Macroporous EH-PEG biomaterials have been created previously with non-water soluble polymers using techniques including porogen-leaching. Macroporosity within hydrogels facilitates both molecular diffusion and cell migration, and thus should promote osteogenic signaling among cells within EH-PEG hydrogels. This study investigates the influence of fibronectin, an extracellular matrix protein, on osteogenic cell signaling and differentiation in porous EH-PEG hydrogels. Human mesenchymal stem cells (hMSCs) were loaded in porous EH-PEG hydrogels with fibronectin concentrations of 0, 10, and 100 µg/mL gel. At days 1, 4, 8, and 12 total RNA was isolated and reverse transcribed. Quantitative real-time PCR was completed on BMP-2 and BMP-2 receptors to analyze osteogenic signal expression, as well as alkaline phosphatase and osteocalcin to analyze differentiation; corresponding protein expression was analyzed by enzyme based assays as well as immunohistochemistry. Results show increased cell attachment and spreading with fibronectin concentration throughout the study. Nevertheless, the inclusion of macropores within a hydrogel does impact the strength of the biomaterial. As the purpose of the orbital floor is to maintain the orbital contents, approximately 43 grams, our scaffold must provide the necessary mechanical support. Therefore, studies were completed to assess the material strength of porous EH-PEG hydrogels. To improve the strength of the construct, a thin layer of crosslinked EHD was bound in the center of the construct creating a three-layer scaffold. Compression testing was performed in addition to three-point bending, which emulates physiological orbital stresses, using an Instron 5565 mechanical tester. Results demonstrate improved strength with the addition of the EHD layer. Overall, this work develops both the biomolecular and mechanical properties of a novel EH-PEG hydrogel for orbital bone regeneration.
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