Nanming Zhao

Properties and biocompatibility of chitosan films modified by blending with PEG.

Chitosan (beta-1,4-D-glucosamine), a polysaccharide with excellent biological properties, has been widely used in biomedical fields, but many barriers still exist to its broader usage due to its chemical and physical limitations. Further work is needed to improve these properties, but changes of the chemical and physical properties will influence its biocompatibility, so the biological attribute of modified chitosan must be evaluated. In this study, the biocompatibility of chitosan modified by several methods was carefully evaluated at the cellular and protein levels using different physical and biological methods. The results provide a theoretical basis for screening biomaterials. We studied the properties of five kinds of materials made by blending chitosan with different types of polyethylene glycol (PEG). The properties included physical and chemical properties, such as mechanical strength, static contact angle, spectroscopy, thermodynamic attributes and so on. The mechanical properties were slightly improved with the proper amount of PEG, but the improvement was not obvious and was destroyed by the wrong proportion of PEG. Cultures of the cells and amounts and structures of the adsorbed proteins on different materials showed that the PEG effectively improved the biocompatibility of the materials. The PEG enhanced the protein adsorption, cell adhesion, growth and proliferation, but the effects were impaired by excessive PEG. The experiments also demonstrated that the optimum PEG concentration helped to maintain the natural structure of the protein adsorbed on the materials and that maintaining the natural structure benefited cell growth. Analysis of the results based on the intramolecular and intermolecular interaction forces leads to a basic theory for the modification of biomaterials.

Studies on nerve cell affinity of biodegradable modified chitosan films

Chitosan, a natural polysaccharide that has excellent biocompatibility and biodegradability,can be used as nerve conduit material. The purpose of this work was to study the ability of chitosan and some chitosan-derived materials to facilitate nerve cell attachment, differentiation and growth. The biomaterials studied were chitosan, poly-L-lysine-blended chitosan (CP), collagen-blended chitosan (CC) and albumin-blended chitosan (CA), with collagen control material. Culture of PC12 cells and fetal mouse cerebral cortex (FMCC) cells on these biomaterials was used to evaluate their nerve cell affinity. The composite materials, including CP, CC and CA, had significantly improved nerve cell affinity compared to chitosan, as established by increasing attachment, differentiation and growth of PC12 cells. FMCC cells could also grow better on composite materials than on chitosan. CP exhibited the best nerve cell affinity among these three types of composite material. CP is an even better material in promoting neurite outgrowth than collagen, a substrate that is widely used in tissue engineering, suggesting that CP is a promising candidate material for nerve regeneration.

Physical, mechanical and degradation properties, and schwann cell affinity of cross-linked chitosan films.

Three kinds of cross-linked chitosan films were prepared with hexamethylene diisocyanate (HDI), epichlorohydrin (ECH) and glutaraldehyde (GA) as cross-linking agents, respectively. The physical and mechanical properties, biodegradability and Schwann cell affinity of the cross-linked films were investigated. A significant decrease in the degradation rate in lysozyme solution and a large change in the mechanical properties were observed compared with non-cross-linked chitosan films. The protein adsorption on chitosan films was determined by means of enzyme-linked immunosorbent assay (ELISA). In comparison with the non-cross-linked films, the chitosan films cross-linked with HDI showed a significant increase (up to 40–50%) in both fibronectin and laminin adsorption, while the protein adsorption on the other two kinds of cross-linked films was similar to that on non-cross-linked films. In addition, cell culture revealed that the HDI cross-linked chitosan films enhanced the spread and proliferation of Schwann cells while the other cross-linked films delayed the cell proliferation. These results suggest that HDI cross-linking of chitosan films provides a combination of physical properties, biodegradability and Schwann cell affinity suitable for peripheral nerve regeneration.

Physical properties and biocompatibility of a porous chitosan-based fiber-reinforced conduit for nerve regeneration

Porous fiber-reinforced chitosan nerve conduits were fabricated from chitosan yarns and a chitosan solution by combining an industrial braiding method with a mold casting/lyophilization technique. The conduits were permeable to molecules ranging in molecular size from 180xa0Da (glucose) to 66,200xa0Da (BSA). The compressive load of the reinforced conduits was significantly higher than that of a non-reinforced control conduit at equal levels of strain. The tensile strength of the reinforced conduits was also increased from 0.41xa0±xa00.17 to 3.69xa0±xa00.64xa0MPa. An inxa0vitro cytotoxicity test showed the conduits were not cytotoxic to Neuro-2a cells. Preliminary inxa0vivo implantation testing indicated that the conduits were compatible with the surrounding tissue.

Preparation and Characterization of a Multilayer Biomimetic Scaffold for Bone Tissue Engineering

In scaffold based bone tissue engineering, both the pore size and the mechanical properties of the scaffold are of great importance. However, an increase in pore size is generally accompanied by a decrease in mechanical properties. In order to achieve both suitable mechanical properties and porosity, a multilayer scaffold is designed to mimic the structure of cancellous bone and cortical bone. A porous nano-hydroxyapatite—chitosan composite scaffold with a multilayer structure is fabricated and encased in a smooth compact chitosan membrane layer to prevent fibrous tissue ingrowth. The exterior tube is shown to have a small pore size (15—40 μm in diameter) for the enhancement of mechanical properties, while the core of the multilayer scaffold has a large pore size (predominantly 70—150 μm in diameter) for nutrition supply and bone formation. Compared with the uniform porous scaffold, the multilayer scaffold with the same size shows an enhanced mechanical strength and larger pore size in the center. More cells are shown to grow into the center of the multilayer scaffold in vitro than into the uniform porous scaffold under the same seeding condition. Finally, the scaffolds are implanted into a rabbit fibula defect to evaluate the osteoconductivity of the scaffold and the efficacy of the scaffold as a barrier to fibrous tissue ingrowth. At 12 weeks post operation, affluent blood vessels and bone formation are found in the center of the scaffold and little fibrous tissue is noted in the defect site.

Thermal conformational changes of bovine fibrinogen by differential scanning calorimetry and circular dichroism.

The thermal denaturation of bovine fibrinogen has been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Differential scanning calorimetry measurements were carried out while changing the scan-rate. The transition at 57 degrees C was found to be irreversible and highly scan-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. The secondary structural changes at various temperatures were monitored by far-ultraviolet CD spectroscopy. These results show that the DSC transition for the thermal denaturation of bovine fibrinogen can be interpreted in terms of a kinetic process, N --> F, where k is a first-order kinetic constant that changes with temperature according to the Arrhenius equation. An important transition peak was observed at 78.8 degrees C which is attributed to the C-terminal parts of the Aalpha chains of fibrinogen.

Novel biodegradable films and scaffolds of chitosan blended with poly(3-hydroxybutyrate)

In order to develop a novel biomaterial, films of chitosan blended with poly(3-hydroxybutyrate) (PHB) were prepared by an emulsion blending technique and their properties were characterized. Scanning electron microscopy (SEM) showed that PHB microspheres were formed and were entrapped in chitosan matrices, which made the film surface rough. With increasing PHB content, the roughness of the film surface increased, while the swelling capability of the films decreased. In a wet state, the blended films exhibited a lower elastic modulus, a higher elongation-at-break and a higher tensile strength compared with chitosan films. Cell-culture experiments revealed that the blended films had better cytocompatibility than chitosan films. To explore the potential application of the blended material in tissue engineering, the porous blended scaffolds were fabricated and their pore morphology was observed by SEM. The results revealed that not only pore structure but also pore wall morphology of the blended scaffolds could be controlled by selecting the parameters of the fabrication process. These advantageous properties indicate that the blended chitosan/PHB material is promising for tissue engineering applications.

Preparation and evaluation of porous poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) hydroxyapatite composite scaffolds.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and PHBHHx-hydroxyapatite (HAP) composite scaffolds have been prepared by phase separation and subsequent sublimation of the solvent for bone tissue engineering. Scanning electron microscopy (SEM), porosity measurement, mechanical tests, and thermogravimertric analysis (TGA) are used to analyze the physical properties of the scaffolds. The biocompatibility and osteoconductivity are assessed by examining the morphology, proliferation, and differentiation of MC3T3-E1 osteoprogenitor cells seeded on the scaffolds. The PHBHHx-HAP composite scaffolds show better mechanical properties, biocompatibility, and osteoconductivity than the PHBHHx scaffolds. The results suggest that PHBHHx-HAP composite scaffolds can be employed as a promising candidate for bone reconstruction.Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and PHBHHx-hydroxyapatite (HAP) composite scaffolds have been prepared by phase separation and subsequent sublimation of the solvent for bone tissue engineering. Scanning electron microscopy (SEM), porosity measurement, mechanical tests, and thermogravimertric analysis (TGA) are used to analyze the physical properties of the scaffolds. The biocompatibility and osteoconductivity are assessed by examining the morphology, proliferation, and differentiation of MC3T3-E1 osteoprogenitor cells seeded on the scaffolds. The PHBHHx-HAP composite scaffolds show better mechanical properties, biocompatibility, and osteoconductivity than the PHBHHx scaffolds. The results suggest that PHBHHx-HAP composite scaffolds can be employed as a promising candidate for bone reconstruction.

Neural Stem Cell Affinity of Chitosan and Feasibility of Chitosan-Based Porous Conduits as Scaffolds for Nerve Tissue Engineering

Abstract Neural stem cells (NSCs) are currently considered as powerful candidate seeding cells for regeneration of both spinal cords and peripheral nerves. In this study, NSCs derived from fetal rat cortices were co-cultured with chitosan to evaluate the cell affinity of this material. The results showed that NSCs grew and proliferated well on chitosan films and most of them differentiated into neuron-like cells after 4 days of culture. Then, molded and braided chitosan conduits were fabricated and characterized for their cytotoxicity, swelling, and mechanical properties. Both types of conduits had no cytotoxic effects on fibroblasts (L929 cells) or neuroblastoma (Neuro-2a) cells. The molded conduits are much softer and more flexible while the braided conduits possess much better mechanical properties, which suggests different potential applications.

Fiber-Based chitosan tubular scaffolds for soft tissue engineering: Fabrication and in vitro evaluation

Abstract Porous, two-ply tubular chitosan conduits for guided tissue regeneration were fabricated by combining the textile technique (inner layer) with the thermally induced phase separation process (outer layer). A hollow chitosan tube was prepared using an industrial warp knitting process with chitosan yarns. Then, an appropriate diameter mandrel was inserted into the pre-fabricated tube. The tube and the mandrel were dipped into the chitosan solution together, taken out, and freeze-dried. After being neutralized in alkaline solution and dried at room temperature, the mandrel was removed to create the chitosan tubular scaffold. Scanning electron micrographs show that the resulting tubes have a biphasic wall structure, with a fibrous inner layer and a semipermeable outer layer. The swelling properties and the mechanical strength before and after in vitro degradation were investigated. The biocompatibility of the scaffolds was also investigated by co-culturing neuroblastoma cells (N2A, mouse) with the scaffolds. The results suggest that these chitosan tubular scaffolds are useful for the regeneration of tissues requiring a tubular scaffold.