Jixiang Zhu

Coseeded Schwann cells myelinate neurites from differentiated neural stem cells in neurotrophin-3-loaded PLGA carriers

Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury.

Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants.

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4 h at 37 °C, followed by lyophilization at -10 °C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7 ± 4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7 ± 4.6% and 23.01 ± 3.45 m(2)/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5 min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.

Elastic chitosan conduits with multiple channels and well defined microstructure.

Four kinds of chitosan conduits with longitudinal multi-channels and controlled internal microstructures were prepared using a special mold and a freeze-drying method. One of the conduits was fabricated from a chitosan solution (ab NC), while the other three groups were made from a pre-gelled chitosan solution using genipin as a chemical cross-linker (ab gNC), dibasic sodium phosphate as a physical cross-linker (ab pNC) or a combined ionic and covalent co-cross-linker (ab gpNC), respectively. The porosity of the chitosan conduits ranged from 88 to 90%. The gpNC showed highly interconnected and uniformly distributed pores compared to NC, the gNC and pNC. In contrast, the gNC and gpNC showed about 10% of the volume swelling ratio in 37°C PBS solution, although the gpNC scaffolds water uptake was the highest, at more than 17 times its original mass. Compressive tests showed that gpNC had significant elasticity and maintained its physical integrity even after compressing them down to 20% of their original height. The elastic modulus of gpNC reached 80 kPa, which was more than twice that of the other groups. Adhesion and proliferation of PC12 cells on chitosan gpNC scaffolds showed excellent properties by MTT and SEM observation, which indicated the potential of gpNC scaffolds for nerve tissue engineering applications.

Synthesis of Functional Polyester Based on Polylactic Acid and Its Effect on PC12 Cells after Coupling with Small Peptides

Polyesters containing functional groups are a suitable candidate matrix for cell culture in tissue engineering. Three types of semicrystalline copolymer poly( L-lactide-co-β-malic acid) [P(LA-co-BMD)] with pendent carboxyl groups were synthesized in this study. The functional monomer 3(S)-[(benzyloxycarbonyl)methyl]-1,4-dioxane-2,5-dione (BMD) was synthesized using L-aspartic acid. The copolymer P(LA-co-BMD) was then synthesized through ring-opening copolymerization of L-LA and BMD, with dodecanol as initiator and stannous octoate as catalyst. Copolymer structure was characterized by 1H nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analyses. Results of 1H NMR and GPC analyses showed that the copolymers were synthesized successfully. DSC curves showed that the crystal melting peak and enthalpy decreased with increased BMD. The crystallinity of the copolymer was destroyed by the presence of the functional monomer. After deprotection, carboxyl groups were coupled with the isoleucine-lysine-valine-alanine-valine peptide through N-hydroxysuccinimide/dicyclohexylcarbodiimide method. The small peptide was beneficial to the axon growth of PC12 cells.

SYNTHESIS OF POLY[(ε-CL)-co-(GA-alt-L-Asp)] COPOLYMER AND EFFECTS ON CELLS AFTER COUPLING WITH SMALL PEPTIDE

A novel poly[(e-CL)-co-(GA-alt-L-Asp)] with improved hydrophilicity was prepared. The monomer 3(S)-[(benzyloxycarbony)methyl]-1,4-dioxane-2,5-dione (BMD) was prepared from aspartic acid. The polymer was obtained by ring-opening polymerization of e-caprolactone (e-CL) and BMD. Poly[(e-CL)-co-(GA-alt-L-Asp)] was synthesized by deprotection. The structure and properties of the polymer were characterized using 1H nuclear magnetic resonance, gel permeation chromatography, and differential scanning calorimetry. The melting point and melting enthalpy of the polymer decreased with the increase of BMD. The structural regularity of polycaprolactone was destroyed by the BMD. A laminin-derived peptide [i.e., Arg-Gly-Asp (RGD)] was covalently tethered to the carboxyl groups and the peptide-grafted films. Results indicated that the addition of RGD had beneficial effects for cell growth, as shown by scanning electron microscopy.