3D Differentiation of Mesenchymal Stem Cells in Peptide Amphiphile Matrices

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DESCRIPTION (provided by applicant): New materials that are capable of interacting with adult stem cells to direct their differentiation toward a certain terminal cell type will play an important role in the emerging field of regenerative medicine. The proposed work details a strategy for inducing the differentiation of human mesenchymal stem cells (hMSCs) into osteoblastic lineage inside a three dimensional scaffold, with applications in bone regeneration. The proposed scaffold will be comprised of peptide amphiphiles (PAs), which are synthetic materials made entirely from common biological components (peptides and fatty acids). PAs of the family described here are well-known to form high aspect ratio nanofibers in aqueous solution, yielding a fibrous gel that resembles the extracellular matrix in nanostructure. PAs will be outfitted with dexamethasone (Dex) bound to the PA via a hydrolysable hydrazone linkage. Dex is well-known to induce osteogenesis of hMSCs in 2-dimensional culture, but an injectable, 3-dimensional, Dex-releasing gel capable of differentiating hMSCs and supporting their proliferation in vivo has never been reported. Once a Dex-releasing PA has been synthesized, Dex release will be measured from the PA gels into buffer in the absence of cells. Zero-order hydrolysis of the hydrazone bond is expected to release Dex in a slow, sustained manner. After release kinetics have been determined, hMSCs will be cultured in three dimensions inside the proposed Dex-releasing gel, which will be constructed with a diluent PA to provide structural support. Characterization of the gel will be accomplished using electron microscopy, small-angle X-ray scattering, rheology, and circular dichroism. A combination of staining, gene expression, and protein expression will be used to quantify the extent of osteogenesis and mineral production. In parallel with the Dex-releasing PA, a PA that displays a peptide sequence mimicking the growth factor bone morphogenic protein 2 (BMP-2) will also be developed. The proposed BMP-2-mimetic PA may function in synergy with the Dex-releasing PA, eventually yielding faster bone production and healing than either component alone. Several in vitro experiments will be performed to compare the two methods of osteogenesis, which function through different cellular pathways. If successful, the Dex-releasing and BMP-2-mimetic PA scaffolds developed and studied here may be used as injectable materials in conjunction with hMSCs for bone development in humans. Such a treatment may drastically improve the quality of life of patients with severely compromised bone regeneration capacities due to bone defects, removal of cancerous bone, or osteoporotic fractures. PUBLIC HEALTH RELEVANCE: Bone defects due to cancer, trauma, or osteoporosis are an increasing problem in the context of an aging population. The proposed research will investigate an injectable material that when mixed with adult mesenchymal stem cells may be capable of promoting bone regeneration when the body's natural regeneration capacity is compromised. The results of this work will inform future studies aimed at improving the quality of life of patients with severe bone defects.