Human Umbilical Vein Endothelial Cells Form a Network on a Hyaluronic Acid/Gelatin Composite Hydrogel Moderately Crosslinked and Degraded by Hydrogen Peroxide

The study of the capillary-like network formation of human umbilical vein endothelial cells (HUVECs) in vitro is important for understanding the factors that promote or inhibit angiogenesis. Here, we report the behavior of HUVECs on the composite hydrogels containing hyaluronic acid (HA) and gelatin with different degrees of degradation, inducing the different physicochemical properties of the hydrogels. The hydrogels were obtained through horseradish peroxidase (HRP)-catalyzed hydrogelation consuming hydrogen peroxide (H2O2, 16 ppm) supplied from the air, and the degradation degree was tuned by altering the exposure time to the air. The HUVECs on the composite hydrogel with intermediate stiffness (1.2 kPa) obtained through 120 min of the exposure were more elongated than those on the soft (0.4 kPa) and the stiff (2.4 kPa) composite hydrogels obtained through 15 min and 60 min of the exposure, respectively. In addition, HUVECs formed a capillary-like network only on the stiff composite hydrogel although those on the hydrogels with comparable stiffness but containing gelatin alone or alginate instead of HA did not form the network. These results show that the HA/gelatin composite hydrogels obtained through the H2O2-mediated crosslinking and degradation could be a tool for studies using HUVECs to understand the promotion and inhibition of angiogenesis.


Introduction
The capillary network, composed of vascular endothelial cells, is essential for nutrient and gas exchanges between blood and tissue. In addition, capillary network formation is essential for tumor growth [1,2]. Therefore, studies dealing the capillary network formation have been intensively conducted both in vivo and in vitro [3][4][5]. Various hydrogels enabling the network formation of vascular endothelial cells have been developed for studies in vitro [6,7]. Hydrogels are water-swollen three-dimensional networks of polymers. To provide mechanical support as well as the chemical environment for cell adhesion sites, cell remodeling, nutrient support, and cell responsive remodeling, hydrogels are widely used in tissue engineering technologies as transient artificial extracellular matrix substitutes [8,9].
The physicochemical properties of the extracellular matrix (ECM) are known to modulate the cell functions such as adhesion and proliferation [10,11]. Numerous reports have been published on the effect of chemical modifications of the ECM to control endothelial cell behavior. Rumiana et al. reported the angiogenic potential of the endothelial cell on gelatin-based hydrogel by controlling the growth factor release by electrical stimulation [12]. The report by Ying et al. showed that the co-delivery of growth factor by gelatin hydrogel promotes angiogenesis compared with growth factors applied alone [13]. Derek et al. reported that hyaluronidase is stimulated in endothelial colony-forming cells through hyaluronic acid (HA)-specific receptors for cord-like structures on HA hydrogels [14].

Mechanical Property Measurement
The stiffness of the synthesized hydrogels was determined in terms of Young's modulus using a material tester (EZ-SX, Shimadzu, Kyoto, Japan). First, composite hydrogel (HA-Ph/Gelatin-Ph) was obtained by exposing the solution of 3.0 wt% Gelatin-Ph, 0.5 wt% HA-Ph dissolved in PBS (pH 7.4), and HRP (1 U/mL) to air containing 16 ppm H2O2 for 15, 60, and 120 min. Then, each composite hydrogel was compressed at 6 mm/s using an 8 mm probe. Next, Young's modulus was calculated to determine the stiffness of prepared hydrogel using the linear compression strain of 1-10% of the stress-strain curve. The same experimental steps were followed for the preparation and mechanical property measurements for 3.0 wt% Gelatin-Ph/0.5 wt% Alginate-Ph, 3 wt% Gelatin-Ph, and 3.25 wt% Gelatin-Ph hydrogels.

Mechanical Property Measurement
The stiffness of the synthesized hydrogels was determined in terms of Young's modulus using a material tester (EZ-SX, Shimadzu, Kyoto, Japan). First, composite hydrogel (HA-Ph/Gelatin-Ph) was obtained by exposing the solution of 3.0 wt% Gelatin-Ph, 0.5 wt% HA-Ph dissolved in PBS (pH 7.4), and HRP (1 U/mL) to air containing 16 ppm H 2 O 2 for 15, 60, and 120 min. Then, each composite hydrogel was compressed at 6 mm/s using an 8 mm probe. Next, Young's modulus was calculated to determine the stiffness of prepared hydrogel using the linear compression strain of 1-10% of the stress-strain curve. The same experimental steps were followed for the preparation and mechanical property measurements for 3.0 wt% Gelatin-Ph/0.5 wt% Alginate-Ph, 3 wt% Gelatin-Ph, and 3.25 wt% Gelatin-Ph hydrogels.

Enzymatic Degradation
The composite hydrogels (Gelatin-Ph/HA-Ph) were soaked in PBS for one day to reach an equilibrium state. Then, the composite hydrogels were soaked in a mixture of hyaluronidase (1 mg/mL) and collagenase (1 mg/mL) in Dulbecco's Modified Eagle's Medium (DMEM). Next, the time required for the total decomposition of hydrogel was measured.

Molecular Weight Analysis
PBS containing 3.0 wt% Gelatin-Ph and 0.5 wt% HA-Ph were separately exposed to air containing 16 ppm H 2 O 2 for 15 min, 60 min, and 120 min, respectively. The molecular weights of resultant polymers in the solutions were analyzed using high-performance liquid chromatography (HPLC) (LC-20AD, Shimadzu, Kyoto, Japan) based on an intensitytime curve ( Figure S2). The unexposed stock solutions of both polymers were used as the control, and the molecular weight of each sample was calculated from the calibration curve of polyethylene glycol standards.

Scanning Electron Microscope Observation of Freeze-Dried Hydrogels
Specimens of freeze-dried hydrogels were prepared based on the freeze-extraction method [30]. Briefly, 1 mL of PBS containing 3.0 wt% Gelatin-Ph/0.5 wt% HA-Ph and 1 U/mL HRP was added to a polydimethylsiloxane (PDMS) mold (diameter: 8 mm, height: 4 mm). Air containing H 2 O 2 was then exposed for 15, 60, and 120 min. The resultant hydrogel was frozen at −80 • C. The frozen specimens were immersed in 70% and 100% ethanol in sequence at −30 • C for 10 h and subsequently vacuum dried. Then, the cross sections of the resultant specimens coated with a thin layer of gold were observed using a field emission scanning electron microscope (SEM, JCM-6000plus, JEOL, Tokyo, Japan) with an acceleration voltage of 15 kV.

Cell Culture
Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from RIKEN Cell Bank (Ibaraki, Japan). The HUVECs were cultured in MDCB107 medium consisting of fetal bovine serum (10 v/v%) and supplemented with the 10 ng/mL of basic fibroblast growth factor (bFGF) and 10 ng/mL of endothelial growth factor (EGF) through passage 6. Cell culture was conducted in a humidified incubator supplied with 5% CO 2 at 37 • C.

Cell Viability and Adhesion
The composite hydrogel (Gelatin-Ph/HA-Ph) was prepared in a 6 well-plate by exposing the air containing H 2 O 2 for 15-120 min to 1 mL of 3.0 wt% Gelatin-Ph, 0.5 wt% HA-Ph dissolved in PBS and HRP (1 U/mL) solution. The unreacted H 2 O 2 was removed from the hydrogel using the growth medium containing catalase (1 mg/mL). Following overnight incubation in catalase, hydrogels were washed thoroughly several times using both PBS and MDCB107 medium. HUVECs were then cultured on the polystyrene cell culture plate or hydrogel at 4.0 × 10 3 cells/cm 2 . HUVECs viability was analyzed based on our previously reported method [28]. HUVECs adhesion to the substrates was analyzed based on the cell morphological parameters, including aspect ratio (ratio between cell length and width), and area, were calculated for the morphology analysis using ImageJ (1.53f51, NIH, Bethesda, MD, USA). Previous reports in the literature mentioned that calcein-AM, which stained the cell cytoplasm, could be used to analyze the morphological parameters. Therefore, in this study, we used the calcein-AM staining for the morphological analysis [31,32].

HUVECs Network Formation
HUVECs network formation analysis was conducted on the hydrogels according to the previously reported protocol [33]. First, 3 wt% Gelatin-Ph, 0.5 wt% HA-Ph, and HRP (1 U/mL) were mixed in PBS and put into a 6-well plate (1 mL/well). Then, the polymer solution was exposed to the air containing H 2 O 2 (16 ppm) for 15-120 min. Subsequently, 1 mL of catalase dissolved in MCDB107 medium was added to fabricated hydrogel and incubated overnight at 37 • C in humidified incubator supplied with 5% CO 2 . After incubation overnight, the cells with 70% confluent were trypsinized and seeded on the Gelatin-Ph/HA-Ph hydrogel at 4.0 × 10 4 cells/cm 2 [33]. HUVECs were cultured in an MCDB107 medium containing 10 ng/mL bFGF, 10 ng/mL EGF, and 2 v/v% FBS. The HUVECs network formation was observed using an optical microscope (OLYMPUS IX71, Tokyo, Japan) at 20 h of post culture. Parallel experiments were conducted on the 3 wt% Gelatin-Ph/0.5 wt% Alginate-Ph, 3 wt% Gelatin-Ph, 3.25 wt% Gelatin-Ph hydrogel, and cell culture dish following the same experimental procedure.

Statistical Analysis
Microsoft ® Excel ® (Microsoft Corp., Redmond, WA, USA) 2019 version 1808 was used for all data analysis. Statistical analyses were performed using one-way analysis of variance (ANOVA). A post hoc t-test was conducted using Tukey HSD, and a p-value of <0.05 was considered significantly different.

Properties of Gelatin-Ph/HA-Ph Hydrogels
Firstly, we examined the influence of the exposure time to the air containing 16 ppm H 2 O 2 (15, 60, and 120 min) on the stiffness of the hydrogels after confirming the hydrogelation within 1 min of the exposure for all the compositions shown in Table 1. As shown in Figure 2a, Young's modulus increased with H 2 O 2 exposure time from 15 to 60 min, and the values decreased with a further increase of the time to 120 min both in G3 and G3/HA0.5 hydrogels. The values detected for G3-60 and G3/HA0.5-60 after exposure to the air containing H 2 O 2 for 60 min were 1.90 kPa and 2.40 kPa, respectively. These values were about 8-and 2-times larger than those detected for the specimens obtained through 15 and 120 min of exposure, respectively, at the same composition. The air containing H 2 O 2 exposure time-dependent change of the hydrogel mechanical properties is consistent with the results in the literature [26,28]. The hydrogel stiffness increase with an increase of H 2 O 2 exposure time from 15 to 60 min was explained by the increase of the crosslinking between Ph groups through the progression of HRP-catalyzed reaction [27]. On the other hand, the hydrogel stiffness decrease with an increase in the exposure time from 60 to 120 min was explained by the HRP inactivation and polymer degradation by H 2 O 2 [26]. Furthermore, the effect of the air containing H 2 O 2 exposure time on the degradability of the composite hydrogels were evaluated by measuring the time required for total degradation of the G3/HA0.5 hydrogel using hyaluronidase and collagenase as shown in Figure 3. The stiff hydrogel (G3/HA0.5-60) required the longest time (92 min) to be degraded, due to the high crosslinking formation compared to the soft and intermediate stiff hydrogels (G3/HA0.5-15 and G3/HA0.5-120, respectively).
due to the high crosslinking formation compared to the soft and intermediate stiff hydrogels (G3/HA0.5-15 and G3/HA0.5-120, respectively).  As shown in Figure 4, the molecular weights of both Gelatin-Ph and HA-Ph decreased with extending the H2O2 exposure time. The higher stiffness of G3/HA0.5 hydrogels than G3 hydrogels at each exposure time was caused by the denser polymer network due to the higher polymer concentration. The stiffness of the hydrogels prepared from 3.25 wt% Gelatin-Ph solution (G3.25-60) and 3 wt% gelatin/0.5 wt% Alginate-Ph solution after 60 min of exposure to the air containing H2O2 (G3.25-60 and G3.25/A0.5-60, respectively) were comparable to that of G3/HA0.5-60 (Figure 2b). G3.25-60 and G3.25/A0.5-60   As shown in Figure 4, the molecular weights of both Gelatin-Ph and HA-Ph decreased with extending the H2O2 exposure time. The higher stiffness of G3/HA0.5 hydrogels than G3 hydrogels at each exposure time was caused by the denser polymer network due to the higher polymer concentration. The stiffness of the hydrogels prepared from 3.25 wt% Gelatin-Ph solution (G3.25-60) and 3 wt% gelatin/0.5 wt% Alginate-Ph solution after 60 min of exposure to the air containing H2O2 (G3.25-60 and G3.25/A0.5-60, respectively) were comparable to that of G3/HA0.5-60 (Figure 2b). G3.25-60 and G3.25/A0.5-60 As shown in Figure 4, the molecular weights of both Gelatin-Ph and HA-Ph decreased with extending the H 2 O 2 exposure time. The higher stiffness of G3/HA0.5 hydrogels than G3 hydrogels at each exposure time was caused by the denser polymer network due to the higher polymer concentration. The stiffness of the hydrogels prepared from 3.25 wt% Gelatin-Ph solution (G3.25-60) and 3 wt% gelatin/0.5 wt% Alginate-Ph solution after 60 min of exposure to the air containing H 2 O 2 (G3.25-60 and G3.25/A0.5-60, respectively) were comparable to that of G3/HA0.5-60 (Figure 2b). G3.25-60 and G3.25/A0.5-60 hydrogels were prepared for evaluating the effect of the hydrogel stiffness and composition on HUVECs behavior shown in Section 3.3. hydrogels were prepared for evaluating the effect of the hydrogel stiffness and composition on HUVECs behavior shown in Section 3.3.  Figure 5 shows the cross-section images of freeze-dried G3/HA0.5 hydrogels observed using SEM. No remarkable difference in the pore size was found for the specimens obtained through different H2O2 exposure time. This result indicates that the difference in the microstructure in the hydrogels that caused the differences in mechanical property ( Figure 2) and enzymatic degradation ( Figure 3) resulting from the differences in H2O2mediated cross-linking and degradation was not large enough to induce the difference in the ice nucleation and ice crystal growth during freezing the hydrogels. It has been reported that the ice nucleation and ice crystal growth during freezing of hydrogels govern the porous structure of the resultant dried specimens [34][35][36].   Figure 5 shows the cross-section images of freeze-dried G3/HA0.5 hydrogels observed using SEM. No remarkable difference in the pore size was found for the specimens obtained through different H 2 O 2 exposure time. This result indicates that the difference in the microstructure in the hydrogels that caused the differences in mechanical property ( Figure 2) and enzymatic degradation ( Figure 3) resulting from the differences in H 2 O 2 -mediated cross-linking and degradation was not large enough to induce the difference in the ice nucleation and ice crystal growth during freezing the hydrogels. It has been reported that the ice nucleation and ice crystal growth during freezing of hydrogels govern the porous structure of the resultant dried specimens [34][35][36].   Figure 5 shows the cross-section images of freeze-dried G3/HA0.5 hydrogels observed using SEM. No remarkable difference in the pore size was found for the specimens obtained through different H2O2 exposure time. This result indicates that the difference in the microstructure in the hydrogels that caused the differences in mechanical property ( Figure 2) and enzymatic degradation (Figure 3) resulting from the differences in H2O2mediated cross-linking and degradation was not large enough to induce the difference in the ice nucleation and ice crystal growth during freezing the hydrogels. It has been reported that the ice nucleation and ice crystal growth during freezing of hydrogels govern the porous structure of the resultant dried specimens [34][35][36].
Previous studies have reported that the low-molecular-weight HA (LMW-HA) could induce cell elongation [37] and epithelial-to-mesenchymal transition (EMT) [38]. The EMT process involves a physiological transition marked by morphological changes to elongated spindle-like morphology. As shown in Figures 4 and S2b, prolonged exposure to air containing H 2 O 2 could degrade the HA-Ph. The degraded fragments of HA-Ph might interact with cell receptors that induce cell elongation [37]. In our previous study, similar result was obtained with mouse mammary cells cultured on the composite hydrogel (Gelatin-Ph/HA-Ph) obtained through 120 min of exposure to the air containing 16 ppm H 2 O 2 [26]. In general, this result shows that HUVECs adhesion depends on the composition and the mechanical property of the hydrogel.

HUVECs Network Formation
Finally, we investigated the HUVECs network formation on each hydrogel by seeding HUVECs at 4.0 × 10 4 cells/cm 2 . This seeding density was selected considering the successful network formation in previous studies [33]. As shown in Figure 7, HUVECs formed a visible network only on G3/HA0.5-60 hydrogel. The different behavior of the cells on G3/HA0.5-15, -60, and -120 hydrogels suggests the possible effect of hydrogel stiffness on the network formation. The different behavior of the cells on G3-60 and G3/HA0.5-60 also suggests the possible effect of the stiffness. G3/HA0.5-60 had the highest stiffness in G3 and G3/HA0.5 hydrogels (Figure 2a). However, the sole effect of the hydrogel stiffness on the network formation of HUVECs was denied by the non-network formations of the cells on G3.25-60 and G3/A0.5-60 hydrogels having similar stiffnesses with G3/HA0.5-60 ( Figure 2b).
These results demonstrate that the combined effects of hydrogel stiffness and the lowmolecular-weight HA-Ph generated through the degradation of HA-Ph by H 2 O 2 caused the network formation of HUVECs on G3/HA0.5-60 hydrogel. The detailed mechanism of how the HA molecular weight and stiffness of the hydrogel affect the HUVECs behavior is unknown and will be a subject of our future study. However, there are several possible mechanisms. The presence of small fragments of HA, such as those as a product of HA-Ph degradation by air containing H 2 O 2 exposure (Figure 4 and Figure S2), was reported to induce HUVECs capillary-like network formation by interacting with CD44 and RHAMM receptors [39,40]. In addition, HA-CD44 interaction could activate γ-adducin, which plays a role in the HUVECs tube formation [41]. Meanwhile, the interaction between HA and RHAMM induces AP-1 binding to the RHAMM promoter, promoting the capillary-like network formation of HUVECs [41,42].
In addition to the chemical signaling of ECM components, ECM stiffness plays an essential role in the capillary-like network formation of HUVECs through mechanical signaling. Joseph et al. reported the effect of substrate mechanics and matrix chemistry on endothelial cell network assembly using polyacrylamide substrates derivatized with type I collagen. They investigated that stiff hydrogel (2.5-10 kPa) with low cell-substrate adhesiveness could form a capillary-like network, while soft substrate (0.2-1 kPa) retained a round morphology without network formation [43]. Therefore, the combination of both substrate stiffness and ECM components is vital for the capillary-like network formation of HUVECs. Taken together, our studies demonstrate that the effect of H 2 O 2 on inducing polymer crosslinking and degradation could modulate the behavior of HUVECs. From the translational perspective, these investigations are useful in various tissue engineering applications. Several studies have reported that the alteration in HA molecular weight and amount are characteristic of pathological conditions. The accumulation of HA in cancer stroma is a marker of malignancy for different kinds of tumors [44]. In addition, angiogenesis is essential for invasive tumor growth and constitutes a crucial point in controlling tumor progression [45]. In cancer therapy, inhibition of angiogenesis may be a valuable new approach. Therefore, our findings could be used as a tool for the in vitro study of cancer therapy through factors affecting inhibition and stimulation of angiogenesis. In addition to the chemical signaling of ECM components, ECM stiffness plays an essential role in the capillary-like network formation of HUVECs through mechanical signaling. Joseph et al. reported the effect of substrate mechanics and matrix chemistry on endothelial cell network assembly using polyacrylamide substrates derivatized with type I collagen. They investigated that stiff hydrogel (2.5-10 kPa) with low cell-substrate adhesiveness could form a capillary-like network, while soft substrate (0.2-1 kPa) retained a round morphology without network formation [43]. Therefore, the combination of both substrate stiffness and ECM components is vital for the capillary-like network formation of HUVECs. Taken together, our studies demonstrate that the effect of H2O2 on inducing polymer crosslinking and degradation could modulate the behavior of HUVECs. From

Conclusions
In this study, we investigated the influence of the composition and mechanical properties of the hydrogels on the behavior of HUVECs. We tuned the degree of polymer degradation and hydrogel stiffness by simply controlling the exposure time to air containing H 2 O 2 during the hydrogel preparation. HUVECs showed different responses depending on the degree of polymer degradation and the stiffness of the hydrogel. HU-VECs cultured on Gelatin-Ph/HA-Ph hydrogel showed higher elongation on prolonged exposure to air containing H 2 O 2 , a phenomenon that was not observed in the absence of HA-Ph. More importantly, HUVECs network formation was observed only on stiff Gelatin-Ph/HA-Ph hydrogels, while cells on hydrogels composed of Gelatin-Ph alone or mixed with Alginate-Ph instead of HA-Ph showed no network formation regardless of the stiffness. Taken together, these results showed that HA-Ph addition to the Gelatin-Ph and the mechanical properties played an important role in governing the HUVECs behavior. Therefore, we believe that our findings could be useful for the fabrication of the hydrogels for the studies using HUVECs, including the biofabrication of artificial tissues and for in vitro studies to understand the factors that promote or inhibit angiogenesis.  Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files.