Gilson Khang

Interaction of cells on chargeable functional group gradient surfaces

Functional group gradient surfaces where the surface density of grafted functional groups changes gradually along the sample length were prepared on low density polyethylene (PE) sheets by corona discharge treatment with gradually increasing power and graft copalymerization of acrylic acid (AA), sodium p-styrene sulphonate (NaSS), and N,N-dimethyl aminopropyl acrylamide (DMAPAA). AA and NaSS are negatively chargeable and DMAPAA is positively chargeable in phosphate buffered saline or cell culture medium at pH 7.3-7.4. The functional group gradient surfaces were characterized by the measurement of water contact angle, Fourier transform infrared spectroscopy in the attenuated total reflectance mode, and electron spectroscopy for chemical analysis. All these measurements indicated that the functional groups were grafted on the PE surfaces with gradual increase of their density. The interaction of Chinese hamster ovary cells with the functional group gradient surfaces along the sample length was investigated. The cells that had adhered and grown on the surfaces were counted and observed by scanning electron microscopy. It was observed that a greater quantity of the cells had adhered and grown onto the positions with moderate density of the functional groups. This may be related to the hydrophilicity of the surface. The DMAPAA-grafted surface showed a large amount of cell attachment probably owing to the positive charge character, while the AA-grafted surface, which is negatively charged, showed poor cell attachment, as expected. The NaSS-grafted surface which is also negatively charged showed a large amount of cell attachment. This may be closely associated with the existence of an aromatic ring close to the ionizable group in NaSS. It seems that surface functional groups and their charge character as well as wettability play important roles for cell adhesion, spreading, and growth.

The effect of surface wettability on induction and growth of neurites from the PC-12 cell on a polymer surface.

Surface properties of polymeric devices that are used to regenerate nervous damage are a point to be considered for axon regeneration in nerve system. In our previous studies, we prepared a wettability gradient on polyethylene (PE) surfaces using a corona discharge treatment from a knife-type electrode whose power increases gradually along the sample length. The PE surfaces were oxidized gradually with increasing power. The effect of surface wettability on the different types of cells has an important role for cell adhesion and proliferation. The purpose of this study is to investigate neurite formation on polymer surfaces with different wettability. Induction and growth of neurites from the rat pheochromocytoma (PC-12) cells attached on the polymer surfaces with different hydrophilicity were investigated using the wettability gradient PE surfaces prepared by a corona discharge treatment. Neurites were investigated for number and length of neurites in terms of surface wettability. It was observed that neurite formation of PC-12 cells was increased more onto the positions with moderate hydrophilicity of the wettability gradient surface than onto the more hydrophobic or hydrophilic positions. From those results, it could be assumed that initial adhesion of PC-12 cells was caused by more calf serum (CS) protein than nerve growth factor (NGF), whereas the neurite formation of PC-12 cells was caused by more NGF than CS protein. It follows from what has been said thus far that PC-12 cells are a differentiated neuronal phenotype with a long neurite at around the position 2.5 cm (water contact angle of about 55 deg). In conclusion, surface wettability plays an important role for neurite formation on the polymer surfaces for axon regeneration.

Nifedipine controlled delivery by sandwiched osmotic tablet system

The sandwiched osmotic tablet system (SOTS), which is composed of a sandwiched osmotic tablet core surrounded by a cellulose acetate membrane with two orifices on both side surfaces, has been successfully prepared with the purpose of delivering nifedipine. The sandwiched osmotic tablet core consists of a middle push layer and two attached drug layers. Influences of tablet formulation variables, orifice size and membrane variables on nifedipine release of SOTS have been studied. It was found that potassium chloride amount of push layer and polyethylene oxide amount of drug layer had markedly positive effects on nifedipine release. A push layer/drug layer co-controlled osmotic delivery mechanism and the optimal core formulation have been proposed. The appropriate orifice size was observed in the range of 0.50-1.41 mm. It was also found that the drug release rate of SOTS could be increased by incorporating hydrophilic plasticizer in the membrane, whereas it decreased with hydrophobic plasticizer. It has been observed that the SOTS gives fairly comparable in vitro release features as that of commercialized push-pull osmotic tablet system, such as an approximately constant rate up to 24 h and independence of release media and agitation rate. Exempting side identification before drilling, it is easier to prepare the SOTS than the push-pull osmotic tablet system.

Reduction of surface-induced inflammatory reaction on PLGA/MPC polymer blend

Poly(lactide-co-glycolide) (PLGA) has been believed to be a good biocompatible material for tissue engineering due to its biodegradability and non-toxicity of the monomer. However, the inflammatory reaction of adherent cells on the surface has not been discussed sufficiently. We hypothesized that the inflammatory reaction of adherent cells on PLGA might occur and could be reduced by blending a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer (PMEH) with the PLGA. PLGA/PMEH blend membranes were prepared by a solvent evaporation technique. The thermal properties of the PLGA/PMEH membrane were determined using a differential scanning calorimeter. The glass transition temperature of the PLGA/PMEH membranes was slightly decreased compared to that of a PLGA membrane. X-ray photoelectron spectrum analysis revealed that the MPC unit was exposed on the PLGA/PMEH membrane and that the surface concentration of the MPC unit on the membrane was increased with an increase in the concentration of the PMEH in the blended membrane. NIH-3T3 mouse fibroblast cells were cultured on the PLGA/ PMEH membrane for 2 days. The number of adherent cells on the PLGA/PMEH membrane was decreased with an increase in the concentration of the PMEH. Using the RT-PCR method, the amount of an inflammatory cytokine, IL-1beta, mRNA expressed from adherent human premyelocytic leukemia cells on PLGA and PLGA/PMEH membranes were determined. On a PLGA/PMEH membrane containing 0.2 wt% of PMEH, the expression of IL-1beta mRNA was significantly lower than that on PLGA, but no difference in the number of adherent cells was found. Therefore, the MPC polymer was a useful additive for reducing the inflammatory reaction of adherent cells on PLGA.

Monolithic osmotic tablet system for nifedipine delivery.

The monolithic osmotic tablet system, which is composed of a monolithic tablet coated with cellulose acetate (CA) membrane drilled with two orifices on both side surfaces, has been described. The influences of tablet formulation variables including molecular weight (MW) and amount of polyethylene oxide (PEO), amount of potassium chloride (KCl), and amount of rice starch as well as nifedipine loading have been investigated. The optimal tablet formulation and the osmotic-suspending co-controlled delivery mechanisms have been proposed. Orifice size and membrane variables including nature and amount of plasticizers as well as thickness on drug release have also been studied. The in vitro release profiles of the optimal system have been evaluated in various release media and different agitation rates, and compared with commercialized conventional capsule and push-pull osmotic tablet. It was found that PEO with MW of 300000 g/mol was suitable to be thickening agent, both amount of KCl and amount of PEO had comparable and profoundly positive effects, while nifedipine loading had a strikingly negative influence on drug release. It could be found that the optimal orifice size was in the range of 0.25-1.41 mm. It has also been observed that hydrophilic plasticizer polyethylene glycol (PEG) improved drug release, whereas hydrophobic plasticizer triacetin depressed drug release when they were incorporated in CA membrane. The monolithic osmotic tablet system was found to be able to deliver nifedipine at the rate of approximate zero-order up to 24 h, independent of both environmental media and agitation rate, and substantially comparable with the push-pull osmotic tablet. The monolithic osmotic tablet system was simple to be prepared as exempting from push layer and simplifying in the orifice drilling compared with the push-pull osmotic tablet. The monolithic osmotic tablet system may be used in drug controlled delivery field, especially suitable for water-insoluble drugs.

Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma.

Corneal transplantation is a common transplant procedure performed to improve visual acuity by replacing the opaque or distorted host tissue by clear healthy donor tissue. However, its clinical utility is limited due to a lack of high quality donor corneas. Bioengineered neo-corneas, created using an expandable population of human donor-derived corneal endothelial cells (HCEC), could address this current shortage. The objectives of this study were to establish HCEC isolation and culture protocols and to investigate the feasibility of bioengineering corneal tissue constructs by seeding the cells on decellularized human corneal stroma. HCECs were removed from the discarded corneas of eye donors by enzymatic digestion. Cells were expanded and evaluated for their expression of Na(+)/K(+)-ATPase and zona occludens-1 (ZO-1). Donor corneal stromas were cut to 120-200 microm thickness slices using a microtome and then decellularized. Extracellular matrix components and mechanical properties of the scaffolds were measured after decellularization. To engineer neo-corneas, 130 HCEC/mm(2) were seeded on decellularized human corneal stromas. The resulting constructs were placed in growth medium for 14 days and then analyzed using scanning electron microscopy (SEM), histology, and immunocytochemistry. Seeded cells retain expression of the functional markers Na(+)/K(+)-ATPase and ZO-1 and constructs have biomechanical properties similar to those of normal corneas. These results indicate that construction of neo-corneas, using HCECs derived from discarded donor corneas and decellularized thin-layer corneal stromas, may create a new source of high quality corneal tissue for transplantation.

Reduction of inflammatory reaction of poly(d,l-lactic-co-glycolic Acid) using demineralized bone particles.

Poly(lactide-co-glycolic acid) (PLGA) has been widely applied to tissue engineering as a good biocompatible material because of its biodegradability and nontoxic metabolites, but how the inflammatory reaction of PLGA on the surrounding tissue in vivo is reduced has not been discussed sufficiently. We hypothesized that the cells neighboring the PLGA implant might have an inflammatory response that could be reduced by impregnating demineralized bone particles (DBPs) into the PLGA. We manufactured five different ratios of DBP/PLGA hybrid materials, with each material containing 0, 10, 20, 40, and 80 wt% of DBPs of PLGA. For biocompatibility test, NIH/3T3 mouse fibroblasts were cultured on the DBP/PLGA scaffold for 3 days. The inflammatory potential of PLGA was evaluated using messenger ribonucleic acid expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) on a human acute promyelocytic leukemic cell (HL-60). The in vivo response of DBP/PLGA film was compared with that of PLGA film implanted subcutaneously; the local inflammatory response was observed according to histology. The DBP/PLGA scaffold had no adverse effect on NIH/3T3 initial cell attachment and did not affect cell viability. DBP/PLGA films, especially PLGA films containing 80% DBP, elicited a significantly lower expression of IL-1beta and TNF-alpha from HL-60 cells than PLGA film alone. In vivo, DBP/PLGA film demonstrated a more favorable tissue response profile than PLGA film, with significantly less inflammation and fibrous capsule formation as below only 20% of DBP in PLGA film during implantation. This study shows that application of DBPs reduces the fibrous tissue encapsulation and foreign body giant cell response that commonly occurs at the interface of PLGA.

Platelet adhesion onto chargeable functional group gradient surfaces

Functional group gradients were prepared on low-density polyethylene (PE) sheets. The surface density of grafted functional groups was gradually changed along the sample length by way of corona discharge treatment with gradually increasing power following graft copolymerization of acrylic acid (AA), sodium p-styrene sulfonate (NaSS), or N,N-dimethyl aminopropyl acrylamide (DMAPAA). AA and NaSS are negatively chargeable and DMAPAA is positively chargeable in phosphate-buffered saline or plasma solution at pH 7.3-7.4. The prepared functional group gradient surfaces were characterized by measurement of the water contact angle, by electron spectroscopy for chemical analysis, and by Fourier transform infrared spectroscopy in the attenuated total reflectance mode. All these measurements indicated that the functional groups were grafted onto the PE surfaces with gradually increasing density. The platelets adhered to the functional group gradient surfaces along the sample length were counted and observed by scanning electron microscopy. It was observed that the platelet adhesion to the gradient surfaces decreased gradually with the increasing surface density of functional groups. This may be related to the hydrophilicity of the surfaces. The DMAPAA-grafted surface showed a large amount of platelet adhesion, probably due to its positive charge character, while the AA-grafted surface, which is charged negatively, showed poor platelet adhesion. However, the NaSS-grafted surface, which is also charged negatively, showed a relatively large amount of platelet adhesion. This may be associated with the existence of an aromatic ring close to the ionizable group in NaSS. It seems that surface functional groups and their charge character, as well as wettability, play important roles for platelet adhesion.

Preparation and characterization of fentanyl-loaded PLGA microspheres: in vitro release profiles

We developed several kinds of fentanyl-loaded poly(L-lactide-co-glycolide) (PLGA) microspheres (FMS) for sustained release of fentanyl. FMS were prepared by an emulsion solvent-evaporation method. In this study, the influences of several preparation parameters, such as initial drug loading, polymer concentration, and solvent volume on the release patterns of fentanyl were investigated. Furthermore, it has been well noted that the detection of fentanyl is extremely difficult because its clinical dose level is very low, about 1-3 ng/ml, in cancer-patient treatment. Therefore, we also developed a rapid and sensitive determination method for fentanyl in systemic circulation by employing gas chromatography (GC) system. Fentanyl was slowly released from FMS over 15 days with a quasi-zero order property. From the results, our FMS may be good formulations to deliver the analgesics and suitable for the treatment of severe pain over long periods.

Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity.

Surface topography appears to be an important but often neglected factor in implant performance. In this study, fibroblasts were cultured on a range of porous polycarbonate (PC) membranes with well defined surface topography (track-etched micropores, 0.2-8.0 microm in diameter) and wettability gradients. The wettability gradient on the PC membrane surfaces was produced by treating the surfaces with corona from a knife-type electrode whose power increased gradually along the sample length. The PC membrane surfaces were characterized by scanning electron microscopy (SEM) and the water contact angle measurement. Fibroblasts were cultured on the corona-treated PC membrane surfaces with different micropore sizes for 1 and 2 days. The cells attached on the membrane surfaces were examined by SEM and the cell density on the surfaces was estimated by counting the number of attached cells along the wettability gradient. It was observed that the cells were adhered and grew more on the hydrophilic positions of the membrane surfaces than the more hydrophobic ones, regardless of micropore size. It was also observed that cell adhesion and growth decreased gradually with increasing micropore size of the membrane surfaces. It seems that the cell adhesion and growth were progressively inhibited as the membrane surfaces had micropores with increasing size, probably due to surface discontinuities produced by tract-etched pores. On the membrane surfaces with smaller micropore sizes, the cells seemed to override these surface discontinuities.