VEGF-delivering PEG hydrogels promote vascularization in the porcine subcutaneous space

Abstract

For cell therapies, the subcutaneous space is an attractive transplant site due to its large surface area and accessibility for implantation, monitoring, biopsy, and retrieval. However, its poor vascularization has catalyzed research to induce blood vessel formation within the site to enhance cell revascularization and survival. Most studies focus on the subcutaneous space of rodents which does not recapitulate important anatomical features and vascularization responses of humans. Herein, we evaluate biomaterial-driven vascularization in the porcine subcutaneous space. Additionally, we report the first use of cost-effective fluorescent microspheres to quantify perfusion in the porcine subcutaneous space. We investigate the vascularization-inducing efficacy of vascular endothelial growth factor (VEGF)-delivering synthetic hydrogels based on 4-arm poly(ethylene) glycol macromers with terminal maleimides (PEG-4MAL). We compare three groups: a non-degradable hydrogel with a VEGF-releasing PEG-4MAL gel coating (Core+VEGF gel); an uncoated, non-degradable hydrogel (Core-only), and naïve tissue. After 2 weeks, Core+VEGF gel has significantly higher tissue perfusion, blood vessel area, blood vessel density, and number of vessels compared to both Core-only and naïve tissue. Furthermore, healthy vital signs during surgery and post-procedure metrics demonstrate the safety of hydrogel delivery. We demonstrate that VEGF-delivering synthetic hydrogels induce robust vascularization and perfusion in the porcine subcutaneous space.

INTRODUCTION

Cell therapies represent transformative approaches to treat or cure various diseases.^1–3 Hydrogel technologies can further enhance the retention, survival, and efficacy of therapeutic cells by functioning as delivery vehicles, instructional templates, and immunoprotecting devices.⁴ Indeed, cell-based therapies delivered in biomaterial constructs have emerged as promising platforms to treat many diseases. Such applications include but are not limited to myocardial infarction,^5,6 muscular dystrophy,⁷ degenerative disorders of the central nervous system,⁸ inflammatory bowel syndrome,⁹ implant infection,¹⁰ and type 1 diabetes.^11–13

The subcutaneous space is an attractive transplant site for biomaterial-delivered cell therapies because of its large surface area for non-marginal transplant volumes, surgical accessibility, minimal invasiveness, ease of monitoring, retrievability, and replenishment.^{14 15} However, the poor vascularization of the subcutaneous space results in less-than-optimal oxygen levels, which can lead to cellular dysfunction and death within implants. This is especially true for highly metabolically active cells such as insulin-producing cells for the treatment of type 1 diabetes.^16,17 Enhancing the vascular connectivity of the cellular graft can increase the survival of cells that require external-sensing inputs for function. Indeed, enhancing the connection between insulin-producing cells and host vasculature has been shown to help the delivered cells sense glucose, transport therapeutic paracrine/endocrine factors, and secrete insulin on-demand.¹⁸ In addition to increasing the survival of delivered cells, vascularizing the subcutaneous space enhances medication absorption; Steyn et al. found that implantable, vascularizing microchambers rapidly increased exogenous insulin uptake compared to conventional subcutaneous injections.¹⁹ As such, strategies to increase vascularization in the subcutaneous space have been pursued and are summarized in the subsequent paragraph. To prioritize clinical translation, it is imperative that any vascularization strategy minimize technical, safety, and regulatory burden.

A promising strategy to induce vascularization in transplant sites is the biomaterial-mediated delivery of angiogenic factors. Growth factors such as vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF) can be encapsulated within materials for soluble release or chemically tethered to biomaterials for sustained, local release.^20–26 Other strategies include the co-delivery of therapeutic cells with pro-angiogenic supporting cells such as mesenchymal stromal cells,²⁷ fibroblasts,²⁸ and endothelial cells.²⁹ However, different cell types must compete for nutrients, and regulatory burden increases with multiple cell types in a single graft. Another strategy is pre-vascularization, a two-step approach that requires an initial surgery to create a cavity for vessel formation and then a second surgery to introduce cellular grafts in the transplant site.³⁰ While pre-vascularization has shown success^31,32, the need for multiple procedures may be a disadvantage.

Our group has engineered a synthetic hydrogel platform based on 4-arm poly(ethylene) glycol with terminal maleimide (PEG-4MAL) macromers to release VEGF in a sustained, on-demand fashion.^{12,25,26,33,34} Specifically, VEGF and cysteine-containing adhesive peptides are conjugated to the PEG-4MAL macromer via reaction with the maleimide group to produce a functionalized macromer. This functionalized precursor is then crosslinked into a network by reacting with a cysteine-flanked crosslinker peptide susceptible to cleavage by proteases including matrix metalloproteinases. Infiltrating host cells remodel the gel via proteolytic degradation within 2–4 weeks in rodents. For this VEGF-delivering hydrogel, the resultant vasculature was functional and connected to the host vasculature, did not leak, and persisted for at least 6 months.^25,26,33,34

Unlike free-radical polymerization and other Michael-type addition chemistries, the PEG-4MAL platform generates structurally defined hydrogels with stoichiometric incorporation of ligands, improved crosslinking efficiency, and excellent in vitro and in vivo cytocompatibility.^35,36 Furthermore, PEG-4MAL exhibits minimal inflammation and toxicity, and the degradation products are rapidly excreted (<24 h) via the urine.³⁵ We previously demonstrated that these VEGF-delivering hydrogels induce vascularization in multiple transplant sites within rats and mice^12,26,33,34 and, within the context of treating type 1 diabetes, facilitate the return to stable, long-term euglycemia.²⁶

While we recognize the advances made with rodents (i.e., small animal models), we also acknowledge the preclinical motivations for large animal models. Transplant volumes must be scaled up from rodents to humans. For example, for type 1 diabetes, using assumptions such as ~10,000–12,000 IEQ/kg^37,38 and 5–10% packing volume,³⁹ the necessary volume of therapeutic cargo for insulin independence is – at a minimum −15 mL for humans, 300 times greater than the necessary volume our lab has used to achieve normalized blood glucose levels in rodents.²⁶

Pigs are a promising large animal model. Anatomically and physiologically similar to humans, pigs have already served as human-adjacent models for cutaneous wound healing,⁴⁰ diabetes,⁴¹ cardiovascular disease,⁴² and neurological disease.⁴³ Most relevant to this work, pigs have similarities to the human skin and cutaneous layers: a relatively thick epidermis and dense elastic fibers within the dermis,⁴⁴ as well as comparable immune cell profiles⁴⁵ and collagen biochemical structure.⁴⁶ While rodent skin is loose and slides over the subcutaneous fascia, porcine skin functions like human skin by adhering to underlying structures.^{40 47} Taken together, the similarities between pig and human skin translate to comparable wound healing processes and reactions to therapies within the subcutaneous space. In this study, we built upon our previous rodent work by developing a more clinically relevant animal model. Using the Yucatan minipig, we evaluated the ability of VEGF-delivering PEG-4MAL hydrogels to promote vascularization in the porcine subcutaneous space.

MATERIALS AND METHODS

Synthetic hydrogel fabrication

The nondegradable synthetic hydrogel (used as an implant core for tracking purposes) was fabricated with 5.0% (weight/volume) 4-arm, amide-linked poly(ethylene) glycol macromer functionalized with norbornene terminal groups (PEG-4aNB, 20 kDa, 98% end-group functionalization, JenKem), 1.0 mM RGD adhesive peptide (GRGDSPC, Genscript), 4.17 mM dithiothreitol (DTT, Invitrogen UltraPure), and 1.0 mM lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate photoinitiator (LAP, TCI America). Following our prior work,⁴⁸ hydrogel precursor solution was cast into a cylindrical polydimethylsiloxane (PDMS) mold and crosslinked with UV light (λ = 365 nm, I₀ = 10 mW cm⁻²) for 20 s before swelling in PBS overnight at 20 °C. The cylindrical PDMS mold was manufactured with a diameter of 22 mm and a height of 4 mm, to accommodate 1.5 mL of hydrogel precursor solution. PEG-4aNB-based nondegradable hydrogels, which served as implant cores in the animal study, were fabricated 24 h prior to the surgery and stored in an incubator under standard cell culture conditions (5% CO₂, 20% O₂, 37°C, and 95% relative humidity).

The VEGF-releasing hydrogel coating (1.25 mL) was prepared as previously described¹² with the following specifications: 4.5% (weight/volume) 4-arm maleimide-end functionalized PEG macromer (PEG-4MAL, 20 kDa, >95% end-group functionalization, Laysan Bio), 10 μg mL⁻¹ recombinant human VEGF-A165 (Invitrogen), and 1.0 mM RGD peptide (GRGDSPC). The resultant functionalized macromer was then crosslinked with 3.61 mM VPM bi-cysteine peptide (GCRDVPMSMRGGDRCG, Genscript), which was calculated for stoichiometric balance (1:1 ratio between the cysteine groups on VPM and the residual maleimide groups on the PEG-4MAL macromer following functionalization with VEGF and RGD).³⁶

Animals

Five Yucatan miniature pigs (minipigs) were purchased from Premier BioSource (Rensselaer, IN, USA). All procedures were approved and conducted in accordance with the Translational Testing and Training Laboratories (T3 Labs) and Georgia Tech Animal Care and Use Committees. Animal housing and procedures took place at T3 Labs (Atlanta, GA, USA), a Georgia Tech-affiliated facility that complies with the following animal welfare standards: United States Agriculture (USDA) registration, Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) Accreditation, Animal Welfare Act Compliance, and Division of Public Health Services (PHS) Approved Animal Welfare Assurance (Office of Laboratory Animal Welfare).

Anesthesia

Prior to all procedures, animals were fasted a minimum of 12 hours overnight. Animals were then sedated with a combination of ketamine (10 mg kg⁻¹) and xylazine (1 mg kg⁻¹) delivered intramuscularly. To maintain anesthesia, animals were intubated and mechanically ventilated with isoflurane (2–3%) and oxygen (2 O₂ L min⁻¹). Heart rate was monitored via Doppler probe placement on the ventral tail artery. Blood pressure was measured directly through a peripheral vein catheter. Oxygen saturation and end-tidal CO₂ were recorded using pulse oximetry and capnometry, respectively. Rectal temperature was recorded with a digital thermometer. Vitals were recorded every 15 minutes. All procedures were performed under the supervision of a veterinarian. Animals recovered from anesthesia with no complications. During the 2 weeks prior to the terminal procedures, animals were fed and observed daily. No signs of pain and discomfort were recorded. No interventions were required.

Implantation of hydrogel samples

All gels were implanted in the ventral subcutaneous space of nondiabetic Yucatan minipigs (n=5, bodyweight: 43.7 ± 4.0 kg, age: 7–10 months). As the effect of sex has been reported in Yucatan minipig models for surgical grafts⁴⁹ and vasculature differences⁵⁰, we used a combination of males (n=2, pigs 1 and 4) and females (n=3, pigs 2, 3, and 5).

Experimental implants and naïve, untouched tissue sites (4 Core-only, 4 Core+VEGF gel, and 4 naïve sites) were evenly distributed in two lengthwise columns along the ventral subcutaneous space. The locations of experimental samples were randomized in every minipig. As the Core-only gel measured 22 mm in diameter, each anesthetized minipig received semicircle-shaped incisions each approximately 25 mm in length. A 20-mm buffer on either side of a sample separated it from adjacent samples. All sides of the implants were surrounded by subcutaneous tissue (i.e., not the skin or muscle layers). Control gel sites only received pre-cast nondegradable gel (Core-only). For the Core+VEGF gel group, the VEGF-releasing hydrogel coating was delivered with gelation in situ. Within the surgical pocket, to coat the bottom surface of the transplant site, 420 μL (1/3) of the VEGF-releasing hydrogel coating was first deposited. To integrate the coating with the core, the nondegradable hydrogel core was then immediately inserted (i.e., within 15 seconds) atop the bottom coating. The remaining 830 μL (2/3) of the VEGF-releasing hydrogel coating was delivered to coat the top and sides of the nondegradable hydrogel core. The placement of the core atop the bottom coating and the placement of the top coating each occurred within 15 seconds, well within the complete polymerization of the overall coating. No cells were encapsulated in any gel.

Fluorescent microsphere injection and quantification

At 2 weeks post-implantation, local tissue perfusion for transplant sites and reference native tissue was measured using fluorescent microspheres as previously described.^51,52 Each minipig was prepared for fluorescent microsphere injection and sample explantation with sedation and anesthesia performed as above. Two 5-Fr pigtail catheters were inserted through femoral sheaths into the left ventricle and the descending abdominal aorta (near the central vertebrae, in the center of the implant sites) for microsphere injection and withdrawal of the reference blood sample, respectively. Fluorescent microspheres (1–3 × 10⁷, 15-μm diameter, yellow-green [505/515], Molecular Probes) were injected uniformly over 30 seconds via syringe pump in tandem with withdrawal of a reference blood sample (7.5 mL min⁻¹, 3 min duration). Pigs were euthanized with intravenous administration of pentobarbital sodium and phenytoin sodium (Euthasol). Next, gels and their immediate surrounding tissue (30 mm × 30 mm × 30 mm) were explanted. Each sample weighed between 1–3 grams. Naïve tissue was also explanted to serve as a reference. Tissue samples from each explant were processed concurrently with reference blood samples as described.^51,52 In short, tissue samples underwent autolysis for 2 weeks, followed by grounding, digestion using a 50°C bacterial shaker, and fluorescent dye extraction using 2-ethoxyethylacetate. Quantification of tissue perfusion was achieved through fluorimetry readings following digestion via a multi-mode microplate reader (BioTek Synergy H1).⁵¹ The perfusion data of the Core-only and Core+VEGF groups were not normalized to the naïve control.

Histology and image analysis

Tissue samples for each implant group and naïve tissue were fixed in neutral-buffered formalin, processed for paraffin, cut into 5-μm thick sections, and stained for hematoxylin and eosin (H&E), Masson’s Trichrome, or immunostaining. Stains for DAPI, CD31, and α-SMA were achieved using the following reagents and antibodies: DAPI (Invitrogen D1306, 1:500), anti-CD31 rabbit IgG (Abcam ab28364, 1:50), anti-α-SMA mouse IgG (Abcam ab7817, 1:250), goat anti-rabbit IgG-AlexaFluor 488 (Invitrogen A11034, 1:250), and goat anti-mouse IgG-AlexaFluor 633 (Invitrogen A21050, 1:250). H&E- and Masson’s Trichrome-stained sections were imaged on a Zeiss Axios Observer microscope using both a 5X objective (0.16 numerical aperture, working distance 12.1 mm) and a 10X objective (0.3 numerical aperture, working distance of 5.2 mm). Immunostained sections were imaged on a Zeiss 700 confocal microscope using a 20X objective (0.8 numerical aperture, working distance of 0.55 mm, field of view of 25 mm). From immunostained sections, vascular structures adjacent to fibrous capsules were quantified for blood vessel metrics as previously described.⁵³ For quantification of capsule thickness, 2 H&E sections per experimental group were analyzed. Each section was then input on ImageJ to obtain a total of ten thickness measurements for averaging.

Statistical analyses

Microsphere perfusion was analyzed by nested one-way ANOVA followed by Tukey’s multiple comparisons test. Fibrous capsule thickness was analyzed by paired t test. Quantified blood vessel metrics (area, area by percentage, density, and number of structures per confocal image area) were analyzed by nested one-way ANOVA followed by Tukey’s multiple comparisons test. Comparisons of vascularization metrics between sexes were performed by ordinary two-way ANOVA followed by uncorrected Fisher’s least significance difference multiple comparisons test. Correlation between microsphere perfusion and quantified blood vessel metrics was analyzed via Spearman’s correlation. Statistical significance was defined as p<0.05. All analyses were carried out with GraphPad Prism (version 9).

RESULTS

Design of synthetic PEG hydrogel implants

We have previously characterized our PEG-VEGF gels via rheology and found no difference in storage modulus (~150 Pa) between them and PEG controls.⁵⁴ For protein stability during formulation, we have validated the conjugation of VEGF to PEG-MAL via gel electrophoresis^34,54. The in vitro release kinetics^34,54 as well as in vivo release profile for PEG-VEGF gels in the subcutaneous space²⁵ and the transabdominal area have been reported.³⁴ To indicate that tethered VEGF retains bioactivity and the PEG macromer does not interfere with VEGF-dependent signaling, we have run endothelial cell metabolic assays and assessed network formation of endothelial cells and demonstrated no loss in bioactivity.⁵⁴ For non-immunogenicity/toxicity purposes, we have run biodistribution studies of analog degradation products, conducted blood serum biochemistry, and analyzed organ histopathology with no adverse effects or toxicities noted.³³

For the subcutaneous space, a major limitation for cell therapies is insufficient vascularization to deliver necessary oxygen and nutrients to therapeutic cargo. We have previously shown the efficacy of our standalone VEGF-delivering hydrogel to induce vascularization in various extrahepatic sites and, as a result, promote graft survival in diabetic mice.²⁶ Herein, we sought to further extend the application of our system as a coating. Particularly, we aimed to evaluate the efficacy of a degradable, VEGF-delivering hydrogel coating to promote vascularization in the porcine subcutaneous space.

Experiments were designed to conduct parallel comparisons of implants without and with a VEGF-delivering hydrogel coating (groups identified as Core-only and Core+VEGF gel, respectively). Between the two types of implants, the only difference was the outer hydrogel coating of the Core+VEGF gel platform (Figure 1). The hydrogel coating was crosslinked with the dithiol protease-cleavable peptide degradable crosslinker VPM (GCRDVPMSMRGGDRCG) and, therefore, engineered to release VEGF upon in vivo proteolytic, including matrix metalloproteinase-1 and −2, degradation.^26,33 Both Core-only and Core+VEGF gel platforms featured a hydrogel core crosslinked by the nondegradable crosslinker DTT. A nondegradable system was chosen to model an implanted device and to facilitate the location of the implant sites at explantation.

Figure 1.

Chemical structures of (a) PEG-4MAL and (b) PEG-4NB. Schematic of synthetic PEG hydrogels. (c) Protease-degradable, VEGF-releasing PEG-4MAL hydrogel coating. (d) Non-degradable PEG-4NB core.

Porcine ventral subcutaneous implantation model

This work aims to establish a baseline model for future studies focused on vascularizing islets delivered to the subcutaneous space of diabetic pigs. Thus, we selected the Yucatan minipig given its extensive use as a preclinical model for diabetes^55–57 and its anatomical similarities in anatomy and vasculature to humans.⁵⁸ We chose the ventral subcutaneous space among others (e.g., dorsal) to mimic the standard implant location of continuous glucose monitors.^59,60 In addition, this location reduced the ability of the animal to perturb the implant by rubbing against the cage or floor. We tested two implant groups, (i) Core-only and (ii) Core+VEGF gel, and we included naïve tissue as a reference (Figure 2a). The naïve tissue is untouched porcine subcutaneous tissue (no defects or incisions were made to mark the naïve tissue; at takedown, to ensure that similar tissue volumes of naïve tissue were analyzed compared to the experimental groups, each tissue sample measured 30 mm × 30 mm × 30 mm and weighed between 1–3 g). In individual minipigs, each implant group occupied 4 sites plus 4 naïve tissue sections, totaling to 12 sites under investigation per animal. Transplant/naïve tissue sites were randomized across all 5 animals (Figure 2b). After 2 weeks, minipigs received microsphere injection for quantification of tissue perfusion before euthanasia. For each subject, samples of each implant group and reference tissue were either explanted for autolysis prior to microsphere digestion (3 gels/samples) or histology/immunostaining (1 gel/sample) (Figure 2c).

Figure 2.

Experimental set-up. (a) Implant groups and reference group, denoted by color. The “Core + VEGF gel” group differs from the “Core only” group only by its outer, degradable VEGF-delivering gel coating (shown in purple). Implant types have nondegradable, inner gels (cores). The core measured 22 mm in diameter and 1.5 mL in volume. The PEG-VEGF coating was 1.25 mL in volume. Naïve tissue = no gel. (b) Porcine subcutaneous model: healthy, nondiabetic Yucatan miniature pigs. 4 transplant sites per implant group or reference group, randomized in each of the five pigs in the ventral subcutaneous space. (c) Analyses to evaluate vascularization: (top) fluorescent polystyrene microsphere injection through the left ventricle and (bottom) quantification of vascular markers CD31 and α-SMA via immunohistochemistry.

Delivery of PEG hydrogels is not detrimental to animal health

To confirm that the gel implantation procedure did not impact minipig health, we tracked the vital signs of all animals throughout the duration of the surgery and monitored their eating, weight-gain, and walking habits until the terminal procedure 2 weeks later. The implantation procedure ranged from 75–120 minutes. All gel implants were well tolerated with no outward signs of inflammation at the local site during, immediately following, and 14 days following the initial implantation procedure (Supplementary Figure 1).

To monitor the stress levels of anesthetized animals during the gel implantation procedure, we tracked vital signs (Figure 3). A previous study focused on the heart vitals of female Yucatan minipigs (16–30 kg, 4–6 months) cited normal heart vitals to be 135 ± 17 bpm for heart rate, 99 ± 9.2 mmHg for mean blood pressure, 80 ± 9.4 mmHg for diastolic blood pressure, and 128 ± 12.6 mmHg for systolic blood pressure.⁶¹ Healthy body temperature for pigs has been reported at 98.6–100.4°F.⁶² In one study focused on testing oxygen saturation in pig models for post-cardiac arrest, the arterial oxygen partial pressure for the healthy control group approximated 80–100 mmHg.⁶³ A historical healthy target range for end-tidal CO₂ is 0–45 mmHg.⁶⁴ With the exception of the blood pressure vitals, most metrics of all subjects stayed within healthy physiological ranges, as shaded in green in Figure 3. It is well-established that for all animals, heart rate is species-specific and varies with age up to maturity.⁶⁵ Specifically, for pigs, heart rates are much higher in young pigs than in adults.⁶⁵ As our minipigs were approximately 1–4 months older and weighed nearly twice as much as the Yucatan pigs in the aforementioned study, we concluded that such differences accounted for the data discrepancies.

Figure 3.

Monitoring of pig health during implantation procedure. Vitals – namely, heart rate, mean blood pressure, diastolic pressure, systolic pressure, body temperature, oxygen saturation, and end-tidal carbon dioxide – were maintained during the implantation procedure for all five pigs. Each green box represents the “healthy” physiological range for pigs.

Following the implantation procedure, all animals regained full mobility and demonstrated no impediments to eating (1.8 ± 1.2 kg of bodyweight gained at 14 days following the procedure). This follows the general trend in healthy Yucatan minipigs that have a typical growth rate of 3–5 kg per month.⁶⁶ Altogether, the data suggested the surgical delivery of PEG hydrogels did not negatively impact pig health.

VEGF-delivering PEG hydrogels increase blood perfusion to subcutaneous tissue sites

Our previous work employing our VEGF-delivering hydrogels in rodents has demonstrated an increase in total vessel infiltration and length and perfusion, as measured by lectin perfusion.²⁶ Given that the necessary lectin amount for such quantitative analyses depends on animal bodyweight, we considered the technique impractical and prohibitively expensive for our large animal model. As such, we sought to employ the microsphere perfusion technique as a quantitative proxy to our previous studies. This present study will inform our future studies in diabetic pigs, wherein we will introduce cells to test the function of islet-loaded grafts with our VEGF-delivering gels.

In the present study, at 14 days post-transplant, fluorescent microspheres were introduced into each subject through the heart. In previous work that evaluated perfusion in hind limb muscles, the distal aorta was selected for the site of the withdrawal catheter.⁵¹ Here, we selected the descending abdominal aorta for blood collection given its proximity to the experimental sites in the subcutaneous space. Immediately following systemic microsphere injection, hydrogels with surrounding tissue and naïve tissue were explanted, digested, processed, and analyzed by fluorimetry using validated digestion and plate reader techniques.⁵¹ Microspheres were introduced into the blood circulation through the heart and were then entrapped in capillaries and small arterioles based on size. Thus, local perfusion could be calculated following microsphere recovery from the tissue of interest and a reference blood sample withdrawn at a constant rate. The Core+VEGF gel group exhibited significantly higher local blood perfusion than the control hydrogel implant (no VEGF-delivering coating) and naïve tissue (Figure 4). Interestingly, there was no significant difference in perfusion between naïve tissue and the Core-only group. Strikingly, in addition to increased perfusion for Core+VEGF gel sites compared to naïve tissue (p < 0.0001), we observed significant differences in blood perfusion between the Core-only and Core+VEGF gel groups (p < 0.01), indicating the increase of perfusion was directed by VEGF-delivering hydrogel coating and not simply a response to implantation trauma or the presence of a device (e.g., foreign-body response). Finally, we observed differences in tissue perfusion between males and females for the Core+VEGF gel group (Supplementary Figure 2). These results demonstrate that a VEGF-delivering PEG hydrogel coating increases local perfusion in the porcine subcutaneous space.

Figure 4.

VEGF-delivering gels induce perfusable, functional vasculature as measured by microsphere analysis. For each experimental group within a pig, 3 tissue samples were processed and quantified. Analyzed by nested one-way ANOVA followed by Tukey’s multiple comparisons test. **** p < 0.0001. **p < 0.01.

Histological analyses show enhanced presence of vascular structures by VEGF-delivering PEG gel

To complement the tissue perfusion findings, we gathered explants to analyze tissue sections for host cell infiltration and key vascular markers. As we wanted to capture tissue adjacent to PEG platforms, we explanted samples with dimensions of 30 mm × 30 mm × 30 mm. The implants each measured 22 mm in diameter, and therefore, we captured approximately an additional 5 mm of adjacent tissue on all sides. As shown in Figure 5a, macroscopic images of the two implant groups and reference tissue presented distinct morphological differences. Compared to naïve tissue, the adjacent tissue of both the Core-only and Core+VEGF gel implants showed increased blood presence.

Figure 5.

Histological analyses. (a) Gross images of implant sites and naïve tissue following implant removal. Nondegradable gels were removed from “Core only” and “Core + VEGF gel” groups. Scale bar = 1 cm. (b) Hematoxylin and eosin staining. Subcutaneous tissue is labeled in the naïve group (in contrast to muscle). In gel-bearing groups, subsequent analysis via immunohistochemistry focused on tissue adjacent to fibrous capsules. Scale bar = 1000 μm. (c) Hematoxylin and eosin staining of all groups. Scale bar = 200 μm. Vascular structures denoted by arrows.

With the goal to investigate vasculogenic potential, we focused our histological analyses in the tissue adjacent to collagen capsules (Figure 5b–c). Masson’s Trichrome staining revealed the presence of collagenous capsules around both Core-only and Core+VEGF gel implants (Figure 6), as confirmed by the presence of nuclei and connective tissue. We found no differences in fibrous capsule thickness between Core+VEGF gel and Core-only implants (Supplementary Figure 3).

Figure 6.

Masson’s trichrome staining of all groups (a) Naïve tissue, (b) Core only, and (c) Core + VEGF gel. Scale bar = 1 mm.

For immunohistochemical analysis, we selected CD31 and α-smooth muscle actin (α-SMA) as they are, respectively, markers for endothelial cells⁶⁷ and blood vessel maturation⁶⁸. In the Core+VEGF gel group, CD31⁺ structures were surrounded by α-SMA⁺ cells, indicating mature vessels (Figure 7a). In the Core-only group, colocalization of said structures was observed but qualitatively at lower levels. In contrast, naïve tissue samples exhibited minimal positive cells for CD31 and α-smooth muscle actin. We attribute the increase of α-SMA⁺ structures to both increased vascularization and fibrotic responses to the non-degradable hydrogel core.

Figure 7.

VEGF-delivering gels increase counts and populations of key vascular markers. (a) Representative confocal images of (i) naïve tissue, (ii) Core only, and (iii) Core + VEGF gel. Nuclei marker DAPI and vascularization markers CD31 and α-SMA. Scale bar: 50 μm. (b) for CD31+ structures and (c) for α-SMA+ structures: Blood vessel metrics for experimental groups: (i) area, (ii) density, (iii) area by percentage and (iv) number of structures / field of view. Each point = confocal image from stained sections. For each experimental group within a pig, a total of 9–12 images were captured (3–4 images per slide, for 3 stained slides). Analyzed by nested one-way ANOVA. *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001.

Images of stained sections (CD31⁺ and α-SMA⁺ structures) were analyzed for blood vessel area, blood vessel density, vessel area by percentage, and number of vessels per field of view (25 mm) using a published image-processing technique for blood vessels.⁵³ (Figure 7b–c). Three separate sections from each experimental group were stained, and at least three different fields of view per section were analyzed. For both CD31⁺ and α-SMA⁺ structures, the Core+VEGF gel group exhibited higher values across all four metrics compared to naïve tissue (p < 0.0001) and the Core-only group (for CD31⁺ structures: p < 0.0001 for density and number of structures per field of view and p < 0.001 for area and area by percentage; for α-SMA⁺ structures: p < 0.0001 for area and area by percentage and p < 0.01 for density and number of structures per field of view). Notably, for both CD31⁺ and α-SMA⁺ structures, no significant difference was observed between the naïve tissue and Core-only group for area and area by percentage. Consistent with the tissue blood perfusion data, we observed differences in vascularization metrics between males and females for the Core+VEGF gel group (Supplementary Figure 4). Together, these histological analyses substantiate our functional perfusion data and indicate that the VEGF-delivering coating increases the presence of vascular structures in the porcine subcutaneous space.

Next, we sought to determine whether the tissue blood perfusion results correlated with the blood vessel metrics obtained from imaging of immunostained sections. As shown in Figure 8, we detected strong positive significant correlations between microsphere perfusion readings and all quantified blood vessel metrics except for CD31⁺ area by percentage (p = 0.0565). These analyses demonstrate excellent agreement between the tissue perfusion and histological measurements showing that the VEGF-delivering hydrogel enhances local tissue vascularization and blood perfusion.

Figure 8.

Correlation between perfusion and quantified metrics based on (a) CD31 and (b) α-SMA. For each correlation, ⍴ for Spearman’s correlation is located on the top of the legend, and the p-value (bolded and colored purple) is located at the bottom. Each point represents one of the experimental groups within a pig. Spearman’s correlation.

DISCUSSION

Large animal model with clinical translation relevance

We established an affordable, pre-clinical large animal model to evaluate vascularization strategies in the subcutaneous space. Porcine skin is more similar to human skin than rodent skin based on structure, thickness, permeability, lipid content, and wound healing properties.^{69 70} Nonhuman primates remain the gold standard for large animal studies,⁷¹ but present exorbitantly higher upkeep and costs compared to pigs.^65,72 Our pigs fully recovered from the implantation procedure, and all implants were well-tolerated with no adverse reactions for the two-week duration of the study. Moreover, in each animal, we could test the localized vascularization effects of multiple implant conditions.

Fluorescent microsphere perfusion for porcine subcutaneous tissue

To our knowledge, we report the first application of the fluorescent microsphere perfusion technique to assess local vascularization in the subcutaneous space. This technique has been applied to organs such as the brain⁵² and hindlimb muscles.⁵¹ To orient the reader, reported values for perfusion of Yorkshire cross swine hamstring and distal posterior limb muscles are 45.9±32.1 and 71.4±56.1 mL/min*100g, respectively,⁵¹ while our values for perfusion of the unmodified Yucatan mini-pig subcutaneous space averaged 6.2±9.4 mL/min*100g. The stark differences in perfusion of the subcutaneous space compared to its organ counterparts emphasizes the relative avascularity of the subcutaneous space and further warrants investigation in its vascularization potential for cell therapies.

Historically, gathering perfusion measurements within the subcutaneous space has remained a challenge. In mice and rats, lectin perfusion is a common terminal procedure to analyze blood perfusion.^73–75 However, given the financial cost of lectin and how lectin delivery is traditionally based on bodyweight,²⁶ a more cost-effective procedure for large animal models is desired. Photoacoustic imaging,⁷⁶ ultrasound imaging,^{77 78} and positron emission topography⁷⁹ are noninvasive techniques that have been employed for the longitudinal tracking of organ blood flow and angiogenesis in the subcutaneous space of rodents,^80,81 although equipment costs and access, low imaging depth, and low spatial resolution are common constraints.⁸² The excellent correlation between our microsphere perfusion data and quantified immunostaining validates the utility of this terminal fluorescent microsphere perfusion technique in the subcutaneous space.

Functional perfusion to corroborate vascularization

Whereas histological analysis for vascular structures remains the hallmark strategy in evaluating vascularization by biomaterials, it alone does not imply robust vascularization. Indeed, multiple studies report the presence of CD31-positive and α-SMA-positive cells in biomaterial constructs, but report no increase in perfusion^82,83 or VEGF-induced dysfunctional, leaky vessels.⁸⁴ Thus, functional studies must augment immunohistochemistry findings to rule out dysfunctional vasculature. In the present study, we detected significant differences in local tissue perfusion between the Core+VEGF gel group and other groups and no significant differences between the Core-only group and naïve tissue. These findings are consistent with our work in rodents, wherein we employed lectin perfusion to evaluate the efficacy of our VEGF-delivering coating on a macroencapsulation device in rats¹² and evaluated the perfusion of VEGF-delivering microgels in mice.⁸⁵ Lee et al. and Layman et al. reported similar trends in perfusion results for their pro-angiogenic implants in a mouse skin flap model⁸⁶ and a murine critical limb ischemic model,⁸⁷ respectively. Our present findings also align with relevant work in pigs, wherein a gelatin methacrylate hydrogel that incorporated VEGF-mimicking peptide enhanced wound healing in a pig skin wound model.⁸⁸

Importance of vascularization-inducing, yet clinically relevant materials and models

Enhancing local vascularization has been shown to prolong the longevity of cellular grafts for many tissue types, including heart tissue for ischemic diseases (myocardial infarction⁸⁹ and peripheral artery disease^90,91), skeletal muscle,^92,93 pancreatic tissue,^18,29,39,94 neural tissue,^95,96 and skin.^97–99 Vascularization also promotes graft survival at defect sites following trauma to facilitate self-repair.¹⁰⁰ For cell therapies, increased vascularization at the transplant site boosts crosstalk between therapeutic cargo and host cells. It also reduces diffusional barriers between implanted cells and necessary host nutrients for survival, such as oxygen and glucose.^101,102

With clinical translation as a priority, we focused on a synthetic hydrogel system with reduced regulatory burden - our hydrogel platform is highly customizable and presents a wide array of delivery modes. Namely, the mechanical and biochemical properties of it can be tuned to cater to the specific needs of delivered cells.¹⁰³ Building upon our previous work in this study, we showed that our biomaterial can serve as a nondegradable/degradable implant/coating delivered via injection/implantation.^12,26,33

Potential applications of cell therapy with our animal model

Future work could focus on analyzing the synergistic effects among multiple implants of the same type in different locations of the subcutaneous space. This type of work would be helpful for type 1 diabetes, for example, as more preclinical research alludes to the requirement of multiple implants in a single patient, given the high nutritional demands of relevant cells and the required cell yields for hyperglycemia reversal.^104,105

Limitations of the current study

The present study focused on local vascularization and tissue responses at 2 weeks post-implantation. It is generally accepted in the field that soluble VEGF results in an acute (<7 days) increase in dysfunctional vessels that quickly regress as the VEGF is cleared. We have shown that this is not the case when VEGF is delivered via sustained release from PEG gels.^{12,25,26,34,35} In our prior rodent work, we observed that PEG hydrogel-mediated release of VEGF results in rapid (<14 days) functional vascularization that persists for least 6 months.^26,34 In a mouse model of peripheral limb ischemia, laser doppler perfusion imaging conducted on day 7 demonstrated that animals receiving PEG+VEGF gels had a 50% increase in leg perfusion and 100% increase in feet perfusion compared to untreated subjects.²⁵ Furthermore, using transgenic mice to control the timing and duration of VEGF expression, Dor et al. showed that VEGF-induced vessels regress when VEGF is expressed for one week but are stabilized when VEGF is expressed for two weeks.¹⁰⁶ Taken together, these results support the evaluation of vascularization at two weeks post-delivery. We did not investigate immune cell profiles, nor the mechanistic differences between the foreign body response and angiogenesis. Future work may include both cellular and tissue investigations on immune responses to these implants. Finally, we reported no crosstalk between neighboring implants in mice that underwent a similar procedure in the subcutaneous space.¹⁰⁷ Certainly, the distances between implants in the mice were shorter than those in pigs. Additionally, in rodents, we showed that controlled delivery of VEGF from degradable hydrogels did not increase leukocyte infiltration compared to control hydrogels.^12,26

A limitation of this study is the lack of a control group consisting empty (no VEGF) hydrogel-coated implants in order to establish that VEGF release is the only factor contributing to differences between Core-only and Core+VEGF. We note that the cost and ethical considerations regarding large animals preclude the examination of all potential experimental groups. We therefore compared two implant groups: non-degradable core vs. VEGF-gel coating applied to non-degradable core. This decision is based on the translational application of the VEGF-gel as a coating for implantable devices. As such, our conclusions focus on the effects of the VEGF-gel coating and not on VEGF alone. In our prior rodent work,^25,26,34,35 we conclusively demonstrated that the enhanced vascularization is due to VEGF release.

We found significant differences in perfusion between Core+VEGF gel and Core-only; this finding alludes to mechanistic differences between the effects of the VEGF-releasing coating and the foreign body response to the hydrogel implants. It is well-known that the mechanisms between foreign body response and angiogenesis remain unclear.^77,108,109 The foreign body response has been linked to an immediate increase in localized angiogenesis that then declines over time.^108,110 Future work could delve into the mechanistic differences. Our findings support the notion that, nevertheless, our VEGF-delivering hydrogel can still produce perfusable tissues.

CONCLUSION

We established a large animal model to evaluate the vasculogenic capabilities of engineered biomaterials. We demonstrated that a VEGF-delivering PEG-4MAL hydrogel coating induces vascularization and local perfusion in the porcine subcutaneous space. No detrimental effect on pig health was observed. Compared to rodents, the proposed animal model better recapitulates the architecture and wound healing processes of the human subcutaneous space.