Qun-Yuan Xu

Culture of neural cells on silicon wafers with nano-scale surface topograph.

The adherence and viability of central neural cells (substantia nigra) on a thin layer of SiO(2) on Si wafers with different surface roughness were investigated. Variable roughness of the Si wafer surface was achieved by etching. The nano-scale surface topography was evaluated by atomic force microscopy. The adherence and subsequent viability of the cells on the wafer were examined by scanning electron microscopy (SEM) and fluorescence immunostaining of tyrosine hydroxylase (TH). It is found that the surface roughness significantly affected cell adhesion and viability. Cells survived for over 5 days with normal morphology and expressed neuronal TH when grown on surfaces with an average roughness (Ra) ranging from 20 to 50 nm. However, cell adherence was adversely affected when surfaces with Ra less than 10 nm and rough surfaces with Ra above 70 nm were used as the substrate. Such a simple preparation procedure may provide a suitable interface surface for silicon-based devices and neurones or other living tissues.

The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin.

The hyaluronic acid (HA) hydrogels modified with laminin were used for implantation in rat brain in present study, in order to investigate its effects in reparation of injury in the CNS. Cross-linked HA hydrogels were synthesized and their characteristics were analyzed. Laminin, an extracellular matrix protein, which participates in neuronal development and survival, was immobilized on the backbone of the hydrogels. Hydrogels unmodified and modified with laminin were implanted into cortical defects mechanically created in rats and their ability to improve tissue reconstruction was then evaluated. After 6 and 12 weeks of implantation, sections of brains were processed with Nissl and Glees staining for revealing neural cell bodies and fibers, with DAB histochemistry for detecting the blood vessels, as well as with immunocytochemistry for recognizing GFAP. The sections were also taken to SEM and TEM for ultrastructral examination. The results showed that the HA hydrogels synthesized had mechanical properties and rheological behavior similar to the brain tissue. After being implanted into the lesion of the cortex, the porous hydrogels created a scaffold, which could support cell infiltration and angiogenesis, and simultaneously inhibit the formation of glial scar. In addition, HA hydrogels modified with laminin could promote neurite extension. It seems possible that the tissue engineering technique may pave the way to repair injury in the CNS as suggested by the results in present study.

Viability and differentiation of neural precursors on hyaluronic acid hydrogel scaffold

The traditional notion that injured neurons are unable to regenerate in the adult mammalian brain and spinal cord has long been a concern. This view has led to methodology designed to overcome this problem, most recently by advancements in tissue engineering. Here, neural precursor cells (NPCs) and the Nogo receptor antibody (NgR‐Ab) or poly‐L‐lysine (PLL) were tested in concert with hyaluronic acid hydrogel scaffolds (HA). In particular, we wished to optimize viability and differentiation of NPCs within HA hydrogel scaffolds. Our results show that HA hydrogels can be modified physically or chemically to improve NPCs attachment on the scaffolding doped with NgR‐Ab or PLL. Both the HA hydrogels and their modifications support the viability of NPCs. NPCs were also able to differentiate into neurons and glial cells on HA hydrogels, although this was affected by the different modifications. Immunofluorescence showed that fewer β‐III‐tubulin antibody and antineurofilament antibody‐positive cells were found on HA‐PLL hydrogel compared with HA or HA NgR‐Ab hydrogels. This indicates that the PLL‐modified HA hydrogels may inhibit differentiation of NPCs, whereas modification by NgR‐Ab had no such effect. Finally, the NgR‐Ab‐modified HA scaffold can be used as not only a NPC delivery system but also a bioactive factor transportation system for CNS repair.

Hyaluronic acid hydrogels with IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain.

A biocompatible hydrogel of hyaluronic acid with the neurite-promoting peptide sequence of IKVAV was synthesized. The characterization of the hydrogel shows an open porous structure and a large surface area available for cell interaction. Its ability to promote tissue repair and axonal regeneration in the lesioned rat cerebrum is also evaluated. After implantation, the polymer hydrogel repaired the tissue defect and formed a permissive interface with the host tissue. Axonal growth occurred within the microstructure of the network. Within 6 weeks the polymer implant was invaded by host-derived tissue, glial cells, blood vessels and axons. Such a hydrogel matrix showed the properties of neuron conduction. It has the potential to repair tissue defects in the central nervous system by promoting the formation of a tissue matrix and axonal growth by replacing the lost tissue.

Hyaluronic acid hydrogel modified with nogo-66 receptor antibody and poly-L-lysine to promote axon regrowth after spinal cord injury.

The biomaterials used for central nervous system injury require not only interacting with specific cell adhesion but also specific growth factor receptors to promote nerve regeneration. In this study, hyaluronic acid (HA)-based hydrogels modified with poly-L-lysine (PLL) and nogo-66 receptor antibody (antiNgR) (HA-PLL/antiNgR) were administered to rats after lateral hemisection of the spinal cord. Anti-neurofilament positive axons were found to extend into the HA-PLL/antiNgR hydrogel at 8 weeks after implantation, which shows significant difference compared with HA-PLL or blank control group. Electron micrographs of implanted hydrogels showed that there were more cells and normal axons with myelin in the HA-PLL/antiNgR implant than that of HA-PLL hydrogel. The antiNgR grafted on HA hydrogels could be detected for 8 weeks after transplantation in vivo. All of these properties may facilitate HA-PLL/antiNgR hydrogels to become a promising scaffold for repairing spinal cord injury. Nevertheless, both two kinds of modified hydrogels (HA-PLL/antiNgR and HA-PLL) showed remarkable advantages in supporting angiogenesis, and simultaneously inhibiting the formation of glial scar.

The enhancement of cell adherence and inducement of neurite outgrowth of dorsal root ganglia co-cultured with hyaluronic acid hydrogels modified with Nogo-66 receptor antagonist in vitro

Hyaluronic acid hydrogels modified with polyclonal anti-Nogo-66 receptor antibody were developed in order to promote regeneration in the injured CNS. These modified hydrogels were intended not only to deliver antibodies, but also to serve as a scaffold for neural regeneration following their implantation into injured tissue. Since unmodified hyaluronic acid-hydrogels do not support cell attachment, the gels were modified with polyclonal anti-Nogo-66 receptor with the aim of altering the surface properties of the gels in such a way as to improve neuronal adherence and survival. After evaluating the immobilization efficiency of the system, chicken dorsal root ganglia and dorsal root ganglia cells were planted on the surface of the modified gels to determine cell viability. Dorsal root ganglia were also cultured close to the gels in order to assay the inducement of neurite outgrowth. In dorsal root ganglia and cell viability assay, dorsal root ganglia and neuron cells could adhere to the modified hydrogels and survive well, but it did not happen to unmodified hydrogels. After 72 h, these attached cells were stained positively with immuno-staining for neurofilament. Neurite outgrowth inducement assay showed that the number and length of dorsal root ganglia neurites on the side toward modified hydrogels were significantly more than that on the opposite side (both P<0.01). The results reveal that hyaluronic acid-hydrogels modified with anti-Nogo-66 receptor can support neural cell attachment and survival in vitro. Furthermore, this system can greatly induce neurite outgrowth. The results also indicate that this modified hydrogels have potential to repair injury in the CNS.

An experimental test of stroke recovery by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model

The objective of the study was to determine the effects of a hyaluronic-acid-based (HA-based) hydrogel implant, carrying a polyclonal antibody to the Nogo-66 receptor (NgR), on adult rats that underwent middle cerebral artery occlusion (MCAO). Behavioral tests of a forelimb-reaching task suggested that the disabled function of the impaired forelimb in this stroke model was ameliorated by the implant to a certain extent. These behavioral findings were correlated with immunohistochemical results of investigating the distribution of NgR antibody, neurofilaments (NF) and neuron-specific class III beta-tubulin (TuJ1) in the brain sections. The porous hydrogel functioned as a scaffold to deliver the NgR antibody, support cell migration and development. In addition, it was found NF-positive and TuJ1-positive expressions were distributed in the implanted hydrogel. Collectively, the results demonstrate the promise of the HA hydrogel as a scaffold material and the delivery vehicle of the NgR antibody for the repair of defects and the support of neural regeneration in the brain.

Development of an artificial neuronal network with post-mitotic rat fetal hippocampal cells by polyethylenimine.

The selection of appropriate surface materials that promote cellular adhesion and growth is an important consideration when designing a simplified neuronal network in vitro. In the past, extracellular matrix proteins such as laminin (LN) or positively charged substances such as poly-l-lysine (PLL) have been used. In this study, we examined the ability of another positively charged polymer, polyethyleneimine (PEI), to promote neuronal adhesion, growth and the formation of a functional neuronal network in vitro. PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by micro-contact printing. Post-mitotic neurons from the rat fetal hippocampus were cultured on the different polymers and the viability and morphology of these neurons under serum-free culture conditions were observed using fluorescent microscopy and atomic force microscopy (AFM). We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LN-coated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions, whereas most neurons cultured on the LN-coated grids died after 1 week. Using AFM, we observed some neurosynapse-like structures near the neuronal soma on PEI-coated coverslips. These findings indicate that PEI is a suitable surface for establishing a functional neuronal network in vitro.

Hyaluronic Acid/Polylysine Hydrogel as a Transfer System for Transplantation of Neural Stem Cells

Graft integration and survival are major factors that limit cell therapy for diseases and injury in the central nervous system. Efforts to improve graft survival and integration have focused on the development of biocompatible scaffolds to support neural cells. In this study, rat neural stem cells (NSC), including neurospheres and single cells, were seeded into hyaluronic acid/polylysine hydrogels. Confocal microscopy was used to noninvasively investigate the key cell phonotypes. After culture for 5 days, single NSC had survived and differentiated into neurons and astrocytes, while neurosphereforming cells had migrated from their original aggregate and maintained the NSC phonotype. These studies, carried out in the absence of serum, identified HA/polylysine hydrogels as potential synthetic cell carriers for transplantation of NSC.

Hyaluronic Acid Hydrogel Modified with Nogo-66 Receptor Antibody and Poly(L-Lysine) Enhancement of Adherence and Survival of Primary Hippocampal Neurons

Hyaluronic acid (HA) hydrogel was modified with poly(L-lysine) (PLL) and Nogo-66 Receptor antibody (antiNgR) to enhance the repair of central nervous system (CNS) injuries. The immobilization of PLL was characterized by X-ray photoelectron spectroscopy (XPS) and the immobilization of antiNgR was studied by immunofluorescence. The cytocompatibility of this modified hydrogel was analyzed by culturing primary hippocampal neurons. The quantity and morphology of the neurons were influenced by different modifications; the primary hippocampal neurons cultured with modified HA hydrogel exhibited multipolar and bipolar morphology were compared with unmodified hydrogel cultures. The number of neurons obtained by culturing with HA hydrogel modified with both PLL and antiNgR was almost twice the number of neurons cultured with HA modified with only PLL or antiNgR. This phenomenon was attributed to the collaborative effect of PLL and antiNgR on the neurons. The characteristics of this new hydrogel system, including pore structure, water absorption, hydrolysis degradation did not change much when compared with the hydrogel modified with PLL or antiNgR, respectively. It is expected that this modified HA hydrogel has potential as a CNS tissue engineering material.