Yandao Gong

Study on physical properties and nerve cell affinity of composite films from chitosan and gelatin solutions

A series of chitosan-gelatin composite films was prepared by varying the ratio of constituents. FT-IR and X-ray analysis showed good compatibility between these two biopolymers. Differential scanning calorimetry (DSC) analysis indicated that the water take-up of chitosan film increased when blended with gelatin. Composite film exhibited a lower Youngs modulus and a higher percentage of elongation-at-break compared with chitosan film, especially in wet state. All composite films were hydrophilic materials with water contact angles ranging from 55 degrees to 65 degrees. The results obtained from ELISA indicated the adsorption amount of fibronectin on composite films was much higher than on chitosan film. PC12 cells culture was used to evaluate the nerve cell affinity of materials. The cells cultured on the composite film with 60wt% gelatin differentiated more rapidly and extended longer neurites than on chitosan film. The results suggest that the soft and elastic complex of chitosan and gelatin, which has better nerve cell affinity compared to chitosan, is a promising candidate biomaterial for nerve regeneration.

Inhibition of γ-secretase activity reduces Aβ production, reduces oxidative stress, increases mitochondrial activity and leads to reduced vulnerability to apoptosis : Implications for the treatment of Alzheimer's disease

It has been argued that gamma-secretase should be considered as a pharmacological target, as there are few mechanism-based experimental and clinical studies on gamma-secretase treatment. In this study, we found that N2a cells bearing APP695 or its Swedish mutant exhibited increased basal levels of ROS, nitric oxide (NO), protein carbonyls, MDA and intracellular calcium, as well as reduced level of the mitochondrial membrane potential and ATP. When the activity of gamma-secretase was inhibited by expression of the D385A PS1 variant, cells (N2a/Swe.D385A) showed reduced basal levels of ROS, nitric oxide (NO), protein carbonyls, MDA and intracellular calcium, as well as increased mitochondrial membrane potential and ATP level. In addition, N2a/Swe.D385A cells showed reduced vulnerability to H(2)O(2)-induced apoptosis. The Bcl-2 and JNK/ERK pathways were proven to be involved in the change of vulnerability to H(2)O(2)-induced apoptosis. Moreover, we discovered that inhibition of gamma-secretase by DAPT would lead to a reduction of ROS levels and stabilization of mitochondrial function in APP (N2a/APP695) and APP Swedish mutant (N2a/APPswe) transfected cells. At last, it was shown that Abeta antibody and antiserum prevented increase of ROS and reduction of mitochondrial membrane potential in N2a/Swe.DeltaE9 cells but not in N2a/Swe.D385A cells, which indicated that reduced formation of Abeta was the reason for reduction of ROS formation and increase of mitochondrial membrane potential when PS-1 activity was impaired in N2a/Swe.D385A cells. We concluded that neurotoxicity was positively correlated with the activity of gamma-secretase, which suggested inhibition of gamma-secretase is a rational pharmacological target for Alzheimers disease treatment.

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.

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.

Synergistic Effect of Neural Stem Cells and Olfactory Ensheathing Cells on Repair of Adult Rat Spinal Cord Injury

Spinal cord injury (SCI) is a common clinical disease that places a heavy burden on families and society. Cellular therapy provides a method of giving a supplement of cells lost in the injury and promoting functional recovery after SCI. Neural stem cells (NSCs) and olfactory ensheathing cells (OECs) are two most promising cell types. NSCs have the potential of differentiating into neurons and glial cells, and OECs could help the axons of neurons pass through the glial scar to promote functional recovery. NSCs were isolated from the cortices of fetal rats on days 12–14 of embryonic development and OECs were isolated from the olfactory bulbs of adult rats. In vitro coculture studies demonstrated OECs could promote NSCs to differentiate into neurons. Four groups of rats that had been 3/4 spinal cord transectioned at T9 were injected with DMEM/F12 solution, NSCs, OECs, and NSCs + OECs, respectively, 7 days post-SCI. Twelve weeks postoperation, the hindlimb locomotor function of rats in the cotransplantation group was significantly improved compared with that in the other three groups. Histological observation and immunohistochemical staining of NF-200 both showed new nerve fibers across the injured region. Cotransplantation of NSCs and OECs might have a synergistic effect on promoting neural regeneration and improving the recovery of locomotion function. Cotransplantation of NSCs and OECs was better than a single graft of either NSCs or OECs. These findings have provided a new way of thinking in the treatment of SCI.

The effect of topology of chitosan biomaterials on the differentiation and proliferation of neural stem cells

Neural stem cells (NSCs) are capable of self-renewal and differentiation into three principle central nervous system cell types under specific local microenvironments. Chitosan films (Chi-F), chitosan porous scaffolds (Chi-PS) and chitosan multimicrotubule conduits (Chi-MC) were used to investigate their effects on the differentiation and proliferation of NSCs isolated from the cortices of fetal rats. In the presence of 10% fetal bovine serum most NSCs cultured on Chi-F differentiated into astrocytes, NSCs cultured on Chi-MC showed a significant increase in neuronal differentiation, while Chi-PS somewhat promoted NSCs to differentiate into neurons. However, in serum-free medium with 20 ng ml(-1) basic fibroblast growth factor NSCs cultured on Chi-F showed the greatest proliferation, NSCs cultured on Chi-MC showed moderate cell proliferation, but NSCs cultured on Chi-PS exhibited the least cell proliferation. These observations indicate that chitosan topology can play an important role in regulating differentiation and proliferation of NSCs and raise the possibility of the utilization of chitosan in various structural biomaterials in neural tissue engineering.

Intracerebral transplantation of adipose-derived mesenchymal stem cells alternatively activates microglia and ameliorates neuropathological deficits in Alzheimer's disease mice.

Recent studies suggest that transplantation of mesenchymal stem cells might have therapeutic effects in preventing pathogenesis of several neurodegenerative disorders. Adipose-derived mesenchymal stem cells (ADSCs) are a promising new cell source for regenerative therapy. However, whether transplantation of ADSCs could actually ameliorate the neuropathological deficits in Alzheimers disease (AD) and the mechanisms involved has not yet been established. Here, we evaluated the therapeutic effects of intracerebral ADSC transplantation on AD pathology and spatial learning/memory of APP/PS1 double transgenic AD model mice. Results showed that ADSC transplantation dramatically reduced β-amyloid (Aβ) peptide deposition and significantly restored the learning/memory function in APP/PS1 transgenic mice. It was observed that in both regions of the hippocampus and the cortex there were more activated microglia, which preferentially surrounded and infiltrated into plaques after ADSC transplantation. The activated microglia exhibited an alternatively activated phenotype, as indicated by their decreased expression levels of proinflammatory factors and elevated expression levels of alternative activation markers, as well as Aβ-degrading enzymes. In conclusion, ADSC transplantation could modulate microglial activation in AD mice, mitigate AD symptoms, and alleviate cognitive decline, all of which suggest ADSC transplantation as a promising choice for AD therapy. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

The behavior of MC3T3-E1 cells on chitosan/poly-L-lysine composite films: Effect of nanotopography, surface chemistry, and wettability

In the present work, a series of composite films were produced from chitosan/poly-L-lysine blend solutions. The surface topography, chemistry, and wettability of composite films were characterized by atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle assay, respectively. For all composite films, blending with poly-L-lysine induced changes in surface chemistry and wettability. Interestingly, it was also found that increasing poly-L-lysine weight fraction in blend solutions could result in different nanoscaled surface topographic features, which displayed particle-, granule-, or fiber-dominant morphologies. MC3T3-E1 osteoblast-like cells were cultured on all composite films to evaluate the effects of surface nanotopography, chemistry, and wettability on cell behavior. The observations indicated that MC3T3-E1 cell behavior was affected by surface topography, chemistry, and wettability simultaneously and that cells showed strong responses to surface topography. On fiber-dominant surface, cells fully spread with obvious cytoskeleton organization and exhibited significantly higher level of adhesion and proliferation compared with particle- or granule-dominant surfaces. Furthermore, fiber-dominant surface also induced greater expression of mature osteogenic marker osteocalcin and higher mineralization based on RT-PCR and von Kossa staining. The results suggest that topographic modification of chitosan substratum at the nanoscale may be exploited in regulating cell behavior for its applications in tissue engineering.

The redox state of plastoquinone pool regulates state transitions via cytochrome b6f complex in Synechocystis sp. PCC 6803

The effects of benzoquinone analogues, phenyl‐1,4‐benzoquinone (PBQ) and 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐1,4‐benzoquinone (DBMIB), on state transitions in Synechocystis sp. PCC 6803 were investigated. PBQ induced a transition from state 2 to state 1 in the absence of actinic light whereas DBMIB caused a state 2 transition. 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethyl urea could not eliminate the effects of PBQ and DBMIB. These results imply that the redox state of the plastoquinone pool controls the state transitions in vivo and cytochrome b 6 f complex is involved in this process. As a working hypothesis, we propose that the occupancy of the quinol oxidation site and the movement of the Rieske protein may be pivotal in this regulation.