Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord.

Severe spinal cord injury (SCI) causes loss of neural connectivity and permanent functional deficits. Re-establishment of new neuronal relay circuits after SCI is therefore of paramount importance. The present study tested our hypothesis if co-culture of neurotrophin-3 (NT-3) gene-modified Schwann cells (SCs, NT-3-SCs) and TrkC (NT-3 receptor) gene-modified neural stem cells (NSCs, TrkC-NSCs) in a gelatin sponge scaffold could construct a tissue engineering neural network for re-establishing an anatomical neuronal relay after rat spinal cord transection. Eight weeks after transplantation, the neural network created a favorable microenvironment for axonal regeneration and for survival and synaptogenesis of NSC-derived neurons. Biotin conjugates of cholera toxin B subunit (b-CTB, a transneuronal tracer) was injected into the crushed sciatic nerve to label spinal cord neurons. Remarkably, not only ascending and descending nerve fibers, but also propriospinal neurons, made contacts with b-CTB positive NSC-derived neurons. Moreover, b-CTB positive NSC-derived neurons extended their axons making contacts with the motor neurons located in areas caudal to the injury/graft site of spinal cord. Further study showed that NT-3/TrkC interactions activated the PI3K/AKT/mTOR pathway and PI3K/AKT/CREB pathway affecting synaptogenesis of NSC-derived neurons. Together, our findings suggest that NT-3-mediated TrkC signaling plays an essential role in constructing a tissue engineering neural network thus representing a promising avenue for effective exogenous neuronal relay-based treatment for SCI.

Graft of the gelatin sponge scaffold containing genetically-modified neural stem cells promotes cell differentiation, axon regeneration, and functional recovery in rat with spinal cord transection.

Biological materials combined with genetically-modified neural stem cells (NSCs) are candidate therapy targeting spinal cord injury (SCI). Based on our previous studies, here we performed gelatin sponge (GS) scaffold seeded with neurotrophin-3 (NT-3) and its receptor TrkC gene modifying NSCs for repairing SCI. Eight weeks later, compared with other groups, neurofilament-200 and 5-hydroxytryptamine positive nerve fibers were more in the injury site of the N+T-NSCs group. Immunofluorescence staining showed the grafted NSCs could differentiate into microtubule associated protein (Map2), postsynaptic density (PSD95), and mouse oligodendrocyte special protein (MOSP) positive cells. The percentage of the Map2, PSD95, and MOSP positive cells in the N+T-NSCs group was higher than the other groups. Immuno-electron microscopy showed the grafted NSCs making contact with each other in the injury site. Behavioral analysis indicated the recovery of hindlimbs locomotion was better in the groups receiving cell transplant, the best recovery was found in the N+T-NSCs group. Electrophysiology revealed the amplitude of cortical motor evoked potentials was increased significantly in the N+T-NSCs group, but the latency remained long. These findings suggest the GS scaffold containing genetically-modified NSCs may bridge the injury site, promote axon regeneration and partial functional recovery in SCI rats.

A comparative study of gelatin sponge scaffolds and PLGA scaffolds transplanted to completely transected spinal cord of rat.

This study sought to investigate whether gelatin sponge (GS) scaffold would produce less acidic medium in injured spinal cord, as compared with poly(lactic-co-glycolic acid) (PLGA) scaffold, to determine which of the two scaffolds as the biomaterial is more suitable for transplantation into spinal cord. GS scaffold or PLGA scaffold was transplanted into a transected spinal cord in this study. Two months after transplantation of scaffolds, acid sensing ion channel 1a (ASIC1a) positive cells expressing microtubule associated protein 2 (Map2) were observed as well as expressing adenomatous polyposis coli (APC) in spinal cord. GFAP positive cells were distributed at the rostral and caudal of the injury/graft area in the GS and PLGA groups. Western blot showed ASIC1a and GFAP expression of injured spinal cord was downregulated in the GS group. The number of CD68 positive cells was fewer and NF nerve fibers were more in the GS group. Nissl staining and cell counting showed that the number of survival neurons was comparable between the GS and PLGA groups in the pyramidal layer of sensorimotor cortex and the red nucleus of midbrain. However, in the Clarke's nucleus at L1 spinal segment, the surviving neurons in the GS group were more numerous than that in the PLGA group. H&E staining showed that the tissue cavities in the GS group were smaller in size than that in the PLGA group. The results suggest that GS scaffold is more suitable for transplantation to promote the recovery of spinal cord injury compared with PLGA scaffold.

Bone marrow mesenchymal stem cells in a three-dimensional gelatin sponge scaffold attenuate inflammation, promote angiogenesis, and reduce cavity formation in experimental spinal cord injury.

Three-dimensional (3D) gelatin sponge (GS) scaffolds were constructed by ensheathing GS with a thin film of poly-(lactide-co-glycolide) (PLGA). Rat bone marrow-derived mesenchymal stem cells (MSCs) were isolated, cultured, and then seeded to the scaffolds. Distribution of cells and cell growth, survival, and proliferation within the scaffolds were then determined. Immunofluorescence and Western blot analysis were employed to detect the deposition of fibronectin to the scaffolds on day 3 and day 7 of culture. Scaffolds with or without MSCs were then transplanted into the transected rat spinal cord. One or 8 weeks following transplantation, cavity areas, activated macrophages/microglia, expression of TNF-α and IL-1ß, and neovascularization within the grafts were examined and quantified. Deposition of fibronectin (FN) and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) as potential inducing factors for angiogenesis were also examined. Results showed that 3D GS scaffolds allowed MSCs to adhere, survive, and proliferate and also FN to deposit. In vivo transplantation experiments demonstrated that these scaffolds were biocompatible, and MSCs seeded to the scaffolds played an important role in attenuating inflammation, promoting angiogenesis, and reducing cavity formation. Therefore, the GS scaffolds with MSCs may serve as promising supporting transplants for repairing spinal cord injury.

Transplantation of artificial neural construct partly improved spinal tissue repair and functional recovery in rats with spinal cord transection.

Delivery of cellular and/or trophic factors to the site of injury may promote neural repair or axonal regeneration and return of function after spinal cord injury. Engineered scaffolds provide a platform to deliver therapeutic cells and neurotrophic molecules. To explore therapeutic potential of engineered neural tissue, we generated an artificial neural construct in vitro, and transplanted this construct into a completely transected spinal cord of adult rats. Two months later, behavioral analysis showed that the locomotion recovery was significantly improved compared with control animals. Immunoreactivity against microtubule associated protein 2 (Map2) and postsynaptic density 95 (PSD95) demonstrated that grafted cells had a higher survival rate and were able to differentiate toward neuronal phenotype with ability to form synapse-like structure at the injury site; this was also observed under the electron microscope. Immunostaining of neurofilament-200 (NF-200) showed that the number of nerve fibers regrowing into the injury site in full treatment group was much higher than that seen in other groups. Furthermore, Nissl staining revealed that host neuron survival rate was significantly increased in rats with full treatments. However, there were no biotin dextran amine (BDA) anterograde tracing fibers crossing through the injury site, suggesting the limited ability of corticospinal tract axonal regeneration. Taken together, although our artificial neural construct permits grafted cells to differentiate into neuronal phenotype, synaptogenesis, axonal regeneration and partial locomotor function recovery, the limited capacity for corticospinal tract axonal regeneration may affect its potential therapy in spinal cord injury.

Synaptic transmission of neural stem cells seeded in 3-dimensional PLGA scaffolds.

To explore therapeutic potential of engineered neural tissue, we combined genetically modified neural stem cells (NSCs) and poly(lactic acid-co-glycolic acid) (PLGA) polymers to generate an artificial neural network in vitro. NSCs transfected with either NT-3 or its receptor TrkC gene were seeded into PLGA scaffold. The NSCs were widely distributed and viable in the scaffold after culturing for 14 days. Immunoreactivity against Map2 was detected in >70% of these grafted cells, suggesting a high rate of differentiation toward neurons. Immunostaining of synapsin-I and PSD95 revealed formation of synaptic structures, which was also observed under electron microscope. Furthermore, using FM1-43 dynamic imaging, synapses in these differentiated neurons were found to be excitable and capable of releasing synaptic vesicles. Taken together, our artificial PLGA construct permits NSCs to differentiate toward neurons, establish connections and exhibit synaptic activities. These findings provide a biological basis for future application or transplantation of this artificial construct in neural repair.

Caspase-3 antisense oligodeoxynucleotides inhibit apoptosis in gamma-irradiated human leukemia HL-60 cells.

To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODNs) on apoptosis, we designed four ASODNs targeting different regions of caspase-3 mRNA and transfected them into human leukemia HL-60 cells. The transfected cells were given 10 Gy gamma-irradiation followed by incubation for 18 h and measurement of apoptosis and caspase-3 expression. Our results showed that ASODN-2 targeting the 5' non-coding region of sites -62 to -46, and ASODN-3 targeting the 5' coding region of sites -1 to 16, both reduced apoptosis measured by gel electrophoresis and flow cytometry. Hoechst 33258 staining and TUNEL assay revealed that apoptotic indexes in the ASODN-2 and ASODN-3 groups were significantly lower than those in the untransfected and mismatched oligodeoxynucleotide (MODN) groups. Immunocytochemistry, Western blotting and RT-PCR showed that expression levels of caspase-3 protein and mRNA in both ASODN-2 and ASODN-3 groups were decreased compared with those in the untransfected and MODN groups. In conclusion, caspase-3 mRNA ASODNs can inhibit gamma-radiation-induced apoptosis of HL-60 cells and reduce expression of caspase-3 protein and mRNA. The results suggest that antisense approach may be useful for therapeutic treatment of certain neurodegenerative diseases in which apoptosis is involved.