"Introduction: In the last decade many advancements have been made in engineering tissues that function as replacement for malfunctioning or damaged tissues and organs(1–3). However, current methods are limited to either thin tissues such as skin and bladder(4), or small sized tissues used in small rodent models(2). Scaling these tissues up towards clinical relevant sizes currently remains a major unresolved challenge in tissue engineering. Specifically, increasing tissue size associates with progressively severe oxygen and nutrient diffusion limitations, which causes cellular necrosis and ultimately implant failure(1–3). Currently, lack of oxygen is considered the main factor leading to necrotic core formation. As a result a variety of solutions to oxygenate engineered tissues have been developed, often using peroxides to generate oxygen in situ (5, 6). However, the effect of nutrient availability under diffusion limit-induced anoxic conditions has remained largely unstudied. Therefore, we have investigated the effect of various metabolites on cell survival under anoxic conditions.
Methodology: Human mesenchymal stem cells (hMSCs) were seeded at a density of 4000 cells/, and cultured without media change for seven days in serum-free and metabolite-free chemically defined media, except for the single type of metabolite as the experimental condition. These metabolites included various sugars, lipids, amino acids and vitamins. Cell viability and functionality were determined using standard cell culture techniques. The glucose release system was engineered by casting polycaprolactone-glucose melt into discs, which were then embedded into gelatin-methacryloyl hydrogels containing 3x hMSCs/ml. Glucose release was determined using a glucose oxidase-DAB assay.
Results: Sugars consistently allowed for high percentage of hMSC viability, while other conditions resulted in massive cell death. This confirms that nutrient (e.g., sugar) depravation rather than anoxia induces cell death. Glucose was then chosen as the model sugar and was used in a metabolite release system, which was able to maintain viability and metabolic activity in 3D cell laden hydrogel constructs. Importantly, the high cell viability combined the anoxic environment resulted secretion of high amounts of pro-angiogenic factors, such as VEGF. This resulted in the vascularization and therefore integration of a subcutaneously implanted tissue. Moreover, unlike commonly explored oxygen generating peroxides, glucose achieved this survival and vascularization in the absence of cytotoxic radical formation.
Conclusion: Metabolism supporting biomaterials are introduced as a novel method to support implant survival and realize functional integration within a host. Specifically, a glucose is identified as a key metabolite maintain cell survival under anoxic conditions, and controlled release of glucose allows for the survival and vascularization of living implants.
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