Preventing hypoxia-induced cell death in beta cells and islets via hydrolytically activated, oxygen-generating biomaterials

Abstract

A major hindrance in engineering tissues containing highly metabolically active cells is the insufficient oxygenation of these implants, which results in dying or dysfunctional cells in portions of the graft. The development of methods to increase oxygen availability within tissue-engineered implants, particularly during the early engraftment period, would serve to allay hypoxia-induced cell death. Herein, we designed and developed a hydrolytically activated oxygen-generating biomaterial in the form of polydimethylsiloxane (PDMS)-encapsulated solid calcium peroxide, PDMS-CaO(2). Encapsulation of solid peroxide within hydrophobic PDMS resulted in sustained oxygen generation, whereby a single disk generated oxygen for more than 6 wk at an average rate of 0.026 mM per day. The ability of this oxygen-generating material to support cell survival was evaluated using a β cell line and pancreatic rat islets. The presence of a single PDMS-CaO(2) disk eliminated hypoxia-induced cell dysfunction and death for both cell types, resulting in metabolic function and glucose-dependent insulin secretion comparable to that in normoxic controls. A single PDMS-CaO(2) disk also sustained enhanced β cell proliferation for more than 3 wk under hypoxic culture conditions. Incorporation of these materials within 3D constructs illustrated the benefits of these materials to prevent the development of detrimental oxygen gradients within large implants. Mathematical simulations permitted accurate prediction of oxygen gradients within 3D constructs and highlighted conditions under which supplementation of oxygen tension would serve to benefit cellular viability. Given the generality of this platform, the translation of these materials to other cell-based implants, as well as ischemic tissues in general, is envisioned.

Fig. 1.

Oxygen-generating material. (A) Schematic of oxygen-generating biomaterial, fabricated using PDMS-CaO₂. Water diffusion is hindered by the hydrophobicity of the PDMS, whereas oxygen, generated via hydrolytic reaction with calcium peroxide, quickly diffuses out of the PDMS material. (B) Photograph of PDMS-CaO₂ disk (10-mm diameter; 1-mm height). (C) Oxygen trace measurements of either a PDMS disk containing 25% wt/wt CaO₂ (filled diamonds) or a PDMS-only disk (open diamonds) incubated in buffered saline (n = 3). Measurements were made in an open system incubator set at 0.05 mM oxygen. Disturbances in the oxygen readings due to incubator opening were removed for clarity and are represented by gaps in curves.

Fig. 2.

PDMS-CaO₂ prevents hypoxia-induced cell death for MIN6 β cells. MIN6 β cells (3 × 10⁵ cells per well) were cultured for 24 h at 0.2 mM (normoxic) or 0.01 mM (hypoxic) oxygen tensions with or without a PDMS-CaO₂ disk. Measurements included (A) MTT metabolic activity, (B) total protein, (C) LDH release, and (D) caspase activity. Initial values represent assessment on day 0. Error = SD; n = 3. *P < 0.05; **P < 0.01, Student's t test.

Fig. 3.

PDMS-CaO₂ prevents hypoxia-induced cell death for pancreatic rat islets. Rat islets (1,500 IEQ) were cultured at 0.05 mM (hypoxic) oxygen with or without a PDMS-CaO₂ disk. Assessments included (A) MTT metabolic activity, (B) glucose-stimulated insulin release, (C) LDH release, and (D) caspase activity. Initial values are day-0 measurements. Error = SD, n = 3. *P < 0.05, **P < 0.01, Student's t test. ^#P < 0.05, ANOVA. (E) Representative confocal z-stacked images of islets, stained for live/dead (live, green; dead, red), after 6 h of culture at 0.05 mM oxygen without (control) or with a PDMS-CaO₂ disk. (Scale bars, 100 μm.)

Fig. 4.

Enhanced and sustained MIN6 β cell survival over 3 wk under low oxygen conditions when PDMS-CaO₂ disk is present. MIN6 cells (3 × 10⁵ cells per well) were cultured for 24 d under low oxygen (0.05 mM) conditions with or without PDMS-CaO₂ disk and assessed via (A) MTT metabolic activity and (B) total DNA. The PDMS-CaO₂ group was significantly higher than the respective control group from 3 to 24 d for MTT and from 10 to 24 d for DNA (P < 0.001, repeated-measures ANOVA). *Time points where PMDS-CaO₂ disk was removed for selected wells, where dashed lines trace initial and final absorbance readings. (C) Representative confocal images of MIN6 β cells coincubated with (Upper) or without (Lower) a PDMS-CaO₂ disk and stained with live/dead stain (live, green; dead, red). Error = SD, n = 3. (Scale bars, 100 μm.)

Fig. 5.

Increased viability of MIN6 β cells within constructs containing PDMS-CaO₂ disk. (A) Schematic of agarose constructs containing a single 25% wt/wt PDMS-CaO₂ disk. The inner PDMS-CaO₂ disk was 8 mm in diameter and 1 mm in height, and the addition of the outer MIN6/agarose construct resulted in a final construct of 10 mm diameter/3 mm height. (B) Fold changes in MIN6 viability, assessed via MTT metabolic assay, over initial cell loading values after 3 d culture of 25 × 10⁵ MIN6 cells within agarose constructs without or with a PDMS-CaO₂ disk. Fold values above 1.0 represent an increase in cell viability during the culture period. Error = SD, n = 3. *P < 0.05, Student's t test. (C) Representative confocal z-stacked images of MIN6 β cells stained with live/dead (live, green; dead, red) and coincubated with PDMS-only (control, Upper) or PDMS-CaO₂ (Lower) disk at either 0.2 mM (Left) or 0.05 mM (Right) oxygen. (Scale bars, 200 μm.)

Fig. 6.

Effect of oxygen availability on MIN6 β cell viability. (A) Multiphysics simulation models of oxygen gradients within 3D agarose constructs containing either PDMS-only or PDMS-CaO₂ disk. (Dimensions identical to those outlined in Fig. 5A). Models run with 0.05 mM external oxygen are shown (0.2 mM in Fig. S3) at the total MIN6 cell loadings indicated Predicted oxygen range (0.05 to 0 mM) is shown on far right. Black, 0 mM oxygen tension. (B) Experimental outcomes of oxygen availability on MTT metabolic activity. Oxygen availability varied via total MIN6 β cell loading (5–25 × 10⁵ total cells), external oxygen tension (0.2 mM or 0.05 mM), and the presence of a PDMS-CaO₂ disk. Measurements were collected after 3 d culture and expressed as fold increase over control at identical cell loading density and oxygen concentration (fold = treated group with PDMS-CaO₂ disk/control group without the PDMS-CaO₂ disk). Fold increase values greater than 1.0 indicate a positive effect of the PDMS-CaO₂ disk on cell viability, whereas values <1 indicate a detrimental effect. Error = SD; n = 3.