanical strength. The scaffolds were examined for their in vitro cell compatibility by the culture of chondrocytes (human chondrocyte cell line) in the scaffolds and the following analyses by MTT assay and SEM observation. It was observed that the alginate/PVA scaffolds had better cell adhesion and faster growth than the control alginate scaffold. It seems that 30 wt% addition of PVA to alginate in the fabrication of the hybrid scaffolds is desirable for improving their flexibility and cell compatibility.

Novel fabrication of PCL porous beads for use as an injectable cell carrier system.

Injectable polycaprolactone (PCL) porous beads were fabricated for use as cell carriers by a novel isolated particle-melting method (for nonporous beads) and the following melt-molding particulate-leaching method (for porous beads). The prepared beads showed highly porous and uniform pore structures with almost the same surface and interior porosities (porosity, over 90%). The PCL porous beads (bead size, 400-550 microm) with different pore sizes (25-50 and 50-100 microm) were compared for their in vitro cell (human chondrocyte) growth behavior with the nonporous beads. The porous beads showed higher cell seeding density and growth than the nonporous beads. The pore size effect between the porous beads was not significant up to 7 days, but after that time the beads with pore sizes of 50-100 microm showed significantly higher cell growth than those of 25-50 microm. To evaluate the tissue compatibility of the PCL porous beads, the beads were dispersed, uniformly, in cold Pluronic F127 solution and injected into hairless mice, subcutaneously, in the gel state of Pluronic F127 at room temperature, leading to the homogeneous bead delivery. The histological findings confirmed that the PCL porous beads in Pluronic F127 gel are biocompatible: surrounding tissues gradually infiltrated into the porous beads for up to 4 weeks with little inflammatory response. The PCL porous beads with highly porous and uniform pore structures fabricated in this study can be widely applicable as cell carriers.

Asymmetrically porous PLGA/Pluronic F127 membrane for effective guided bone regeneration

Porous guided bone regeneration (GBR) membranes with selective permeability, hydrophilicity and adhesiveness to bone were prepared with PLGA and Pluronic F127 using an immersion precipitation method. The porous PLGA/Pluronic F127 membranes were fabricated by immersing the PLGA/Pluronic F127 mixture solution (in tetraglycol) in a mold into water. The PLGA/Pluronic F127 mixture was precipitated in water by the diffusion of water into PLGA/Pluronic F127 mixture solution. It was observed that the membrane has an asymmetric column-shape porous structure. The top surface of the membrane (water contact side) had nano-size pores (approx. 50 nm) which can effectively prevent from fibrous connective tissue invasion but permeate nutrients, while the bottom surface (mold contact size) had micro-size pores (approx. 40 μm) which can improve adhesiveness with bone. From the investigations of mechanical property, water absorbability, model nutrient permeability and preliminary in vivo bone regeneration, the hydrophilized porous PLGA/F127 (5 wt%) membrane seems to be a good candidate as a GBR membrane for the effective permeation of nutrients and osteoconductivity, as well as good mechanical strength to maintain a secluded space for bone regeneration.

Hydrophilized polycaprolactone nanofiber mesh-embedded poly(glycolic-co-lactic acid) membrane for effective guided bone regeneration.

A novel guided bone regeneration (GBR) membrane was fabricated by an immersion precipitation of poly (glycolic-co-lactic acid) (PLGA)/Pluronic F127 solution impregnated in an electrospun polycaprolactone (PCL)/Tween 80 nanofiber mesh. The prepared PCL/Tween 80 nanofiber mesh-embedded PLGA/Pluronic F127 membrane (hydrophilized PCL/PLGA hybrid membrane) had nano-size pores on the top side (which can prevent from fibrous connective tissue infiltration but allow permeation of oxygen and nutrients) and micro-size pores on the bottom side (which can improve adhesiveness with bone). From the comparisons of mechanical properties (tensile and suture pullout strengths), model nutrient (FITC-labeled bovine serum albumin) permeability, and bone regeneration behavior using a rat model (skull bone defect) of the hybrid membrane with those of PLGA/Pluronic F127 membrane (asymmetrically porous, hydrophilized PLGA membrane), PCL/Tween 80 nanofiber mesh (electrospun, hydrophilized PCL nanofiber mesh), and a commercialized GBR membrane, Bio-Gide (collagen type I/III membrane), it was observed that the PCL/PLGA hybrid membrane seems to be highly desirable as a GBR membrane for the selective permeability caused by its unique morphology and osteoconductivity provided by several tens micro-size pores of the bottom side as well as the excellent mechanical strengths by the hybridization of porous PLGA membrane and PCL nanofiber mesh.

Therapeutic Effect of Adipose-Derived Stem Cells and BDNF-immobilized PLGA Membrane in a Rat Model of Cavernous Nerve Injury

INTRODUCTION Cavernous nerve injury is the main reason for post-prostatectomy erectile dysfunction (ED). Stem cell and neuroprotection therapy are promising therapeutic strategy for ED. AIM To evaluate the therapeutic efficacy of adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF) immobilized Poly-Lactic-Co-Glycolic (PLGA) membrane on the cavernous nerve in a rat model of post-prostatectomy ED. Methods.  Rats were randomly divided into five groups: normal group, bilateral cavernous nerve crush injury (BCNI) group, ADSC (BCNI group with ADSCs on cavernous nerve) group, BDNF-membrane (BCNI group with BDNF/PLGA membrane on cavernous nerve) group, and ADSC/BDNF-membrane (BCNI group with ADSCs covered with BDNF/PLGA membrane on cavernous nerve) group. BDNF was controlled-released for a period of 4 weeks in a BDNF/PLGA porous membrane system. MAIN OUTCOME MEASURES Four weeks after the operation, erectile function was assessed by detecting the ratio of intra-cavernous pressure (ICP)/mean arterial pressure (MAP). Smooth muscle and collagen content were determined by Massons trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. Phospho-endothelial nitric oxide synthase (eNOS) protein expression and cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by Western blotting and cGMP assay, respectively. RESULTS In the ADSC/BDNF-membrane group, erectile function was significantly elevated, compared with the BCNI and other treated groups. ADSC/BDNF-membrane treatment significantly increased smooth muscle/collagen ratio, nNOS content, phospho-eNOS protein expression, and cGMP level, compared with the BCNI and other treated groups. CONCLUSIONS ADSCs with BDNF-membrane on the cavernous nerve can improve erectile function in a rat model of post-prostatectomy ED, which may be used as a novel therapy for post-prostatectomy ED.

Degradation Behavior of 3D Porous Polydioxanone-b-Polycaprolactone Scaffolds Fabricated Using the Melt-Molding Particulate-Leaching Method

Recently, polydioxanone (PDO) and polycaprolactone (PCL) have been applied in applications for tissue engineering owing to their flexibility, as well as biocompatibility and biodegradability, even though their degradation rates are usually either too fast or too slow for many applications. In this study, we synthesized poly(dioxanone-b-caprolactone) co-polymers (PDOCLs) with different DO/CL ratio (0:10–10:0) by ring-opening polymerization. The synthesized co-polymers were characterized by 1H-NMR, the measurement of inherent viscosity (IV), GPC and DSC. PDOCL scaffolds with different DO/CL ratio were fabricated by a melt-molding particulate-leaching method without using any organic solvents during the scaffold fabrication process. The degradation behavior (in vitro) of the PDOCL scaffolds was evaluated in PBS at 37°C for up to 56 days by the changes in molecular weight, mechanical strength, gross weight and pH. It was observed that the degradation rate of PDOCL scaffolds could be controlled by adjusting the DO/CL ratio of the co-polymers (increasing CL composition leads to slower degradation rate). The PDOCL scaffolds did not lead to a significant drop in pH during the degradation, not even for the PDO-dominant PDOCL scaffolds showing a fast degradation rate, indicating the formation of a small amount of acidic by-products compared to the PLGA scaffolds. From the results, it was expected that the PDOCLs can be a new flexible scaffolding material with different degradation rate for various tissue-engineering applications.