Utilization of GelMA with phosphate glass fibers for glial cell alignment

Glial cell alignment in tissue engineered constructs is essential for achieving functional outcomes in neural recovery. While gelatin methacrylate (GelMA) hydrogel offers superior biocompatibility along with permissive structure and tailorable mechanical properties, phosphate glass fibers (PGFs) can provide physical cues for directionality of neural growth. Aligned PGFs were fabricated by a melt quenching and fiber drawing method and utilized with synthesized GelMA hydrogel. The mechanical properties of GelMA and biocompatibility of the GelMA-PGFs composite were investigated in vitro using rat glial cells. GelMA with 86% methacrylation degree were photo-crosslinked using 0.1%wt photo-initiator (PI). Photocrosslinking under UV exposure for 60 s was used to produce hydrogels (GelMA-60). PGFs were intro-duced into the GelMA before crosslinking. Storage modulus and loss modulus of GelMA-60 was 24.73 ± 2.52 and 1.08 ± 0.23 kN/m 2 , respectively. Increased cell alignment was observed in GelMA-PGFs compared with GelMA hydrogel alone. These findings suggest GelMA-PGFs can provide glial cells with physical cues neces-sary to achieve cell alignment. This approach could further be used to achieve glial cell alignment in bioengineered constructs designed to bridge damaged nerve tissue.

interactions of these surviving cells can start the CNS repair and regeneration process. Due to the various challenges in neural regeneration processes such as slow axonal regeneration and inhibitory effects of the injured environment, the nerve recovery rate is very slow and can take a significant period of time in this complex biological environment. [2][3][4] Particularly, after major spinal cord injuries (SCI) substantial tissue loss may occur and result in a fluid-filled cavity, astrocyte reactivity, and glial scar formation at the injured site which may even propagate over time and lead to further secondary tissue damage. 5 Over the past few decades, various types of biomaterial scaffolds, processed by methods such as electrospun nanofibers, 6 freeze dried/ solvent cast, 7 self-assembly, gas foaming and hydrogels [8][9][10][11] have been investigated for neural tissue regeneration. These studies suggest that polymer-based biomaterial scaffolds can be used to repair CNS injury, alter the microenvironment of lesions, and promote the recovery of neural function. 12 Recently, hydrogel-based biomaterials have gained significant attention in CNS as well as peripheral nerve system (PNS) regeneration applications due to low cost, easy processing, controlled mechanical properties, permeability, and by serving as carriers for bioactive molecules and cell delivery to provide a permissive environment for regeneration. Moreover, hydrogels can mimic the extracellular matrix (ECM) to provide a niche for cells, support the surrounding neural tissue and also act as a substrate for cell growth, neurite formation, and axon regeneration. 10 The high-water content and porous inner structure help long-term nutrient supply for cells and thus can aid in axon survival. 13 Numerous studies have indicated that hydrogels can promote cell adhesion, axon regeneration, and myelination in neural damage both in vitro and in vivo. [14][15][16] Although hydrogelbased biomaterials possess superior biological properties, there are some drawbacks to natural hydrogels (such as collagen, fibrin, hyaluronic acid, and gelatin) as their mechanical properties are mostly dependent on polymerisation and the crosslinking mechanism and it can be difficult to control the microstructure and reproducibility between experiments. Compared to natural hydrogels, synthetic hydrogels offer more flexibility for closely defining the chemical composition and mechanical properties. Combining natural and synthetic hydrogels as hybrid materials or using naturally derived semi synthetic hydrogels such as Gelatin-methacry(late)/(loyl) (GelMA) hold great potential in tissue engineering applications. GelMA is a semisynthetic hydrogel, which consists of gelatin coupled with methacrylamide (MA) and the methacrylate groups enables the exploitation of the biological signals inherent in the gelatin molecule, while allowing control of mechanical properties. 17,18 Moreover, GelMA has shown important features such as enzymatic degradation in response to matrix metalloproteinases (MMPs), biocompatibility, cell adhesion due to presence of arginine-glycine-aspartic acid sequence (RGD) and controllable biophysical properties. 19 GelMA-based hydrogels have been investigated vastly for potential use in tissue engineering, drug delivery, and 3D bioprinting applications. [20][21][22] Phosphate-based glasses are mainly composed of P 2 O 5 as glass network former, Na 2 O and CaO. 23 Modifying oxides such as SrO, TiO 2 , 24,25 Ag 2 O, 26 Fe 3 O 4 , 27 ZnO, CeO 2 , 28 and CuO 29 have been included to induce the specific properties such as antibacterial, antioxidant, and anti-inflammatory properties, and other biological responses. Phosphate-based glasses have been studied to examine their potential for biomaterial applications mainly in bone tissue engineering, and drug and therapeutic ions such as copper, and silver delivery. 25 Phosphate glass fibers (PGFs) are biocompatible and biodegradable, and their tuneable degradation rate can be easily achieved by altering the compositions of phosphorus pentoxide, sodium oxide and calcium oxide within the glass network. Moreover, PGFs have shown excellent properties as guidance systems especially for the regeneration of outgrowing axons. 27 PGFs can promote highly directional growth of neurites in vitro and three-dimensional (3D) scaffolds with fibers facilitated the rate of directional axonal outgrowth in the in vivo sciatic nerve transection model. 30 Up to date nerve guidance systems have been evaluated on mostly peripheral nerves and also with spinal cord. In more detail, both for peripheral nerves and SCI the outer structure of an implantable scaffold is usually tubular in order to bridge up disconnected tissue, while its inner structures should be designed such that injured axons can migrate into it from both proximal and distal stumps following transection. From the perspective of axonal regeneration, providing physical cues for alignment to the fibrous scaffolds are ideal for surrounding cells' and ultimately axonal guidance. 30 Particularly for CNS glial cells are aroused interest for their crucial roles as supportive cells for neural tissue by providing optimal environment and function for neurons, 31 thus glial cells are subjected into this work as potential treatment elements.
This study aimed to evaluate the combination of PGFs and GelMA and to design an effective 3D scaffold as a potential cell carrier for spinal tissue engineering that potentially can provide directionality and a permissive environment for growth of glial cells.

| Production and characterization of PGFs
The quaternary glass compositions of (50P 2 O 5 -40CaO-5Na 2 O, 5Fe 2 O 3 , mol.%) were produced using precursors chemicals of NaH 2 PO 4 , P 2 O 5 , CaCO 3 , Fe 2 O 3 (Sigma-Aldrich, UK). The chemicals were mixed and then placed in a 200 mL platinum/ 5% rhodium (Pt/5% Rh) crucible (Type 71040, Johnson Matthey, UK) and heated in a furnace (Carbolite, model RHF 1500, UK) in air at 700 C for 30 min and then melted at 1100 C for 1 h. After that, the melted glass was poured onto a steel plate and left to cool to room temperature.
Using this glass, the fibers were produced using a continuous, fiber drawing method as previously reported. 27 For glass compositions, the drum (collector) pulling speed was at 800 m/min. To measure the fiber diameter, a small bundle of parallel-aligned fibers was placed into a polytetrafluoroethylene (PTFE) mould and covered with resin (SpeciFix-20 kit Resin, Struers). The resin was ground and polished to examine under a microscope that attached to a CoolSnap Digital Image Analysis. Image Pro Plus software was used to measure the diameter of the fibers in microns. The mean values and errors were calculated from measurements on 55 fibers. This value was also confirmed via scanning electron microscopy (SEM; Philips XL30 field emission SEM, Netherlands) of the fibers. As an inorganic material, PGFs did not require sample coating.

| Degradation of PGFs and ion release test
Degradation experiments were conducted on the glass fibers. From the same bundle of glass fibers, 1 cm long samples were cut and immersed in 3 mL of ultra-pure water (UPW) (10 mg/mL). All samples were incubated in an incubator at 37 C for 1, 3, 5, 7, 14, 21, 28 days and supernatants of samples were collected to conduct inductively

| GelMA production and characterization
The GelMA synthesis method has been modified from Zhu et al. 20 Briefly, to obtain GelMA, 10% wt/v of gelatin (Sigma-Porcine skin 300 bloom, Type A, UK) was dissolved in 0. loss modulus (E") between GelMA and GelMA-PGFs was carried out.
The aspect ratio of the sample was kept consistent for all hydrogel tests to assure a similar loading mode. Uniaxial compression testing was conducted at room temperature using cyclic sinusoidal load mode to frequency oscillations, which varied from 0.05 to 20 Hz with 1 and 5 Hz intervals for GelMA and GelMA-PGFs. Samples were pre-loaded to 0.001 N force and dynamically tested at low deformation (0.1% strain) compression to ensure that the data collected was repeatable.
To determine the effect of photo-crosslinking time on Young's modulus of GelMA hydrogels (n = 3), static compression mode was used to obtain stress/strain curves with ramp rate of 1 mm/min. Moduli were calculated from the slope of the linear region on the stress/ strain curve using OriginPro 2019 of linear curve fitting.

| Crosslinking studies
The crosslinking time and effect of UV exposure on mechanical and biological performance of GelMA hydrogels was examined at 6 different time periods as 10, 20, 30, 60, 120, 300 s of UV exposure with the same conditions. XYZPrinting UV chamber (Model 3UD10, Taiwan, UV LED λ 375-405 nm, 16 W) was used for photo-crosslinking.
The assessment of mechanical properties was carried out with respect of Young's modulus via DMA, and cell metabolic activity and cell viability (Live/Dead assays) via alamarBlue assay and imaging on an inverted fluorescence microscope (Leica, DM IRB, UK), respectively.
Methodological details for these tests were as described in previous related sections unless otherwise stated.

| Statistical analysis
The data were analysed using one-way and two-way analysis of vari-

| Mass loss and ion release
The PGFs were further characterized for in vitro degradation by mean of mass loss and ion release test. The mass loss of the PGFs during the degradation test were monitored up to 28 days (Figure 1(d). The degradation results showed a linear mass loss for an initial 2 weeks (up to 14 days, $ 40% mass loss) and the next two weeks showed slower degradation (14 days to 28 days, $ 20% mass loss), and a total of 60% mass loss at 28 days was observed. Moreover, the degradation of the PGFs also affects the fibers surface and cracks and breaks

| GelMA characterization
GelMA was synthesized by modification of gelatin using MA to create polymers with the methacrylate substitution. ATR-FTIR spectrum of GelMA (Figure 3(a) exhibits a strong peak for the primary amide (amide I) related C=O stretching groups appears at 1650 cm À1 and changes at the peak vibrations at 1650-1670 cm À1 interval corresponds to C=C, C=O bonds that presents in the backbone of GelMA polymer the interaction between gelatin and methacrylate anhydride. 38 Shifts and changes determined in peaks of GelMA compared to gelatin indicate that gelatin has been successfully modified to GelMA and this has also been proved by comparing commercial GelMA product referred as GelMA-Sigma on Figure 3(a). Moreover on

| Mechanical and biological effects of crosslinking time
As a semi-synthetic material, it has been known that physiochemical properties of GelMA can be easily tailored by altering the degree of methacrylation and photo-crosslinking conditions. 40  The PI concentration is also crucial to optimize cell viability following 24.73 ± 2.52 kN/m 2 and 1.08 ± 0.23 kN/m 2 , respectively, which was considered suitable for application with soft and mild-soft tissues such as nerve and muscle. 42,43 As noticed, for all frequency values, the value of E 0 was higher than the E 00 , which indicates a predominantly elastic behavior rather than viscous behavior in the hydrogel structure. 40 The representative images of GelMA and GelMA-PGFs hydrogels are shown in the related graphs of Figure 5 (c),(d). As it can be seen on Figure 5  In the light of inadequacies of two-dimensional (2D) cell culture systems for allowing mechanical cues to the cells in 3D cell culture with hydrogels have been used in tissue engineering for decades, therefore we have optimized our GelMA hydrogel system for 3D culturing of C6 cells (Figure 4). The SEM image of cell loaded GelMA-60 hydrogel samples cross-sectional area showed it was fully covered with grown cells at day 7 (D7) (Figure 4(b),(c).
Thanks to their superior swelling properties and biocompatibility hydrogels provides a good permissive environment to cells for nutrient and gas exchange for long term cell culture studies. 16 54 The alignment of cells in the injury site when supported with mitigated environmental changes can be associated with functional neural recovery. [51][52][53][54][55] In this study, the results have shown that PGFs degrades by

| CONCLUSIONS
This study showed the development and characterization of GelMA and PGFs for neural tissue engineering applications. PGFs structured GelMA hydrogels have shown potential to promote directional growth of glial cells that can be advantageous to encourage nerve repair in SCIs.
In conclusion, the results from this study offer a promising system combining the benefits of a hydrogel system constructed with GelMA and PGFs which are known to direct and drive neural cell axial growth, making them highly beneficial for SCI. In this sense our system is particularly promising as it provides directionality with glial cells and tuneable mechanical and biological properties via the materials. Further simple casting models have been developed to move to translational studies ( Figure S1). Moreover, a combination of PGFs systems with hydrogels will provide easy usage of fibers for transplantation applications as well as their promising biological properties.
Our future studies will, therefore, explore changing composition and/or diameter of the PGFs and the hydrogels by addressing SCI induced environmental changes for potential translational works.

CONFLICTS OF INTEREST
The authors have no competing financial interests on this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author, [JCK], upon reasonable request ORCID