Anti-VEGF-R2 Aptamer and RGD Peptide Synergize in a Bifunctional Hydrogel for Enhanced Angiogenic Potential

Abstract

Hydrogels have gained interest for use in tissue regeneration and wound healing because of their absorbing and swelling properties as well as their ability to mimic the natural extracellular matrix. Their use in wound healing specifically may be in the form of a patch or wound dressing or they may be administered within the wound bed as a filler, gel in situ, to promote healing. Thiolated hyaluronic acid-polyethylene diacrylate (tHA-PEGDA) hydrogels are ideal for this purpose due to their short gelation times at physiological temperature and pH. But these hydrogels alone are not enough and require added components to gain bioactivity. In this work, RGD adhesion peptides and an anti-vascular endothelial growth factor receptor-2 (VEGF-R2) DNA aptamer are incorporated into a tHA-PEGDA hydrogel to make a bifunctional hyaluronic acid hydrogel. RGD peptides promote attachment and growth of cells while the anti-VEGF-R2 DNA aptamer seems to improve cell viability, induce cell migration, and spur the onset of angiogenesis by tube formation by endothelial cells. This bifunctional hydrogel supports cell culture and has improved biological properties. The data suggests that these hydrogels can be used for advanced tissue regeneration applications such as in wound healing.

Graphical Abstract

In this work, a bifunctional hyaluronic acid-based hydrogel supports endothelial cells culture and shows improved biological properties. The gel is functionalized with both RGD peptides and anti-VEGF-R2 DNA aptamer. The data suggests improved cell viability, cell migration, and the initiation angiogenesis by tube formation by endothelial cells. The data suggests that these hydrogels can be used in wound healing.

1. Introduction

Despite many hydrogels being biocompatible in the human body, they suffer from several challenges. Compared to extracellular matrix (ECM) components, many hydrogels are naked or bioinert- they lack the necessary biochemical cues for cell interaction.^[1] For tissue engineering and regenerative medicine, an ideal scaffold material should not only provide mechanical support and cell adhesion; but like the native cellular microenvironment, a scaffold should provide the biochemical signaling that activates signaling pathways and thus regulates cellular activities.^[2,3] Therefore, hydrogel scaffolds should strive to emulate these more complex cell-ECM interactions.

To achieve this, bioinert hydrogels have been made more hospitable and active for cells by incorporating more diverse ECM components, several bioactive peptide sequences, and soluble/releasable growth factors. For instance, by adding heparan sulfate to collagen meshes enhanced cell proliferation and immune-suppressive potential via receptor-ligand binding.^[4] Adhesion peptide sequences such as arginine-glycine-aspartate (RGD) have been used for their fibronectin and laminin origin. Integrins on the cell’s surface can bind these sequences facilitating cell attachment and spreading.^[5–7] Thiol-modified RGD peptides have been incorporated into thiolated hyaluronic acid (tHA) – polyethylene glycol diacrylate (PEGDA) hydrogels to make a cell-friendly, injectable system.^[8] Introducing soluble growth factors such as, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), have been used to increase both the differentiation of stem cells and the ingrowth of cells into tissue.^[9–11] Hydrogel scaffolds and growth factors that naturally interact with each other have been paired together to resolve challenges in controlling growth factor release such as pairing heparin-hydrogels with VEGF. In this case, heparin binds to growth factors and inhibits their enzymatic breakdown enabling the controlled delivery of the growth factor.^[12] QK peptide is a VEGF-mimicking peptide which has been used in polyethylene glycol (PEG) hydrogels as a substitute for VEGF to induce angiogenesis.^[13]

In the last few decades, aptamers have emerged as another powerful biomolecule for hydrogels due to their excellent targeting properties. DNA aptamers are single-stranded DNA sequences that act like “chemical antibodies” meaning they have very high affinity and selectivity for their target.^[14] Aptamers have been used in a myriad of applications from treating ocular vascular disease to use as sensors.^[15–17] In hydrogel applications, DNA aptamers have been used to either sequester or release species such as growth factors.^[18–23] Chitosan films with immobilized anti-fibronectin aptamers on the surface were shown to help improve cell attachment and function.^[22] Incorporating anti-platelet derived growth factor (PDGF) aptamers enabled the binding and then the release of PDGF in agarose gels. When triggered by the addition of the complimentary nucleotide sequence, the gel gave a slow sustained release profile.^[21] In another instance, an aptamer mimicking ECM adhesion was able to support cell attachment with cell-type specificity.^[24] Multi aptamer gels have added greater functionality by being able to provide signaling from multiple sources. For instance, a dual aptamer fibrin hydrogel was developed to deliver both PDGF and VEGF, which promoted angiogenesis.^[20] Similarly, an RGD and anti-VEGF aptamer functionalized polyethylene glycol (PEG) hydrogel supported enhanced endothelial cell growth and survival.^[19] One of the hallmark challenges of tissue regeneration and the future success of implanted hydrogels is the development of a functioning vasculature. In particular, vascular endothelial growth factors have been used towards these goals because they are instrumental in the growth and differentiation of endothelial cells as well as are responsible for inducing angiogenesis.^[25,26] VEGF proteins stimulate pathways by attaching to vascular endothelial cell receptors on the endothelial cell surface. VEGF-A, which mediates cell processes like angiogenesis and survival, specifically binds to the vascular endothelial growth factor receptor 2 (VEGF-R2). However, the presence of multiple VEGF proteins and receptors leads to cross interactions, which can delay cell processes.^[27] Thus, aptamers and aptamer assemblies that bind and activate the VEGF-R2 receptor have been developed to circumvent these issues.^[28,29]

We sought to investigate whether VEGF-R2 binding aptamers incorporated into a hydrogel would support angiogenic behavior by endothelial cells. To do this, we prepared a bifunctional tHA-PEGDA hydrogel with both immobilized RGD peptides for cell adhesion and anti-VEGF-R2 DNA aptamers for stimulating angiogenesis without the use of exogenous growth factors. Herein, we show that this bifunctional hydrogel outperformed gels with only RGD or only aptamer to enhance endothelial cell attachment, viability, migration, proliferation, and to promote the potential onset of angiogenesis.

2. Results and Discussion

2.1. Hydrogel Synthesis and Optimization

To efficiently incorporate both a thiol-modified RGD peptide and an acrydite-modified DNA aptamer sequence into the hydrogel system requires optimizing the amount of tHA to PEGDA. Our synthesis scheme is performed in three simple steps (Figure 1). First, the tHA is reacted for a short time with an acrydite-modified DNA aptamer sequence to introduce the aptamer. Then the gel is formed by the addition of the PEGDA crosslinker. The PEGDA needs to be added in excess so that there are a number of free acrylate groups remaining for the final step. Lastly, after the gel has partially crosslinked it is incubated in a solution of the thiol-modified RGD peptide to react with the remaining free acrylate groups on the PEGDA. The amount of tHA to PEGDA will influence the amount of free thiols and free acrylates available for reaction with the functionalizing species (RGD peptide and DNA aptamer). The amount of tHA to PEGDA will also influence the crosslinking density of the hydrogel and thus a number of key hydrogel properties. A more crosslinked gel will have reduced functionalization with thiol-tagged species because of the consumption of free acrylates on the PEGDA. This same fact holds true for acrydite-tagged species and consumed free thiols on the thiolated hyaluronic acid. A large amount of information about a hydrogel can be gleaned from a simple measurement of its swelling behavior. Swelling is related to the crosslinking density of a hydrogel. The more crosslinks in the gel, the more resistance there is to volume change from the outward pressure exerted by the imbibed fluid and thus there is less swelling. Each formulation using a ratio of either 3:1, 3:2, or 1:1 tHA to PEGDA reached its equilibrium swelling after 16 hours. As expected, the swelling ratio increased as the ratio of tHA to PEGDA decreased (Figure 2a). At the same time crosslinking density also influences handleability. A poorly crosslinked gel can pose challenges in terms of handling due to its weak mechanical properties. Poor handleability was found for both the 3:1 and 3:2 formulations. Taking all of these factors into consideration, the 1:1 ratio of tHA to PEGDA formulation was chosen for its increased mechanical stability and handleability as well as for its greater degree of free acrylates available for reacting with the thiol-modified RGD peptide following partial crosslinking. Thus, the 1:1 ratio of tHA to PEGDA hydrogel was used for all further experiments. The tHA-PEGDA substrate is advantageous because it can be used as a blank slate to study adhesion with biomolecules. Moreover, it is highly tunable due to the presence of functional groups that can be used to incorporate different molecules. Additionally, for in-wound healing applications, hydrogel systems need to be injectable, gel in situ, and be bioactive by supporting revascularization. The tHA-PEGDA hydrogels are ideal for this purpose because their crosslinking mechanism is a direct nucleophilic reaction that gives short gelation times and it can occur at physiological temperature and pH.^[30,31]

Figure 1.

Bifunctional hydrogel synthesis.

Figure 2.

Swelling ratio was briefly assessed as a measurement to gauge crosslinking density for different formulations of tHA to PEGDA (a). 1 replicate was measured at each time point. The 1:1 ratio of tHA to PEGDA was selected and its degradation in physiological buffer at body temperature was assessed (b). 2 replicates were measured at each time point.

RGD peptides were incorporated to facilitate cell attachment to the tHA-PEGDA hydrogel. From measurement by a BCA assay, we found that ~52% of the RGD peptide was incorporated from the RGD peptide solution into the 1:1 ratio of tHA to PEGDA formulated hydrogel (Supporting Information). This was measured by a change in the peptide solution concentration before and after application of the partially crosslinked hydrogel. The amount of RGD uptake could be increased by increasing the crosslinker PEGDA (providing additional excess free acrylates at the benefit of mechanical stability) or decreasing the partial crosslinking time of the tHA and PEGDA (providing additional excess free acrylates at the expense of mechanical stability). Staining of the aptamer containing hydrogels with a nucleic acid dye produced a confirmatory green fluorescence under blue light (Supporting Information). Because there were no other sources of DNA in the hydrogel, we could infer that the fluorescence was from the DNA aptamer. No fluorescence was observed for hydrogels without DNA aptamer. The hydrogel continued to stain for DNA after subsequent PBS rinses indicating that the aptamer had been covalently incorporated into the hydrogel (data not shown).

Degradability is an important aspect for engineering wound dressings. Importantly, for our thiolated hyaluronic acid hydrogel system, as the crosslinking density decreases, there is a greater propensity for the hydrogel to degrade. This occurs because of the presence of unreacted, dangling thiol groups in the reduced crosslinked gels. Thus, it was necessary to characterize the degradation of the hydrogel under physiological conditions. After 18 days in phosphate-buffered saline kept at 37 °C, the gels had degraded by ~75% by mass (Figure 2b). The degradation profile trended towards a plateau of this value starting around day 12. In vivo degradation of this hydrogel is expected to increase due to enzymatic activity from the likes of endogenous hyaluronidase. Importantly, the degradation rate of an implanted hydrogel should ideally coincide with the rate of new tissue growth at the site. This balance is necessary so that the implant does not disappear before the body has recovered or so that the implant does not remain longer than it needs to in the body and impede healthy tissue function. For wound healing, new tissue formation completes by 2–3 weeks, which coincides with the degradation rate of our hydrogels.^[32]

2.2. Cell Attachment

After validating incorporation of both RGD peptide and the DNA aptamer into the hydrogel, we assessed functional cell properties in response to it. We began by evaluating cell attachment by visual inspection by phase contrast microscopy. As expected, endothelial cells could not attach to the naked HA-PEGDA hydrogel demonstrated by their balling up and agglomerating together (Figure 3). Conversely, the addition of RGD peptide to the hydrogel supported attachment and after 12 hours the endothelial cells took on their characteristic cobblestone morphology (Figure 3). Interestingly, the aptamer containing hydrogel (HAptagel) was unable to support attachment on its own and endothelial cells behaved the same way as they had for the naked hydrogel (Figure 3). Incorporating both the RGD peptide and the DNA aptamer into the hydrogel (RGD-HAptagel), facilitated attachment in the same way as that of the RGD-only condition (Figure 3). This indicated that cells needed RGD peptide to attach and that the DNA aptamer was not sufficient to support attachment on its own. Still remained to be discovered was whether the combination of RGD peptide and the VEGF-R2 targeting DNA aptamer synergized in any way.

Figure 3.

Endothelial cell attachment to the different functionalized gels was visually assessed by phase contrast microscopy after 2, 6, and 12 hours. Scale bars are 200 μm.

2.3. Cytotoxicity and Cell Viability

The RGD peptide and DNA aptamer synergized to promote the least cytotoxicity and greatest cell viability. Paralleling our attachment results, endothelial cells more or less completely perished on the naked hydrogel and the HAptagel. This was because the cells could not attach to these substrates. Likewise, endothelial cells attached and survived on the RGD-only and RGD-HAptagel conditions (Figure 4b). This was observed from LIVE/DEAD imaging in which live cells stain green with calcein AM and dead or dying cells stain red with ethidium bromide homodimer. Following image quantification for relative fluorescent intensity at day 6, we observed a significant difference between the RGD-only and the RGD-HAptagel conditions in which the RGD-HAptagel had the greatest fluorescent intensity of any condition (Figure 4a). To confirm this promising synergy for cell survival between the RGD peptide and the anti-VEGF-R2 aptamer, we measured cell viability using an MTT assay. Quantification of metabolized MTT by endothelial cells after 2 days of culture with the different hydrogel conditions showed the same response as that of the LIVE/DEAD imaging. Viability was less than 20% for the naked hydrogel and the aptamer-only hydrogel with respect to that of a tissue culture polystyrene control. Surprisingly, the RGD-only hydrogel only had a viability of ~65% whereas the RGD-HAptagel had a viability of ~95% (Figure 4c). Phase contrast from each condition mirrored those during the cell attachment experiments and LIVE/DEAD imaging; attached endothelial cells were only observed for the hydrogels with RGD peptide. Interestingly, the RGD-HAptagel condition showed possible tubulogenic structures that were not seen in the other conditions (Figure 4d).

Figure 4.

Cytotoxicity was quantified after 6 days of culture using a LIVE/DEAD assay from the fluorescent intensity of live cells and dead cells stained with calcein AM and ethidium bromide homodimer (a). Representative fluorescence images of the cells stained during the LIVE/DEAD assay after 2 and 6 days of culture (b). Cell viability was quantified using a MTT assay after 2 days of culture (c). Representative phase contrast images of the cells from MTT assay (d). In all images the scale bar is 200 μm. One-way ANOVA with post hoc Tukey HSD tests were used to assess statistical significance between groups. Statistical significance was defined as * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

2.4. Cell Proliferation

Growth curves over 6 days trended with the results of attachment and viability. Cell proliferation was tracked using the cell proliferation dye, Cytopainter at 2-day increments for a total of 6 days. The dye tracks proliferation because it is retained by cells during cell division such that daughter cells contain the dye. As expected, endothelial cells displayed negative growth on both the naked HA-PEGDA hydrogel and the aptamer-only hydrogel; decreasing by ~95% and ~75%, respectively (Figure 5). This was not the case for gels that facilitated cell attachment. Whereas the RGD-only condition more or less maintained cell number until day 6 where there was a slight decrease, the RGD-HAptagel stimulated a consistent increase in cell number over time (Figure 5). This enhanced proliferation encouraged by the RGD-HAptagel could explain the observed increase in metabolic activity from the MTT assay and the increased fluorescence from the LIVE/DEAD assay for endothelial cells cultured with the RGD-HAptagel. This net increase in cells can be especially useful in wound healing where quick proliferation of endothelial cells is necessary for new capillary formation. Overall, cell proliferation also demonstrated a synergy between the effects of the RGD peptide and the anti-VEGF-R2 aptamer functionalized to a HA-PEGDA hydrogel.

Figure 5.

Endothelial cell growth curves over 6 days. Data at each time point represent mean ± standard deviation from 3 replicates.

2.5. Cell Migration

Chemotactic migration to the gel favored gels that were functionalized with the DNA aptamer. To investigate migration to the gels, the gels were loaded with VEGF, a chemoattractant for endothelial cells. All sets of gels had VEGF except for a tHA-PEGDA negative control. Then endothelial cells were seeded into a porous transwell that was placed above the gels (Figure 6a). Cells that migrated to the gels were stained with calcein AM and ethidium bromide homodimer and their fluorescence was quantified. Building on our results thus far as expected, we observed the greatest migration and attachment to the RGD-HAptagel (Figure 6b). This meant that the RGD peptide, VEGF, and aptamer work together to increase migration and adhesion. Surprisingly, the aptamer-only condition had the second most number of migrated cells. This could be due to the involvement of a biochemical pathway being initiated with VEGF and our anti-VEGF-R2 DNA aptamer together, which needs to be investigated in more detail. Thus, we concluded that the aptamer along with VEGF increased cell migration to the gel. Conversely, RGD peptide did not aid in migration to the gel. There was no significant difference in the number of migrated cells between the RGD-only and naked hydrogel loaded with VEGF (positive control). This meant that though RGD peptide enabled attachment to the gel, but it gave no additional attractive benefit to the gel unlike with the aptamer. Little to no migration was observed for the naked hydrogel without VEGF chemoattractant as compared to the other conditions, which was confirmed visually (Figure 6c). We can infer through this experiment that though VEGF has a high chemotactic effect on cells, this effect increases when it is combined with the receptor-ligand specific signaling of an immobilized anti-VEGF-R2 DNA aptamer.

Figure 6.

Schematic of the chemotactic migration assay. (a). Quantification of fluorescent intensity produced by migrated cells stained with calcein AM (b). A one-way ANOVA with post hoc Tukey HSD test was used to assess statistical significance between groups. Statistical significance was defined as * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. Representative fluorescence images of migrated cells stained with calcein AM (live cells) and ethidium bromide homodimer (dying cells) (c). For the HA-PEGDA conditions, (−) indicates no VEGF loading and (+) indicates with VEGF loading. All other conditions were with VEGF loading. For all images, the scale bar is 200 μm.

2.6. Tube Formation

Tube formation is a first step indicator of a matrix that could support angiogenesis. We cultured endothelial cells on the different gels and imaged the cells for their organization after 24 hours. Only the combination of RGD peptide and anti-VEGF-R2 DNA aptamer gave rise to tube formation (Figure 7). For comparison, tube formation has also been seen for endothelial cells on natural matrices of collagen starting after 7 days of culture.^[33] Thus, it was quite remarkable to observe tube formation after 24 hours. In most cases, this rapidity for tube formation has been shown when endothelial cells were encapsulated in 3D Matrigels.^[34] But in our experiment, cells had not been encapsulated, rather they were seeded on top. The blurriness of some tubes could be attributed to the 3D nature of the hydrogel and an inability to focus on all planes. It is important to note the HGM contains 5 ng/mL VEGF. This was used in all conditions and could initiate tube formation. Tube formation incited by the RGD-HAptagel was also seen during the MTT assay in which the formazan crystals highlighted tubules (Figure 4d). VEGF-R2 plays an important role in tube formation by activating the Akt pathway while VEGF-R1 does not play a role in tube formation and angiogenesis.^[35,36] Thus, we can infer that specific targeting of VEGF-R2 might be responsible for inciting tube formation without the cross-interference of VEGF-R1 signaling from VEGF-A and VEGF-B in the HGM. This could explain the tube formation in samples with the anti-VEGF-R2 DNA aptamer while there was no rearrangement into tubes for the RGD-only gels at the end of 24 hours; despite, the low amount of VEGF in the media. Understandably, there was no arrangement by endothelial cells on the naked gel and the HAptagel due to their inability for endothelial cell attachment perpetuated by the absence of an available adhesion peptide.

Figure 7.

Representative fluorescence images of endothelial cells taken during the in vitro tube formation assay. Endothelial cells were seeded on top of the hydrogel and cultured in HGM. After 24 hours, cells were stained with calcein AM. White arrows indicate locations of tubule formation. For all images, the scale bar is 200 μm.

3. Conclusion

We successfully synthesized a bifunctional hyaluronic acid hydrogel with both anti-VEGF-R2 DNA aptamer and RGD peptide that together synergized to yield an enhanced cellular response. The RGD peptide provided regions for cell attachment to the gel that supported adherent cell viability. At the same time, the aptamer enhanced cell migration and long-term cell survival, which could be due to the VEGF-R2 signaling pathway. Especially promising was the formation of tubules that are indicative of the angiogenic potential of this bifunctional hydrogel. The dual functional hydrogel could advantageously circumvent issues of adhesion and migration related to synthetic scaffold materials by using signaling and materials native to natural ECM. Because hyaluronic acid gels are both injectable and form in situ, they can be used as a filler in chronic wounds. When used for this application, our bifunctional hyaluronic acid hydrogel may encourage enhanced migration of endothelial cells to support greater revascularization at the site of injury. Because hydrogels swell, they can be used to elute drugs for delivery at wound sites, which could be additional means to give this bifunctional hydrogel even more potential to stimulate healing. This research paves the way by demonstrating the synergistic ability of rationally chosen biomolecules in natural hydrogels to enhance favorable cell processes. Future studies will look to evaluate the in vivo action of this dual functionalized hydrogel.

4. Experimental Section

Hydrogel Materials:

Thiolated hyaluronic acid (tHA) and polyethylene glycol diacrylate (PEGDA) (average molecular weight: 3400 Da) were purchased from BioTime Inc. (Almeda, CA, USA), now part of Advanced Biomatrix (Carlsbad, CA, USA). Lyophilized acrydite-modified DNA aptamer was purchased from Integrated DNA Technologies (Coralville, IA, USA). Thiol-modified RGD peptide was purchased from Cellendes (Reutlingen, Germany).

Aptamer Preparation:

The VEGF-R2 targeting aptamer was prepared for incorporation with the tHA hydrogel. The anti-VEGF-R2 DNA aptamer sequence was ‘5- GAT GTG AGT GTG TGA CGA GCT ACG ACG TCT GGT GTA ATT TAT AAA GAC ACT GTG TAT ATC AAC AAC AGA ACA AGG AAA GG −3’.^[28] It was purchased from Integrated DNA Technologies with an acrydite group conjugated to the 5’ end of the oligomer. The acrydite group reacts with free thiols on the tHA to form a thioether bond that is stable at both physiological pH and physiological temperature. Under sterile conditions, the aptamer was reconstituted and diluted with sterile deionized water to a stock concentration of 300 μM. To activate the oligomer, 100 μl of the stock solution was boiled to 94 °C for 3 minutes and then cooled back to room temperature.

Hydrogel Preparation:

The hydrogel was prepared in a three-step process by first reacting the tHA with the acrydite functionalized aptamer sequence, then forming a partially crosslinked gel with the PEGDA and finally incorporating the RGD adhesion peptide (Figure 1). Under sterile conditions, lyophilized tHA was reconstituted to 1 w/v% solution with sterile deionized water and mixed on an orbital shaker for 40 minutes to dissolve the material. The activated aptamer was added to the tHA solution to a final concentration of 15 μM and incubated for 10 minutes to initialize the thiol-acrydite reaction. The crosslinker PEGDA was dissolved in sterile deionized water at 1 w/v% solution and vortexed briefly to form a clear solution. PEGDA was added to the tHA and aptamer solution at varying ratios of PEGDA to tHA and vortexed for 5 minutes. The solution was pipetted and incubated for 30 minutes at room temperature. Different volumes and containers were used to create different sized gels from films to droplets. After 30 minutes the gels were partially crosslinked so that some unreacted acrylate groups remained for subsequent reaction with the thiol-modified RGD peptide. The thiol-modified RGD peptide was prepared by reconstituting the lyophilized material in sterile phosphate-buffered saline (PBS). The partially crosslinked hydrogels were immersed in 0.2 mM RGD solution and incubated overnight in a cell culture incubator kept at 37°C, 5% CO₂, and 95% relative humidity. The thiol groups on the RGD sequence reacted with the remaining acrylate groups of the PEGDA to form stable ester bonds. Afterwards, the hydrogels were thoroughly rinsed with PBS to remove any unreacted species. For all imaging experiments, hydrogels were made using 50 μl of solution for 96 well plates or 150 μl for 24 well plates. For migration experiments, UV-sterilized parafilm was placed on the bottom of the wells of a 24 well plate to create a hydrophobic layer for the hydrogel solution to stay in a droplet-shape.

Swelling Ratio:

The swelling ratio of different hydrogel formulations was used as an indirect measure of gel crosslinking density. Swelling ratio was defined as the mass difference of the gel with imbibed water and the dry gel normalized to the mass of the dry gel. Measurements were performed for hydrogels formed using a 3:1, a 3:2, and a 1:1 ratio of tHA to PEGDA. The hydrogels were made without aptamer or RGD to understand the influence of the PEGDA crosslinker. After forming, the gels were air-dried overnight. Dried gels were massed and then immersed in 10 ml of PBS. Over 16 hours at multiple timepoints, the gels were removed, carefully blotted to remove excess PBS, massed, and then returned to incubate in PBS.

Degradability:

Hydrogel degradability was tested using an in vitro assay. Degradation was measured as the percent weight loss over time calculated as the difference between the initial weight and the final weight normalized to the initial weight. First, complete hydrogels were swelled in PBS and massed after blotting excess buffer. The hydrogels were then immersed in PBS and incubated in a cell culture incubator kept at 37°C, 5% CO₂, and 95% humidity. Over 18 days, the gels were removed and massed after blotting excess buffer.

Cell Culture:

Human umbilical vein endothelial cells (HUVECs) were cultured in the hydrogels. HUVECs were purchased from Lifeline Cell Technologies (Oceanside, CA, USA) and used between passages 4 to 7. They were cultured in a cell culture incubator at 37 °C, 5% CO₂, and 95% humidity. Cells were cultured with growth medium (HGM) made with VascuLife® Basal Medium (Lifeline Cell Technologies, Oceanside, CA, USA) supplemented with VascuLife® LifeFactors® kit (Lifeline Cell Technologies, Oceanside, CA, USA) that includes fetal bovine serum (FBS), recombinant human (rh) fibroblast growth factor, rh VEGF, rh insulin-like growth factor-1, hydrocortisone hemisuccinate, ascorbic acid, L-glutamine, heparin sulfate, rh epidermal growth factor, gentamicin and amphotericin B. HUVECs were seeded at 100,000 cells onto the hydrogels.

Cell Attachment:

Cell attachment was assessed for different hydrogel formulations containing only RGD peptide (RGD-HA-PEGDA), only aptamer (HAptagel), both RGD peptide and aptamer (RGD-HAptagel), and without RGD peptide or aptamer (HA-PEGDA). Attachment to the different functionalized gels was observed by phase contrast microscopy after 2, 6, and 12 hours. Gels were imaged using a Nikon TE-2000U inverted phase contrast microscope (Nikon Instruments, Melville, NY, USA).

Cell Viability and Cytotoxicity:

Cytotoxicity was tested using a fluorescent LIVE/DEAD viability assay. Cells were stained with a solution of 2 μM calcein AM and 4 μM ethidium homodimer (Thermo Scientific, Waltham, MA, USA) prepared in sterile PBS. Calcein AM is a green fluorescent stain for live cells while ethidium homodimer is a red fluorescent stain for dead or dying cells. After 2 and 6 days of culture with the gels, the wells were rinsed with serum-free HGM to reduce background fluorescence and the cells were incubated with the staining solution for 45 minutes in a cell culture incubator. Gels were imaged using a Nikon TE-2000U inverted epi-fluorescence microscope (Nikon Instruments, Melville, NY, USA). Fluorescent images were taken at each timepoint and were analyzed using ImageJ (NIH, Bethesda, MD) following a method by Burgess et. al.^[37]

Cell viability was quantified using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (MTT) assay (Abcam, Cambridge, UK). Briefly, in a 96 well plate, 50 μL formed gels were seeded with 30,000 cells per well. 3 wells of the tissue culture plate (TCP) were also seeded as control. After 2 days of culture, MTT reagent was added to the wells and incubated for 4 hours in a cell culture incubator. The cells metabolized the reagent forming purple formazan crystals. In the wells, the crystals were then dissolved overnight at 37°C while on an orbital shaker using a supplied detergent. The resulting solution from each well was transferred to its own well in a black walled 96 well plate. Absorbance was measured at 590 nm using a UV–Vis Synergy H1 plate reader (BioTek, Winooski, VT, USA). For background subtraction, wells with just media and MTT were added. The percentage of viable cells relative to control calculated as,

$$\%\mathit{\text{viable}}\text{cells}=\frac{{\mathit{\text{OD}}}_{\mathit{\text{sample}}}-{\mathit{\text{OD}}}_{\mathit{\text{background}}}}{{\mathit{\text{OD}}}_{\mathit{\text{sample}}}-{\mathit{\text{OD}}}_{\mathit{\text{background}}}}\times 100\%.$$

Cell Proliferation:

Cell proliferation was determined using a cell proliferation stain. Prior to seeding the hydrogels, cells were fluorescently stained with Cytopainter Orange (Abcam, Cambridge, UK), which stains live cells orange at an excitation wavelength of 488 nm and emission wavelength of 542 nm and is incorporated into dividing cells. Cytopainter was reconstituted according to the manufacturer’s instructions. The staining solution was prepared by diluting 1 μL of stain with 500 μL of sterile PBS. HUVECs were trypsinized, pelleted, and resuspended in the staining solution. The cells were incubated in the solution for 30 minutes in a cell culture incubator. The HUVECs were then repelleted, the staining solution removed and rinsed once with PBS, and resuspended in HGM. Then these cells were seeded at 25,000 cells/well onto hydrogels prepared in a black wall, clear bottom 96-well plate. Fluorescent intensity was measured at 2 days, 4 days, and 6 days with a UV–Vis Synergy H1 plate reader (BioTek, Winooski, VT, USA). HGM blanks were used to subtract any background fluorescence due to the cell culture media. Standard curves were prepared by measuring the fluorescence of known numbers of stained cells.

Cell Migration:

Cell migration to the hydrogel was assessed using a chemotactic transwell migration assay. 24-well transwell plate inserts with 8 μm pore size PET membrane were used (Corning, Corning, NY, USA) in conjunction with a 24-well ultra-low attachment plate. In the plate, the droplet shaped gels were incubated in a cell culture incubator overnight with HGM containing 50 ng/mL VEGF. This was to load the hydrogels with VEGF before cell seeding. Following incubation, the inserts were placed in contact with the gels directly underneath them. HUVECs were resuspended in VEGF-free HGM and were seeded onto the top side of the inserts at a seeding density of 100,000 cells per well. The media in the insert was VEGF-free HGM while the media in the wells was supplemented with VEGF except for a HA-PEGDA negative control, which was without VEGF in the well or insert. The cells were then cultured for 7 days to allow their migration through the inserts into the gels. At day seven, the gels were then stained and analyzed in the same way as in the Cell Viability and Cytotoxicity section.

Tube Formation:

In vitro tube formation was assessed using a modified protocol of that commonly employed for endothelial cells cultured on Matrigel.^[38] HUVECs were cultured in HGM and seeded on hydrogel samples at a density of 30,000 cells/well in a 96-well plate. Gels were monitored and upon tubule formation, the cells were stained with 2 μM calcein AM diluted in sterile PBS and were imaged.

Statistical Analysis:

All data is presented as mean ± standard deviation. The number of replicates for each experiment is specified in the text with data presented as individual points. Where applicable, one-way ANOVA with post hoc Tukey HSD tests were used to assess statistical significance between groups. Statistical significance was defined as a p value less than 0.05. All statistical tests were conducted in GraphPad Prism 8