Bioactive scaffolds for redirecting endogenous neural stem cell migration to repair the injured brain

The number of individuals affected by neurological disorders rises rapidly world-wide, however, there is no clinical treatment to restore lost functions. The brain’s attempt at regeneration happens through the re-direction of neural stem cells residing in the stem cell niches towards the injured zones. However, insufficient numbers of migrating cells and an inhibitory environment limits endogenous regeneration after severe damage. The design and synthesis of new biomaterials to provide a defined pathway for neuroblast migration from the subventricular zone (SVZ) towards the lesion site of the brain is essential as a promising therapy for brain repair. This dissertation introduces the design and functionalization of a new class of self-assembling peptide hydrogel to enhance neurogenesis in the injured adult brain. A novel aspect of this work is the design of self-assembling peptide hydrogels solely composed of β-amino acids as a proteolytically stable material.<br> <br>    Amongst the proposed biomaterials for brain tissue engineering, hydrogels based on peptide self-assembly have received the most attention due to their similarity to brain tissue. However, they can degrade rapidly by proteolytic enzymes <i>in vivo</i>, which limits their application to provide long term physical guidance for cellular migration. To address the rapid degradation, peptide hydrogels have been synthesized through a novel self-assembly approach consisting of only β³-amino acids which are inherently stable against proteolytic enzymes. An alkyl chain was laterally attached to the N-acetylated tripeptide to induce self-assembly in physiological conditions and eventually form a stable hydrogel (C₁₄-peptide). The hydrogel showed similar mechanical properties to brain tissue and proved non-toxic to neuronal cells, however, a deposition of serum proteins was essential for cell attachment to the hydrogel. To induce cell attachment, fibronectin-derived RGD was laterally attached to the side chain of the peptide through the incorporation of a novel alloc-protected β-amino acid (RGD-peptide). The bioactive signalling for fibroblast cell attachment and similar stiffness to brain tissue was optimised by mixing the two aforementioned peptides (RGD-peptide: C₁₄-peptide; 5%:95%).<br> <br>    To investigate the feasibility of the synthesized peptide hydrogels to induce neural stem cell migration through the newly defined pathway, the optimum hydrogel consisting of 10% RGD-peptide and 90% C14-peptide was implanted into the brain to disrupt the SVZ. The inflammatory response was minimal and the scaffold integrated with the parenchyma, proving the biocompatibility of the synthesized hydrogel. Most importantly, it was shown that the scaffold was capable of migrating cells along defined pathways to distant regions in the brain. The effect of brain-derived neurotrophic factor (BDNF) on the rate of migrating cells and their survival has also been investigated. By releasing BDNF, the scaffold provides a suitable migratory stream for neuroblasts to migrate in larger numbers. This study represents a promising therapy for the treatment of the injured brain

Phenol Removal by Immobilized Horseradish Peroxidase

Horseradish peroxidase was successfully encapsulated in calcium alginate for phenol removal. The optimum gelation condition was found to be 0.75%w/v of sodium alginate solution and 4.5% w/v of calcium chloride hexahydrate. Upon immobilization, the pH profile of enzyme activity changed as it showed a higher relative value in basic and acidic solutions. It was also observed that enzyme activity retention of encapsulated HRP was independent of enzyme concentration. Besides, for each phenol concentration, there would be an enzyme concentration beyond which it had no significant effect on phenol removal. Investigation of phenol removal with time for both encapsulated and free enzymes showed that the encapsulated enzyme had a lower efficiency compared to the same concentration of the free enzyme; however, the capsules were reusable up to four cycles without any changes in their retention activity. The optimum ratio of hydrogen peroxide/phenol was found to depend on phenol concentration and that it varied from 0.94 to 1.15 for phenol concentrations between 2-10 mM

Neurokinin-1 receptor (NK-1R) antagonists: potential targets in the treatment of glioblastoma multiforme

The current standard of care in glioblastoma multiforme (GBM), as the most morbid brain tumor, is not adequate, despite substantial progress in cancer therapy. Among patients receiving current standard treatments, including surgery, irradiation, and chemotherapy, the overall survival (OS) period with GBM is less than one year. The high mortality frequency of GBM is due to its aggressive nature, including accelerated growth, deregulated apoptosis, and invasion into surrounding tissues. The understanding of the molecular pathogenesis of GBM is, therefore, crucial for identifying, designing, and repurposing potential agents in future therapeutic approaches. In recent decades, it has been apparent that several neurotransmitters, specifically substance P (SP), an undecapeptide in the family of neuropeptides tachykinins, are found in astrocytes. After binding to the neurokinin-1 receptor (NK-1R), the SP controls cancer cell growth, exerts antiapoptotic impacts, stimulates cell invasion/metastasis, and activates vascularization. Since SP/NK-1R signaling pathway is a growth driver in many cancers, this potential mechanism is proposed as an additional target for treating GBM. Following an evaluation of the function of both SP and its NK-1R inhibitors in neoplastic cells, we recommend a unique and promising approach for the treatment of patients with GBM

Graphene functionalized scaffolds reduce the inflammatory response and supports endogenous neuroblast migration when implanted in the Adult Brain

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration

Migration and Differentiation of Neural Stem Cells Diverted From the Subventricular Zone by an Injectable Self-Assembling β-Peptide Hydrogel

Neural stem cells, which are confined in localised niches are unable to repair large brain lesions because of an inability to migrate long distances and engraft. To overcome these problems, previous research has demonstrated the use of biomaterial implants to redirect increased numbers of endogenous neural stem cell populations. However, the fate of the diverted neural stem cells and their progeny remains unknown. Here we show that neural stem cells originating from the subventricular zone can migrate to the cortex with the aid of a long-lasting injectable hydrogel within a mouse brain. Specifically, large numbers of neuroblasts were diverted to the cortex through a self-assembling β-peptide hydrogel that acted as a tract from the subventricular zone to the cortex of transgenic mice (NestinCreERT2:R26eYFP) in which neuroblasts and their progeny are permanently fluorescently labelled. Moreover, neuroblasts differentiated into neurons and astrocytes 35 days post implantation, and the neuroblast-derived neurons were Syn1 positive suggesting integration into existing neural circuitry. In addition, astrocytes co-localised with neuroblasts along the hydrogel tract, suggesting that they assisted migration and simulated pathways similar to the native rostral migratory stream. Lower levels of astrocytes were found at the boundary of hydrogels with encapsulated brain-derived neurotrophic factor, comparing with hydrogel implants alone

Astrocyte morphology and infiltration at different time points following H6, P6, gP6, gH6 scaffolds implantation.

<p>Astrocyte/gP6 scaffolds interaction at Week (A) 1, (B) 3 and (C) 7; Astrocytes/P6 scaffolds interaction at Week (E) 3 and (F) 7; Astrocyte process infiltration into (H) gH6 at Week 3 and (I) H6 at Week 7. (D, G) detailed astrocyte morphology of the dash-box indicated area in (B, I) respectively. Green: GFAP positive astrocytes, blue: DAPI stained nucleus, red: surface functionalized scaffolds. * indicates astrocytes that bridge a gap between two scaffold layers in (D). All brain tissue sections were collected on the transverse plane. Scale bar for (D, G) represents 20 μm; for all other images represents 50 μm.</p

GFAP expression at different time points in the outer layer of scaffolds and in adjacent tissue.

<p>(150 μm from tissue/scaffold boundary) in terms of GFAP⁺ astrocytes occupied pixel percentage (pixel%). * (p<0.05, n = 4) indicate significant difference in GFAP expression between Week 3 and 7, within scaffold or tissue for gP6 implants. Error bar shows standard error of the mean.</p

Microglial response to graphene-free or—inclusive PCL scaffold implantation at Week 1 and Week 3.

<p>(A) Image shows an overview of microglial infiltration into the outer shell of a gP6 scaffold at Week 3, and gP6 at high magnification at (B) Week 1 and (C) Week 3. (D) Graphene-free P6 scaffold at Week 3 (Green: Iba1⁺ microglia cells, blue: DAPI stained nucleus). (E) Microglial profile across the tissue/scaffold interfaces (*** p<0.001, n = 4). All brain tissue sections were collected on the transverse plane. Scale bar for (A) represents 100 μm; Scale bar for (B, C and D) represents 20 μm. Error bar in (E) shows standard error of the mean.</p

Neuroblast migration/integration along/with gP6 implants at Week 3.

<p>Immunostaining images show DCX⁺ neuroblast (red) and nuclei (DAPI, blue) of tissue sections at the transverse plane. Schematic coronal brain section indicates the gP6 implantation track and locations of A-C, D-E tissue sections respectively. gP6 implant boundary is marked by dotted line. (A) gP6 implant in LV, bottom right part of the outermost scaffold layer is in direct contact with SVZ. Insert enlarges the box region showing neuroblast process across the entire scaffold layer. (B) Neuroblasts migrate along the scaffold surface/inter-layer gap and (C) processes infiltrating into the scaffold layers. Neuroblasts are identified in deeper sections of the gP6 implants either (D) within the scaffold or (E) close to implant in the LSV. LV: lateral ventricle, LSI: lateral septal nucleus, intermediate part, LSV: lateral septal nucleus, ventral part. Scale bars represent 20 μm for all images.</p

Microstructure of electrospun PCL microfibers and polyelectrolyte modified fibers with and without graphene.

<p>SEM images showing the microstructure of (A, B) PCL microfibers, (C) P6 and (D) gP6 modified microfibers. Partially aligned smooth microfiber morphology was revealed in all images at different magnifications.</p