A. Lee Miller

Novel biodegradable poly(propylene fumarate)-co-poly(L-lactic acid) porous scaffolds fabricated by phase separation for tissue engineering applications

Scaffolds with intrinsically interconnected porous structures are highly desirable in tissue engineering and regenerative medicine. In this study, three-dimensional polymer scaffolds with highly interconnected porous structures were fabricated by thermally induced phase separation of novel synthesized biodegradable poly(propylene fumarate)-co-poly(l-lactic acid) in a dioxane/water binary system. Defined porous scaffolds were achieved by optimizing conditions to attain interconnected porous structures. The effect of phase separation parameters on scaffold morphology were investigated, including polymer concentration (1, 3, 5, 7, and 9%), quench time (1, 4, and 8 min), dioxane/water ratio (83/17, 85/15, and 87/13 wt/wt), and freeze temperature (-20, -80, and -196 °C). Interesting pore morphologies were created by adjusting these processing parameters, e.g., flower-shaped (5%; 85/15; 1 min; -80 °C), spherulite-like (5%; 85/15; 8 min; -80 °C), and bead-like (5%; 87/13; 1 min; -80 °C) morphology. Modulation of phase separation conditions also resulted in remarkable differences in scaffold porosities (81% to 91%) and thermal properties. Furthermore, scaffolds with varied mechanic strengths, degradation rates, and protein adsorption capabilities could be fabricated using the phase separation method. In summary, this work provides an effective route to generate multi-dimensional porous scaffolds that can be applied to a variety of hydrophobic polymers and copolymers. The generated scaffolds could potentially be useful for various tissue engineering applications including bone tissue engineering.

Functionalized Carbon Nanotube and Graphene Oxide Embedded Electrically Conductive Hydrogel Synergistically Stimulates Nerve Cell Differentiation

Nerve regeneration after injury is a critical medical issue. In previous work, we have developed an oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel incorporated with positive charges as a promising nerve conduit. In this study, we introduced cross-linkable bonds to graphene oxide and carbon nanotube to obtain the functionalized graphene oxide acrylate (GOa) and carbon nanotube poly(ethylene glycol) acrylate (CNTpega). An electrically conductive hydrogel was then fabricated by covalently embedding GOa and CNTpega within OPF hydrogel through chemical cross-linking followed by in situ reduction of GOa in l-ascorbic acid solution. Positive charges were incorporated by 2-(methacryloyloxy)ethyltrimethylammonium chloride (MTAC) to obtain rGOaCNTpega-OPF-MTAC composite hydrogel with both surface charge and electrical conductivity. The distribution of CNTpega and GOa in the hydrogels was substantiated by transmission electron microscopy (TEM), and strengthened electrical conductivities were determined. Excellent biocompatibility was demonstrated for the carbon embedded composite hydrogels. Biological evaluation showed enhanced proliferation and spreading of PC12 cells on the conductive hydrogels. After induced differentiation using nerve growth factor (NGF), cells on the conductive hydrogels were effectively stimulated to have robust neurite development as observed by confocal microscope. A synergistic effect of electrical conductivity and positive charges on nerve cells was also observed in this study. Using a glass mold method, the composite hydrogel was successfully fabricated into conductive nerve conduits with surficial positive charges. These results suggest that rGOa-CNTpega-OPF-MTAC composite hydrogel holds great potential as conduits for neural tissue engineering.

Biodegradable and crosslinkable PPF-PLGA-PEG self-assembled nanoparticles dual-decorated with folic acid ligands and Rhodamine B fluorescent probes for targeted cancer imaging

Novel biodegradable and crosslinkable copolymers of hydrophobic poly(propylene fumarate)-co-poly(lactic-co-glycolic acid) (PPF–PLGA) linked with hydrophilic poly(ethylene glycol) (PEG), namely PPF–PLGA–PEG, were developed and fabricated into core–shell nanoparticles through self-assembly and photocrosslinking. A fluorescent probe, Rhodamine B (RhB), was conjugated to the end of the copolymer chain (PPF–PLGA–PEG–RhB), which allows tracking of the nanoparticles through visualizing the fluorescence probe. Folic acid (FA) ligand was conjugated to another series of chains (PPF–PLGA–PEG–FA) for targeted delivery of the nanoparticles to the tumor sites by binding to the ubiquitously overexpressed FA receptors on tumor cells. Our results showed that PPF–PLGA–PEG nanoparticles incorporated with RhB fluorescence probes and FA tumor binding ligands have specific cancer cell targeting and imaging abilities. These crosslinkable nanoparticles are potentially useful to serve as a platform for conjugation of fluorescence probes as well as various antibodies and peptides for cancer targeted imaging or drug delivery.

Covalent crosslinking of graphene oxide and carbon nanotube into hydrogels enhances nerve cell responses

Healing of nerve injuries is a critical medical issue. Biodegradable polymeric conduits are a promising therapeutic solution to provide guidance for axon growth in a given space, thus helping nerve heal. Extensive studies in the past decade reported that conductive materials could effectively increase neurite and axon extension in vitro and nerve regeneration in vivo. In this study, graphene oxide and carbon nanotubes were covalently functionalized with double bonds to obtain crosslinkable graphene oxide acrylate (GOa) sheets and carbon nanotube poly(ethylene glycol) acrylate (CNTpega). An electrically conductive reduced GOa-CNTpega-oligo(polyethylene glycol fumarate) (OPF) hydrogel (rGOa-CNTpega-OPF) was successfully fabricated by chemically crosslinking GOa sheets and CNTpega with OPF chains followed by in situ chemical reduction in l-ascorbic acid solution. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging showed homogenous distribution of GOa/CNTpega carbon content in the rGOa-CNTpega-OPF composite hydrogel, resulting in a significant increase of electrical conductivity compared with neutral OPF without carbon content. Cell studies showed excellent biocompatibility and distinguished PC12 cell proliferation and spreading on the rGOa-CNTpega-OPF composite hydrogel. Fluorescent microscopy imaging demonstrated robustly stimulated neurite development in these cells on a conductive rGOa-CNTpega-OPF composite hydrogel compared with that on neutral OPF hydrogels. These results illustrated a promising potential for the rGOa-CNTpega-OPF composite hydrogel to serve as conduits for neural tissue engineering.

Tunable tissue scaffolds fabricated by in situ crosslink in phase separation system

Three-dimensional (3-D) scaffolds with intrinsic porous structures are desirable in various tissue regeneration applications. In this study, a unique method that combines thermally induced phase separation with a photocrosslinking process was developed for the fabrication of 3-D crosslinked polymer scaffolds with densely interconnected porous structures. Biodegradable poly(propylene fumarate)-co-poly(L-lactic acid) with crosslinkable fumarate bonds were used as the structural polymer material and a dioxane/water binary system was applied for the phase separation. By altering the polymer composition (9, 5 and 3 wt%), different types of scaffolds with distinct morphology, mechanical strength, degradation rate, cell growth and morphology, and extracellular matrix production were fabricated. These crosslinked 3-D porous scaffolds with tunable strength and biological responses show promise for potential applications in regenerative therapies, including bone and neural tissue engineering.

Hydrolysable core crosslinked particles for receptor-mediated pH-sensitive anticancer drug delivery

Biodegradable micelle systems with both extracellular stabilities and specific targeting properties are highly desirable for anti-cancer drug delivery. Here, we report a biodegradable and crosslinkable poly(propylene fumarate)-co-poly(lactide-co-glycolide)-co-poly(ethylene glycol) (PPF-PLGA-PEG) copolymer conjugated with folate (FA) molecules for receptor-mediated delivery of doxorubicin. Micelles with folate ligands on surface and fumarate bonds within the core were self-assembled and crosslinked, which exhibited better stability against potential physiological conditions during and after drug administration. A pH sensitive drug release profile was observed showing robust release at acidic environment due to the ester hydrolysis of PLGA (50:50). Further, micelles with folate ligands on surface showed strong targeting ability and therapeutic efficacy through receptor-mediated endocytosis, as evidenced by efficacious cancer killing and fatal DNA damage. These results imply promising potential for ligand-conjugated core crosslinked PPF-PLGA-PEG-FA micelles as carrier system for targeted anti-cancer drug delivery.

Novel porous poly(propylene fumarate‐co‐caprolactone) scaffolds fabricated by thermally induced phase separation

Scaffolds with porous structures are highly applicable for tissue engineering and regenerative medicine. In the present study, 3-dimensional poly(propylene fumarate-co-caprolactone) [P(PF-co-CL)] scaffolds were fabricated from a P(PF-co-CL)-dioxane-water ternary system through thermally induced phase separation (TIPS). Cloud points of P(PF-co-CL) in dioxane-water solutions increased with increased solute concentration, but increased dioxane composition decreased cloud point. Among 3 polymer concentrations (4, 8, and 12 wt%), 8 wt% P(PF-co-CL) scaffolds exhibited the best pore interconnectivity, with large, regular sized pores. Scaffolds were formed in 3 solutions with different dioxane-water ratios (74/26, 78/22, and 82/18 wt/wt); the 78/22 wt/wt scaffold had finger-shaped patterns with better interconnectivity than scaffolds from the other two ratios. Higher dioxane-water ratios resulted in a larger contact angle and thus less wettability for the fabricated scaffold, while scaffolds fabricated from higher concentrations of P(PF-co-CL) or high dioxane-water ratios had better biomineralization after soaking in simulated body fluid. In vitro cell viability testing showed the scaffolds had good biocompatibility with both bone and nerve cells. The results indicate that the polymer concentration and solvents ratio significantly affect the formation of porous structures, and optimum processing parameters were found to be 8% polymer concentration and 22% to 24% water content. These porous P(PF-co-CL) scaffolds fabricated via TIPS may be useful in various tissue engineering applications

Electrically conductive nanocomposite hydrogels embedded with functionalized carbon nanotubes for spinal cord injury

More than a million people are suffering from spinal cord injury (SCI) worldwide. However, currently there are no therapies that could ameliorate the neurological impairments, and the long-term SCI survival rate is far from satisfactory. Electrical stimulation and electrically conductive conduits are emerging as promising strategies with positive results showing increased neurite and axon growth in vitro and in vivo. In this study, we functionalized carbon nanotubes (CNTs) with hydrophilic poly(ethylene glycol) (PEG) chains and further introduced crosslinkablility with double bonds. The final carbon-nanotube-poly(ethylene glycol)-acrylate (CNTpega) material was embedded within oligo(poly(ethylene glycol) fumarate) (OPF) at varying concentrations to form conductive hydrogels with modulable conductivities. The morphological properties, mechanical strengths, conductivities, and CNT distributions within the hydrogels were characterized. In vitro neural cell adhesion and proliferation as well as neurite development after nerve growth factor (NGF) induction showed significant enhancement in the conductive hydrogels. Various types of nerve conduits were then successfully fabricated using an injection molding technique. With many desired properties, these conductive nerve conduits have promising potential for promoting axon guidance and enhancing nerve recovery in spinal cord injury patients.

Three-dimensional porous poly(propylene fumarate)-co-poly(lactic-co-glycolic acid) scaffolds for tissue engineering: 3D POROUS PPF-co-PLGA SCAFFOLDS

Three-dimensional structural scaffolds have played an important role in tissue engineering, especially broad applications in areas such as regenerative medicine. We have developed novel biodegradable porous poly(propylene fumarate)-co-poly(lactic-co-glycolic acid) (PPF-co-PLGA) scaffolds using thermally induced phase separation, and determined the effects of critical parameters such as copolymer concentration (6, 8, and 10 wt %) and the binary solvent ratio of dioxane:water (78/22, 80/20, 82/18 wt/wt %) on the fabrication process. The cloud-point temperatures of PPF-co-PLGA changed in parallel with increasing copolymer concentration, but inversely with increasing dioxane content. The compressive moduli of the scaffolds increased with greater weight composition and dioxane:water ratio. Scaffolds formed using high copolymer concentrations and solvent ratios exhibited preferable biomineralization. All samples showed biodegradation capability in both accelerated solution and phosphate-buffered saline (PBS). Cell toxicity testing indicated that the scaffolds had good biocompatibility with bone and nerve cells, which adhered well to the scaffolds. Variations in the copolymer concentration and solvent ratio exercised a remarkable influence on morphology, mechanical properties, biomineralization, and biodegradation, but not on the cell viability and adhesion of the cross-linked scaffolds. An 8 to 10 wt % solute concentration and 80/20 to 82/18 wt/wt dioxane:water ratio were the optimum parameters for scaffold fabrication. PPF-co-PLGA scaffolds thus possess several promising prospects for tissue engineering applications.

Cross-linkable graphene oxide embedded nanocomposite hydrogel with enhanced mechanics and cytocompatibility for tissue engineering: CROSS-LINKABLE GO EMBEDDED NANOCOMPOSITE HYDROGEL FOR TISSUE ENGINEERING

Graphene oxide (GO) is an attractive material that can be utilized to enhance the modulus and conductivities of substrates and hydrogels. To covalently cross-link graphene oxide sheets into hydrogels, abundant cross-linkable double bonds were introduced to synthesize the graphene-oxide-tris-acrylate sheet (GO-TrisA). Polyacrylamide (PAM) nanocomposite hydrogels were then fabricated with inherent covalently and permanently cross-linked GO-TrisA sheets. Results showed that the covalently cross-linked GO-TrisA/PAM nanocomposite hydrogel had enhanced mechanical strength, thermo stability compared with GO/PAM hydrogel maintained mainly by hydrogen bonding between PAM chains and GO sheets. In vitro cell study showed that the covalently cross-linked rGO-TrisA/PAM nanocomposite hydrogel had excellent cytocompatibility after in situ reduction. These results suggest that rGO-TrisA/PAM nanocomposite hydrogel holds great potential for tissue engineering applications.