Delivery of prolyl hydroxylase inhibitors to MSC spheroids for enhanced angiogenic factor secretion

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Lassahn, Katy Ann
McDevitt, Todd C.
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Mesenchymal stromal cells (MSCs) are of clinical interest due to their ability to differentiate towards musculoskeletal lineages, modulate inflammatory responses, and promote new blood vessel formation. Angiogenesis is an important aspect of both tissue engineering and wound healing because it is critical to deliver essential nutrients and oxygen in order to facilitate tissue repair and regeneration. In hypoxic environments, the pro-angiogenic effects of MSCs are enhanced through stimulation of the HIF-1α pathway. A class of small molecules termed prolyl hydroxylase inhibitors (PHDi), stabilize HIF-1α through inhibition of the enzyme that degrades HIF-1α in the presence of oxygen. Thus, a chemically induced hypoxic cell response could be engineered to enable greater control over the pro-angiogenic secretory response of transplanted cells through varying the duration and dosage of exposure to PHDi. Treatment of MSCs with PHDi has been shown to enhance cell survival, improve bone regeneration, and increase new vessel formation in vivo. In addition to treatment with PHDi, the culture format of MSC growth can affect the angiogenic properties of MSCs. The culture of MSCs as spheroids has been shown to promote secretion of angiogenic growth factors such as VEGF. Thus, delivery of PHDi to MSC spheroids may have a greater effect on the angiogenic properties of MSCs than monolayer treatment. Sustained delivery of PHDi may be achieved within spheroids via biomaterial based microparticle incorporation. The microparticle delivery of PHDi within spheroids may allow for localized delivery of PHDi in order to reduce potential off target effects if delivered in vivo. The objective of this project is to determine the effect of sustained delivery of PHDi on the angiogenic properties of MSC spheroids. It is hypothesized that sustained delivery of a PHDi in MSC spheroids via MP incorporation will enhance the angiogenic factor secretion of the MSC spheroids compared to spheroid culture alone. To address this hypothesis, three candidate PHDi were screened to determine appropriate dosage based on VEGF secretion and efficiency of encapsulation within MPs. IOX2 was chosen to be encapsulated in poly lactic-co-glycolic acid (PLGA) microparticles (MPs) as a vehicle for sustained delivery within the spheroids. The effect of PHDi delivery on pro-angiogenic factor secretion was assessed by measuring expression of HIF-1α, secretion of angiogenic growth factors such as VEGF, and HUVEC migration assays. While soluble PHDi treatment of MSC spheroids had a significant effect on pro-angiogenic factor secretion, delivery of PHDi via PLGA MPs was unsuccessful. The ability to modulate the hypoxia response of MSC spheroids through PHDi delivery may prolong and enhance the pro-angiogenic effects of hypoxic environments on MSCs, thus alternative biomaterials should be investigated in the future for efficient PHDi delivery.
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