(52c) An in Vitro Platform Using DNA Handles to Spatiotemporally Control Multiple Bioactive Peptides

INTRODUCTION: Musculoskeletal injuries and diseases are a significant health concern in the United States with autographs as the primary standard; however, some disadvantages include donor site injury and morbidity, chronic pain, and a limited tissue supply. A potential solution is to use natural polymers, like hyaluronic acid (HA), combined with bioactive peptides to create tissue-engineered scaffolds to promote osteogenesis. However, during bone regeneration, there are complex molecular signaling cascades where multiple soluble factors are expressed at precise times and locations [1]. This makes the design of biomaterials that are capable of recapitulating the natural spatiotemporal signaling cascade challenging, but essential for promoting functional tissue regeneration. Due to DNA’s high specificity, we combined the use of complementary single stranded DNA (ssDNA) for temporal control of bioactive peptide presentation with a photosensitive HA hydrogel for spatial control to develop an in vitro platform that can independently and reversibly control the spatiotemporal display of multiple bioactive peptides.

METHODS: Norbornene-modified HA (NorHA) hydrogels were synthesized by dissolving NorHA in a photoinitiator and crosslinked with nondegradeable DL-dithiothreitol (DTT) when exposed to UV light. The total molar equivalence with DTT was set to 20% leaving the remaining 80% of the reaction sites available for tethering ssDNA; the hydrogels were soaked in a ssDNA/photoinitiator solution followed by photoconjugation using a photomask for spatial control and rinsed. A complementary fluorescently labeled biomolecule strand was added, rinsed, and imaged. For temporal control, the biomolecule strands and the complementary displacement strands were designed with a toe-hold region allowing for the removal of the biomolecule strands from the hydrogel surface. Using this approach, DNA handles were used to spatiotemporally tether peptides of interest, including RGD for cell-matrix adhesion, HAVDI for cell-cell adhesion, and OGP for osteogenesis. Cell behavior on these hydrogel surfaces were assessed using a phalloidin stain to analyze cell morphology and alkaline phosphatase staining as an early marker of osteogenesis.

RESULTS AND DISCUSSION: To spatially and temporally control bioactive peptide delivery for improved control over cell behavior, ssDNA was bound to the surface of NorHA hydrogels through photoconjugation using a photomask with 200 μm thick lines. When complementary biomolecule strands were added, 200 μm thick green fluorescent lines were observed demonstrating that DNA was spatially bound to the surface. After adding a complementary displacement strand, no fluorescent lines were detected demonstrating complete removal of the biomolecule strand from the surface. Mismatched (non-complementary) biomolecule strands, mismatched displacement strands, and having no DNA bound to the hydrogel surface served as controls and resulted in no fluorescent activity. Hydrogels were also made with two different ssDNA handles photopatterned to the surface resulting in two different complementary biomolecule strands spatially arranged in a crosshatch pattern, Figure 1. One of the two biomolecule strands was removed and re-added to show precise temporal control. This work demonstrates that using NorHA as the platform allows for spatial control, ssDNA handles are required to be bound to the hydrogel surface for biomolecule attachment, and that the correct DNA sequence on the biomolecule and displacement strands is required for proper temporal control. Fluorescent molecules were used as a model while ongoing work is using complementary peptide-conjugated ssDNA strands to evaluate the complicated interplay between cell adhesion (both cell-cell and cell-matrix) and osteogenesis.

REFERENCES: [1] C. Gao, et. al. Bone Res. 2017, 5, 59.