M. Brett Runge

The Development of Electrically Conductive Polycaprolactone Fumarate-Polypyrrole Composite Materials for Nerve Regeneration

Electrically conductive polymer composites composed of polycaprolactone fumarate and polypyrrole (PCLF-PPy) have been developed for nerve regeneration applications. Here we report the synthesis and characterization of PCLF-PPy and in vitro studies showing PCLF-PPy materials support both PC12 cell and dorsal root ganglia (DRG) neurite extension. PCLF-PPy composite materials were synthesized by polymerizing pyrrole in preformed PCLF scaffolds (M(n) 7,000 or 18,000 g mol(-1)) resulting in interpenetrating networks of PCLF-PPy. Chemical compositions and thermal properties were characterized by ATR-FTIR, XPS, DSC, and TGA. PCLF-PPy materials were synthesized with five different anions (naphthalene-2-sulfonic acid sodium salt (NSA), dodecylbenzenesulfonic acid sodium salt (DBSA), dioctyl sulfosuccinate sodium salt (DOSS), potassium iodide (I), and lysine) to investigate effects on electrical conductivity and to optimize chemical composition for cellular compatibility. PCLF-PPy materials have variable electrical conductivity up to 6 mS cm(-1) with bulk compositions ranging from 5 to 13.5 percent polypyrrole. AFM and SEM characterization show microstructures with a root mean squared (RMS) roughness of 1195 nm and nanostructures with RMS roughness of 8 nm. In vitro studies using PC12 cells and DRG show PCLF-PPy materials synthesized with NSA or DBSA support cell attachment, proliferation, neurite extension, and are promising materials for future studies involving electrical stimulation.

Development of Electrically Conductive Oligo(polyethylene Glycol) Fumarate-Polypyrrole Hydrogels for Nerve Regeneration

Electrically conductive hydrogel composites consisting of oligo(polyethylene glycol) fumarate (OPF) and polypyrrole (PPy) were developed for applications in nerve regeneration. OPF-PPy scaffolds were synthesized using three different anions: naphthalene-2-sulfonic acid sodium salt (NSA), dodecylbenzenesulfonic acid sodium salt (DBSA), and dioctyl sulfosuccinate sodium salt (DOSS). Scaffolds were characterized by ATR-FTIR, XPS, AFM, dynamic mechanical analysis, electrical resistivity measurements, and swelling experiments. OPF-PPy scaffolds were shown to consist of up to 25 mol % polypyrrole with a compressive modulus ranging from 265 to 323 kPa and a sheet resistance ranging from 6 to 30 × 10(3) Ohms/square. In vitro studies using PC12 cells showed OPF-PPy materials had no cytotoxicity and PC12 cells showed distinctly better cell attachment and an increase in the percent of neurite bearing cells on OPF-PPy materials compared to OPF. The neurite lengths of PC12 cells were significantly higher on OPF-PPyNSA and OPF-PPyDBSA. These results show that electrically conductive OPF-PPy hydrogels are promising candidates for future applications in nerve regeneration.

Material properties and electrical stimulation regimens of polycaprolactone fumarate–polypyrrole scaffolds as potential conductive nerve conduits

The mechanical and electrical properties of polycaprolactone fumarate-polypyrrole (PCLF-PPy) scaffolds were studied under physiological conditions to evaluate their ability to maintain the material properties necessary for application as conductive nerve conduits. PC12 cells cultured on PCLF-PPy scaffolds were stimulated with regimens of 10 μA of either a constant or a 20 Hz frequency current passed through the scaffolds for 1h per day. PC12 cellular morphologies were analyzed by fluorescence microscopy after 48 h. PCLF-PPy scaffolds exhibited excellent mechanical properties at 37 °C which would allow suturing and flexibility. The surface resistivity of the scaffolds was 2 kΩ and the scaffolds were electrically stable during the application of electrical stimulation (ES). In vitro studies showed significant increases in the percentage of neurite bearing cells, number of neurites per cell and neurite length in the presence of ES compared with no ES. Additionally, extending neurites were observed to align in the direction of the applied current. This study shows that electrically conductive PCLF-PPy scaffolds possess the material properties necessary for application as nerve conduits. Additionally, the capability to significantly enhance and direct neurite extension by passing an electrical current through PCLF-PPy scaffolds renders them even more promising as future therapeutic treatments for severe nerve injuries.

Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: In vitro characterization and application in polycaprolactone fumarate nerve conduits

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator. Controlled release of such stimulators may enhance and guide the vascularization process, and when applied in a nerve conduit may play a role in nerve regeneration. We report the fabrication and in vitro characterization of poly-lactic-co-glycolic acid (PLGA) microspheres encapsulating VEGF and the in vivo application of nerve conduits supplemented with VEGF-containing microspheres. PLGA microspheres containing VEGF were prepared by the double emulsion-solvent evaporation technique. This yielded 83.16% of microspheres with a diameter <53 μm. VEGF content measured by ELISA indicated 93.79±10.64% encapsulation efficiency. Release kinetics were characterized by an initial burst release of 67.6±8.25% within the first 24h, followed by consistent release of approximately 0.34% per day for 4 weeks. Bioactivity of the released VEGF was tested by human umbilical vein endothelial cell (HUVEC) proliferation assay. VEGF released at all time points enhanced HUVEC proliferation, confirming that VEGF retained its bioactivity throughout the 4 week time period. When the microsphere delivery system was placed in a biosynthetic nerve scaffold robust nerve regeneration was observed. This study established a novel system for controlled release of growth factors and enables in vivo studies of nerve conduits conditioned with this system.

Cross-linking characteristics and mechanical properties of an injectable biomaterial composed of polypropylene fumarate and polycaprolactone co-polymer.

In this work, a series of co-polymers of polypropylene fumarate-co-polycaprolactone (PPF-co-PCL) were synthesized via a three-step polycondensation reaction of oligomeric polypropylene fumarate (PPF) with polycaprolactone (PCL). The effects of PPF precursor molecular weight, PCL precursor molecular weight and PCL fraction in the co-polymer (PCL feed ratio) on the maximum cross-linking temperature, gelation time and mechanical properties of the cross-linked co-polymers were investigated. The maximum cross-linking temperature fell between 38.2 ± 0.3 and 47.2 ± 0.4°C, which increased with increasing PCL precursor molecular weight. The gelation time was between 4.2 ± 0.2 and 8.5 ± 0.7 min, and decreased with increasing PCL precursor molecular weight. The compressive moduli ranged from 44 ± 1.8 to 142 ± 7.4 MPa, with enhanced moduli at higher PPF precursor molecular weight and lower PCL feed ratio. The compressive toughness was in the range of 4.1 ± 0.3 and 17.1 ± 1.3 kJ/m3. Our data suggest that the cross-linking and mechanical properties of PPF-co-PCL can be modulated by varying the composition. Therefore, the PPF-co-PCL co-polymers may offer increased versatility as an injectable, in situ polymerizable biomaterial than the individual polymers of PPF and PCL.

Incorporation of phosphate group modulates bone cell attachment and differentiation on oligo(polyethylene glycol) fumarate hydrogel

In this work, we have investigated the development of a synthetic hydrogel that contains a negatively charged phosphate group for use as a substrate for bone cell attachment and differentiation in culture. The photoreactive, phosphate-containing molecule, bis(2-(methacryloyloxy)ethyl)phosphate (BP), was incorporated into oligo(polyethylene glycol) fumarate hydrogel and the mechanical, rheological and thermal properties of the resulting hydrogels were characterized. Our results showed changes in hydrogel compression and storage moduli with incorporation of BP. The modification also resulted in decreased crystallinity as recorded by differential scanning calorimetry. Our data revealed that incorporation of BP improved attachment and differentiation of human fetal osteoblast (hFOB) cells in a dose-dependent manner. A change in surface chemistry and mineralization of the phosphate-containing surfaces verified by scanning electron microscopy and energy dispersive X-ray analysis was found to be important for hFOB cell attachment and differentiation. We also demonstrated that phosphate-containing hydrogels support attachment and differentiation of primary bone marrow stromal cells. These findings suggest that BP-modified hydrogels are capable of sustaining attachment and differentiation of both bone marrow stromal cells and osteoblasts that are critical for bone regeneration.

Effect of Calcium Phosphate Coating and rhBMP-2 on Bone Regeneration in Rabbit Calvaria Using Poly(propylene fumarate) Scaffolds

Various calcium phosphate based coatings have been evaluated for better bony integration of metallic implants and are currently being investigated to improve the surface bioactivity of polymeric scaffolds. The aim of this study was to evaluate the role of calcium phosphate coating and simultaneous delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the in vivo bone regeneration capacity of biodegradable, porous poly(propylene fumarate) (PPF) scaffolds. PPF scaffolds were coated with three different calcium phosphate formulations: magnesium-substituted β-tricalcium phosphate (β-TCMP), carbonated hydroxyapatite (synthetic bone mineral, SBM) and biphasic calcium phosphate (BCP). In vivo bone regeneration was evaluated by implantation of scaffolds in a critical-sized rabbit calvarial defect loaded with different doses of rhBMP-2. Our data demonstrated that scaffolds with each of the calcium phosphate coatings were capable of sustaining rhBMP-2 release and retained an open porous structure. After 6weeks of implantation, micro-computed tomography revealed that the rhBMP-2 dose had a significant effect on bone formation within the scaffolds and that the SBM-coated scaffolds regenerated significantly greater bone than BCP-coated scaffolds. Mechanical testing of the defects also indicated restoration of strength in the SBM and β-TCMP with rhBMP-2 delivery. Histology results demonstrated bone growth immediately adjacent to the scaffold surface, indicating good osteointegration and osteoconductivity for coated scaffolds. The results obtained in this study suggest that the coated scaffold platform demonstrated a synergistic effect between calcium phosphate coatings and rhBMP-2 delivery and may provide a promising platform for the functional restoration of large bone defects.

Nanocomposite bone scaffolds based on biodegradable polymers and hydroxyapatite

In tissue engineering, development of an osteoconductive construct that integrates with host tissue remains a challenge. In this work, the effect of bone-like minerals on maturation of pre-osteoblast cells was investigated using polymer-mineral scaffolds composed of poly(propylene fumarate)-co-poly(caprolactone) (PPF-co-PCL) and nano-sized hydroxyapatite (HA). The HA of varying concentrations was added to an injectable formulation of PPF-co-PCL and the change in thermal and mechanical properties of the scaffolds was evaluated. No change in onset of degradation temperature was observed due to the addition of HA, however compressive and tensile moduli of copolymer changed significantly when HA amounts were increased in composite formulation. The change in mechanical properties of copolymer was found to correlate well to HA concentration in the constructs. Electron microscopy revealed mineral nucleation and a change in surface morphology and the presence of calcium and phosphate on surfaces was confirmed using energy dispersive X-ray analysis. To characterize the effect of mineral on attachment and maturation of pre-osteoblasts, W20-17 cells were seeded on HA/copolymer composites. We demonstrated that cells attached more to the surface of HA containing copolymers and their proliferation rate was significantly increased. Thus, these findings suggest that HA/PPF-co-PCL composite scaffolds are capable of inducing maturation of pre-osteoblasts and have the potential for use as scaffold in bone tissue engineering.

Reformulating polycaprolactone fumarate to eliminate toxic diethylene glycol: effects of polymeric branching and autoclave sterilization on material properties.

Polycaprolactone fumarate (PCLF) is a cross-linkable derivative of polycaprolactone diol that has been shown to be an effective nerve conduit material that supports regeneration across segmental nerve defects and has warranted future clinical trials. Degradation of PCLF (PCLF(DEG)) releases toxic small molecules of diethylene glycol used as the initiator for the synthesis of polycaprolactone diol. In an effort to eliminate this toxic degradation product we present a strategy for the synthesis of PCLF from either propylene glycol (PCLF(PPD)) or glycerol (PCLF(GLY)). PCLF(PPD) is linear and resembles the previously studied PCLF(DEG), while PCLF(GLY) is branched and exhibits dramatically different material properties. The synthesis and characterization of their thermal, rheological, and mechanical properties are reported. The results show that the linear PCLF(PPD) has material properties similar to the previously studied PCLF(DEG). The branched PCLF(GLY) exhibits dramatically lower crystalline properties resulting in lower rheological and mechanical moduli, and is therefore a more compliant material. In addition, the question of an appropriate Food and Drug Administration approvable sterilization method is addressed. This study shows that autoclave sterilization of PCLF materials is an acceptable sterilization method for cross-linked PCLF and has minimal effect on the PCLF thermal and mechanical properties.

Gait cycle analysis: parameters sensitive for functional evaluation of peripheral nerve recovery in rat hind limbs.

BackgroundVideo-assisted gait kinetics analysis has been a sensitive method to assess rat sciatic nerve function after injury and repair. However, in conduit repair of sciatic nerve defects, previously reported kinematic measurements failed to be a sensitive indicator because of the inferior recovery and inevitable joint contracture. ObjectiveThis study aimed to explore the role of physiotherapy in mitigating joint contracture and to seek motion analysis indices that can sensitively reflect motor function. MethodsData were collected from 26 rats that underwent sciatic nerve transection and conduit repair. Regular postoperative physiotherapy was applied. Parameters regarding step length, phase duration, and ankle angle were acquired and analyzed from video recording of gait kinetics preoperatively and at regular postoperative intervals. ResultsStride length ratio (step length of uninjured foot/step length of injured foot), percent swing of the normal paw (percentage of the total stride duration when the uninjured paw is in the air), propulsion angle (toe-off angle subtracted by midstance angle), and clearance angle (ankle angle change from toe off to midswing) decreased postoperatively comparing with baseline values. The gradual recovery of these measurements had a strong correlation with the post–nerve repair time course. ConclusionsAnkle joint contracture persisted despite rigorous physiotherapy. Parameters acquired from a 2-dimensional motion analysis system, that is, stride length ratio, percent swing of the normal paw, propulsion angle, and clearance angle, could sensitively reflect nerve function impairment and recovery in the rat sciatic nerve conduit repair model despite the existence of joint contractures.