Microstructure and properties of nano-fibrous PCL-b-PLLA scaffolds for cartilage tissue engineering.

Nano-fibrous scaffolds which could potentially mimic the architecture of extracellular matrix (ECM) have been considered a good candidate matrix for cell delivery in tissue engineering applications. In the present study, a semicrystalline diblock copolymer, poly(epsilon-caprolactone)-block-poly(L-lactide) (PCL-b-PLLA), was synthesized and utilized to fabricate nano-fibrous scaffolds via a thermally induced phase separation process. Uniform nano-fibrous networks were created by quenching a PCL-b-PLLA/THF homogenous solution to -20 degrees C or below, followed by further gelation for 2 hours due to the presence of PLLA and PCL microcrystals. However, knot-like structures as well as continuously smooth pellicles appeared among the nano-fibrous network with increasing gelation temperature. DSC analysis indicated that the crystallization of PCL segments was interrupted by rigid PLLA segments, resulting in an amorphous phase at high gelation temperatures. Combining TIPS (thermally induced phase separation) with salt-leaching methods, nano-fibrous architecture and interconnected pore structures (144+/-36 mm in diameter) with a high porosity were created for in vitro culture of chondrocytes. Specific surface area and protein adsorption on the surface of the nano-fibrous scaffold were three times higher than on the surface of the solid-walled scaffold. Chondrocytes cultured on the nano-fibrous scaffold exhibited a spherical condrocyte-like phenotype and secreted more cartilage-like extracellular matrix (ECM) than those cultured on the solid-walled scaffold. Moreover, the protein and DNA contents of cells cultured on the nano-fibrous scaffold were 1.2-1.4 times higher than those on the solid-walled scaffold. Higher expression levels of collagen II and aggrecan mRNA were induced on the nano-fibrous scaffold compared to on the solid-walled scaffold. These findings demonstrated that scaffolds with a nano-fibrous architecture could serve as superior scaffolds for cartilage tissue engineering.

Nano-fibrous and ladder-like multi-channel nerve conduits: Degradation and modification by gelatin.

We recently fabricated multi-channel PLLA nerve conduits (NCs, conduits diameter: ~3mm, channels diameter: ~200µm) with nano-fibrous microstructure (NNCs) and ladder-like microstructure (LNCs), and found the nanofibers in the NNCs promote differentiation of nerve stem cells (NSCs) into neurons. In the present study, we evaluated the degradation profile of NNCs and LNCs, and observed that NNCs degraded too fast to implant. To delay the degradation and retain the nano-scale effect of NNCs, we used gelatin to wrap (2% w/v gelatin) or embed (8% w/v gelatin) NNCs and LNCs via vacuum infusion and chemical cross-linking with genipin. NNCs-wrapped maintained their original nano-fibrous microstructure, but NNCs-embedded presented a porous morphology without nanofibers appearing. Incorporation of gelatin did not change their compressive moduli, but increased the creep recovery ratios of LNCs and NNCs. In vitro degradation revealed that integrity was maintained and the mass loss was <5% for NNCs-wrapped after 10weeks, in comparison with 15% mass loss and collapsed structure of the pure NNCs after 4weeks. Meanwhile, there were no obvious changes in the degradation of LNCs with modification. Nerve stem cells (NSCs) were then seeded onto the six NCs represented as: NNCs, NNCs-wrapped, NNCs-embedded, LNCs, LNCs-wrapped, and LNCs-embedded. Immunocytochemistry analysis demonstrated that gelatin coating enhanced the adhesion and proliferation of NSCs, and the NNCs-wrapped scaffold promoted the differentiation proportion of NSCs into neurons from 25.8% (on pure NNCs) to 53.4% after 14days of seeding. On the other hand, only 14.3% of neurons were derived from the differentiation of the seeded NSCs on the NNCs-embedded. NNCs-wrapped would be a good choice for future studies in nerve injury repair in vivo due to its appropriate degradation rate, flexibility, and nano-scale effect.

Biocompatible zwitterionic phosphorylcholine polymers with aggregation-induced emission feature.

Two novel zwitterionic phosphorylcholine polymers (MTP1 and MTP2) with aggregation-induced emission (AIE) feature were prepared through reversible addition fragmentation chain transfer polymerization between an AIE monomer with vinyl end group and a zwitterionic phosphorylcholine monomer. The synthesized copolymers were characterized and confirmed by 1H NMR, FT-IR, and X-ray photoelectron spectra. By introduction of the zwitterionic phosphorylcholine component, the synthesized copolymers showed amphiphilic properties and tended to self-assemble into fluorescent polymeric nanoparticles (FPNs) in water. The dynamic light scattering results indicated the size distribution of the MTP1 FPNs was 345±22nm, and that of the MTP2 FPNs was 147±36nm. The transmission electron microscopy results demonstrated spherical nanoparticle morphology for the FPNs. The high dispersibility of the FPNs in water was proved by the UV-vis absorption study with high transmittance of the solution. Fluorescent spectra of the prepared FPNs revealed bright green fluorescence with high fluorescence quantum yield of 45% for MTP1 and 34% for MTP2. More importantly, the FPNs showed excellent particle stability with low critical micelle concentration of 0.008mgmL-1 for MTP1 and 0.007mgmL-1 for MTP2. The cytotoxicity evaluation confirmed high cytocompatibility of the prepared FPNs at different concentrations, and demonstrated excellent biocompatibility for cell imaging. In virtue of the high-performance MTP1 and MTP2 FPNs, including high water dispersion, good particle stability, and excellent cytocompatibility, this work would inspire more researches about high-performance biocompatible fluorescent polymers for biomedical application.

Fabrication and evaluation of PLLA multichannel conduits with nanofibrous microstructure for the differentiation of NSCs in vitro.

Nerve conduits (NCs) with multiple longitudinally aligned channels, being mimicking the natural nerves anatomical structure, have been attracted more and more attentions. However, some specific structural parameters of a conduit that would be beneficial for further improvement of neural tissue regeneration were not comprehensively considered. Using a systematized device and combining low-pressure injection molding and thermal-induced phase separation, we fabricated 33-channel NCs (outer diameter 3.5 mm, channel diameter 200 µm) with different well-defined microscopic features, including NCs with a nano-fibrous microstructure (NNC), NCs with microspherical pores and nano-fibrous pore walls (MNC), and NCs with a ladder-like microstructure (LNC). The porosities of these NCs were ∼90% and were independent of the fine microstructures, whereas the pore size distributions were clearly distinct. The adsorption of bovine serum albumin for the NNC was a result of having the highest specific surface area, which was 3.5 times that of the LNC. But the mechanical strength of NNC was lower than that of two groups because of a relative high crystallinity and brittle characteristics. In vitro nerve stem cells (NSCs) incubation revealed that 14 days after seeding the NSCs, 31.32% cells were Map2 positive in the NNC group, as opposed to 15.76% in the LNC group and 23.29% in the MNC group. Addition of NGF into the culture medium, being distinctive specific surface area and a high adsorption of proteon for NNC, 81.11% of neurons derived from the differentiation of the seeded NSCs was obtained. As a result of imitating the physical structure of the basement membrane of the neural matrix, the nanofibrous structure of the NCs has facilitated the differentiation of NSCs into neurons.