Date of Completion
Yusuf Khan, Lakshmi Nair, Barry Carter, Jon Goldberg
Field of Study
Master of Science
Regenerative engineering is a developing field that strongly relies on two strategies to promote the repair of target tissue— the implantation of constructs that: 1) contain cultures of relevant cells, such as adult stem cells, that can participate in tissue formation and influence the surrounding microenvironment, and/or 2) promote the activity of host cells via physical and/or chemical cues introduced by the selected biomaterial. To this aim, poly(lactide-co-glycolide)/calcium peroxide (PLAGA/CPO) microsphere scaffolds have been fabricated, characterized, and shown to have potential in directing human adipose derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) towards bone and blood vessel formation in vitro due to the observed release of calcium ions, hydrogen peroxide molecules, oxygen molecules, and hydroxyl ions. The incorporation of CPO at 0.5% and 1% did not affect exterior microsphere smoothness, however led to the concentration-dependent formation of interior oxygen-containing reservoirs. Calcium ions and hydrogen peroxide molecules were released over a 3 day period, and in the presence of phosphate groups, calcium ions reprecipitated to scaffold surfaces to form a biomineralized layer. Enhanced oxygenation was observed as early as 1 hour post incubation, and the pH levels were maintained at or slightly above the control conditions despite the acidic by-products from polymer degradation. In culture with HUVECs, CPO scaffolds were effective in promoting cell adhesion, proliferation, and the formation of microcapillary-like structured as early as 3 days, as determined by scanning electron microscopy (SEM). Furthermore, key markers in angiogenesis, such as angiogenin and vascular endothelial growth factor (VEGF) were upregulated or secreted in enhanced quantities due to the released by-products of CPO decomposition. hADSCs seeded on CPO scaffolds adhered, spread, and underwent osteogenic differentiation up to 28 days, as shown by SEM, alkaline phosphatase activity, alizarin red staining, and elevated levels of osteocalcin, a late stage bone matrix protein. Interestingly, the addition of catalase, an enzyme that catalyzes the decomposition of hydrogen peroxide to oxygen, accelerated bone formation as well as promoted the induction of hADSCs towards the endothelial phenotype, as observed by changes in cellular morphologies as well as the formation of microcapillary-like structures. This work details the first use of CPO as an inexpensive, stable, chemical agent capable of delivery from biodegradable polymeric microsphere scaffolds to promote the angiogenic behaviors of endothelial cells as well as osteo- and angiogenic behaviors of adult stem cells, and provides a strong foundation for future work exploring the molecular mechanisms regulating the observed behaviors as well as potential success in clinically relevant settings as a bone graft substitute.
Ashe, Keshia, "Design and Fabrication of Osteogenic and Angiogenic Composite PLAGA/CPO Sintered Microsphere Scaffolds for Bone Tissue Engineering" (2016). Master's Theses. 867.