Every year, over twelve million people suffer from chronic wounds characterized by impaired tissue formation and remodeling. Previous treatments for acute wounds have failed to address the complexity of chronic wounds. In order to construct an appropriate biomaterial for the healing of chronic wounds, the material must be able to support successful tissue repair and regeneration, which is partially determined by the viscoelastic properties of the hydrogel [1]. Therefore we propose to use thiol functionalized hyaluronic acid (HA-DTPH) conjugated with recombinant fibronectin functional domains (rFNfd) as a scaffold to facilitate the healing of chronic wounds [2]. The thiol groups are covalently crosslinked with poly(ethylene glycol) diacrylate. By varying the ratio between the number of free thiols and acrylate groups of the crosslinkiner molecule, the rigidity of the hydrogel can be controlled. Thus we derived three crosslinking densities: 2 to 1, 6 to 1, and 12 to 1. The functional response of adult human dermal fibroblasts (AHDFs) was investigated as a function of crosslinker density using six assays: migration, proliferation, tractional force, cell rigidity via AFM, spreading and arrangement of actin cytoskeleton.
In the migration assay, a relationship was seen that AHDF migration was enhanced as the substrate stiffness increased. Additionally, the proliferation assay demonstrated that AHDFs prefer the 2-1 hydrogels rather than the 6-1 or 12-1. This was verified by cell counting after 1, 2, 3 and 4 day incubation period. Tractional force applied by the AHDFs was quantified using the "DISC" method; images of spread and relaxed AHDFs used for DISC analysis were acquired using a Leica Confocal microscope as a function of hydrogel deformation. Extent of hydrogel deformation was determined through the movement of florescent 40 nm beads embedded in the HA hydorgel. Levels of isometric tension within the living AHDFs were measured using AFM under the Shear Modulation Force Microscopy (SMFM). In the cell spreading assay the AHDFs had optimal spreading on the more rigid substrate indicating that the cells prefer a stiffer substrate. Arrangement of actin cytoskeleton was determined viewing the organization of the Actin fibers under the C onfocal microscope. Data from all the functional assays indicate that increasing mechanical properties is important for the AHDFs to retain their normal morphology and function. Ultimately, this data will provides insight into the optimal mechanical properties of hyaluronic acid hydrogels capable of sustaining invasive cell migration in chronic wounds.

Figure 1-Chart of Human Dermal Fibroblast Migration