Oxygen Generating Biomaterial Mitigates Hypoxia Induced Apoptosis on Pancreatic Islets
Maria Coronel1,2,3,4, Eileen Pedraza1,2,3,4, Camillo Ricordi1,2,3,4, Cherie Stabler1,2,3,4
1Department of Biomedical Engineering, University of Miami, MIAMI, FL, United States; 2Diabetes Research Institute, University of Miami, Miami, FL, United States; 3Department of Surgery, University of Miami, Miami, FL, United States; 4Department of Medicine, University of Miami, Miami, FL, United States
Introduction:Cellular transplantation of tissue engineering constructs is an attractive approach for the replacement or repair of damage tissue. Nevertheless, the success of engineered tissues has been hindered by poor oxygenation upon transplantation, due to the inevitable delay in the vascularization process. Inadequate oxygen delivery of cellular constructs leads to a necrotic core of cells and a dysfunctional, pro-inflammatory environment. This is particularly challenging in constructs comprising highly metabolically active cells, such as pancreatic islets. To mitigate this damage, we have developed an oxygen generating biomaterial capable of in situ oxygenation of cellular implants1. Herein, we report on the effect of this oxygen generating biomaterial on alleviating hypoxia-induced pancreatic islets apoptosis.
Methods: Oxygen generating disks (CaO2-PDMS) were made as previously reported1. Rat pancreatic islets were co-incubated with a blank PDMS disk (control) or with CaO2-PDMS disk (treated) at normoxic (20%) or hypoxic (1%) oxygen tensions and incubated for short (8 hr) and long-term (24 hr) assessment. Analysis of the HIF-1α target genes using RT-PCR was performed, as well as protein assessment, western blotting, and live dead imaging.
Results: Islets cultured in hypoxic conditions for long-term incubation periods show stabilization and accumulation of HIF-1α protein in control groups, compared to treated, as seen by western blotting. Results for the expression of HIF-1α target genes in hypoxic controls show an up-regulation of glycolytic enzymes such as, phosphoglycerate kinase 1, pgk1, Lactate dehydrogenase A,ldhA, Glucose transporter 1, GLUT1, as well as the DNA damage protein, Ddit-4, when compared to the treated hypoxic group in both the short and long-term incubations. This up-regulation represents an increase in oxidative stress, a shift to anaerobic metabolism, and induced apoptosis in response to DNA damage. For pro-angiogenic markers, the expected up-regulation of the vascular endothelial growth factor A, VEGFA, in the hypoxic controls compared to the treated was observed in the short-term (8 hr). Furthermore, there was a down-regulation of pro-apoptotic genes BCL2-antagonist/killer 1, BAK, and BCL-2 associated X protein, BAX, in the hypoxic controls compared to treated, suggesting a shift to BNIP3 mediated apoptosis, which resembles more a necrotic pathway of cell death. Live/dead imaging of hypoxic controls presented fragmented islets with extensive dead cells. In contrast, treated hypoxic group showed large, morphologically intact islets comparable to normoxic controls.
Conclusions: Herein, we have illustrated the ability of our oxygen generator to mitigate the effects of hypoxia and HIF-1α involvement on pancreatic islet viability. Thus, this platform can be advantageous to improve early graft loss due to limited oxygen supply for cellular based tissue engineered constructs.
E Pedraza, M M Coronel, C A Fraker, C Ricordi, C L Stabler: Enhancing Beta-Cell Viability via Hydrolytically Activated, Oxygen Generating Biomaterials.
PNAS, 109(11), pp. 4245-50, 2012