Enzyme-Mediated Alleviation of Peroxide Toxicity in Self-Oxygenating Biomaterials

Abstract

Oxygen releasing biomaterials can facilitate the survival of living implants by creating environments with a viable oxygen level. Hydrophobic oxygen generating microparticles (HOGMPs) encapsulated calcium peroxide (CPO) have recently been used in tissue engineering to release physiologically relevant amounts of oxygen for several weeks. However, generating oxygen using CPO is mediated via the generation of toxic levels of hydrogen peroxide (H₂O₂). The incorporation of antioxidants, such as catalases, can potentially reduce H₂O₂ levels. However, the formulation in which catalases can most effectively scavenge H₂O₂ within oxygen generating biomaterials has remained unexplored. In this study, three distinct catalase incorporation methods are compared based on their ability to decrease H₂O₂ levels. Specifically, catalase is incorporated within HOGMPs, or absorbed onto HOGMPs, or freely laden into the hydrogel entrapping HOGMPs and compared with control without catalase. Supplementation of free catalase in an HOGMP-laden hydrogel significantly decreases H₂O₂ levels reflecting a higher cellular viability and metabolic activity of all the groups. An HOGMP/catalase-laden hydrogel precursor solution containing cells is used as an oxygenating bioink allowing improved viability of printed constructs under severe hypoxic conditions. The combination of HOGMPs with a catalase-laden hydrogel has the potential to decrease peroxide toxicity of oxygen generating tissues.

1. Introduction

Engineering viable clinically-sized living implants has remained a major challenge.^[1,2] The metabolism of implanted engineered tissues depends on oxygen and nutrient diffusion, as well as the rate of host-to-tissue vascularization, which creates chronic severe hypoxic core in larger implants where oxygen becomes scar. Prolonged severe hypoxia leads to cell death followed by necrosis, inflammation, infection, and ultimately to implant failure.^[1] Numerous strategies to vascularization have been explored to provide the implant access to oxygen and nutrients present in the host’s vascular system. However, this process is limited by the maximal vascularization rate, which is too slow to prevent the failure of larger implants.^[3]

To bridge the survival of living implants during the prevascular phase, several oxygen releasing biomaterials (i.e., perfluorocarbons and hemoglobin-based carriers) have been incorporated to facilitate oxygen delivery and diffusion.^[4-9] Unfortunately, these platforms are unable to provide sufficient amounts of oxygen over the period of time required to bridge the prevascular phase.^[10] In contrast, oxygen generating compounds such as solid peroxides can store sufficient amounts of oxygen to enable cell survival during the prevascular phase of implants as well as offer straightforward chemical catalysis that happens via hydrolysis.^[11-15] As the hydrolysis of solid peroxides in hydrogels is a fast-paced process (e.g., based on hydrogel degradation rate), it is essential to engineer systems that slow down the hydrolysis rate, resulting in a controlled oxygen release. This can be achieved by the encapsulation of these peroxides in hydrophobic polymers to create hydrophobic oxygen generating microparticles (HOGMPs).^[14] The most commonly explored solid peroxide for oxygen generation is calcium peroxide (CPO).^[10] In presence of water CPO generates oxygen via the intermediate formation of hydrogen peroxide (H₂O₂), which is governed by the following reaction sequence^[16]

$${\text{CaO}}_2+2{\mathrm{H}}_2\mathrm{O}\to \text{Ca}{(\text{OH})}_2+{\mathrm{H}}_2{\mathrm{O}}_2$$ (1)

$$2{\mathrm{H}}_2{\mathrm{O}}_2\to 2{\mathrm{H}}_2\mathrm{O}+{\mathrm{O}}_2$$ (2)

The intermediate generation of H₂O₂ has limited the clinical application of solid peroxides due to its cytotoxic nature at higher concentrations.^{[11,14,17-19]} Exposure to higher concentrations of H₂O₂ (mM range) causes damage to cells and irreversible DNA damage through oxidation of Fe(II) to Fe(III) via Fenton’s reaction.^[20] However, lower (10-100 μM range) concentrations of H₂O₂ can lead to the formation of free radicals that can cause cell death.^[21-25] One way to reduce CPO-induced cytotoxicity is to lower reduce the solid peroxide concentration.^[1,11] However, this would prevent the CPO to generate relevant amounts of oxygen.

Cells can protect themselves from being damaged by overproduction of H₂O₂ via producing various antioxidants and scavenging enzymes such as catalase, glutathione peroxidase, and superoxide dismutase.^[26,27] Catalases specifically hydrolyze H₂O₂ into water and oxygen^[28,29]

$${\mathrm{H}}_2{\mathrm{O}}_2+\text{Fe}(\text{III})-\mathrm{E}\to {\mathrm{H}}_2\mathrm{O}+\mathrm{O}=\text{Fe}(\text{IV})-\mathrm{E}(.+)$$ (3)

$${\mathrm{H}}_2{\mathrm{O}}_2+\mathrm{O}=\text{Fe}(\text{IV})-\mathrm{E}(.+)\to {\mathrm{H}}_2\mathrm{O}+\text{Fe}(\text{III})-\mathrm{E}+{\mathrm{O}}_2$$ (4)

where $\text{Fe}()\text{-}\mathrm{E}$ represents the iron center of the heme group attached to the enzyme and $\text{Fe}(\text{IV})\text{-}\mathrm{E}(.+)$ is the incompletely oxidized mesomeric form of $\text{Fe}(\mathrm{V})\text{-}\mathrm{E}$. With these enzymes and ferritins, cells can create a homeostasis to survive higher concentrations of H₂O₂ over several hours.^[21] Prior groups have incorporated catalase into their culture media to mitigate CPO-induced H₂O₂ toxicity.^[1,11] However, has an exceeding short functional window at 37 °C (i.e., half-life of 30 min),^[1,11,17,30] requiring the catalase to be refreshed on a regular basis. On the contrary, in order to protect the oxygen generating implant, catalase activity is required for multiple days to several weeks.^[31] Prior studies have shown an increased stability of catalase when encapsulated in a hydrogel.^[32-36] Thus, reducing H₂O₂ levels with catalase in engineered implanted constructs represents a biomimetic approach to reduce H₂O₂ cytotoxicity in CPO-based oxygen generating strategies. To the best of our knowledge, there is no study in which the function of catalase and its suitable concentration are optimized to efficiently decrease H₂O₂-induced cytotoxicity in self-oxygenating engineered tissues. Consequently, a functional catalase-based strategy, which has the potential to engineer 3D tissue constructs by advanced microfabrication techniques (e.g., bioprinting), to protect oxygen generating engineered tissues from H₂O₂-induced cytotoxicity has remained wanted.

Here, we investigate and optimize three distinct methods to incorporate catalase in the oxygen-generating biomaterial (i.e., control with no catalase (HOGMP), catalase in HOGMPs (CITP), catalase on HOGMPs (COTP), or catalase in proximity of HOGMPs (CITG)) and map their impacts on cell survival within HOGMP-laden engineered tissues. Additionally, specific attention was given to the stability of catalase in each method to provide insight into the extended release of oxygen while maintaining H₂O₂ at sub-toxic levels (≈<10 × 10⁻⁶ m).^[37-40] The addition of catalase in proximity of HOGMPs (i.e., dispersed into the hydrogel containing HOGMPs) most potently decreased H₂O₂ levels, resulting in the highest cell viability and improved morphology of C2C12 myoblasts encapsulated within the engineered tissues. Subsequently, we investigate the effect of this method on the survival and printability of a C2C12-laden oxygenating bioink, and demonstrate the printability and cell survival of the bioprinted constructs significantly increases compared to previously reported CPO-laden bioinks.^[30] In short, we anticipate that the combination of HOGMPs with catalase-laden hydrogels has the potential to improve implant survival by decreasing the H₂O₂ production to sub-toxic levels. In addition to improving cell viability in the hydrogels, it offers superior 3D bioprinting of oxygen generating tissues, which remain viable for prolonged periods in the injured area.

2. Results and Discussion

2.1. Characterization of HOGMP and HOGMP-Laden Hydrogels

We performed a comparative analysis of the various strategies in which catalase could be integrated into oxygen generating biomaterials for engineered tissues. Specifically, we investigated the following conditions: 1) a control in which HOGMPs were incorporated into gelatin methacryloyl (GelMA) hydrogels without catalase supplementation (HOGMP), 2) catalase incorporated within HOGMPs that were encapsulated in GelMA hydrogel (CITP), 3) catalase coated on the surface of HOGMPs that were encapsulated within GelMA hydrogel (COTP), and 4) catalase and HOGMPs separately encapsulated within GelMA hydrogel (CITG) (Figure 1A and Figure S1, Supporting Information). Prior studies have shown that one unit of catalase can decompose 1× 10⁻⁶ m of H₂O₂ per minute at 25 °C.^[41] Based on this, an excess of catalase (1:3, 1:5 and 1:10 mole CPO:unit catalase) was added to several of the HOGMP-containing conditions (CITP, COTP, and CITG) to ensure H₂O₂ released by the HOGMPs would be decomposed before reaching toxic concentrations leading to cell injury. The catalase-laden and -coated HOGMPs were synthesized by using the double emulsion method. As proteins are known to affect emulsion stability,^[42] we expected that catalase may have interfered in the emulsification process in terms of particle size. While pristine HOGMPs demonstrated a smooth surface with a spherical shape, both catalase-laden and -coated HOGMPs demonstrated a larger, rough, and fibrous surface (Figure 1B). HOGMPs were produced at a consistently yet moderately smaller size than the catalase-laden HOGMPs with 3.14 ± 1.32 and 3.43 ± 1.30 μm, respectively (Figure 1C). The oxygen generating ability of HOGMPs was measured in Dulbecco’s Phosphate Buffered Saline (DPBS) for 14 days, with higher HOGMP concentrations leading to increased total accumulation of oxygen, as expected (Figure 1D). To place these results into perspective, we compared the oxygen release from HOGMPs to the oxygen consumption per cell as determined in previous research.^[43] The authors showed that one cell consumes 2.5 x 10⁻¹⁸ moles of oxygen per second, meaning one of our constructs (≈1 x 10⁵ cells) would consume 22 nmoles of oxygen per day (i.e., ≈0.31 μmoles of oxygen for two weeks; Figure 1D). Calculations revealed that the HOGMPs released >0.31 μmoles over 14 days when concentrations of 0.5% (w/v) HOGMP or more were used (0.10 μmoles (≈7.1 nmoles per day), 0.26 μmoles (≈18.6 nmoles per day), 0.47 μmoles (33.6 nmoles per day) and 0.65 μmoles (46.4 nmoles per day) of oxygen corresponding to 0.1%, 0.25%, 0.5%, and 1% (w/v) HOGMP in DPBS, respectively).

Figure 1.

Engineering various types of catalase-mediated oxygen generating biomaterials and their physical and structural properties. A) Schematic illustration of reactions underlying oxygen generation via CPO hydrolysis, and H₂O₂ decomposition by catalase in various types of oxygen generating hydrogels (HOGMP, CITP, COTP, and CITG). B) SEM images of the synthesized HOGMPs, CITPs, and COTPs. Scale bar: 4 μm. C) Size distribution of the HOGMP (n = 3, number of microparticles = 664) and CITP (n = 3, number of microparticles = 481). D) Oxygen release and accumulative oxygen release kinetics of the various concentrations (0.1–1% (w/v)) of HOGMPs dispersed in deoxygenated DPBS under severe hypoxic conditions (n = 3). E) SEM images of the pristine GelMA, HOGMPs, CITP, COTP, and CITG hydrogels. Scale bar: 20 μm. F) Equilibrium swelling properties of the GelMA hydrogels with CPO, HOGMP, and catalase after 24 h in DPBS at 37 °C. All samples were nonsignificant Kruskal–Wallis ANOVA with Dunn’s post hoc test (n = 3). G) Degradation profile of GelMA hydrogels with CPO, HOGMP, and catalase in DPBS with or without 0.5 U mL⁻¹ collagenase type II at 37 °C (n = 3). All data in the figure are depicted as mean ± SD.

Multiple types of HOGMP- and catalase-laden hydrogels were fabricated using UV photocrosslinkable GelMA solutions, containing three different combinations of HOGMPs and catalase (Figure 1A). Mechanical properties and morphological structures of various types of oxygen generating hydrogels were characterized. No major changes in the porous structure of the hydrogels were observed by adding HOGMPs and catalase (Figure 1E). Moreover, HOGMPs were observed within the hydrogels (Figure S2, Supporting Information). From a mechanical point of view, the presence of HOGMPs and catalase did not alter the stiffness of the hydrogel, which remained around 10 kPa for all hydrogel conditions (Figure S3, Supporting Information). Although previous studies on multiphase polymeric microcomposites have shown an increase in construct stiffness when polymeric micromaterials were incorporated,^[44,45] it is likely that the low concentrations of HOGMPs in the hydrogel allowed for unaltered and consistent mechanical properties in our microcomposite hydrogels.

The swelling capability and the rate of degradation are important parameters for a hydrogel, as they indicate how the hydrogel will behave in vivo. To put the results in perspective, HOGMP-laden hydrogels (1% (w/v); with and without catalase; 1:5 mole CPO:unit catalase ratio) were compared to plain GelMA and CPO-laden (also with and without catalase) hydrogels for both the swelling and degradation studies. HOGMP only had minor and non-significant effects on the swelling property of the hydrogel when compared to pristine GelMA hydrogel, irrespective of the presence of catalase (Figure 1F). Interestingly, the addition of CPO increased the swelling properties of the hydrogel, which indicated that the hydrophilic nature of CPO played a significant role in the absorption and retention of aqueous solutions.^[46]

Biodegradation of hydrogels was simulated by immersing various hydrogels in DPBS or DPBS supplemented with 0.5 U/mL collagenase type II at 37 °C for seven days (Figure 1G). As expected, the HOGMP-laden hydrogels degraded at a similar rate to the plain GelMA hydrogels after approximately five to six days, irrespective of the addition of catalase. In line with the swelling properties, the CPO-laden hydrogels degraded at a faster rate compared to the plain GelMA and HOGMP-laden hydrogels, and fully decomposed within two to three days. Despite the longer exposure to UV-light for the CPO-laden hydrogels, it is likely that the hydrogels are crosslinked to a lesser degree, and thus degrade at a faster rate.^[46]

2.2. Oxygen and H₂O₂ Release Kinetics from HOGMP- and Catalase-Laden Hydrogels

As expected, the HOGMP concentration in the GelMA hydrogels positively correlated with accumulative oxygen release (Figure 2A). All investigated HOGMP concentrations released oxygen for at least 14 days, which would create a window of opportunity to control stem cell fate.^[47-50] Interestingly, we observed that HOGMP oxygen generation rate was significantly higher in solution (i.e., DPBS) compared to HOGMPs encapsulated within hydrogels (Figure 2B; accumulative oxygen release of 0.63 ± 0.03 versus 0.39 ± 0.01 μmoles oxygen after 14 days for the 1% (w/v) groups, respectively). Similar oxygen release profiles were observed for HOGMP-laden alginate and PEGDA hydrogels, which showed average accumulative oxygen releases of 0.36 ± 0.04 and 0.33 ± 0.04 μmoles oxygen after 14 days for the 1% (w/v) groups, respectively.

Figure 2.

Characterization of oxygen release and H₂O₂ decomposition kinetics of the catalase-mediated oxygen generating hydrogels. A) Accumulative oxygen release kinetics of the various concentrations of HOGMP hydrogels in deoxygenated DPBS under severe hypoxic conditions for up to 14 days (n = 3). B) Comparison of accumulative oxygen release of GelMA, alginate, and PEDGA hydrogels containing 1% (w/v) HOGMPs in deoxygenated DPBS, and directly dispersed 1% (w/v) HOGMPs in deoxygenated DPBS (no hydrogel) under severe hypoxic conditions for up to seven days (n = 3). C) Decomposition of 150 × 10⁻⁶ m H₂O₂ by adding catalase into DPBS and into the GelMA hydrogel for seven days (n = 3). D,E) H₂O₂ release kinetics of the HOGMP, CITP, COTP, and CITG hydrogels containing various concentrations of microparticles (n = 3). In the CITP, COTP, and CITG-laden hydrogels, the catalase was used to make a 1:5 mole CPO:unit catalase ratio. The dotted line indicates the noncytotoxic level of H₂O₂ (10 × 10⁻⁶ m). All data in the figure are depicted as mean ± SD.

Following this observation, we then investigated whether the encapsulation of catalase into GelMA hydrogels also affected the catalase’s ability to decompose H₂O₂ over time. Several studies have shown an increased stability of catalase when encapsulated in a hydrogel.^[32-36] To test this, we quantified the catalase-induced hydrolysis of 150 × 10⁻⁶ m H₂O₂ (a cytotoxic concentration) in DPBS (with a noncatalase laden GelMA hydrogel) and a catalase-laden GelMA hydrogel for a period of seven days (Figure 2C). Catalase in solution became inactive after two days, caused most likely by a relatively short half-life of catalase, as was also reported in previous literature.^[42] As expected, catalase encapsulated in GelMA hydrogels maintained its ability to decompose H₂O₂ throughout the entire duration of the experiment. The half-life for the catalase in solution and in hydrogel were determined to be 33 min and 19 h, respectively. Earlier work showed a similar half-life for catalase in solution (≈35 min at 40 °C).^[31] In line with prior literature,^[32-36] this observation suggests that simply adding catalase into a hydrogel can increase the stability of catalase, which offers protection of implanted cells and host tissue from oxidative stresses (i.e., as generated by HOGMPs) for a prolonged period of time.

In order to gain insight into what type of material design (i.e., GelMA hydrogel or hydrophobic microparticle) catalase would act most efficiently in terms of H₂O₂ reduction, we measured H₂O₂ release from various oxygen generating GelMA hydrogels containing catalase in different physical locations (i.e., HOGMP, CITP, COTP, and CITG) and at multiple distinct concentrations of HOGMPs (i.e., 0% to 0.5% (w/v)) for all CPO:catalase ratios. This revealed that COTP and CITG hydrogels effectively decomposed H₂O₂, which allowed the engineered constructs to release below 10 × 10⁻⁶ m of H₂O₂ (cytotoxic threshold) for seven days (Figures 2D,E and Figure S4, Supporting Information). On the other hand, HOGMP and CITP hydrogels with higher concentrations (>0.25% (w/v)) generated cytotoxic levels of H₂O₂. Previous studies reported catalase encapsulation efficiencies of 50–60% within poly(lactic acid) (PLA)^[51,52] and hydrogel microspheres.^[53] Although our study also demonstrated that catalase was able to decrease the H₂O₂ concentration, COTP and CITG proved significantly more effective at lowering the H₂O₂ concentration within our oxygen generating hydrogels.

2.3. Biological Response of C2C12 Myoblasts within HOGMP- and Catalase-Laden Hydrogels

To investigate the effect of catalase on the cytocompatibility of HOGMPs, C2C12 myoblast-laden GelMA hydrogels containing HOGMP, CITP, COTP, and CITG (1:5 mole CPO:unit catalase) were compared on C2C12 survival under severely hypoxic (≈1.0% O₂) conditions. In a previous study we demonstrated that host-tissue vascularization already occurs after seven days when using oxygen-generating biomaterials in GelMA hydrogels.^[46] Thus, viability studies were performed for up to seven days. In line with the measured H₂O₂ levels, C2C12 myoblasts in hydrogel containing CITG demonstrated a significantly higher viability (≈70% for all HOGMP tested concentrations) compared to all other catalase formulations (Figure 3A,B). Indeed, CITP and COTP had, at best, marginal effects on cell survival compared to HOGMP. Similar results were observed for the 1:3 and 1:10 mole CPO:unit catalase hydrogels (Figure S5, Supporting Information). Furthermore, encapsulated C2C12 cells demonstrated limited proliferation over time (Figure S6, Supporting Information). This is most likely due to the hypoxic microenvironment, which is known to decrease cell proliferation for many cell types due to a lack of oxygen.^[46,54] Moreover, an initial low viability (≈70%) was noticed for most of the samples. In line with the literature, this phenomenon could stem from the photo-crosslinking process to fabricate hydrogels and cell culture under hostile environmental conditions (severe hypoxia).^[46,55-57] Although other byproducts, such as Ca²⁺ and calcium hydroxide (Ca(OH)₂), have been shown to be cytotoxic, the released amount of these molecules in this study is below what is physiologically relevant.^[58-61] Furthermore, C2C12 cells encapsulated in the hydrogel containing CITG consistently adopted an elongated and less round morphology, as confirmed by the length and sphericity of the cultured cells (Figure 3C,D). As damaged encapsulated cells typically display increased sphericity and decreased spreading, our findings suggest that CITG considerably decreased the cytotoxic effects of HOGMP.

Figure 3.

Comparison of viability and morphology for C2C12 myoblasts encapsulated in the catalase-mediated H₂O₂ hydrolysis of oxygen generating hydrogels under severe hypoxic conditions. A,B) Representative fluorescence images of live/dead staining and semi-quantified viability for C2C12 myoblasts encapsulated in the HOGMP, CITP, COTP, and CITG hydrogels containing 0.5% (w/v) microparticles and a 1:5 mole CPO:unit catalase ratio for seven-days post-encapsulation (n = 4). Pristine GelMA hydrogel was used as control. Scale bar: 250 μm. C) Average length and D) sphericity of C2C12 myoblasts encapsulated in the HOGMP, CITP, COTP, and CITG GelMA hydrogels containing 0.5% (w/v) microparticles and a 1:5 mole CPO:unit catalase ratio for seven-days post-encapsulation (n = 3, number of cells: 100). *p < 0.05 and ***p < 0.0005 as determined by Kruskal–Wallis ANOVA with Dunn’s post hoc test. All data in the figure are depicted as mean ± SD.

2.4. Optimization of CPO:Catalase Ratio and HOGMP Concentration

To more accurately determine the optimal catalase concentration required to prevent the cytotoxic effects of oxygen generating hydrogels using the CITG formulation, various ratios of CPO:catalase (1:3, 1:5, and 1:10 (mole:unit )) were tested in C2C12-laden hydrogels containing increased concentrations (1% and 2% (w/v)) of HOGMPs under severely hypoxic (≈1.0% O₂) conditions for seven days. The viability data was normalized to the control (0% HOGMP; day one). In the absence of catalase, the cytotoxic effect of the released H₂O₂ was evident as virtually all cells underwent cell death after a week in culture (Figure 4A,B, and Figures S7 and S8, Supporting Information). In contrast, all cultures containing catalase maintained a viable population of cells with CITG (mole CPO:unit catalase 1:5) showing the highest normalized viability after one week of culture (i.e., ≈0.91 A.U. and ≈0.77 A.U. survival for 1% and 2% of HOGMP conditions, respectively). Interestingly, further increasing the catalase concentration correlated with decreased levels of cell viability. Although it has not been described in literature that an overload of catalase may be detrimental to cells, this phenomenon might be explained by previous research that demonstrated that low subtoxic concentrations of H₂O₂ play an important role in the regulation of different cellular functions.^[62,63] For example, reducing H₂O₂ signaling too strongly with catalase has been reported to result in aberrant p38 mitogen-activated protein kinase activation and tumor necrosis factor-α expression.^[64] Although these studies were demonstrated with different cell types, it might be possible that high catalase activity severely impacted the intracellular H₂O₂ signaling, resulting in lower viability. Regardless, a CITG formulation with a 1:5 mole CPO:unit catalase ratio was considered to yield optimal outcomes in terms of cell viability and was used for further studies.

Figure 4.

Optimization of CPO:catalase ratio and HOGMP concentration. A,B) Representative fluorescence images of live/dead staining and normalized semi-quantified viability for C2C12 cells encapsulated in the HOGMP and CITG hydrogels containing two different concentrations of HOGMPs under severe hypoxic conditions for seven days post-encapsulation. Various ratios of mole CPO:unit catalase (1:3, 1:5, and 1:10) were used for the CITG hydrogels and the pristine GelMA hydrogel was used as a control (n ≥ 3). Scale bar: 250 μm. C) H₂O₂ release from the HOGMP and CITG hydrogels containing various concentrations of HOGMPs (0–1% (w/v)) without (left) and with C2C12 cells (right) for 14 days post-encapsulation. The CITG hydrogel has a 1:5 mole CPO:unit catalase ratio (n = 3). D) Difference in H₂O₂ release (H₂O₂ without cells minus H₂O₂ with cells) among the pristine GelMA hydrogel, and the HOGMP and CITG hydrogels containing 1% (w/v) HOGMP presented in C). (n = 3). The dotted line indicates the noncytotoxic level of H₂O₂ (10 × 10⁻⁶ m). N.S.: nonsignificant, *p < 0.05 and **p < 0.01 as determined by a one-tailed paired t-test to the control (0% (w/v)). E) Metabolic activity of the C2C12 cells encapsulated in the pristine GelMA hydrogel, and the HOGMP and CITG hydrogels containing 0.5% (w/v) HOGMP (n = 3). N.S.: nonsignificant, *p < 0.05 and **p < 0.01 as determined by a one-tailed paired t-test to the control (0% (w/v)). All data in the figure are depicted as mean ± SD.

To confirm that the generated H₂O₂ was able to react with cells and thus cause HOGMP-induced cytotoxicity, HOGMP-containing GelMA hydrogels with and without C2C12 cells and with (i.e., CITG) and without catalase were quantitated on their H₂O₂ levels over a period of 14 days. Although catalase and HOGMP-containing hydrogels (CITG) accumulated similar levels of H₂O₂ in the presence and absence of cells, HOGMP-laden hydrogels with cells were associated with significantly lower amounts of H₂O₂ accumulation compared to HOGMP-laden hydrogels without cells and in the absence of catalase (Figures 4C,D and Figure S9, Supporting Information). This indicated that the HOGMPs generated H₂O₂ that reacted with the myoblasts, which could explain the HOGMP induced cytotoxicity. Indeed, catalase-induced reduction of H₂O₂ levels was consistently associated with higher levels of viability, increased metabolic activity, and improved cellular morphology.

The metabolic activity of C2C12 myoblasts encapsulated in GelMA hydrogels containing HOGMPs (0-1% (w/v)) and catalase (1:5 mole CPO:unit catalase ratio) was assessed by MTS assay over a period of 10 days. Compared to cells in noncatalase and control conditions, catalase containing HOGMP-laden hydrogels demonstrated significantly increased metabolic activity for on the tenth day post-encapsulation (Figure 4E and Figure S10, Supporting Information). In particular, the absence of catalase is associated with a decrease in metabolic activity, compared to control hydrogels. However, an unexpected decrease in cell viability and metabolic activity was noticed on day 3 and 5, respectively, when compared to day one for almost all CITG conditions (Figure 4A,B,E, and Figures S7, S8, and S10, Supporting Information). A recent study has shown that hyperoxia damages the cell membrane of C2C12 cells due to lipid oxidation and a disrupted cytoskeletal structure, resulting in decreased cell viability and proliferation.^[65] The initial oxygen release from our HOGMP was high in the first few days compared to later timepoints (Figure 1D). Thus, it seems plausible that the C2C12 cells in the CITG conditions were exposed to high oxygen levels (e.g., hyperoxia) in the first few days after encapsulation into the GelMA hydrogel, resulting in suppressed cell viability and metabolic activity. Nevertheless, the data suggests that the addition of catalase not only protects oxygen generating tissues from HOGMP-induced H₂O₂ accumulation, but also augments the metabolic activity of engineered constructs.

2.5. Bioprinting with the Oxygenating Bioinks

Recently, CPO has been used to create an oxygenating bioink for bioprinting applications.^[30] However, both high levels of oxygen and H₂O₂ are capable of adversely affecting crosslinking, which reduces the printability of the material system.^[66-69] We therefore investigated whether a printable GelMA bioink based on CITG formulation would result in improved printability and functional performance compared to the previously reported CPO-based approach by reducing the levels of both oxygen and H₂O₂. To this end, oxygen-generating bioinks composed of 5% (w/v) GelMA containing 1% (w/v) HOGMPs or 0.3% (w/v) CPO (equivalent of 1% CPO in HOGMPs) were formulated. All bioinks contained catalase (1:5 mole CPO:unit catalase). Microscopic analysis of the bioprinted oxygenating constructs revealed that the CITG-laden bioinks were associated with significantly reduced filament spreading and improved printability (a printability of 1 is related to a perfect square shape),^[70] compared to CPO-based formulations with 500 and 1000 mm min⁻¹ printing speeds, resulting in the best printed structures (Figure 5A-C). To explain the observed differences in printability and spreading ratio, various rheological measurements were performed. A temperature ramp (from 25 to 4 °C) revealed that pristine GelMA bioink and HOGMP-laden bioink gelled at temperatures of 18.9 ± 0.10 °C and 16.6 ± 0.43 °C, respectively (Figure 5D, left). Interestingly, the CPO-laden bioink remained liquid during the entire measurement. Moreover, the storage (G′) and loss (G″) moduli were measured at 4 °C for 10 min to determine the respective bioink crosslinking times (Figure 5D, right). The GelMA and HOGMP bioinks gelled after 22.0 ± 3.5 and 40.5 ± 3.0 s, respectively. However, the CPO-laden bioink remained in the liquid phase for up to 600 s. It is likely that the burst release of oxygen, hydrogen peroxide, and calcium hydroxide by nonencapsulated CPO influenced the temperature and rate of polymerization. Importantly, CITG-based formulations rescued this adverse effect and largely restored GelMA’s ability to crosslink, which correlated with a notable improvement in the printability of self-oxygenating engineered tissues. Moreover, the versatility of bioprinting self-oxygenating tissues was shown by printing structures of different shapes, including a honeycomb (Figure 5E), and the molecular formula of oxygen (O₂; Figure 5F).

Figure 5.

3D bioprinting of cell-laden oxygen generating bioinks. A) Microscopic images of the printed constructs in a grid-like pattern using 1% (w/v) CPO- and HOGMP-laden GelMA bioinks at a printing speed of 500 mm min⁻¹. Scale bar is 500 μm. B) Filament spreading ratio of the printed fibers using 1% (w/v) CPO- and HOGMP-laden GelMA bioinks at various printing speeds (n = 3, number of printed fibers ≥ 36). N.S.: nonsignificant and ****p < 0.0001 as determined by Kruskal–Wallis ANOVA with Dunn’s post-hoc test. C) Printability of the printed fibers using 1% (w/v) CPO- and HOGMP-laden GelMA bioinks at various printing speeds (n = 3, number of printed fibers ≥ 18). ***p < 0.001 and ****p < 0.0001 as determined by two-way ANOVA with Bonferroni’s post hoc test. D) Storage (G′) and Loss (G″) modulus for the pristine GelMA, CPO-laden, and HOGMP-laden GelMA bioinks at various temperatures (left) and at 4 °C (right) (n = 3). Different printed structures with E) a honeycomb and F) a molecular oxygen structure using the 1% (w/v) HOGMP-laden GelMA bioink to show bioprinting versatility. Scale bar is 1 mm. G) Representative fluorescence images of live/dead staining for C2C12-laden printed constructs with a grid-like structure using pristine GelMA, CPO-laden, and HOGMP-laden GelMA bioinks after seven days of printing in severe hypoxia. Scale bar is 500 μm. H) Semi-quantified viability for C2C12 cells in the core and shell of printed constructs after seven days of culture in severe hypoxia (n = 3). N.S.: nonsignificant, *p < 0.05 and **p < 0.01 as determined by a one-tailed paired t-test to the control (0% (w/v)). I) Average length and J) sphericity of C2C12 cells in the various printed constructs after 10 days of culture in hypoxia (n = 3, #cells ≥125). ****p < 0.0001 as determined by Kruskal–Wallis ANOVA with Dunn’s post hoc test for all conditions. All data in the figure are depicted as mean ± SD.

Oxygen-laden bioinks composed of 1% (w/v) CPO or HOGMPs, catalase (1:5 mole CPO:unit catalase), and C2C12 myoblasts were used to print self-oxygenating tissues, which were assessed based on their viability and morphology over a 10-day period under severe hypoxic conditions. Nonoxygen generating control tissues with the C2C12-laden GelMA bioink were characterized by the formation of a dead core (<30% cell survival) and a shell of viable cells (≈70% cell survival) when cultured in severely depleted oxygen environment (Figures 5G,H), which most likely represented the adverse impacts of oxygen diffusion limitations on cell viability. Self-oxygenating tissues based on CPO incorporation were associated with decreased cell survival (<40% cell survival for core and shell). Although previous literature reported that CPO-based bioinks could improve cell survival in the short term, this was at substantially lower CPO concentrations (0.1% to 0.5% (w/v)), which offer only minimal oxygen release durations and quantities (≈10 × 10⁻⁶ m per mg CPO of released oxygen on day seven).^[30] In contrast, CITG-based bioinks (1% (w/v) HOGMPs) created self-oxygenating tissues that were characterized by significantly increased viability of cells cultured in both hypoxic and normoxic conditions, compared to CPO and control tissues (Figures 5G,H, and Figures S11A,B, Supporting Information), with CITG-based tissues reaching viabilities of >70% in both the core and shell of the construct. The data also reveals the detrimental impact of CPO even when used with catalase, but without a hydrophobic encapsulant, such as PCL. Moreover, fluorescence microscopy revealed a significant difference in cellular morphology between the GelMA, CPO- and HOGMP-laden tissues , indicated by significantly increased cell length (Figure 5I and Figure S11C, Supporting Information) and reduced cellular sphericity (Figure 5J and Figure S11D, Supporting Information). Together, these observations demonstrate that CITG-based bioinks can improve the printing and functional performance of self-oxygenating engineered tissue constructs.

3. Conclusion

This study demonstrated that the dispersion of catalase into a HOGMP-laden hydrogel constructs improves cellular viability, metabolic activity, and morphology when cultured in severe hypoxic conditions by offering a safe conversion of H₂O₂ into water and oxygen for at least seven days. Specifically, we demonstrated that pristine HOGMPs release toxic levels of H₂O₂ due to CPO hydrolysis for prolonged periods of time. In contrast, the addition of catalase significantly lowered the H₂O₂ concentration. Moreover, we revealed that the stability of free catalase is remarkably improved by encapsulation within hydrogels, which significantly increased the viability and metabolic activity of C2C12 cells for HOGMP concentrations of 0.25% (w/v) or higher. The addition of HOGMPs and catalase notably improved bioink printability when compared to CPO-laden bioinks, and increased the cellular survival and morphology of C2C12-laden printed constructs. A major advantage of using a combination of HOGMPs and catalase is the capability in bioprinting self-oxygenating engineered tissues, without the formation of cytotoxic H₂O₂ concentrations, that otherwise decreases the enthusiasm of using peroxides as oxygenating source in biomaterials. HOGMPs in combination with catalase therefore function as a promising steppingstone towards the fabrication of viable clinically-sized tissues.

4. Experimental Section

Synthesis of Calcium Peroxide-Laden Poly(caprolactone) Microparticles (HOGMP):

A double emulsion synthesis method of water-in-oil-in-water was used to prepare CPO-laden poly(caprolactone) (PCL) microparticles (Figure S1, Supporting Information). Briefly, 10% (w/v) PCL (MW = 50 000, kind donation from Perstorp) was prepared in dichloromethane (DCM, CH₂Cl₂, Sigma Aldrich). The desired concentration 3% (w/v) of CPO (Sigma Aldrich) dispersed in distilled water was subsequently added to this solution. Ultrasonication (QSonica) for 2 min with a 2 s on/off pulse at 60% amplitude was used to obtain an emulsion. The PCL-CPO emulsion was added to 1% (w/v) gelatin from porcine skin (Type A, Sigma Aldrich) dissolved in distilled water and ultrasonicated for an additional 4 min with a 2 s on/off time pulse at 60% amplitude. During the ultrasonication steps, the solution was placed on ice. Afterwards, the solution was stirred overnight to dry off the solvents at room temperature. The particles were concentrated by centrifugation at 5000 rpm for 20 min. The microparticle pellet was washed twice with methanol, ethanol, and distilled water to remove any residual additives. The resultant pellet was flash-frozen using liquid nitrogen, lyophilized, and stored in a dry place for further use.

To produce catalase- and CPO-containing PCL microparticles (CITP), the double emulsion method to produce HOGMP was used, with the only distinction being that catalase was mixed into the PCL-CPO emulsion. To produce catalase-coated HOGMPs (COTP), catalase was incubated in a HOGMP solution for 1 h at 4 °C under slight agitation. Subsequently, the HOGMP-catalase solution was centrifuged for 20 min at 15 000 rpm at 4 °C. The supernatant was drained and the particles were thoroughly resuspended at the desired concentration into a prepolymer solution.

Gelatin Methacryolyl (GelMA) Synthesis:

Synthesis was performed as reported previously.^[71] 10% (w/v) of powdered gelatin type A from porcine skin was dissolved in DPBS (Sigma Aldrich) at 50 °C and stirred at 240 rpm until completely dissolved. 1.25% (v/v) of methacrylic anhydride (Sigma-Aldrich) was added dropwise while constantly stirred at 240 rpm for 2 h at 50 °C, which resulted in a methacryloyl substitution degree of 60%.^[72] One volume of pre-heated (50 °C) DPBS was subsequently added to the solution. The solution was then dialyzed using 12–14 kDa cut-off dialysis membranes (Thermo Fisher) against deionized water at 40 °C. The deionized water was refreshed twice a day for a week, after which the solution was filtered, frozen at −80 °C, and lyophilized.

Fabrication of Oxygen-Generating Hydrogels:

Different oxygen generating hydrogels were prepared according to the polymers used. For “CITG” hydrogels, 5% (w/v) GelMA pre-polymer solutions were prepared by dissolving freeze-dried GelMA in DPBS. HOGMPs were added to the GelMA pre-polymer solutions at various concentrations (0%, 0.1%, 0.25%, 0.5%, 0.75%, 1% and 2% (w/v)) and catalase was added at a mole CPO:unit catalase ratio of 1:0, 1:3, 1:5 or 1:10. To initiate photocrosslinking by UV light, 0.25% (w/v) 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator (PI), Irgacure 2959; Sigma Aldrich) was added to all solutions and then dissolved at 80 °C. HOGMPs-laden GelMA hydrogels were fabricated by UV crosslinking (Omnicure 2000, Excelitas, operated at 12.5 mW cm⁻²) for 30 s, flipping the sample half-way. Three other types of oxygen-generating hydrogels were fabricated following an identical procedure (Figure S1, Supporting Information), but with only one variance for each type: 1) the catalase- and CPO-containing PCL microparticles were added in the GelMA pre-polymer solution, which was crosslinked to produce “CITP” hydrogels, 2) the catalase-coated HOGMPs were added in the GelMA pre-polymer solution, which was crosslinked using an identical procedure to produce “COTP” hydrogels, 3) GelMA containing pristine HOGMPs (without catalase) was crosslinked to produce “HOGMP” hydrogels, which acted as controls.

HOGMP-laden alginate hydrogels were prepared by dissolving 4% (w/v) sodium alginate salt (Sigma) in DPBS at 50 °C for 2 h following addition of 1% (w/v) HOGMPs to the alginate pre-polymer solution. 100 × 10⁻³ m CaCl₂ was used to crosslink the HOGMP-laden alginate prepolymer solutions in 1.5 mL Eppendorf tube caps.

15% (w/v) poly(ethylene glycol) diacrylate (PEGDA; Esibio) was dissolved in DPBS along with 0.25% (w/v) PI. HOGMPs were then added to PEGDA pre-polymer solutions at 1% (w/v). The UV light was applied to crosslink HOGMP-laden PEGDA hydrogels at 12.5 mW cm⁻² for 30 s.

Microscopic Analyses of HOGMPs and Hydrogels:

Microstructures of lyophilized samples of HOGMPs and CITP microparticles and hydrogels were evaluated using scanning electron microscopy (SEM: Zeiss). The samples were sputter coated with a 5 nm coating of Pt/Pd and analyzed for their size and structure. Data analysis was done using ImageJ.

Characterization of Mechanical Properties:

Mechanical properties of acellular hydrogels were measured via a compression test. Acellular hydrogels with 0% to 1% (w/v) HOGMPs with and without catalase were synthesized according to protocol. Samples were placed in DPBS for one day, thereby allowing the hydrogels to swell. A mechanical tester (Force Transducer Model SM-250, ADMET) was used to compress the samples to obtain the stress-strain curve at the rate of 10 mm min⁻¹. The elastic modulus was determined as the slope in the linear region corresponding to 10–20% strain.

Hydrogel Swelling Analysis:

Various GelMA, HOGMP-laden (1% (w/v)), and CPO-laden (0.3% (w/v); equivalent of CPO concentration in 1% (w/v) HOGMPS) hydrogels (with and without catalase; 1:5 mole CPO:unit catalase)) were fabricated according to protocol. CPO-laden hydrogels were exposed to UV light for twice as long (60 s) to ensure full crosslinking. Hydrogels were incubated in DPBS at 37 °C for 24 h after which they were removed from the DPBS, dried using tissue paper, and weighed to obtain their wet weight $(W_{\mathrm{w}})$. The hydrogels were subsequently flash-frozen using liquid nitrogen, lyophilized, and weighed again to obtain the dry weight $(W_{\mathrm{d}})$. The swelling ratio was obtained using the following formula

$$\text{Swelling ratio}(\%)=({\mathrm{W}}_{\mathrm{w}}-{\mathrm{W}}_{\mathrm{d}})∕({\mathrm{W}}_{\mathrm{d}})\ast 100\%$$ (5)

Enzymatic Degradation of the Hydrogels:

Various GelMA, HOGMP-laden (1% (w/v)), and CPO-laden (0.3% (w/v)) hydrogels (with and without catalase; 1:5 mole CPO:unit catalase) were fabricated according to protocol. Again, the CPO-laden hydrogels were exposed to UV light for twice as long (60 s) to ensure full crosslinking. The hydrogels were either incubated in DPBS or DPBS with 0.5 U mL⁻¹ collagenase type II (Worthington) and incubated for 0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, and 144 h. At the different timepoints, the hydrogels were washed twice, flash-frozen with liquid nitrogen, lyophilized, and weighed. The loss of mass was calculated using the following formula

$$\text{Loss of mass}(\%)=(W_0-W_{\mathrm{t}})∕W_{\mathrm{t}}\ast 100\%$$ (6)

where $W_0$ is the dry weight at 0 h and $W_{\mathrm{t}}$ is the dry weight at a specific timepoint.

C2C12 Myoblast Culture:

C2C12 myoblasts were cultured in C2C12 proliferation medium, which was composed of high glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1× antibiotic-antimycotic (Gibco), in a humidified incubator at 37 °C with 21% O₂, 5% CO₂, and 74% N₂. The cells were detached using 0.25% (w/v) Trypsin-EDTA (Gibco) at 37 °C when the cells reached ≈70% confluence, and were subsequently subcultured or used for experimentation.

Fabrication of C2C12-Laden GelMA Hydrogels:

HOGMPs were added to the GelMA solution (dissolved in DPBS) at various concentrations (0%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, and 2% (w/v)) and catalase was added at mole CPO:unit catalase ratios of 1:0, 1:3, 1:5 or 1:10. C2C12 myoblasts were then added into a HOGMP-laden 5% (w/v) GelMA solution to a final concentration of 2 × 10⁶ cells mL⁻¹. All samples were subsequently UV-crosslinked for 30 s, flipping the samples half-way through the process. The constructs were cultured in C2C12 proliferation medium under normoxic (≈21% oxygen) and severe hypoxic (≈1% oxygen) conditions.

Quantification of Oxygen Generation:

400 μL of HOGMP-laden hydrogel (450 μm height and ≈16 mm in radius; 0%, 0.1%, 0.25%, 0.5%, and 1% (w/v) HOGMPs) was placed inside a cryovial with 600 μL of anoxic DPBS. Anoxic DPBS was obtained by placing DPBS inside a hermetically sealed glove box overnight, which was kept at anoxic levels (≈0.1–0.5% oxygen as measured with an Opto-F1 Uni-Amp optic sensor (Unisense)) by continuously flushing with nitrogen gas. Measurements were performed under anoxic conditions, by flushing the glove box prior to measuring. The level of dissolved oxygen on a daily basis for ≈5–10 min per condition using the optic sensor. The oxygen release was determined from the average over the measured time. The cryovials with samples were closed in between the daily measurements. Similar experiments were performed on an equal amount of microparticles (compared to cell-laden hydrogels) dispersed in DPBS.

The oxygen consumption per construct was based on previous research,^[43] where the authors determined a cell’s oxygen consumption (2.5 x 10⁻¹⁸ moles per cell per second). For one construct (≈1 x 10⁵ cells) this equals a consumption of 22 nmoles per day (i.e., 0.15 μmoles for seven days). The oxygen consumption of the construct was also added to the oxygen release graphs as a threshold.

Quantification of Hydrogen Peroxide Generation:

To monitor the release of H₂O₂ under acellular conditions, HOGMP-laden hydrogel samples (50 μL) were placed in 800 μL of DPBS inside an Eppendorftube (Eppendorf) and agitated continuously in the dark at room temperature. 20 μL of each sample was collected every alternate day for each condition for a period of seven days. Samples were stored at −80 °C until analysis was performed. To determine the effect of the presence of cells on the release of H₂O₂, hydrogel constructs (GelMA containing 0.5-1% (w/v) HOGMPs and mole CPO:unit catalase 1:5) with and without C2C12 myoblasts were analyzed on the released levels of H₂O₂. The samples were placed in 800 μL of C2C12 growth medium and 20 μL solution was taken on days 1, 3, 7, and 14 for analysis. The hydrogels were placed in a hypoxic chamber (≈1% oxygen at 37 °C) and media was refreshed every third day. Samples were stored at −80 °C until analysis was performed. An Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (ThermoFisher Scientific) was used to measure the concentration of H₂O₂ in DPBS or growth medium following the manufacturer’s protocol. Briefly, 10 μL of sample was added to 40 μL of 1× Reaction Buffer and 50 μL of 100 × 10⁻⁶ m Amplex Red reagent containing 0.2 U mL⁻¹ horseradish peroxidase in a 96-well plate. The solutions were incubated for 30 min, protected from light, and fluorescence was measured (excitation 530 nm, emission 590 nm) using a 96-well plate reader (SpectraMax Paradigm, Molecular Devices).

Stability of Catalase Function Over Time:

The stability and enzyme function of catalase inside a hydrogel and aqueous solution were investigated either by adding eight units of catalase to a 150 × 10⁻⁶ m H₂O₂ solution (with a pristine GelMA hydrogel in the solution), or by adding 8 units per hydrogel construct that was placed inside a 150 × 10⁻⁶ m H₂O₂ solution. Solution samples were collected daily for seven days. For each time-point, the H₂O₂ concentration was measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit.

Viability, Proliferation, and Metabolic Activity of the C2Cl2 Cells over Time:

Live and dead C2C12 myoblasts were visualized using calcein-AM and ethidium homodimer (ThermoFisher Scientific) and photographed using an inverted fluorescent microscope (Nikon Eclipse Ti-S) over a period of seven days. Viability, length, and sphericity of the C2C12 cells were quantified using ImageJ. Proliferation was semi-quantified by counting the number of live cells over time. CellTiter 96s Aqueous One Solution Cell Proliferation Assay (MTS; Promega) was used to evaluate the C2C12 myoblast metabolic activity after 1, 5, and 10 days of culture. 60 μL of MTS solution was added to 300 μL of cell culture media for 1 h and 100 μL of the resulting solution was subsequently added to a 96-well plate (Corning). The absorbance at 490nm was recorded using a 96-well plate reader.

Printability of Oxygen Generating Bioinks:

To assess the printability and effect of catalase and encapsulation of CPO within PCL on the release of H₂O₂, three different bio-inks were made: GelMA (5% (w/v), control), 1% (w/v) HOGMPs in catalase-laden GelMA, and 0.3% (w/v) CPO (equivalent of CPO concentration in 1% (w/v) HOGMPs) in catalase-laden GelMA. Quantification of printing was performed as described by Ouyang et al.^[70] Printability (Pr) was defined from the circularity of the enclosed area of the square-formed prints, which is defined as

$$\mathsf{C}=4\pi {\mathsf{AL}}^2$$ (7)

where $A$ is the area and $L$ is the perimeter. The shape is defined as a circle when $C$ is close to 1. For a square shape, circularity is equal to $\pi ∕4$, and therefore printability of a square shape is defined as

$$\text{Pr}=\pi ∕(4\mathsf{C})={\mathsf{L}}^2∕(16\mathsf{A})$$ (8)

When the interconnected channels of the construct show a perfect square shape, printability is defined as 1. To create cell-laden printed constructs, C2C12 cells were mixed in the bioink solutions at a concentration of 2 × 10⁶ cells mL⁻¹. 3D bioprinting was performed using a Cellink Inkredible bioprinter. A CAD file was sliced using Slic3r software to generate g-codes readable by the bioprinter. A nozzle speed of 500 mm min⁻¹ was used to print the final constructs. A 3 mL syringe and 30-gauge needle (Fisnar) were used for the printing all inks. The extrusion of the ink from the nozzle was done via nitrogen gas fed at ≈40 kPa pressure. UV crosslinking was induced using an OmniCure S2000 machine. The stage was adjusted to 8 cm distance from the light guide and the UV intensity was calibrated to 12.5 mW cm⁻². All constructs were crosslinked for 30 s, flipping the construct carefully half-way through the process. Viable and dead C2C12 myoblasts were visualized using calcein-AM and ethidium homodimer, and photographed using an inverted fluorescent microscope. Viability of the cells was divided into two groups: inside and edge (until 200 μm into the printed tissue). The length and sphericity of the C2C12 cells were quantified using ImageJ.

Rheological Measurements:

Prior to the measurements, various 5% (w/v) GelMA solutions were made (GelMA, GelMA with 1% (w/v) CPO and GelMA with 1% (w/v) HOGMPs) and kept at 37 °C. Warm hydrogel precursor solutions (i.e., non-crosslinked) were deposited onto a 4 °C rheometer plate (8 mm plate-plate geometry) with a 1 mm gap. A 10 min oscillatory time sweep was directly performed at a frequency of 1 Hz with 0.5% strain (linear elastic regime) using an Anton Paar-Physica MCR 301 Rheometer (Anton Paar, GmbH, Germany). Afterwards, the plates, and thereby the solution, were rapidly heated to 25 °C. The temperature was kept at 25 °C for 10 min to ensure homogeneous melting. Subsequently, a temperature ramp from 25 to 4 °C was performed at a rate of 1 °C min⁻¹ to observe the crossover temperature point by oscillatory measurements at a frequency of 1 Hz and 0.5% strain. Temperature control was achieved with a Peltier heating system.

Statistical Analysis:

All experiments were performed in triplicate. Data were statistically expressed as mean ± standard deviation. The normal distribution was first tested by Shapiro–Wilk (n < 50). A paired one-sided t-test was used for small sample sizes (n < 9). For large sample sizes (n > 9), a one-way or two-way analysis of variance (ANOVA) or Kruskal–Wallis ANOVA was used for analysis of quantitative values, and a post hoc test was used for all pair-wise comparisons among groups. p-values were deemed significant below p < 0.05, and indicated with asterisks (e.g., *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). The Prism software package was used to perform the analysis.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.