Development of Conjugated Kefiran-Chondroitin Sulphate Cryogels with Enhanced Properties for Biomedical Applications.

Hydrogels based on natural polysaccharides can have unique properties and be tailored for several applications, which may be mainly limited by the fragile structure and weak mechanical properties of this type of system. We successfully prepared cryogels made of newly synthesized kefiran exopolysaccharide-chondroitin sulfate (CS) conjugate via carbodiimide-mediated coupling to overcome these drawbacks. The freeze-thawing procedure of cryogel preparation followed by lyophilization is a promising route to fabricate polymer-based scaffolds with countless and valuable biomedical applications. The novel graft macromolecular compound (kefiran-CS conjugate) was characterized through 1H-NMR and FTIR spectroscopy-which confirmed the structure of the conjugate, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)-which mirrored good thermal stability (degradation temperature of about 215 °C) and, finally, gel permeation chromatography-size exclusion chromatography (GPC-SEC)-which proved an increased molecular weight due to chemical coupling of kefiran with CS. At the same time, the corresponding cryogels physically crosslinked after the freeze-thawing procedure were investigated by scanning electron microscopy (SEM), Micro-CT, and dynamic rheology. The results revealed a prevalent contribution of elastic/storage component to the viscoelastic behavior of cryogels in swollen state, a micromorphology with micrometer-sized open pores fully interconnected, and high porosity (ca. 90%) observed for freeze-dried cryogels. Furthermore, the metabolic activity and proliferation of human adipose stem cells (hASCs), when cultured onto the developed kefiran-CS cryogel, was maintained at a satisfactory level over 72 h. Based on the results obtained, it can be inferred that the newly freeze-dried kefiran-CS cryogels possess a host of unique properties that render them highly suitable for use in tissue engineering, regenerative medicine, drug delivery, and other biomedical applications where robust mechanical properties and biocompatibility are crucial.

Manganese-Labeled Alginate Hydrogels for Image-Guided Cell Transplantation.

Cell transplantation has been studied extensively as a therapeutic strategy for neurological disorders. However, to date, its effectiveness remains unsatisfactory due to low precision and efficacy of cell delivery; poor survival of transplanted cells; and inadequate monitoring of their fate in vivo. Fortunately, different bio-scaffolds have been proposed as cell carriers to improve the accuracy of cell delivery, survival, differentiation, and controlled release of embedded stem cells. The goal of our study was to establish hydrogel scaffolds suitable for stem cell delivery that also allow non-invasive magnetic resonance imaging (MRI). We focused on alginate-based hydrogels due to their natural origin, biocompatibility, resemblance to the extracellular matrix, and easy manipulation of gelation processes. We optimized the properties of alginate-based hydrogels, turning them into suitable carriers for transplanted cells. Human adipose-derived stem cells embedded in these hydrogels survived for at least 14 days in vitro. Alginate-based hydrogels were also modified successfully to allow their injectability via a needle. Finally, supplementing alginate hydrogels with Mn ions or Mn nanoparticles allowed for their visualization in vivo using manganese-enhanced MRI. We demonstrated that modified alginate-based hydrogels can support therapeutic cells as MRI-detectable matrices.

Advances in bioinks and in vivo imaging of biomaterials for CNS applications.

Due to increasing life expectancy incidence of neurological disorders is rapidly rising, thus adding urgency to develop effective strategies for treatment. Stem cell-based therapies were considered highly promising and while progress in this field is evident, outcomes of clinical trials are rather disappointing. Suboptimal engraftment, poor cell survival and uncontrolled differentiation may be the reasons behind dismal results. Clearly, new direction is needed and we postulate that with recent progress in biomaterials and bioprinting, regenerative approaches for neurological applications may be finally successful. The use of biomaterials aids engraftment of stem cells, protects them from harmful microenvironment and importantly, it facilitates the incorporation of cell-supporting molecules. The biomaterials used in bioprinting (the bioinks) form a scaffold for embedding the cells/biomolecules of interest, but also could be exploited as a source of endogenous contrast or supplemented with contrast agents for imaging. Additionally, bioprinting enables patient-specific customization with shape/size tailored for actual needs. In stroke or traumatic brain injury for example lesions are localized and focal, and usually progress with significant loss of tissue volume creating space that could be filled with artificial tissue using bioprinting modalities. The value of imaging for bioprinting technology is advantageous on many levels including design of custom shapes scaffolds based on anatomical 3D scans, assessment of performance and integration after scaffold implantation, or to learn about the degradation over time. In this review, we focus on bioprinting technology describing different printing techniques and properties of biomaterials in the context of requirements for neurological applications. We also discuss the need for in vivo imaging of implanted materials and tissue constructs reviewing applicable imaging modalities and type of information they can provide. STATEMENT OF SIGNIFICANCE: Current stem cell-based regenerative strategies for neurological diseases are ineffective due to inaccurate engraftment, low cell viability and suboptimal differentiation. Bioprinting and embedding stem cells within biomaterials at high precision, including building complex multi-material and multi-cell type composites may bring a breakthrough in this field. We provide here comprehensive review of bioinks, bioprinting techniques applicable to application for neurological disorders. Appreciating importance of longitudinal monitoring of implanted scaffolds, we discuss advantages of various imaging modalities available and suitable for imaging biomaterials in the central nervous system. Our goal is to inspire new experimental approaches combining imaging, biomaterials/bioinks, advanced manufacturing and tissue engineering approaches, and stimulate interest in image-guided therapies based on bioprinting.

Biomaterials Developments for Brain Tissue Engineering.

The Central Nervous System (CNS) is a highly complex organ that works as the control centre of the body, managing vital and non-vital functions. Neuro-diseases can lead to the degeneration of neural tissue, breakage of the neuronal networks which can affect vital functions and originate cognitive deficits. The complexity of the neural networks, their components and the low regenerative capacity of the CNS are on the basis for the lack of recovery, having the need for therapies that can promote tissue repair and recovery. Most brain processes are mediated through molecules (e.g. cytokines, neurotransmitters) and cells response accordingly and to surrounding cues, either biological or physical, which offers molecule administration and/or cell transplantation a great potential for use in brain recovery. Biomaterials and in particular, of natural-origin are attractive candidates owed to their intrinsic biological cues and biocompatibility and degradability. Through the use of biomaterials, it is possible to protect the cells/molecules from body clearance, enzymatic degradation while maintaining the components in a place of interest. Moreover, by means of combining several components, it is possible to obtain a more targeted and controlled delivery, to image the biomaterial implantation and its degradation over time and tackling simultaneously occurring events (cell death and inflammation) in brain diseases. In this chapter, it is reviewed some brain-affecting diseases and the current developments on tissue engineering approaches for a functional recovery of the brain from those diseases.

Combining neuroprotective agents: effect of riluzole and magnesium in a rat model of thoracic spinal cord injury.

BACKGROUND CONTEXT: Damage to the spinal cord can result in irreversible impairments or complete loss of motor, sensory, and autonomic functions. Riluzole and magnesium have been widely investigated as neuroprotective agents in animal models of spinal cord injury. As these drugs protect the injured spinal cord through different mechanisms, we aimed to investigate if their neuroprotective efficacy could be cumulative. PURPOSE: This study aimed to investigate the neuroprotective efficacy of combined administration of riluzole and magnesium chloride in a contusive model of thoracic spinal cord injury. STUDY DESIGN: An in vivo experiment was set using female Wistar Han rats that underwent a thoracic spinal cord contusion (T8) using a weight drop method. An hour after injury, animals were randomly distributed to receive (1) saline, (2) riluzole (2.50 mg/kg), (3) magnesium chloride (24.18 mg/kg) in a polyethylene glycol formulation, or (4) a combined treatment (riluzole and magnesium). Subsequent treatments were given in four intraperitoneal injections (spaced 12 hours apart). METHODS: The Basso, Beattie, and Bresnahan locomotor rating scale, an activity box test, and a swimming test were used to evaluate behavioral recovery over a 4-week period. Histologic analysis of the spinal cords was performed to measure the extent and volume of the lesion, axonal preservation, serotonergic and glutamatergic fiber sparing, motor neuron survival, and inflammation. RESULTS: Our results show that only the riluzole treatment significantly improved behavioral recovery up to 4 weeks after injury when compared with saline controls (6.2±1.8), with animals achieving weight-supported stepping (9.1±1.2). Riluzole also promoted tissue sparing with significant differences achieved from 200 to 600 µm (caudally to the lesion epicenter), and reduced lesion volume, with animals presenting a significantly smaller lesion (3.23±0.26 mm(3)) when compared with the saline-treated group (4.74±0.80 mm(3)), representing a 32% decrease in lesion volume. Riluzole treatment induced significant axonal preservation, as well as serotonergic fiber sparing, caudally to the injury epicenter. CONCLUSIONS: Our results suggest that the combined treatment, although simultaneously targeting two excitotoxic-related mechanisms, did not further improve behavioral and histologic outcome when compared with riluzole given alone.