Stiffness of photocrosslinkable gelatin hydrogel influences nucleus pulposus cell propertiesin vitro

A key early sign of degenerative disc disease (DDD) is the loss of nucleus pulposus (NP) cells (NPCs). Accordingly, NPC transplantation is a treatment strategy for intervertebral disc (IVD) degeneration. However, in advanced DDD, due to structural damage of the IVD and scaffold mechanical properties, the transplanted cells are less viable and secrete less extracellular matrix, and thus, are unable to efficiently promote NP regeneration. In this study, we evaluated the encapsulation of NPCs in a photosensitive hydrogel made of collagen hydrolysate gelatin and methacrylate (GelMA) to improve NP regeneration. By adjusting the concentration of GelMA, we prepared hydrogels with different mechanical properties. After examining the mechanical properties, cell compatibility and tissue engineering indices of the GelMA‐based hydrogels, we determined the optimal hydrogel concentration of the NPC‐encapsulating GelMA hydrogel for NP regeneration as 5%. NPCs effectively combined with GelMA and proliferated. As the concentration of the GelMA hydrogel increased, the survival, proliferation and matrix deposition of the encapsulated NPCs gradually decreased, which is the opposite of NPCs grown on the surface of the hydrogel. The controllability of the GelMA hydrogels suggests that these NPC‐encapsulating hydrogels are promising candidates to aid in NP tissue engineering and repairing endogenous NPCs.


| INTRODUC TI ON
Degenerative disc disease (DDD) is a major cause of neck pain, low back pain and disability in elderly individuals that is detrimental to their quality of life and ability to work. 1 Recently, with the ageing of the population, DDD has become a major public health issue and economic burden. 2 Current treatment strategies, including conservative and surgical treatments, do not resolve the underlying mechanisms and can cause complications. 3 Therefore, there is an urgent need to develop more effective treatments for DDD that address the causal mechanisms.
The intervertebral disc (IVD) has a typical multi-scale structural organization, consisting of three components: the highly hydrated gelatinous nucleus pulposus (NP) in the centre, the surrounding elastic annulus fibrosus (AF), and the upper and lower cartilaginous endplates. 4 The NP plays an important role in the axial loading of the spine, as it can distribute hydraulic pressure evenly between adjacent vertebral bodies. 5 However, in DDD, the NP is in a dehydrated state and cannot achieve fluid pressurization, which increases the axial compression of the NP and AF, resulting in an uneven stress distribution on the IVD and the destruction of the surrounding AF.
NP changes are early signs of DDD and include a decreased cell count in the NP, inflammation, reduced synthesis of extracellular matrix (ECM) components (such as type II collagen [Col II] and proteoglycans), and fibrosis. 6,7 After the onset of DDD, due to the lack of vascular tissue, NP cells (NPCs) suffer nutrient deficiency and difficulty in regeneration, thus limiting endogenous repair. 8 Tissue engineering and regenerative medicine have provided solutions to this problem. Hydrogels are considered the most promising candidate for NP tissue engineering because they have similar characteristics and functions to those of natural NP tissues, such as a high water content and fluid pressurization effect. 9,10 Many types of hydrogels have been evaluated for NP tissue engineering, including alginate, 11,12 fibrin/fibrinogen, 13,14 collagen, 15,16 carboxymethyl cellulose, 17,18 gellan gum, 19,20 chitosan, 21,22 and composite hydrogels. 23,24 Ideally, these self-healing hydrogels should have certain biological properties, such as an appropriate gel fraction, injectability, the ability to support encapsulated cell proliferation and ECM secretion, high mechanical strength, degradability, biocompatibility, and no side effects after implantation. 25 More importantly, in advanced DDD, due to the damaged IVD structure, the NP will bear a greater axial compression force, and appropriate mechanical properties will help maintain the 3D structure of the implanted NP at an early stage. 26 Furthermore, the hydrogel must facilitate the survival, growth, and migration of NPCs. Most hydrogels developed to date do not fulfil these criteria; therefore, they cannot support the longterm regeneration of the NP.
Gelatin methacrylate (GelMA) is a photosensitive hydrogel that not only retains the unique properties of gelatin but can also photocoagulate from liquid to solid via chemical cross-linking methacrylamide groups. 27 With the right photoinitiator, the hydrogel can be cross-linked to form a solid within a few minutes or even a few seconds, thereby minimizing cytotoxicity. 28 Moreover, since the GelMA hydrogel is transparent, it is easy to observe cells that are encapsulated or those on the surface. Notably, the mechanical properties, degradability, and biological properties of the GelMA hydrogel can be modified by altering the ratio of methacrylamide or the photopolymerization time, 27,29 making it possible to precisely control its performance. The 3D GelMA hydrogel system has characteristics similar to those of natural NP tissues, including a high water content and porous structure, thereby facilitating the penetration of nutrients and oxygen as well as providing a good local microenvironment for growing NPCs. 30 GelMA hydrogel scaffolds have been utilized for bone repair, cardiovascular disease, and skin regeneration. [31][32][33] However, their application to NP tissue engineering has yet to be explored.
In this study, we used GelMA hydrogel for NPC transplantation to support NP regeneration. To determine the optimal hydrogel concentration for NP regeneration, we adjusted the concentration of GelMA hydrogel, thus modifying its mechanical properties and cell compatibility in a controlled manner. We first evaluated the mechanical properties of GelMA hydrogels at different concentrations.
Following this, we examined their microstructures and hydration properties. Finally, we assessed the ability of NPCs to survive, extend, and deposit ECM in the 3D GelMA hydrogel system.

| Preparation and characterization of GelMA hydrogels
According to the literature, 27 was added to the gelatin solution at a rate of 0.5 mL/min and allowed to react for 2 hours. The mixture was packed into dialysis bags (Mw: 8000-14 000) to remove unbound methacrylic acid and other small molecular impurities. After a week of continuous dialysis, replacing the deionized water twice daily, the dialysate was heated to 60°C and then filtered using a 0.22 μm filtration membrane. The filtrate was then freeze-dried to a white foam and collected for use.
Thereafter, 5%, 10%, and 15% GelMA (w/v) were dissolved with 0.05% of the photoinitiator lithium aryl phosphonate in PBS, and a hydrogel unit was prepared by irradiating it under a UV light for 12 seconds ( Figure 1A).

| Aperture size evaluation
The three hydrogels with different concentrations were frozen at −80°C overnight and then freeze-dried at −40°C. The sample surface was sputter-coated with gold (SC7620, Quorum Technologies, UK) and then observed and photographed using a scanning electron microscope (SEM; Hitachi, Kyoto, Japan). The average pore sizes of the samples were measured using ImageJ software (National Institute of Health, Bethesda, MD, USA).

| Hydrogel swelling analysis and water contact angles
After freeze-drying, the weight of the hydrogel was recorded as W 0 , and the weight of hydrogel soaked in PBS overnight was recorded as W 1 . Following this, the scaffold was maintained at room temperature (25℃). According to different time points, the weight of the hydrogel was recorded as W t after water was sucked off its surface.
The percentage of water retention (W R ) at the different periods was recorded using the following equation: The wettability of hydrogels was assessed at 25°C via the water contact angle (WCA) and droplet shape. 2 U/mL of collagenase II at 37ºC.

| Hydrogel degradation
The weight of the freeze-dried hydrogel was denoted as W 0 . The hydrogel was then soaked in 2 U/mL of collagenase II (Sigma-Aldrich, USA) in a 37°C, constant temperature water bath. When taken out at different time points, the weight after freeze-drying was recorded as W t . The percent degradation rate (W D ) of the sample was denoted as W D = W t /W 0 × 100%.

| Mechanical testing
Hydrogel scaffolds with a diameter of 8 mm were prepared and then immersed in PBS overnight. The compression modulus of the hydrogels of different concentrations was determined using mechanical testing instruments (Shanghai Heng Yi Precision Instrument Co. Ltd., China) at a 20% strain rate.

| Isolation and culture of NPCs
All animal feeding and surgical procedures were approved by the were killed via an intraperitoneal injection of pentobarbital sodium.
The intervertebral discs in the tail were collected, and subsequently, the NP tissue was separated, cut into pieces under sterile conditions, and then treated with 0.2% collagenase II in an incubator at 37°C for 1 hour. After washing and centrifuging, NPCs were cultured in DMEM/F12 (Gibco, USA) complete medium containing 10% foetal bovine serum (Hyclone, China) and 100 U/mL penicillin-streptomycin (Invitrogen, USA). The cells were cultured at 37°C in 5% CO 2 . The complete medium was replaced every two days and the cells were used at the third passage.

| NPC adhesion
The GelMA hydrogel was injected into a 96-well plate, irradiated under ultraviolet light, and plated with 2 × 10 3 NPCs in each well.
The cultures were washed with PBS after 4 and 7 days, the cells were

| NPCs encapsulated in hydrogels
A total of 5 × 10 6 cells/ml NPCs were suspended and mixed with the GelMA hydrogel. Then, the suspension was injected into a circular mould with a diameter of 5 mm and a height of 2 mm, and the cell-encapsulated hydrogel was prepared using ultraviolet irradiation. The cell-encapsulated hydrogel scaffold was immediately placed in a complete medium and then cultured in a humidified incubator at 37°C and 5% CO 2 . The medium was changed every other day.

| Immunofluorescence analysis
The samples were washed with PBS and fixed with 4% paraformaldehyde at 4°C for 30 minutes. The scaffolds were permeated with 0.5% Triton X-100 for 30 minutes, sealed with 5% BSA (Invitrogen) for 1 hour, and then incubated with the primary antibodies anti-aggrecan (Affinity Biosciences, USA) and anti-Col II (Affinity Biosciences, USA) at 4°C overnight. Next, the primary antibodies were collected and the scaffolds were washed with PBS. Next, the scaffolds were incubated with Alexa Fluor 488-conjugated Goat anti-rabbit (Proteintech, USA) and Cy3-conjugated Goat anti-rabbit (Proteintech, USA) for 1 hour.
Finally, the scaffolds were stained with DAPI for 15 minutes and were observed and photographed using LSCM.

| Histological analysis
Histological analysis was performed after 7 and 14 days of cell encapsulation. The samples were washed with PBS and fixed with 4% paraformaldehyde for 1 day. Then OCT (Sakura Finetek, Japan) and ethanol were used to dehydrate 5% and 10%-15% of the samples, respectively. The samples were embedded with OCT and cut into 9 μm-thick slices in a cryogenically frozen slicer (Leica, Germany).
The samples were stained in the following order: haematoxylin and eosin (H&E), Masson and toluidine blue. The sections were observed using an inverted microscope.

| Western blotting analysis
The hydrogel-encapsulated scaffolds were repeatedly suspended in the GelMA lysate (Suzhou Intelligent Manufacturing Research Institute, China) for 1 hour until the GelMA dissolved completely.
After that, the protein was extracted and analysed using Western blotting with primary antibodies (anti-aggrecan and anti-Col II) and the secondary antibody HRP-conjugated goat anti-rabbit (Proteintech, USA). Finally, Image J software was used to measure the density of the immunoreactive bands, and quantify and normalize them.

| Statistical analysis
All experimental data are presented as mean ± standard deviation (SD) and were statistically analysed using ANOVA with GraphPad Prism 5.00 software (La Jolla, CA, USA).

| Characteristics of the GelMA hydrogel
Pore size is attributed to the cross-linking distance within the GelMA hydrogel that is mainly affected by the cross-link density. As determined using SEM, all cross-sections of the GelMA hydrogels at different concentrations showed a porous honeycomb structure, which can help to facilitate nutrient transport and provide space for proliferating and expanding encapsulated cells ( Figure 1B-D).
The elasticity of the GelMA hydrogel and resistance to deformation are critical parameters for the long-term structural stability of the NP. To determine the relationship between the concentration of the GelMA hydrogel and mechanical strength, we performed compression tests using 5%, 10%, and 15% (w/v) GelMA hydrogels.
The compression modulus was the lowest for the 5% (46.78 kPa) hydrogel and the highest for the 15% (82.05 kPa) hydrogel. Therefore, there was a positive correlation between the hydrogel concentration and compression modulus ( Figure 1G).
Biodegradability is a key characteristic of hydrogel used in tissue engineering. As shown in Figure 1H-I, the degradation rate decreased gradually as the concentration of the hydrogel increased. At 2.5 hours, the 5% GelMA hydrogel completely dissolved ( Figure 1I), while the 10% and 15% GelMA hydrogels reduced in volume, with degradation rates of 30% and 38.6%, respectively ( Figure 1H). This As high water content can facilitate the transport of nutrients, the W R capacity and WCA are important parameters. In our experiment, the W R capacity of the 5% GelMA hydrogel was consistently higher than those of the 10% and 15% GelMA hydrogels. This may be due to the low cross-link density of the 5% GelMA hydrogel, which enabled more water to penetrate ( Figure 1J). The WCA for the 5%, 10%, and 15% GelMA hydrogels were 27.50 ± 1.44°, 38.75 ± 1.11°, and 44.75 ± 0.48°, respectively ( Figure 1K). Therefore, the 5% GelMA hydrogel had the highest hydrophilicity and wettability, that is, parameters that are the most useful in facilitating cell survival.

GelMA hydrogel
Cell-scaffold adhesion is critical for the survival and growth of cells.
We found that NPCs had stronger adhesion to the surface of the 15% GelMA hydrogel than to the surface of the 5% and 10% GelMA hydrogels (Figure 2). At 4 days after cell transplantation, the NPCs adhered to the surfaces of the GelMA hydrogels at all concentrations. Based on confluence (ie the percentage of adherent cells), NPC extension and migration were the greatest on the surfaces of the 10% and 15% GelMA hydrogels, whereas cells on the 5% GelMA hydrogel were mostly round and had no extension (Figure 2A). After 7 days, compared with cells on the 10% and 15% GelMA hydrogels, cells on the 5% GelMA hydrogel showed extension but still showed less confluence (Figure 2A). Moreover, confluence increased substantially on the 10% and 15% GelMA hydrogels, with values two-to three-fold higher than those at 4 days ( Figure 2B). Confluence on the 5% GelMA hydrogel did not show a considerable change over time and was significantly different from that of the 10% and 15% GelMA hydrogels (P < .01). The cell density (based on nuclear DAPI staining in a target area) after 7 days also showed significant differences concerning hydrogel concentration, consistent with the results for confluence (P < .01; Figure 2C).
For overall NPC morphology, the 5% GelMA hydrogel showed cell aggregation and expansion, and the degree of confluence increased as the concentration of the hydrogel increased. The relationship between confluence and concentration of hydrogel was not simply a reflection of increases in extension. The difference in morphology was also related to increases in cell quantity and aggregation. This difference may be due to the increased hardness of the hydrogel with an increase in the hydrogel concentration, resulting in a denser structure and improved bioactivity of the seeded surface cells.

| Cell encapsulation in the 3D GelMA hydrogel
To detect the proportion of live cells, live/dead cell staining was performed 7 days after the encapsulation of NPCs in the GelMA hydrogel ( Figure 3A-C). As visualized using fluorescence imaging, most of the cells encapsulated in the hydrogels were alive, and almost no dead cells were detected for all three concentrations; the proportion of dead cells was the lowest in the 5% GelMA hydrogel (P < .01; Figure 3G). These results indicate that NPCs had high viability on 7 days after encapsulation, and the hydrogels had no cell toxicity and exhibited good cell compatibility. In the 5% GelMA hydrogel, NPCs grew outward, migrated, and connected with the surrounding cells ( Figure 3D). However, in the 10% and 15% GelMA hydrogels, cells maintained a rounded shape ( Figure 3E-F).
To determine whether the three concentrations of hydrogels have a positive effect on the proliferation of NPCs, CCK-8 assays were conducted after 1, 4, and 7 days ( Figure 3H). After 1 day, there was no difference in cell growth rates among the three concentrations of hydrogels. In contrast, after 4 and 7 days, the proliferation of encapsulated NPCs significantly decreased at the higher hydrogel concentrations, and the difference was the highest after 7 days (P < .01).
Aggrecan and Col II are important components of the NP.
Therefore, immunofluorescence was used to evaluate the growth and protein expression in the encapsulated NPCs after 7 days (Figures 4-5). There was an extensive mesh structure in the 5% GelMA hydrogel, consisting mainly of NPCs connected through the pores within the hydrogel. In contrast, cells in the 10% and 15% GelMA hydrogels remained rounded ( Figure 4A). Based on quantitative measurements of the optical density, we found that the expression levels of aggrecan were significantly higher when cells were embedded in the 5% GelMA hydrogel than in the 10% and 15% GelMA hydrogels (P < .01; Figure 4B).
We also examined the expression of Col II. Compared with the densities of cells encapsulated in the 5% GelMA hydrogel, those of cells encapsulated in the 10% and 15% GelMA hydrogels were significantly lower, with no extension ( Figure 5A). Based on optical densities, Col II expression patterns were consistent with those of aggrecan, with the highest expression levels in NPCs cultured within the 5% GelMA hydrogel (P < .01; Figure 5B).

| Histological analysis
To observe the histological characteristics of NPCs encapsulated within the GelMA hydrogel, H&E staining was performed on sections of the hydrogels after cells were cultured for 7 and 14 days The mesh structure of the hydrogel scaffold was also observed in the H&E sections. The 5% GelMA hydrogel exhibited a sparse, highly porous mesh structure ( Figure 6). In contrast, the 10% GelMA hydrogel exhibited a uniform structure with small pores, whereas the 15% GelMA hydrogel had thick, cord-like matrices and a dense, mesh-like overall structure.

| ECM deposition in hydrogels
Masson trichrome and toluidine blue were used to detect the accumulation of Col II and aggrecan in the three hydrogels after culturing embedded NPCs for 7 and 14 days ( Figure 6). After 7 days, the 5% and 10% GelMA hydrogels had more Col II and aggrecan deposits around the encapsulated cells than the 15% GelMA hydrogel, and their expression levels were the highest in NPCs cultured within the 5% GelMA hydrogel. Similarly, in the 5% GelMA hydrogel, a large number of proteoglycans could be observed, with dark staining around the encapsulated cells. After 14 days, the cells encapsulated within the 5% GelMA hydrogel secreted significantly higher amounts of Col II and aggrecan compared to NPCs embedded within the 10% and 15% GelMA hydrogels, as indicated by the sparse ECM.
We quantified the expression of the ECM-related proteins Col II and aggrecan in the hydrogels using Western blotting ( Figure 7A).

| D ISCUSS I ON
In this study, we generated a NPC-encapsulating 3D hydrogel scaf-   53 In this study, the 5% GelMA hydrogel had a low cross-linking density and large mesh size, and its mechanical and hydration properties were very similar to those of natural NP tissues.
Thus, the encapsulated cells aggregated and formed cell clusters. It is possible that the extensive binding sites within the GelMA hydrogel can increase cell adhesion at early stages, and its highly porous structure can facilitate cell growth, migration, and connection at later stages. In contrast, aggregated cell clusters did not form in the 15% GelMA hydrogel, and cell growth was poor after 14 days; thus, the hydrogel could not support the formation of NP tissues.
A good 3D microenvironment increases the viability of NPCs and improves the spatial distribution of ECM components. 15 The low-density 5% GelMA hydrogel had a significantly higher percentage of viable cells after 7 days than the 10% and 15% GelMA hydrogels. ECM deposition also increased gradually with time. In contrast, the small mesh size of the hydrogels with high cross-linking densities limited cell extension and migration as well as hindered the deposition of aggrecan and Col II, as confirmed using 3D immunofluorescence and Western blotting. As shown via Masson's trichrome and toluidine blue staining, aggrecan and Col II were evenly distributed in the 5% GelMA hydrogel, indicating that the two ECM components of different molecular weights can be effectively transported in the hydrogel. Combined, our results broaden the potential applications of GelMA in tissue engineering. The GelMA hydrogel allows cells to adhere, grow, and migrate. At the same time, cells encapsulated within the GelMA hydrogel can extend, form connections, and express ECM proteins. Our findings indicate that the 5% GelMA hydrogel is optimal for cell migration and connections in a 3D space and is suitable for supporting NP regeneration and producing ECM.

| CON CLUS ION
In this study, we synthesized a photocrosslinked GelMA hydrogel with adjustable mechanical and hydration properties, providing an ideal scaffold for NP regeneration. By changing the ratio of methacrylate, the physical and biological properties of the GelMA hydrogel could be effectively controlled to meet the requirements for NP regeneration. The 5% GelMA hydrogel showed the best performance for cell proliferation, cell morphology, and aggrecan and Col II expression. These data indicate that GelMA hydrogels are a promising scaffold for NP regeneration in the treatment of DDD.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request from the authors.