Neural tissue engineering

Traumas and deficits in the human central nervous system (CNS), such as traumatic brain injuries (TBI), spinal cord injuries (SCI), Parkinson’s disease, Alzheimer’s, Multiple Sclerosis, Retinitis Pigment, and Age-related Macular degeneration, may have a permanent effect on the functionality of the patient, and the prognosis in many cases is poor. At the same time, human CNS suffers from a low inbuilt regenerative capacity, which makes healing with traditional medicine (drugs and surgical procedures) insufficient. [7] As a result, TE or more specifically regenerative medicine is considered to be a potential treatment for CNS deficits because it aims to restore normal functionality by enhancing the regeneration of tissue or by replacing the damaged parts with engineered biological transplants. One such strategy is cell therapy combined with a supportive biomaterial scaffold, such as hydrogel.

The central nervous system (CNS) (Fig. 2.19 (a)) is a part of the nervous system,

and it comprises the brain, spinal cord, and retina. [7, 144]. The skull and vertebrae protect the brain and spinal cord, respectively. The nervous system is composed of neurons, and non-neuronal glial cells (e.g., astrocytes, oligodentrocytes) that mainly support and protect neurons. The cell body of a neuron is called a soma (plural somata) (Fig. 2.19 (b)). A neuron also consists of some processes, i.e., dendrites (extend the receiving surface of neuron) and an axon (conducts nerve impulses).

Many axons are surrounded by a myelin sheath. The nervous tissue can be divided into white and gray matter. White matter contains axons and no neuronal somata (color due to myelin), whereas gray matter contains somata and dendrites. Both white and gray matter also contain glial cells. [144] Hyaluronan, proteoglycans (lecticans) and tenascins are the main constituents of healthy brain tissue [8]. HA is a major glycosaminoglucan component in the ECM of the brain. Since HA increases the hydration of brain tissue, it has been hypothesized to be the reason for the low stiffness of brain tissue (compressive moduli for brain tissue ranges from 2 kPa to 5 kPa, whereas for spinal cord it is around 8 kPa [137]). [8] HA also plays a vital role in the development of the CNS. During the differentiation of the spinal cord, HA surrounds the immature neurons, whereas it is also abundant in the fetal brain [137]. The CNS has limited spontaneous regenerative capacity [7, 145].

Hydrogels should fulfill specific criteria when they are used for neural TE. For example, they should have similar mechanical properties to those of the brain or spinal cord, since soft matrices (stiffness < 1 kPa) have been shown to improve axon length and cell attachment and survival [8, 148]. In addition, hydrogels should also allow the infiltration of cells and axons and enable the transportation of nutrients and metabolites (suitable porosity and pore size, and diffusion properties). Hydrogels should also integrate with the host tissue so that there is no inflammatory reaction or glial scar formation. [148] A suitable degradation rate together with non-toxic degradation products are also desired [8, 148]. Optical clarity might be needed for the imaging of cell cultures. The electrical conductance of materials would be beneficial in order to deliver an electrical current to the cells. [8] Injectability, together with a suitable gelation time, would allow minimally invasive surgery [145]. More general requirements for the hydrogel material have already been discussed in Section 2.4.2.

Based on the polymer-basis of the hydrogels used in this thesis, only examples of studies related to HA- and AL-based hydrogels used for neural TE (especially for the 3D in

Brain Spinal cord (a) (b) Dendrite Nucleus Soma Myelin sheath Axon terminal Axon

Figure 2.19: (a) The central nervous system (CNS) and (b) the anatomy of a neuron. Modified

from [146] and [147].

vitro culture of neural lineage cells) were chosen to be listed in Table 2.1. The role of

HA in the native CNS was discussed earlier. In addition, there are also many favorable properties discussed in Section 2.2.1.1 that explain why HA-based hydrogels are widely used for neural applications. AL-based hydrogels are also used for neural applications. AL is structurally similar to the ECM. As a natural hydrogel, it has many favorable properties (Section 2.2.1.1), which make it suitable for these applications. In addition, collagen type-I, Matrigel (mixture of ECM proteins), PuraMatrix (synthetic peptide hydrogel), chitosan, fibrin, IKVAV/RADA16 self-assembling peptides, and polyethylene glycol (PEG) hydrogels have all been successfully used for 3D in vitro culture of neural lineage cells, as reviewed by Murphy et al. [149].

The primary aim of this thesis was to find optimal hydrazone crosslinked hydrogel material for soft TE applications, especially for corneal and neural applications, and to investigate their detailed properties using suitable characterization methods. It was hypothesized that the chemical, physical and mechanical properties of the hydrogel would affect their usability in different biological applications. Further, it was hypothesized that by choosing mainly natural-based gel components as well as a mild and simple crosslinking method, bio-mimicking materials with suitable characteristics for the soft TE applications could be achieved.

The specific objectives of the studies were:

Study I:

• To create GG-HA-based hydrogels that would be suitable for soft TE applications based on their physical and mechanical properties, and replace the traditional crosslinking methods of GG-based hydrogels with hydrazone crosslinking to improve their properties as well as enable the injectability of the hydrogel, and to improve the method used to analyze the mechanical compression test data.

Study II:

• To create transparent HA-based hydrogels (with suitable physical and mechanical properties) that would be suitable for the delivery of hASCs for regeneration of the corneal stroma, to incorporate collagen I into these hydrogels in order to promote hASC attachment and survival, and to use an organ culture model with excised porcine corneas to show the clinical relevance of the HA-based hydrogels for hASC delivery to stromal defects.

Study III:

• To create injectable HA- and AL-based hydrogels (with suitable physical and mechanical properties) that would serve as a 3D supportive and biomimicking material for neural cell culture (human pluripotent stem cell-derived neuronal cells).

Study IV:

• To evaluate the microstructure of previously studied hydrazone crosslinked HA-, AL-, and GG-based hydrogels (Studies I-III) using rheology- and FRAP-based methods in order to show their suitability as biomaterial for soft TE applications, and to show how different gel parameters have an effect on the viscoelastic and diffusion properties, and further on the microstructure of these hydrogels.

4.1

Materials

Hyaluronic acid sodium salt (HA, Mw= 1.5 × 105g/mol) was purchased from Lifecore

(Chaska, MN, USA). Acetic acid, adipic acid dihydrazide (ADH), alginic acid sodium salt from brown algae (AL, low viscosity), 3-amino-1,2-propanediol, boric acid, car- bodihydrazide (CDH), 1,1’- carbonyldiimidazole (CDI), collagen type I from human placenta, deuterium oxide (99.9 atom % D, contains 0.05 wt.% 3-(trimethylsilyl)-propionic- 2,2,3,3-d4 acid, sodium salt), dimethyl sulfoxide (DMSO), 1-Ethyl-3-[3-(dimethylamino) propyl]carbodiimide (EDC), ethylene glycol, gellan gum (GG, GelzanT M_{, M}

w= 1,000

kg/mol), glycine ethyl ester hydrochloride, hyaluronic acid sodium salt from streptococ- cus equi (HA, Mw= 1.5-1.8 × 106 g/mol), hyaluronidase from bovine testes (Type I-S,

400-1000 units/mg solid), hydrazine solution (35 wt.% in H2O), 1-hydroxybenzotriazole

(HOBt), hydroxylamine hydrochloride, picrylsulfonic acid solution (5-% (w/v) in H2O,

TNBS), polyvinyl alcohol (PVA, Mw= 27000 g/mol, 98.0-98.8% hydrolyzed), sodium

acetate, sodium cyanoborohydride (NaBH3CN), sodium periodate, sodium tetraborate

decahydrate, sucrose and t-butyl carbazate (TBC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride and triethylamine were purchased from J.T. Baker (The Netherlands). Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ac- etate buffer was prepared from sodium acetate and acetic acid, and borate buffer was prepared from sodium tetraborate decahydrate and boric acid. All solvents used were of analytical quality. Milli-Q water was used in synthesis and determinations. Dialysis mem- branes (Spectra/Por cut-off 1000, 3500, 12000-14000 and 25000 g/mol) were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA).

FITC-dextrans with average molecular weight of 20, 150, 500 and 2000 kDa were purchased from TdB Consultancy AB (Uppsala, Sweden). Table 4.1 shows the hydrodynamic radii of the FITC-dextrans given by the supplier and those calculated based on the equation provided by Hadjiev and Amsden [150],

Rh= 0.0163Mw0.52, (4.1)

where Rhis the hydrodynamic radius, and Mwis the molecular weight of solute (dextran).