A bioinspired scaffold for rapid oxygenation of cell encapsulation systems

Materials

Poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP, Mw = 455 kDa), Tris hydrochloride (Tris-HCl), sodium hydroxide, dopamine hydrochloride, sodium chloride (NaCl), calcium chloride dihydrate (CaCl₂·2H₂O), barium chloride dihydrate (BaCl₂·2H₂O), calcium sulfate dihydrate (CaSO₄·2H₂O), Nile Red, gelatin and D-glucose were purchased from Sigma-Aldrich. Poly(lactic acid) (PLA) filament was purchased from PRUSA. Ultrapure sodium alginate (Pronova SLG100) was purchased from NovaMatrix. Water was deionized to 18.2 MΩ cm with a Synergy UV purification system (Millipore Sigma).

Animals

Male C57BL/6 J mice (2 months old) were purchased from The Jackson Laboratory. The mice were maintained at a temperature of 70–72 °F with 30–70% humidity under a 14 h light/10 h dark cycle. Male Sprague-Dawley rats (weight of ~300 g) were purchased from Charles River Laboratories. All animal procedures were approved by the Cornell Institutional Animal Care and Use Committee and complied with relevant ethical regulations.

Characterizations

High-resolution X-ray computer tomography (Nano-CT) scanning was conducted on a 3D X-ray microscope (ZEISS Xradia 520 Versa). Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) element mapping were performed using a field emission scanning electron micro-analyzer (LEO 1550). Contact angle images were taken using a contact angle goniometer (Rame-Hart 500). Optical and fluorescent microscope images were taken using a digital microscope (EVOS FL). H&E staining images were taken using an Aperio Scanscope (CS2). Stereo microscope images were taken by a stereomicroscope (Olympus SZ61). Immunofluorescence images were taken using a confocal microscope (ZEISS LSM 710). OriginPro 8.5.1 software and GraphPad Prism 8 software were used for data plotting.

Fabrication of the SONIC scaffold

PVDF-HFP was dissolved in acetone at a concentration of 15 wt% under heat in a sealed glass vial. After cooling to room temperature, the PVDF-HFP solution was filled into a 3D printed PLA mold (Original Prusa i3 MK2S) and immersed in a water/ethanol (V/V = 1/1) bath for a phase separation process, and then transferred to a water bath for a solidification process. Next, the solidified PVDF-HFP was immersed in ethanol and hexane for two dehydration processes, followed by air drying at ambient temperature. Finally, the SONIC scaffold was obtained after the selective extraction of the PLA mold with chloroform. (The SONIC scaffolds for in vivo studies were sterilized by autoclave.)

Nano-CT imaging of the mealworm and SONIC scaffold

To prepare a mealworm specimen for Nano-CT scanning, a 2 cm-long mealworm was loaded in a 1 mL pipet tip (Supplementary Fig. 1) and sacrificed by freezing at −20 °C. 6 individual scans were performed on different sections of the mealworm using an “oversize scan” option to get a full image of the specimen. During the scans, the X-ray source was set to a voltage of 100 kV, and the scanning resolution was set as 5.19 µm per pixel under a binning mode of 2 × 2. Subsequently, a 3D reconstruction of the obtained images was performed using Avizo software (version 8.1.1). A segmentation process was conducted to visualize the tracheal system of the mealworm based on the different absorption contrasts between the respiratory gases and mealworm tissues.

To prepare a SONIC scaffold specimen for the Nano-CT scanning, a small piece of scaffold (~1 mm³) was cut and attached on a tip. During the scan, the X-ray source was set to a voltage of 100 kV, and the scanning resolution was set as 0.268 µm per pixel under a binning mode of 2 × 2. Subsequently, 3D reconstruction of the obtained images was performed using Avizo software. Network connectivity on the polymeric and the porous regions of the scaffold was performed using ImageJ.

Electron paramagnetic resonance (EPR) for O₂ mapping

O₂ mapping was performed on a 25 mT EPR imager (JIVA-25, O2M Technologies, LLC). The JIVA-25 operates at 720 MHz using electron paramagnetic resonance oxygen imaging (EPROI) principles and utilizes oxygen sensitive electron spin-lattice relaxation rates (T₁) of trityl radical probe OX063-D24 (methyl-tris[8-carboxy-2,2,6,6-tetrakis[(2-hydroxyethyl]benzo[1,2-d:4,5-d’]bis[1,3]dithiol-4-yl]- trisodium salt) for reporting pO₂.

A SONIC scaffold or control scaffold was fixed at the bottom of a glass tube (VWR, 10 × 75 mm) using a dental vinyl polysiloxane impression material. 1 mL gelatin solution (1 wt%) containing spin probe (1 mM OX063-d24) was filled into the container with the top end of the scaffold exposed above the gelatin. First, the system was deoxygenated using N₂ to reduce pO₂ close to 0 mmHg. After deoxygenation, the system was exposed to a gas mixture containing 5% O₂ and 95% N₂, and the pO₂ change in gelatin was continuously monitored until a steady-state (40 mmHg) was approached.

Average pO₂ measurements were performed using inversion recovery electron spin-echo (IRESE)⁶¹ sequence with the following parameters: pulse lengths 60 ns, 16 phase cycles scheme with FID suppression, spin-echo delay 500 ns, 80 logarithmically spaced delays from 400 ns–65 μs, 100 us repetition time. The curves were fitted using single exponential recovery to extract R₁ (1/T₁) values that were converted to pO₂ (Fig. 3c). pO₂ imaging was performed using IRESE sequence with the following parameters: pulse lengths 60 ns, 16 phase cycles scheme with FID suppression, spin-echo delay 400 ns, equal solid angle spaced 654 projections, 67 baselines, 1.5 G/cm gradient, 10 time delays from 410 ns–40 μs, 35 μs–59 μs repetition time, overall 10 min image duration. Images were reconstructed using filtered back-projection in isotropic (64,times 64,times 64) cube with 0.66 mm voxel linear size.

Fabrication of the SONIC device

The SONIC scaffold was immersed into a dopamine solution (2 mg/mL in 10 mM tris buffer, pH 8.5) overnight to create a hydrophilic polydopamine coating on the scaffold surface. Subsequently, CaSO₄ was deposited onto the scaffold surface by dipping it into a CaSO₄ saturated solution (0.24 wt% in water) and then drying it at 60 °C, leaving CaSO₄ crystals on the scaffold surface. Next, the scaffold was inserted into a glass tubing mold with sodium alginate (2%) solution. Alginate cross-linking then occurred by the Ca²⁺ ions diffused from the CaSO₄. Finally, the SONIC device was pushed out from the tubing mold into a cross-linking buffer (95 mM CaCl₂ + 5 mM BaCl₂), leaving the device in a buffer around 4 min for further cross-linking. Constructs that contained INS-1 cells or islets were fabricated by premixing the alginate solution with the cells before application onto the scaffolds.

To fabricate control devices without the SONIC scaffold, a tubing mold was prepared by rolling a dialysis membrane (Spectra/Por^®, MWCO 3500) into a tube with an inner diameter of ~4 mm and sealing one end with a PDMS cap. Then, INS-1 cells or islets alginate solution were loaded into the mold and immersed in the buffer (95 mM CaCl₂ + 5 mM BaCl₂) for cross-linking by the Ca²⁺ and Ba²⁺ ions which diffused through the dialysis membrane. Next, the tubing mold was unrolled to leave the alginate in the buffer for around 4 min for further cross-linking.

For the devices in mice studies, 500 IEQ of rat islets distributed in approximately 170 µL alginate were incorporated in each cylindrical (4.2 mm in diameter, 20.4 mm in length) SONIC device (Fig. 5b) and 500 IEQ of rat islets distributed in approximately 280 µL alginate were incorporated in each corresponding cylindrical scaffold-free control device (Supplementary Fig. 14); 500 IEQ of rat islets distributed in approximately 160 µL alginate were incorporated in each cubic (6.6 × 6.6 × 6.6 mm) SONIC device (Fig. 6h).

In vitro cell viability study

INS-1 cells were purchased from Sigma-Aldrich and cultured with RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 10 mM HEPES (Gibco), 2 mM glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 50 µM β-mercaptoethanol (Gibco), and 1% penicillin/streptomycin (Gibco). Trypsin-dissociated INS-1 cells were suspended in alginate solution at a density of 2.5 million cells/mL and incorporated into SONIC devices or control devices, and then were incubated in the above-mentioned medium in a hypoxic incubator with 5% O₂, 5% CO₂ at 37 °C. After 48 h, the cells in devices were stained with a LIVE/DEAD™ viability/cytotoxicity kit (Invitrogen).

Rat islet isolation and purification

Sprague-Dawley rats were used for harvesting islets. The rats were anesthetized using 3% isoflurane in O₂ throughout the whole surgery. Briefly, the pancreas was distended with 10 mL 0.15% Liberase (Roche) in M199 media (Gibco) through the bile duct. The pancreas was digested at 37 °C circulating water bath for ~28 min (digestion time varied slightly for different batches of Liberase). The digestion was stopped by adding cold M199 media with 10% FBS (Gibco). After vigorously shaking, the digested pancreases were washed twice with media (M199 + 10% FBS), filtered through a 450 μm sieve, and then suspended in a Histopaque 1077 (Sigma)/M199 media gradient and centrifuged at 1700 RCF with 0 breaks and 0 acceleration for 17 min at 4 °C. This gradient centrifugation step was repeated for higher purity. Finally, the islets were collected from the gradient and further isolated by a series of gravity sedimentations, in which each top supernatant was discarded after 4 min of settling. Islet equivalent (IEQ) number of purified islets was counted by reported IEQ conversion factors⁶². Islets were then washed once with islet culture media (RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, and 1% penicillin/streptomycin) and cultured in this medium overnight before further use.

Implantation and retrieval in mice

C57BL/6 J mice were administered an intraperitoneal injection of freshly prepared STZ solution (22.5 mg/mL in 100 mM sodium citrate buffer, pH 4.5) at a dosage of 150 mg STZ/kg mouse to induce diabetes one week before device implantation. Only mice with non-fasted blood glucose levels above 350 mg/dL were considered as diabetic. The diabetic mice were anesthetized with 3% isoflurane in O₂ and the abdomen aera was shaved and sterilized using betadine and 70% ethanol. A small skin incision (~8 mm) was made along the midline of the abdomen, and then a following incision was made along the linea alba. The device was introduced into the peritoneal cavity through the incision. The peritoneal wall was closed using 5-0 absorbable polydioxanone (PDS II) sutures and the skin incision was closed using 5-0 nylon sutures.

For retrieval, the mice were treated with the same procedures as above. Then, the device was located and pulled out from the peritoneal cavity using a tweezer. The incisions were sutured and keep the mice alive for following BG monitoring after device retrieval.

Morphology and immunohistochemistry of islets in retrieved devices

The retrieved devices were fixed with 10% formalin, embedded in paraffin, and sectioned into 5 µm sections. Hematoxylin and eosin (H&E) staining was performed by Cornell’s Histology Core Facility. For immunofluorescent insulin and glucagon staining, paraffin-embedded sections were deparaffinized in xylene and sequentially rehydrated in 100% ethanol, 95% ethanol, 75% ethanol, and PBS. Slides were then boiled in citric acid buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 30 min for antigen retrieval. After blocking with 5% donkey serum, primary rabbit anti-rat insulin (Abcam, ab63820, 1:200) and mouse anti-rat glucagon (Abcam, ab10988, 1:200) antibodies were applied and incubated overnight at 4 ˚C. After washing with PBS, Alexa Fluor 594-conjugated goat anti-rabbit IgG (Thermofisher, A11037, 1:400) and Alexa Fluor 488-conjgated donkey anti-mouse IgG (Thermofisher, A21202, 1:400) were applied and incubated for 60 min. Finally, slides were washed with PBS, applied with antifade/DAPI, and covered with glass coverslips.

BG monitoring & intraperitoneal glucose tolerance test (IPGTT)

Mouse BG levels were measured by a commercial glucometer (Contour Next EZ, Bayer) with a drop of blood collected from the tail vein. For the IPGTT, mice were fasted for 16 h and administered an intraperitoneal injection of 20% glucose solution at a dosage of 2 g glucose/kg mouse. BG levels were measured at 0, 15, 30, 60, 90, and 120 min following the injection.

Ex vivo static glucose-stimulated insulin secretion (GSIS) assay

Krebs Ringer Bicarbonate (KRB) buffer was prepared according to the following formula: 98.5 mM NaCl, 4.9 mM KCl, 2.6 mM CaCl₂·2H₂O, 1.2 mM MgSO₄·7H₂O, 1.2 mM KH₂PO₄, 25.9 mM NaHCO₃, 0.1% BSA (all from Sigma-Aldrich), and 20 mM HEPES (Gibco). The retrieved devices were incubated in the KRB buffer for 2 h at 37 ˚C, 5% CO₂. Devices were transferred and incubated in KRB buffer supplemented with 3.3 mM glucose, then 16.7 mM glucose for 75 min each. The buffer was collected after each incubation step, and insulin concentration was measured using an ultrasensitive rat insulin ELISA kit (ALPCO).

Computational modeling

Five general finite element models were created to calculate theoretical O₂ profiles in SONIC-enabled constructs and corresponding controls. In all models, O₂ tension (pO₂) was related to the concentration of O₂ (({{{{{{rm{c}}}}}}}_{{{{{{{rm{O}}}}}}}_{2}})) by the Bunsen solubility, or equilibrium concentration of O₂ in a material i at 37 °C, ({{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{i}}}}}}}):

In other words, O₂ partitioning was governed by Henry’s law where, in Eq. 1, ({{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{i}}}}}}}) represents the inverse of Henry’s constant. Each model is described below.

Model 1 (Fig. 3h, i and Supplementary Fig. 5) simulated time-dependent oxygenation of the in vitro acellular test. Two domains were considered: a rectangular prism representing the SONIC scaffold or PLA control (2 × 2 × 23 mm), and a surrounding cylinder (8.6 mm diameter, 17 mm length), representing gelatin, with the scaffold located in the center of the gelatin (Supplementary Fig. 5a, b). To emulate the experimental set up, boundary conditions at the top face of the gelatin and exposed faces of the scaffold were implemented at a constant pO₂ of 40 mmHg, while no flux conditions were implemented on all other faces due to the O₂ impermeability of the glass test tube and fixing resin containing the system (Supplementary Fig. 5c). Transient pO₂ transport in each domain was governed by Fick’s second law:

here, ({{{{{{rm{D}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{i}}}}}}}) represents diffusivity of O₂ in domain i (i.e., SONIC scaffold, PLA, or gelatin) at 37 °C. As it was confirmed that a bicontinuous porous structure was maintained throughout the SONIC scaffold (Fig. 1j, k, Supplementary Fig. 3, and Supplementary Movie 2), this domain was modeled as a gaseous air phase. Therefore, ({{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{SONIC}}}}}}}=3.9,times {10}^{-4}) mol/(m³ Pa) rather represents the equilibrium O₂ concentration in air and was calculated by the ideal gas law. Solubility and diffusivity values for gelatin varied widely in the literature, thus values for alginate hydrogel were used, given their physical and chemical similarity. The remaining solubilities were thus implemented as ({{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{gelatin}}}}}}}={{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{alginate}}}}}}}=9.3,times {10}^{-6},)mol/(m³ Pa)⁵⁹, and ({{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{PLA}}}}}}}=4.5,times {10}^{-5},)mol/(m³ Pa)⁶³, each obtained from the literature. Likewise, ({{{{{{rm{D}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{SONIC}}}}}}}=1.8,times {10}^{-5},)m²/s⁶⁴, ({{{{{{rm{D}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{gelatin}}}}}}}={{{{{{rm{D}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{alginate}}}}}}}=2.7,times {10}^{-9},)m²/s^59,65, ({{{{{{rm{D}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{PLA}}}}}}}=1.6,times {10}^{-12},)m²/s⁶³, also obtained from the literature, were implemented for O₂ diffusivities in the respective materials. A time-dependent study simulated over 80 h was performed with a time step of 5 mins.

Model 2 simulated steady-state O₂ transport in cylindrical constructs (4.2 mm diameter, 20.4 mm length) containing alginate-encapsulated INS-1 cells with cell densities from 1.0–8.0 million cells per mL alginate (Fig. 4a–d and Supplementary Fig. 7). O₂ profiles in a construct featuring the ladder-like SONIC scaffold were compared to those in a scaffold-free control (Supplementary Fig. 7a). The alginate and encapsulated cells were modeled as one composite domain, with diffusivity and solubility coefficients of that of alginate. A constant pO₂ of 40 mmHg was implemented at all external boundaries (Supplementary Fig. 7b). Steady-state pO₂ profiles were obtained by solving the diffusion-reaction mass balance equation:

Above, ({{{{{{rm{R}}}}}}}_{{{{{{{rm{O}}}}}}}_{2}}) represents O₂ consumption by the encapsulated INS-1 cells, modeled using Michaelis-Menten kinetics and a step-down function⁶⁶:

In Eq. 4, ({{{{{{rm{V}}}}}}}_{{{{{{rm{INS}}}}}}-1}=5.0,times {10}^{-17}) mol/(m³ s cell) represents the literature-retrieved INS-1 cellular O₂ consumption rate⁶⁷, ({{{{{{rm{K}}}}}}}_{{{{{{rm{m}}}}}}}=0.81) mmHg represents the half-maximum constant derived from studies on mitochondrial respiration⁶⁸, and ({{{{{rm{rho }}}}}}) represents the cell density which was implemented at 2.5 million cells/mL to match experimental conditions (Fig. 4a–f) but also varied between 1–8 million cells/mL to explore the effect of varying cell density (Supplementary Fig. 7d, e). Below the threshold of ({{{{{rm{p}}}}}}{{{{{{rm{O}}}}}}}_{2}=0.08{{{{{rm{mmHg}}}}}}), O₂ consumption is set to zero^48,69, representing the lack of respiration in necrotic cells as in models described elsewhere.

Model 3 simulated steady-state O₂ transport in all cylindrical constructs (4.2 mm in diameter, 20.4 mm or 6.4 mm in length) containing alginate-encapsulated rat islets (Fig. 4g–l, Fig. 5k, m, Supplementary Fig. 8–11, Supplementary Fig. 13, and Supplementary Fig. 18; 500 IEQ rat islets per device) or human islets with cell densities from 2.74–8.04% v/v in devices (4.2 mm in diameter, 2.2 mm in length) (Supplementary Fig. 12). In all cases, islets were implemented as perfect spheres and seeded randomly in the alginate domain. As a default, islet diameters, d, were randomly selected from a size distribution. Rat islet diameters were selected from a lognormal distribution (Supplementary Fig. 8b), with the probability density function given by:

where ({{{{{rm{alpha }}}}}}=0.40) and ({{{{{rm{beta }}}}}}=112.6) (these values were obtained empirically and were found to be similar to distributions observed in other animal islet sources)⁷⁰. Human islet diameters were selected from Weibull distribution⁶² (Supplementary Fig. 12c), with the probability density function given by:

where ({{{{{rm{alpha }}}}}}=1.5) and ({{{{{rm{beta }}}}}}=105). In specified cases (Fig. 4k, l and Supplementary Fig. 10), islets were instead all generated with ({{{{{rm{d}}}}}}=150) μm.

Steady-state pO₂ profiles were obtained by solving the diffusion-reaction mass balance equation (Eq. 3). Solubility and diffusivity in the islets were given by ({{{{{{rm{alpha }}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{islets}}}}}}}=7.3,times {10}^{-6}) mol/(m³ Pa) and ({{{{{{rm{D}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{islets}}}}}}}=2.0,times {10}^{-9}) m²/s respectively^57,64,65. In this model, ({{{{{{rm{R}}}}}}}_{{{{{{{rm{O}}}}}}}_{2},{{{{{rm{i}}}}}}}) was only defined in the islet domains according to the following:

where ({{{{{{rm{V}}}}}}}_{{{{{{rm{islets}}}}}}}=0.0340) mol/(m³ s) represents the O₂ consumption rate in rat islets⁷¹ and ({{{{{{rm{V}}}}}}}_{{{{{{rm{islets}}}}}}}=0.0134) mol/(m³ s) in human islets⁵⁰, respectively. Model 3 was used to predict islet oxygenation and necrosis in cylindrical constructs implanted intraperitoneally in mice (Fig. 4g–j), whereby a constant pO₂ of 40 mmHg was implemented on all external boundaries (Supplementary Fig. 8c). The effect of variable external boundary pO₂ was explored in Supplementary Fig. 13. This model was also used to test the hypothetical impact of alternative scaffold compositions, including PLA, solid PVDF-HFP, or porous PVDF-HFP/alginate in constructs (Supplementary Fig. 8d, e). Solubility and diffusivity coefficients^72,73 for the alternative scaffold materials are listed in Table S1. Model 3 was also used to predict the influence of partial fibrosis in the same constructs (Fig. 5k, m and Supplementary Fig. 18), modeled by a no flux condition implemented on one half of the exterior of a cylindrical device (SONIC versus scaffold-free control, each 20.4 mm length) containing 500 IEQ rat islets to imitate a severe case of blockage by a fibrotic layer (Fig S17a). Oxygenation of 500 IEQ rat islets in cylindrical constructs of variable lengths (6.4 mm vs. 20.4 mm) and therefore cell densities was also evaluated with this model (Supplementary Fig. 11). Finally, Model 3 was used to evaluate human islet oxygenation and necrosis in cylindrical devices (SONIC versus scaffold-free) at variable densities (Supplementary Fig. 12).

Model 4 simulated steady-state O₂ transport in two thick cubics (6.6 × 6.6 × 6.6 mm) devices, each containing 500 IEQ rat islets (Fig. 6a–f, and Supplementary Fig. 19): a scaffold-free control device and a SONIC device. A constant pO₂ of 40 mmHg was applied to all exterior boundaries. All physics implementations of Model 4 were identical to those of Model 3, except for the dimensions, which are defined in Supplementary Fig. 19a, b.

Model 5 simulated steady-state O₂ transport in the SONIC spiral device and empty control containing variable loading densities of human islets. A constant pO₂ of 40 mmHg was imposed on the top and bottom boundaries whereas a no-flux condition was imposed on the lateral face, as the modeled geometry is intended to represent only the central region of a device which would be extruded radially. All other physics implementations were identical to those of Model 3, except for the dimensions, which are defined in Supplementary Fig. 25a.

All models were solved in COMSOL Multiphysics or COMSOL Livelink for MATLAB. In Models 3–5, all calculations were repeated for at least 3 iterations, whereby the islets were reselected and repositioned at random each time. For all calculations, a mesh was implemented using COMSOL’s “Free Tetrahedral” program with the following settings: maximum element size of 100 µm, minimum element size of 1 µm, curvature factor of 0.3, resolution of narrow domains of 3.3, and maximum growth rate of 1.25. It was ensured that all results were independent of the mesh.

Statistics

All results are expressed as raw data or as mean ± SD. Data from random BG measurements (Fig. 5c) were analyzed via a one-way analysis of covariance (ANCOVA) where device treatment (e.g., control device or SONIC device) was considered a discrete factor and time was considered a continuous covariate. Here, data from two-month SONIC device-treated mice (pink) were compared to data from control-treated mice (black) between days 4 and 61, while data from 6-month SONIC device-treated mice (red) were compared to data from control-treated mice (black) between days 4 and 181. Data from IPGTT studies (Fig. 5d, e and Fig. 6j) were analyzed via two-way analysis of variance (ANOVA) where both time and treatment (e.g., diabetic control mice, healthy control mice, control device-treated mice, and SONIC device-treated mice) were considered discrete factors, followed by a Sidak’s post hoc p-value adjustment for multiple comparisons. Data from the GSIS test (Fig. 5f) was analyzed via a paired two-tailed students t-test. Average population pO₂ and necrotic fraction data from modeling studies (Fig. 4i, j) were analyzed via an unpaired two-sided students t-test. Data from modeling studies of the thick hydrogel (Fig. 6c, d), variable scaffold compositions (Supplementary Fig. 11d, e), variable density studies (Fig. 7c, d and Supplementary Fig. 12), and variable external pO₂ (Supplementary Fig. 13) were analyzed via a two-way ANOVA followed by Sidak’s post hoc p-value adjustment for multiple comparisons. Statistical significance was concluded at p < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.