Introduction
Stroke is a degenerative injury of the central nervous system (CNS) and is one of the major causes of long-term disabilities in humans as well as the third largest cause of death in the world [
1]. By grafting neural stem cells to the CNS it may be possible to treat stroke. Neural stem cells can be used for regeneration because they have the capability to release neurotrophic factors and differentiate into all kinds of neural cells [
2,
3]. Using stem cells often requires a three-dimensional scaffold for attachment and to provide an appropriate environment for cell survival and differentiation.
Hydrogel scaffolds have been shown to be a good physiological model of the extracellular matrix, and they are good candidates for encapsulating neural cells since they can be modified to have structural, mechanical and degradation properties similar to biological tissues. Additionally, exogenous cells and axons are able to move into hydrogels due to the size of their micropores [
4‐
6]. Hydrogels that consist of only one polymer are normally not able to provide the mechanical and biological characteristics required for tissue engineering purposes. There are different ways to resolve this problem, for example, by designing composite hydrogels of two polymers, adding nanomaterials such as inorganic, organic, metallic, magnetic and carbonic nanomaterials or making hybrid hydrogels such as interpenetrating polymer networks (IPNs) or semi-interpenetrating polymer networks (semi-IPNs) [
7,
8]. Recently, researchers were able to resolve brain tissue engineering-related difficulties by designing and synthesizing IPNs using different polymers with tunable physical, biochemical and mechanical properties based on the requirements of the regeneration progress [
9].
The ECM of CNS tissue consists of a broad range of fibrous proteins, such as collagen, fibronectin, laminin, vitronectin and hyaluronic acid (HA). The amount of HA in the extracellular matrix is high, and it has a key role in the growth and migration of fetal cells and mature tissue regeneration. The majority of the ECM in brain is HA; after birth, the ECM in the brain is composed of 25% HA. Assessing HA hydrogels in vitro has been shown to help cell migration, as a hydrogel can be adjusted to have a loose matrix with a high water absorption capability [
10]. It has also been shown that HA has the potential to be an appropriate matrix for neural stem cell encapsulation [
11].
HA is a nonsulfated glycosaminoglycan containing D-glucuronic acid and D-
N-acetyl glucose amine. HA has positive characteristics such as degradability, chemical and mechanical sustainability, low immune response and high potential for angiogenesis [
9]. The molar mass of HA in brain tissue is high and hinders inflammation, vascularization and neuronal differentiation. When a stroke occurs in the brain, enzymatic degradation of the high molar mass HA begins, generating a low molar mass HA that in turn induces the proliferation of stem cells. Furthermore, low molar mass HA is also effective in the differentiation and migration of neural stem cells. HA with a low molar mass then activates the immune response in the body, leading to endothelial cell proliferation, tubulization and angiogenesis [
7]. In addition, HA fibers in natural ECM have the ability to guide the migration of neural cells in the brain by providing physical cues which lead to stem cell proliferation and differentiation [
11]. Thermoresponsive hydrogels that form hydrogels after injected in the body are compelling in tissue engineering for different reasons, for example because of the minimally invasive injection procedure. Poly(
N-isopropylacrylamide) (pNIPAAm) is a thermosensitive polymer which has the ability to form a gel at 32 °C. Adding pNIPAAm to polymers and hydrogels designed for tissue engineering purpose, leads to thermoresponsive scaffolds with more complex and appealing properties [
12].
Another important substance for successful brain tissue engineering is Puramatrix™ (RADA), a 16-amino-acid synthetic peptide that is resuspended in water. This is a self-assembling nanofibrous hydrogel scaffold that is assembled through the ionic self-complementarity of β sheet oligopeptides under physiological conditions (
T = 37 °C and pH = 7). A range of concentrations is known to have excellent effects on reknitting the lesion site in the spinal cord and brain after injury. RADA has been applied in a variety of neural tissue engineering applications and demonstrates a positive effect on neuronal growth, differentiation and synapse formation. One of the distinguishing properties of RADA is the ability to provide a fibrous 3D matrix with a fiber size similar to ECM and the ability to fill the injury site with a self-assembling, potent hydrogel [
13‐
16].
Our starting hypothesis was that the mechanical properties of an injectable IPN hydrogel based on HA and RADA can be adjusted for the mechanical properties required of neural tissue. We also hypothesized that an aligned porous structure would lead to the growth and alignment of endogenous and exogenous neural stem cells. Such IPNs would be of use for brain tissue regeneration. We therefore developed IPN hydrogels based on high molar mass HA and RADA, combining the HA hydrogel and RADA peptide, tuning the thermosensitivity, mechanical properties and fibrous structure. Thermosensitive HA was first synthesized by attaching N-isopropylacrylamide (NIPAAm) to HA, which in turn was combined with RADA in different ratios. The IPN was formed by increasing the temperature, and these thermosensitive injectable IPNs were carefully characterized and evaluated for brain tissue engineering. The morphological, chemical and mechanical properties of the hydrogels were assessed, and the cell–material interactions were evaluated by following the viability and morphology of cells encapsulated in the hydrogels.
Discussion
As discussed before, HA fibers in natural ECM are able to guide the migration of neural cells in the brain as they provide physical cues for them which lead to stem cell proliferation and differentiation [
11]. Also, RADA with 3D fibrous structure formation in which the fiber size is similar to natural ECM could have a positive effect on neuronal growth, differentiation and synapse formation [
35]. Thus, the IPN platform has the potential to provide neural stem cell growth and differentiation toward mature neural cells in the lesion site if injected
in situ.
To achieve a successful and functional tissue engineering scaffold, physical properties such as morphology, porosity, swelling ratio, degradation rate as well as mechanical properties such as rheological behavior and cellular behavior must match tissue properties in which it would be applied. In IPN hydrogels, a broad range of physical and mechanical properties can be attained by changing the type and quantity of the components. Here we developed an IPN hydrogel based on HA and Puramatrix with different concentration of each in order to optimize the scaffold. The IPN can be formed by increasing the temperature, since both the HA and Puramatrix are temperature sensitive. This kind of IPN is easy to form as the only factor controlling the formation is temperature, so that there is no need for cytotoxic crosslinking agents or UV [
36,
37]. Moreover, the temperature-responsive IPN is a very easily applicable and low invasive scaffold where the components can be added to each other and then injected in to the body, where crosslinking and entangling of both components take place at body temperature. Adding Puramatrix to HA-1 has been shown to change the structure of the hydrogel to that of an aligned porous structure with less swelling ratio in both PBS and medium, which can be effective in directing the migration and growth of the neural cells. Directing the growth of the neural stem cells, along with their attachment and guidance, is critical in neural tissue regeneration. It has been shown that a composite of electrospun polymeric nanofibers inside collagen hydrogel can have a great effect on spinal cord regeneration as the electrospun fibers provide a suitable surface for cell attachment, growth and migration [
38]. The effect of aligned nanofibers composed of PCL, fibrin and carbon nanotubes on growth guidance and differentiation of neural stem cells in neural tissue regeneration has been shown [
39]. These composite nanofibers were encapsulated in gelatin methacrylate hydrogel which was crosslinked using UV, after which the composite nanofibers and the crosslinked hydrogel were crosslinked together again with UV. This composite hydrogel with great similarity to the natural 3D neural tissue was efficient in aligned growth and differentiation of neural stem cells. Here, adding Puramatrix to the thermosensitive hydrogel as a nanofibrous structure provided a surface for cell attachment and growth. Also, Puramatrix is a self-assembling peptide with functional groups so there is no necessity for further functionalization of the hydrogel which is advantageous in tissue regeneration and vascularization.
Conclusion
A thermoresponsive injectable IPN hydrogel based on HA and Puramatrix™ with mechanical and physical properties similar to brain ECM has been designed. IPN hydrogels were synthesized using two different HA-to-NIPAAm ratios and three different Puramatrix™ concentrations and named HA-1-RADA-1, HA-1-RADA-5, HA-1-RADA-10, HA-2-RADA-1, HA-2-RADA-5 and HA-2-RADA-10. We demonstrated the effect of adding Puramatrix™ to HA on morphology and rheological behavior. Adding Puramatrix™ to modified HA changed the structure of the hydrogel to an aligned porous structure with a lower swelling ratio in both PBS and cell culture medium in addition to a lower storage modulus, which can be effective in directing the migration and growth of neural cells. Due to its mechanical properties and hydrogel structure as well as its injectability, it is suggested that the synthesized IPN is a material worthy of further study for the treatment of CNS degenerative injuries such as stroke or trauma.
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