**4. Regeneration**

Regenerative medicine is a dynamically developing area of medicine whose mission is to restore damaged tissue. Although different tissues and organs have different recovery capabilities, there are diseases and CNS injuries that have limited regeneration and, unfortunately, they cannot be treated by conventional therapies. One of the innovative regenerative medicinal approaches is the use of stem cells and biomaterial-based treatments in order to replace damaged tissue or to supplement missing trophic factors in various CNS diseases [3].

The twenty-first century resonates with the rapid development of regenerative medicine, where methods of isolation and processing of stem cells and the use of highly compatible biodegradable materials and nanotechnologies directed to the treatment of SCI patients has been improved [74]. However, successful cell therapy is influenced by various factors such as: (i) selection and processing of stem cells (adult, induced pluripotent stem cells), (ii) delivery strategies (local, systemic), (iii) dosage (single, continuous), and (iv) appropriate timing of administration (acute, chronic phase of SCI). Selection of stem cells is important for their compatibility with host tissue. For this reason, in clinical studies, stem cells obtained from the tissues of the patient are preferred. Autologous stem cell transplantation obtained from the bone marrow and adipose tissue of a patient is used in the treatment of hematopoietic diseases, in the regeneration of bone tissue and cartilage, and possibly also in spinal trauma [74]. At present, an autologous transplantation of the so-called induced pluripotent stem cells (iPKB) derived from adult somatic cell patients has also been considered. By new procedures, we can reprogram a fully differentiated somatic cell (fibroblast) toward a cell with primitive pluripotent origin that is derived into the desired cell population [75]. In other cases, allogenic stem cells that meet the compatibility criteria (ABO, HLA) may still be used, but patients must still receive immunosuppressive therapy for a lifetime. In addition, stem cells are a major tool for gene therapy when they can produce some trophic factors and other molecules that are necessary for the regeneration of injured nerve tissue.

Among different **mono-therapies**, more **complex cellular therapy** has reached considerable attention due to targeting multiple aims, such as bridging the cavities or cysts, replacing dead cells, and creating a favorable environment allowing axonal regeneration [76].

#### **4.1. Regenerative approaches toward biomaterials**

action on immune cells [72]. Furthermore, the immunosuppressive action of FK506 was proven

**Riluzole** is commonly used in the treatment of amyotrophic lateral sclerosis (ALS) to protect against nerve cell degeneration. The possible mechanism of action of riluzole is blocking sodium channels as well as glutamate excitotoxicity. The deleterious effect of glutamate overproduction during CNS damage can be reduced by both reducing the synthesis and preventing its release into the synaptic cleft. In the case of riluzole, its mechanism of action is most likely thought to be the reduction of glutamate synthesis and thereby its release into the presynaptic region of the neuron. In a recent study, 155 patients were randomized to riluzole treatment (100 mg/day) or to placebo. The patients were monitored for 12–21 months [48]. Survival was significantly longer in the riluzole-treated group compared to placebo-treated patients. The median survival time was 17.7 months for riluzole compared to 14.9 months for placebo [4]. Additionally, there was a significant improvement in motor function in patients with cervical SCI receiving 50 mg

Regenerative medicine is a dynamically developing area of medicine whose mission is to restore damaged tissue. Although different tissues and organs have different recovery capabilities, there are diseases and CNS injuries that have limited regeneration and, unfortunately, they cannot be treated by conventional therapies. One of the innovative regenerative medicinal approaches is the use of stem cells and biomaterial-based treatments in order to replace

The twenty-first century resonates with the rapid development of regenerative medicine, where methods of isolation and processing of stem cells and the use of highly compatible biodegradable materials and nanotechnologies directed to the treatment of SCI patients has been improved [74]. However, successful cell therapy is influenced by various factors such as: (i) selection and processing of stem cells (adult, induced pluripotent stem cells), (ii) delivery strategies (local, systemic), (iii) dosage (single, continuous), and (iv) appropriate timing of administration (acute, chronic phase of SCI). Selection of stem cells is important for their compatibility with host tissue. For this reason, in clinical studies, stem cells obtained from the tissues of the patient are preferred. Autologous stem cell transplantation obtained from the bone marrow and adipose tissue of a patient is used in the treatment of hematopoietic diseases, in the regeneration of bone tissue and cartilage, and possibly also in spinal trauma [74]. At present, an autologous transplantation of the so-called induced pluripotent stem cells (iPKB) derived from adult somatic cell patients has also been considered. By new procedures, we can reprogram a fully differentiated somatic cell (fibroblast) toward a cell with primitive pluripotent origin that is derived into the desired cell population [75]. In other cases, allogenic stem cells that meet the compatibility criteria (ABO, HLA) may still be used, but patients must still receive immunosuppressive therapy for a lifetime. In addition, stem cells are a major tool for gene therapy when they can produce some trophic factors and other molecules that are

damaged tissue or to supplement missing trophic factors in various CNS diseases [3].

necessary for the regeneration of injured nerve tissue.

by the prevention of graft rejection following spinal cord ischemia and SCI [44].

riluzole twice a day for 14 days after injury, compared to the control group [73].

**4. Regeneration**

12 Essentials of Spinal Cord Injury Medicine

SCI results in cysts or cavities at the site of the lesion, which gradually expand in the caudal direction. From this point of view, cell therapy alone for such a progressive pathological process as SCI is insufficient. Therefore, it is recommended to combine the administration of stem cells with biodegradable biomaterials that fill the cavities. The main objective is to optimize mechanical properties, cell adhesion, and biodegradability of synthetic or natural materials and develop new methods to deliver cells to the lesion site. One of the most important features for the successful integration of the implant into damaged tissue of the spinal cord is its optimal mechanical strength. If the biomaterial is too rigid, it can cause compression of regenerating axons and the formation of additional secondary cavities between the implant and surrounding spinal tissue. Therefore, it is preferable to use an injectable biomaterial that can properly adapt to the lesion [63, 77].The stem cells with which the implant should colonize also require the presence of growth factors that help them to survive in the unfavorable environment of the injured spinal cord. Chen and his scientific group compared the regenerative capabilities of several biodegradable multichannel biomaterials with different mechanical properties that were colonized by Schwann cells and implanted into the spinal cord after transection [63]. Compared to the poly-caprolactone fumarate material, which had significantly higher compression modulus values, biomaterials based on hydrogels showed significantly smaller cavities and promoted material vascularization and Schwann cell infiltration [63].

The biomaterial has to be biocompatible; this depends on the properties of the surface of the material and its interactions with cells or proteins [78]. However, we have to be aware of non-specific inflammatory responses of the recipient to the foreign biomaterial, and its extent determines the rate of implant biocompatibility. Interestingly, the acute response of the immune system that is mediated by macrophages or dendritic cells can be neuroprotective and can promote CNS regeneration. Modulation of the inflammatory response by the type of biomaterial surface can therefore be an auxiliary tool for repair mechanisms of the tissue. In principle, the physical properties of biomaterials should simulate the extracellular environment of the central nervous system and thereby ensure the diffusion of neurotrophic factors. Interactions between biomaterial surface and living tissue are usually mediated by a layer of proteins. Most biomaterials have an optimized surface with bioactive molecules or oligopeptide sequences [77]. This guarantees the adhesion of specific cells or their parts (e.g., axons).

**Biodegradable** materials are more desirable than non-degradable ones. Their degradation is most often mediated by hydrolysis and enzymatic cleavage. The rate of degradation can be controlled by various factors, such as molecular weight and polymer structure, crosslinking, and use of copolymers [79]. Of course, degradation products must not cause any immune response and the rate of breakdown of the material must be appropriate to the formation of new tissue. Biomaterials that are used to regenerate nerve tissue usually degrade for weeks or months, depending on the axonal and vascular material growth. Degradation can take place by gradual erosion of the surface of the material while maintaining the structural integrity of the material or by the gradual breakdown of the material structures. The first method is more advantageous because the collapse of the material may stop the regeneration process.

**Acknowledgements**

**Author details**

Milan Cizek1

Michel Salzet2

**References**

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1 University of Veterinary Medicine and Pharmacy in Kosice, Slovakia

\*Address all correspondence to: cizkova.dasa@gmail.com

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15

and

**Alginate materials** are natural and have a significant role because most of them are biodegradable. This group of natural materials also includes collagen, methylcellulose, or hyaluronic acid-based materials. The disadvantage is their natural variability and the risk of immunogenicity. The implantation of lyophilized alginate into the cavity of newborn or young rats stimulated the growth of non-myelinated and myelinated fibers in the hydrogel [80], as well as the formation of functional neuronal connections that have been demonstrated. In another study, the optimal combination of EGF and bFGF was chosen routinely in conventional 2D cultures in order to obtain the desired amount of proliferating cells. The goal was to create a strong but reversible binding of both factors to alginate-sulfate [81], allowing their prolonged and sustained local presentation to neural progenitors in cell culture. This develops an active biomaterial that eliminates the need for external continuous growth factor substitution during cell culture. However, it is crucial to determine the optimal concentration of growth factors that could mimic similar concentrations of bFGF/EGF commonly used in the 2D system culture (10–20 ng/ml for each factor/3 days). In this case, the equilibrium binding constant of the selected factors on alginate-sulfate plays an important role. The initial concentration of both bFGF and EGF factors (200 ng) was shown to be sufficient for their continuous release over 21-day incubation [82]. The concentration of growth factors released within the first week *in vitro* initiated cell proliferation and the formation of typical 3D neurospheres. Consequently, there was a decline in the growth factor concentrations; the cells migrated from the neurosphere and differentiated to neurons, astrocytes and oligodendrocytes. These results confirmed that the 3D alginate biomaterial, which gradually released growth factors, creates optimal conditions for long-term survival and differentiation of neural progenitors *in vitro* [82].

The developed 3D biomaterial was implanted locally into SCI rats. The results confirmed that the optimal bioavailability of growth factors (EGF and bFGF) from the implant stimulated neuroregenerative processes. Enhanced sparing of spinal cord tissue and increased number of surviving neurons (ChAT-cholinacetyltransferase-positive neurons), corticospinal fibers (BDAlabeled), and blood vessels at the site of injury [83] occurred. Inflammatory processes were partially suppressed, but not astrogliosis. These partial results indicate the possible use of active alginate biomaterials enriched with bioactive molecules in the treatment of CNS trauma [83].

Although the biomaterials themselves can affect nerve tissue regeneration by creating a space for cell growth through the lesion, it is increasingly clear that combined therapy has a synergistic effect and leads to better results. Therefore, biomaterials are most often combined with different types of cells or enzymes digesting proteoglycans in glial scars, as known for chondroitinase ABC. The most commonly used cells are MSC, Schwann cells, and neural stem cells that can express Noggin, promoting neurogenesis and suppressing gliogenesis [84]. Biomaterials can also serve to release the biologically active substance, which can then create a gradient that promotes cell growth into the implant. Biologically active agents may be growth factors (EGF, FGF), cytokines, neurotrophins (NT3, NGF, BDNF, and GDNF), neurotransmitters, and anti-axon growth inhibitory antibodies [85].

In conclusion, it is necessary to combine these strategies to further enhance the final effect.
