Synthetic-based hydrogels

Among synthetic non-biodegradable hydrogel used for SCI repair poly(N-(2-hydroxypropyl)-methacrylamide) (PHPMA) is very promising. The research group of Woerly et al. developed a hydrogel made of PHPMA, obtained by a radical polymerization of the monomer HPMA with the use of a divinyl cross-linking agent ( ), functionalized by a synthetic peptide which includes RGD sequence ( ). The implantation of the hydrogel into the neonatal and adult spinal cord reveals a good infiltration of cells and blood vessels and the following implantation of the hydrogel seeded with rat mesenchymal stem cells (MSCs) in rats results in better Basso, Beattie, Bresnahan (BBB) score than the control group without the implantation ( ). Another investigated polymer in nerve tissue regeneration is the biocompatible and hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA). As previously said, the charge and the structure of the hydrogel can influence the behavior of cells and the ingrowth of the new tissue. A study by Hejcl et al. ( ) was conducted to compare the different effects of the surface charge and structure of HPMA and HEMA hydrogels on tissue regeneration. Specifically, four different hydrogels were prepared and seeded with rat MSCs: one HPMA-RGD hydrogel by using heterophase separation (HPMA-HS-RGD), which resulted in a structure characterized by microparticles, two HPMA hydrogels by using a solid porogen, one functionalized by RGD peptide (HPMA-SP-RGD), and one without functionalization (HPMA-SP), which resulted in network structures, and the last hydrogel made by positively charged copolymers of HEMA with [2-(methacryloyloxy)ethyl]trimethylammonium chloride (MOETA +). After successful in vitro studies, hydrogels were implanted into rat SCI hemisection model. The best results in terms of in vitro adhesiveness and in vivo survival of MSC was found in the positively charged HEMA-MOETA + hydrogel, whereas the best results in terms of axonal ingrowth and vascularization was found in the HPMA-SP-RGD hydrogel demonstrating the higher efficacy of the network architecture respect to the globular ones. With respect to the influence of the RGD peptide, it increases the vascularization but has no effect the growth of axons.

Hydrogel functionalization with cell-adhesive peptides

The presence of cell-adhesive peptides on hydrogels, such as the laminin-derived peptide sequence SIKVAV [Ser-Ile-Lys-Val-Ala-Val] and fibronectin-derived peptide RGD [Arg-Gly-Asp], can enhance cell adhesion, migration on the scaffold, proliferation, and differentiation ( ). The research group of Kubinova et al. functionalized a copolymer-based hydrogel of HEMA and 2-aminoethyl methacrylate (AEMA) with the laminin-derived Ac-CGGASIKVAVS-OH peptide by disulfide bridges ( ). The functionalization with SIKVAV and RGD ( ) was made on the same type of hydrogel also through the maleimide-thiol coupling reaction. All the functionalized hydrogels guarantee the adhesion and proliferation of rat MSCs maintaining their multi-lineage potential. In addition, in vivo studies results shown a higher connective tissue and vascularization on fibronectin-modified HEMA hydrogel compared to the non-functionalized one ( ). Other molecules such as serotonin can be used as neurotransmitter and can improve neuronal differentiation of implanted or endogenous neuronal progenitor precursors. Despite promising in vitro results, in vivo model studies on implanted PHEMA functionalized with serotonin showed a migration of seeded neural progenitor out from the polymer leading to a fail in proving a long-term effect on nerve tissue reconstruction ( ).

Porosity orientation

The orientation of fibers is important for tissue regeneration because it provides preferential lines along with the cells growth and proliferation. In addition, adequate porosity and mechanical properties have to support the movements of the organ and the regeneration of the new tissue. Hence, in the case of scaffolds for nervous tissue regeneration, the best one has to be characterized by parallel guiding channels and pores. The group of developed SIKVAV-modified PHEMA hydrogels with parallel oriented pores prepared by a salt-leaching method with ammonium oxalate needle-like crystals, and added 8%, 4%, and 0% (wt%) of [2-(methacryloyloxy)ethoxy]acetic acid (MOEAA) obtaining three hydrogels with 57%–77% porosity, pore diameter of ~ 60 mm, and an elastic modulus of 6.7, 27.4, and 45.3 kPa along the pore axis and 2.9, 3.6, and 11 kPa in a perpendicular direction. After 2 months of implantation the results showed that the softest hydrogel collapses because of the thinness of walls causing a sparse axonal growth inside the hydrogel, whereas the stiffest hydrogel supported axonal ingrowth into the pore guides but cyst formed at the tissue-scaffold interface because of difference in mechanical properties between the two components. The best results in terms of axonal ingrowth, presence of blood vessels and Schwann cells are obtained using the hydrogel with the moderate elasticity modulus of 27.4 kPa along the pores. Unfortunately, the use of the moderate scaffold seeded with MSCs was not able to promote a sufficient axonal growth.

Indeed, the rate of axonal growth resulted very slow and after 6 months from the implantation only few axons were be able to cross the hydrogel and infiltrate the caudal stump ( ). Therefore, other factors are necessary in order to promote axonal regeneration. For example, the presence of MSCs overexpressing of an NT-3 receptor ( ) or brain-derived neutrophic factor (BDNF) ( ) on the scaffold can be added in order to enhance axonal growth and recovery of motor functions.

Natural-based hydrogels

This type of hydrogels may be made by ECM derived components such as collagen or hyaluronic acid. Hyaluronic acid (HA) is a natural biocompatible polymer, biodegradable and non-toxic, but it is does not favor the attachment of cells. A possible overcoming solution is represented by the use the hydroxyphenyl derivative of HA which is able to covalently crosslink in situ, forming a hydrogel in presence of horseradish peroxidase enzyme and hydrogen peroxidase ( ). Moreover, the RGD peptide can be linked to the HA-PH derivative ( ) in order to favor the attachment of cells on (HP-HA) hydrogel. Human Wharton’s jelly derived mesenchymal stem cells (hWJ-MSCs) were encapsulated in the hydrogel, which then was injected in the sub-acute spinal cord hemisection. In situ crosslinking had no cytotoxic effect or negative effect on cells. HA-PH-RGD hydrogel was able to favor axonal ingrowth and the presence of hWJ-MSCs increases the effect. However, there were no improvements of motor function probably due to the low quantity of cells incapsulated inside the gel.

Extracellular matrix-based hydrogels

Another type of natural-based hydrogel is represented by decellularized ECM: it is suitable for tissue regeneration because of its biocompatibility, biomolecular and complex chemical composition which characterize it and distinguish it from other scaffolds. Decellularization is performed by different chemical, physical or enzymatic method and then the decellularized ECM is transformed in a liquid phase using pepsin solubilization at pH < 2 in order to be injected into the site of injury. The physiological temperature and pH favor its crosslinking in situ, leading to its original structure. The research group of Kubinova tried to use ECM-based hydrogels derived from CNS, such as porcine spinal cord (SC-ECM), and non-CNS derived, such as human umbilical cord tissue (UB-ECM) and porcine urinary bladder (UB-ECM). After implantation into injured spinal cord they stimulated nerve tissue regeneration and no differences on biological response were seen between the use of CNS and non-CNS–derived ECM ( ; ). However, a critical problem was represented by the fast degradation rate of the scaffold, which was due to the infiltration of resident cells present in the site of lesion.

Therefore, inadequate structure was provided to the new tissue and a correct regeneration of the tissue was compromise. In order to decrease the rate of degradation it was necessary to increase the number of crosslinks. This can be done using crosslinking agents such as genipin, which is able to bridge free amino groups present in the ECM. Its use on the UC-ECM hydrogel did not increase in vivo inflammatory response ( ), moreover the lack of ethical problems and the allogeneic source leads to consider promising the use of umbilical cord in neural tissue regeneration.

Nanotechnology and nanomedicine

Nanotechnology is the synthesis and characterization of nanosystems and their application in different fields, from the research to the industrial practice. When nanotechnology is applied in medicine and healthcare, it is called nanomedicine. Nanomedicine covers different medical fields such as prevention, diagnosis and treatment. It uses nanomaterials in the range of 10–1000 nm for interacting with biological systems at the molecular level. In addition, the resulting high surface area per unit volume favors a higher number of interactions with biological systems. Thanks to the binding with specific cellular receptors nanosystems can also deliver drugs and molecules in specific site without damaging the surrounding healthy tissue. Nanomedicine developed a lot of structures such as nanoparticles, nanotubes, nanorods, nanogels, quantum dots, etc., but the most used in SCI field are nanoparticles, nanogels, and nanotubes.

Properties of nanomaterials

The treatment of SCI with drugs administrated by oral, intravenous or intra-arterial ways is not effective due to the filtrating action of the blood–spinal cord barrier (BSCB) which prevents the passage of foreign and immunological substance from bloodstream to the SC parenchyma. Nanomaterials can be developed by top-down, bottom-up or hybrid methods. The first method consists in transforming a bulk material to a nanosized material, the second one consists in forming a nanomaterial starting from molecular arrangements and interactions whereas the last method is based on mixing the previous two. Nanocarriers have to correspond to specific size in order to favor their migration across the biological barrier of spinal cord and the target the desired tissue. Smaller particles are more suitable for this purpose and the possible presence of ligands on their surface can bind to receptor molecule of neural cells favoring the activation of specific cellular response. The drawback of using small particles is the limited control on modification in a batch-to-batch synthesis approach ( ). Furthermore, the shape of the nanovectors influences their behavior inside biological environments and their cellular uptake too.

Specifically, in the case of nervous system, nanorods characterized by targeting peptides are considered more able than nanoparticles in accumulating in specific vascular environment without activating immune clearance ( ) or, for example, biconcave nanoparticles enhance the release of drug respect to spherical or tubular particles ( ). Finally, surface charge has a key role with respect to the final aim of particles. In general, positively charged nanoparticles are better internalized by cells ( ; ) but the modification with chemical groups or peptides can change the surface charge leading to a different aim such as to target a specific area or avoid activation of immune systems. As for the composition of hydrogels, also nanomaterials can be made by synthetic or natural materials. Natural nanomaterials are in general biocompatible, non-toxic and very similar in composition and chemical features with the biological environment allowing a weak immune response, but it is difficult to achieve a good reproducibility during their development and production. On the other hand, synthetic nanomaterials guarantee a high reproducibility and possibility to modify their chemical, physical and morphological properties adapting them to the final purpose, but their immunogenicity is higher compared to the natural ones. Natural materials used for developing nanoparticles are collagen, lipids, albumin, fibrin, silicone, alginate, agarose, hyaluronic acid, chitosan, cellulose, heparin and chondroitin sulfate, whereas synthetic materials used are polyethylene glycol (PEG), polyethyleneimine (PEI), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyglycolic derivatives, polymethacrylate, polyacrylates, polycyanoacrylates, and poly(ɛ-caprolactone) (PCL). Fig. 1 represents a summary of natural and synthetic polymers.

Polymers for NPs. Chemical structure of natural and synthetic polymers used for developing NPs.

Nanoparticles

Nanoparticles are colloidal systems made of polymer chains from which is possible to obtain nanospheres or nanocapsules ( Fig. 2 ). Nanosphere are systems with a size of 100–200 nm composed of a solid matrix with physically or chemically entrapped drug ( ). They can be covered at their surface with surfactants or hydrophilic polymers which avoid opsonization and subsequent internalization from immune cells. Nanocapsules are nanosystems composed by an external polymeric layer which surround a lipophilic core.

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