Photo-crosslinkable and biodegradable Pluronic/heparin hydrogels for local and sustained delivery of angiogenic growth factor.

Photo-crosslinkable and biodegradable Pluronic/heparin composite hydrogels were fabricated for local and sustained delivery of basic fibroblast growth factor (bFGF) to induce angiogenesis. Terminally di-acrylated Pluronic F127 and vinyl group conjugated heparin were used as a mixed macromer precursor solution to prepare a photo-crosslinkable hydrogel. An aqueous solution containing the two macromers with different weight ratios was photo-crosslinked in the presence of bFGF to produce in situ formed bFGF loaded Pluronic/heparin hydrogels. Swelling, mass erosion, bFGF release characteristics of Pluronic/heparin hydrogels were thoroughly examined by varying the weight ratio of the two macromers. The incorporation of heparin in the composite hydrogel enabled the controlled release of bFGF over a one month period in a near zero order manner. The prolonged release of bFGF could be attributed to the specific interaction between bFGF and heparin in the hydrogel matrices. The released bFGF fraction from the degradable hydrogels also showed sufficient proliferation activity of human umbilical vein endothelial cell (HUVEC). When the Pluronic/heparin hydrogels were implanted in vivo, a significant extent of neo-vascularization was observed.

Gas foamed open porous biodegradable polymeric microspheres.

Highly open porous biodegradable polymeric microspheres were fabricated for use as injectable scaffold microcarriers for cell delivery. A modified water-in-oil-in-water (W1/O/W2) double emulsion solvent evaporation method was employed for producing the microspheres. The incorporation of an effervescent salt, ammonium bicarbonate, in the primary W1 droplets spontaneously produced carbon dioxide and ammonia gas bubbles during the solvent evaporation process, which not only stabilized the primary emulsion, but also created well inter-connected pores in the resultant microspheres. The porous microspheres fabricated under various gas foaming conditions were characterized. The surface pores became as large as 20 microm in diameter with increasing the concentration of ammonium bicarbonate, being sufficient enough for cell infiltration and seeding. These porous scaffold microspheres could be potentially utilized for cultivating cells in a suspension manner and for delivering the seeded cells to the tissue defect site in an injectable manner.

Fabrication of hyaluronic acid hydrogel beads for cell encapsulation.

Hyaluronic acid (HA) hydrogel beads were prepared by photopolymerization of methacrylated HA and N-vinylpyrrolidone using alginate as a temporal spherical mold. Various fabrication conditions for preparing the hydrogel beads, such as the concentration of methacrylated HA and UV irradiation time, were optimized to control swelling properties and enzymatic degradability. A new concept for cell encapsulation is proposed in this paper. Viable cells were directly injected into the hydrogel beads using a microinjection technique. When bovine articular chondrocytes were injected into HA hydrogel beads and cultivated for 1 week, the cells could proliferate well within the HA beads. HA hydrogel beads could be potentially used as injectable cell delivery vehicles for regenerating tissue defects.

Heparin-immobilized biodegradable scaffolds for local and sustained release of angiogenic growth factor.

Heparin-immobilized porous biodegradable scaffolds were fabricated to release basic fibroblast growth factor (bFGF) in a sustained manner. Heparin was covalently conjugated onto the surface of macroporous PLGA scaffolds fabricated by a gas-foaming/salt-leaching method. Sustained release of bFGF was successfully achieved for over 20 days due to high affinity of bFGF onto the immobilized heparin. It appears that bFGF release rate was regulated by the specific interaction between bFGF and heparin. The bFGF fraction released from the scaffolds maintained its bioactivity, as judged from determining the proliferation extent of human umbilical vein endothelial cells (HUVECs) in vitro. When heparin-immobilized scaffolds loaded with bFGF were implanted subcutaneously in vivo, they effectively induced the formation of blood vessels in the vicinity of the implant site. This study demonstrated that local and sustained delivery of angiogenic growth factor for tissue regeneration could be achieved by surface modification of porous scaffolds with heparin.

Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering.

Hyaluronic acid (hyaluronan, HA) was immobilized onto the surface of macroporous biodegradable poly(D,L-lactic acid-co-glycolic acid) [PLGA] scaffolds to enhance the attachment, proliferation, and differentiation of chondrocytes for cartilage tissue engineering. The PLGA scaffolds were prepared by blending PLGA with varying amounts of amine-terminated PLGA-PEG di-block copolymer. They were fabricated by a gas foaming/salt leaching method. HA was chemically conjugated to the surface-exposed amine groups on the pre-fabricated scaffolds. The amount of surface exposed free amine groups was quantitatively determined by conjugating an amine-reactive fluorescent dye to the PLGA blend films. The extent of HA immobilization was also confirmed by measuring water contact angles. When chondrocytes were seeded within HA modified PLGA scaffolds, enhanced cellular attachment was observed compared to unmodified PLGA scaffolds. Furthermore, glycosaminoglycan and total collagen synthesis increased substantially for HA modified PLGA scaffolds. RT-PCR result and histological examination of the resultant cartilage tissue revealed that HA modified scaffolds excelled in inducing cartilage tissue formation in terms of collagen type II expression and tissue morphological characteristics.

Dexamethasone-releasing biodegradable polymer scaffolds fabricated by a gas-foaming/salt-leaching method.

Dexamethasone, a steroidal anti-inflammatory drug, was incorporated into porous biodegradable polymer scaffolds for sustained release. The slowly released dexamethasone from the degrading scaffolds was hypothesized to locally modulate the proliferation and differentiation of various cells. Dexamethasone containing porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by a gas-foaming/salt-leaching method. Dexamethasone was loaded within the polymer phase of the PLGA scaffold in a molecularly dissolved state. The loading efficiency of dexamethasone varied from 57% to 65% depending on the initial loading amount. Dexamethasone was slowly released out in a controlled manner for over 30 days without showing an initial burst release. Release amount and duration could be adjusted by controlling the initial loading amount within the scaffolds. Released dexamethasone from the scaffolds drastically suppressed the proliferations of lymphocytes and smooth muscle cells in vitro. This study suggests that dexamethasone-releasing PLGA scaffolds could be potentially used either as an anti-inflammatory porous prosthetic device or as a temporal biodegradable stent for reducing intimal hyperplasia in restenosis.

Immobilization of cell adhesive RGD peptide onto the surface of highly porous biodegradable polymer scaffolds fabricated by a gas foaming/salt leaching method.

A cell adhesive peptide moiety, Gly-Arg-Gly-Asp-Tyr (GRGDY), was immobilized onto the surface of highly porous biodegradable polymer scaffolds for enhancing cell adhesion and function. A carboxyl terminal end of poly(D,L-lactic-co-glycolic acid) (PLGA) was functionalized with a primary amine group by conjugating hexaethylene glycol-diamine. The PLGA-NH2 was blended with PLGA in varying ratios to prepare films by solvent casting or to fabricate porous scaffolds by a gas foaming/salt leaching method. Under hydrating conditions, the activated GRGDY could be directly immobilized to the surface exposed amine groups of the PLGA-NH2 blend films or scaffolds. For the PLGA blend films, the surface density of GRGDY, surface wettability change, and cell adhesion behaviors were characterized. The extent of cell adhesion was substantially enhanced by increasing the blend ratio of PLGA-NH2 to PLGA. The level of an alkaline phosphatase activity, measured as a degree of cell differentiation, was also enhanced as a result of the introduction of cell adhesive peptides.

Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function.

Poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres were prepared by an oil/water emulsion solvent evaporation method to use as an injectable microcarrier for cell delivery. Three different kinds of PLGA microspheres having hydrophobic, negatively charged, and positively charged surfaces were prepared. Hydrophobic and negatively charged PLGA microspheres were prepared by using terminally capped and uncapped PLGA polymer, respectively. Positively charged PLGA microspheres were prepared by blending PLGA with PLGA-g-poly(L-lysine) graft copolymer as a surface modifying agent. Bovine chondrocytes were cultured on the three PLGA microspheres under serum conditions to comparatively evaluate cell attachment, cell proliferation, and cell function with respect to surface properties. Positively charged PLGA microspheres showed the highest cell attachment, growth, and function compared to hydrophobic and negatively charged microspheres. Surface-modified PLGA microspheres can potentially be used as an injectable delivery system for cells into a tissue defect site.

Tissue-engineered cartilage on biodegradable macroporous scaffolds: cell shape and phenotypic expression.

OBJECTIVE: The purpose of the study was to establish in vitro culture of chondrocytes on biodegradable, poly(D,L-lactic-co-glycolic acid) [PLGA] scaffolds. STUDY DESIGN: Laboratory experiment using cartilage of rat rib and biodegradable scaffolds. METHODS: Chondrocytes were cultured on a poly-hydroxyethyl methacrylate (poly-HEMA)-coated dish, proliferated, and transferred into the PLGA scaffolds. Phenotypic expression of cells was examined according to the condition of poly-HEMA coating. Morphological, biochemical, and immunohistochemical characteristics of cells cultured within PLGA scaffolds were also examined. RESULTS: Chondrocytes cultured on a poly-HEMA-coated dish aggregated into distinct nodules containing large clusters of spherical cells and showed cartilage-specific phenotype, collagen type II. The results of immunostaining andreverse transcriptase-polymerase chain reaction of cells cultured within PLGA scaffolds showed cartilage-specific morphological appearance and structural characteristics such as lacunae and expression of collagen type II. CONCLUSION: The chondrocytes cultured on a poly-HEMA-coated dish and PLGA scaffolds showed chondrocyte-specific phenotypes and morphological appearance.

Controlling degradation of acid-hydrolyzable pluronic hydrogels by physical entrapment of poly(lactic acid-co-glycolic acid) microspheres.

Chemically crosslinked biodegradable hydrogels based on di-acrylated Pluronic F-127 tri-block copolymer were prepared by a photopolymerization method. Poly(lactic acid-co-glycolic acid) (PLGA) microspheres were physically entrapped within the Pluronic hydrogel in order to modulate the local pH environment by acidic degradation by-products of PLGA microspheres. The PLGA microspheres were slowly degraded to create an acidic microenvironment, which facilitated the cleavage of an acid-labile ester-linkage in the biodegradable Pluronic hydrogel network. The presence of PLGA microspheres accelerated the degradation of the Pluronic hydrogel and enhanced the protein release rate when protein was loaded in the hydrogel.SEM image of photo-crosslinked Pluronic hydrogel entrapping PLGA microspheres.

Galactosylated poly(N-isopropylacrylamide) hydrogel submicrometer particles for specific cellular uptake within hepatocytes.

Poly(N-isopropylacrylamide-co-acrylic acid) hydrogel submicrometer particles were prepared by free radical copolymerization of N-isopropylacrylamide and acrylic acid in the presence of a crosslinker above the lower critical solution temperature (LCST). They exhibited a reversible swelling and deswelling behavior: ca. 200-nm diameter below the LCST and ca. 100-nm diameter above the LCST. The hydrogel particles were tagged with fluorescent dye (FITC) in order to monitor the extent of cellular uptake and were further modified with galactose moieties to evaluate the extent of receptor-mediated endocytosis against HepG2 cells. Flow cytometry and confocal microscopy were used to investigate cellular uptake behaviors of the submicrometer particles. It was found that the extent of cellular uptake of submicrometer particles was far greater above the LCST than below the LCST, suggesting that smaller particles were taken up more readily within cells. When the submicrometer particles were galactosylated, the extent of cellular uptake increased dramatically due to receptor-mediated endocytosis. This study proposes a new possibility of controlling intracellular events such as protein and gene expression by a thermally modulated endocytosis process using thermo-sensitive microgel beads.

Injectable extracellular matrix hydrogel developed using porcine articular cartilage.

This work was first development of a delivery system capable of maintaining a sustained release of protein drugs at specific sites by using potentially biocompatible porcine articular cartilage. The prepared porcine articular cartilage powder (PCP) was easily soluble in phosphate-buffered saline. The PCP suspension easily entrapped bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) in pharmaceutical formulations at room temperature. The aggregation of PCP and BSA-FITC was confirmed by dynamic light scattering. When the BSA-FITC-loaded PCP suspension was subcutaneously injected into rats, it gelled and formed an interconnecting three-dimensional PCP structure that allowed BSA to penetrate through it. The amount of BSA-FITC released from the PCP hydrogel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated sustained release of BSA-FITC for 20 days in vivo. In addition, the PCP hydrogel induced a slight inflammatory response. In conclusion, we showed that the PCP hydrogel could serve as a minimally invasive therapeutics depot.

Cell adhesion and proliferation evaluation of SFF-based biodegradable scaffolds fabricated using a multi-head deposition system.

Scaffolds composed of biodegradable polymers and biocompatible ceramics are being used as substitutes for tissue engineering. In the development of such techniques, scaffolds with a controllable pore size and porosity were manufactured using solid free-form fabrication (SFF) methods to investigate the effects of cell interactions such as cell proliferation and differentiation. In this study, we describe the adhesion of human bone marrow stromal cells (hBMSCs) and proliferation characteristics of various scaffolds, which consist of biodegradable materials, fabricated using a multi-head deposition system (MHDS) that we developed. The MHDS uses novel technology that enables the production of three-dimensional (3D) microstructures. Fabrication of 3D tissue engineering scaffolds using the MHDS requires the combination of several technologies, such as motion control, thermal control, pneumatic control and computer-aided design/computer-assisted manufacturing software. The effects of a polymer and ceramic on a tissue scaffold were evaluated through mechanical testing and cell interaction analysis of various kinds of scaffolds fabricated using polycaprolactone, poly-lactic-co-glycolic acid and tri-calcium phosphate for bone regeneration applications. Based on these results, the feasibility of application to the tissue engineering of SFF-based 3D scaffolds fabricated by the MHDS was demonstrated.

Heparin immobilized porous PLGA microspheres for angiogenic growth factor delivery.

PURPOSE: Heparin immobilized porous poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres were prepared for sustained release of basic fibroblast growth factor (bFGF) to induce angiogenesis. MATERIALS AND METHODS: Porous PLGA microspheres having primary amine groups on the surface were prepared using an oil-in-water (O/W) single emulsion method using Pluronic F-127 as an extractable porogen. Heparin was surface immobilized via covalent conjugation. bFGF was loaded into the heparin functionalized (PLGA-heparin) microspheres by a simple dipping method. The bFGF loaded PLGA-heparin microspheres were tested for in vitro release and in vivo angiogenic activity. RESULTS: PLGA microspheres with an open-porous structure were formed. The amount of conjugated amine group onto the microspheres was 1.93+/-0.01 nmol/mg-microspheres, while the amount of heparin was 95.8 pmol/mg-microspheres. PLGA-heparin microspheres released out bFGF in a more sustained manner with a smaller extent of initial burst than PLGA microspheres, indicating that surface immobilized heparin controlled the release rate of bFGF. Subcutaneous implantation of bFGF loaded PLGA-heparin microspheres in mice significantly induced the formation of new vascular microvessels. CONCLUSIONS: PLGA microspheres with an open porous structure allowed significant amount of heparin immobilization and bFGF loading. bFGF loaded PLGA-HP microspheres showed sustained release profiles of bFGF in vitro, demonstrating reversible and specific binding of bFGF to immobilized heparin. They also induced local angiogenesis in vivo in an animal model.

Adhesion and growth of human umbilical vein endothelial cells on collagen-treated PU/PEGDA IPNs.

For ideal non-thrombogenicity under normal physiologic conditions, we propose endothelialization. Endothelialization means that synthetic biomaterials are seeded by endothelial cells to mimic natural blood vessels. In our study, we synthesized amphiphilic polyurethane (PU)/poly(ethyleneglycol)diacrylate (PEGDA) interpenetrating polymer networks (IPNs) with different levels of surface energy to investigate the effect of adhesion and the growth of human umbilical vein endothelial cells (HUVECs). Collagen with cell-binding molecules was adsorbed on the surface of PU/PEGDA IPNs to enhance the adhesion of HUVECs. The morphology of collagens adsorbed on the IPN surfaces depends highly on the surface energy of the IPNs. As the surface becomes hydrophilic, there is greater aggregation of the adsorbed collagens on the IPN surface. The HUVECs successfully adhere to the collagen-immobilized IPN surface. The morphology of the endothelial cells (ECs) that adhere to IPN 2k-C and IPN 2k after 1 day and after 3 days incubation shows that ECs were successfully spread. The adhesion and the proliferation of ECs increase on non-treated IPN surfaces as the hydrophobicity of the IPNs increases. The surface energy of IPN 2k-C is suitable for the adhesion and proliferation of ECs. Therefore, platelet adhesion is significantly reduced on the EC-hybridized surface of IPNs.

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture.

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.

Photo-crosslinkable, thermo-sensitive and biodegradable pluronic hydrogels for sustained release of protein.

Thermo-sensitive and biodegradable hydrogels based on Pluronic tri-block copolymers were prepared by a photo-polymerization method. Two terminal hydroxyl groups in Pluronic F-127 were acrylated to form a Pluronic macromer. Photo-cross-linked Pluronic hydrogels prepared by UV radiation showed a gradually decreased swelling ratio with increasing temperature and exhibited a thermally-responsive change in the swelling ratio when the temperature was cycled between 10 degrees C and 37 degrees C. These hydrogels degraded slowly due to the cleavage of ester linkage in the acrylated Pluronic terminal end. When lysozyme, a model protein drug, was loaded in the hydrogels, bi-phasic protein release profiles were attained: a burst-free and rapid controlled release profile was initially observed for a one week period and a much slower sustained release was followed thereafter. The release rates could be controlled by varying the amount of Pluronic macromer for photo-polymerization.