**Acknowledgements**

or by the gradual breakdown of the material structures. The first method is more advantageous

**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 condi-

tions for long-term survival and differentiation of neural progenitors *in vitro* [82].

inhibitory antibodies [85].

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

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

because the collapse of the material may stop the regeneration process.

14 Essentials of Spinal Cord Injury Medicine

Supported by APVV 15-0613, ERANET- AxonRepair, INSERM U1003, VEGA 2/0125/15, SK-FR-2015-0018/ Stefanik, EU SF ITMS 26240220008.
