Development of a heterogeneous pro-angiogenic protein mixture encapsulation system

Tissue engineering aims to develop functional organs and tissues, using a therapeutic combination of exogenous cells, 3D scaffolds and growth factors. One of the most significant hurdles limiting organ regeneration is the development of an effective vasculature supply of oxygen and nutrients to cells seeded in the engineered construct. Current approaches have remained unsatisfactory. As a possible solution to this problem, in Cardiovascular Tissue Engineering laboratory of University of Florida, a pro-angiogenic extract from the human placenta, referred to as Human Placental Matrix (hPM), was developed. Despite the observation that the extract induces and modulates the initial stages of angiogenesis, the newly formed networks degrade 5 days after initial treatment. Multiple applications of hPM at discrete time points promoted the formation of a more mature and stable capillary network, thus the use of a controlled release method was hypothesized stabilize network formation over extended periods. In these investigations, hPM was encapsulated using poly(lactic-co-glycolic acid) (PLGA) microparticles to extend the release period, without the use of crosslinking agents. Following optimization of the microparticle preparation phase, microparticle morphological features (size, encapsulation efficiency, porosity) were characterized and the associated protein release profiled. Subsequently, the cellular response to hPM delivered via PLGA microparticles was assessed using 2D and 3D Alginate-based hydrogel culture system with Human Umbilical Vein Endothelial Cells (HUVECs), to assess the angiogenic response. Results from the optimized encapsulation process showed microparticles with an average size of 91.8 ± 2.9 µm, with an encapsulation efficiency of 75%, and a release profile extending over 30 days. 3D angiogenic assay with hPM-loaded PLGA microparticles embedded showed initial stimulation of tubular angiogenic structures after 14 days and formation of a more mature angiogenic network after 21 days of culture, which was maintained stable until 28 days of culture. Although further optimization is required, a sustained angiogenic response over an extended period of 28 days was observed within the 3D hydrogel culture system. This confirmed the effectiveness of the controlled hPM release approach to guide formation and maintain capillary networks.