(WHS-O.02) THE BALANCE BETWEEN CELL CONTRACTILITY AND ADHESION MODULATES PROVISIONAL MATRIX ASSEMBLY DURING WOUND CLOSURE
Thursday, May 16, 2024
6:00 PM – 6:30 PM East Coast USA Time
Upon injury of fibrous tissues, resident fibroblasts migrate into the wound bed, depositing a fibrous provisional matrix and pull the margins of the wound closer to accelerate wound closure. While cellular contractility and cell adhesion are two processes regulating wound healing, it remains unclear how these processes coordinate provisional matrix assembly during stromal closure. To address this question, we manipulated cell contractility and cell-matrix adhesion of Normal Human Dermal Fibroblasts Neonatal (NHDFneos) and Normal Human Lung Fibroblasts (NHLFs) and assessed wound closure in a 3D microtissue model of stromal wound healing. Briefly, NHDFneos and NHLFs were embedded in a type I collagen gel and seeded into μTug devices composed of wells with micropillars to form 3D microtissues. 24 hours after the tissues formed, a Nd:Yag Laser was used to create full-thickness wounds in the center of the tissue. Both NHDFneo and NHLF tissues were treated with contractility or adhesion modulators before injury and wound closure dynamics were assessed for 24 hours post-injury using time-lapse microscopy. Traction force microscopy was performed on the individual cell types seeded on type I collagen coated 5 kPa polyacrylamide gels under different treatment conditions using a Nikon-Eclipse-Ti microscope. Immunofluorescent staining against phosphorylated-paxillin was performed to visualize and quantify size of focal adhesions. In some experiments, staining against cellular fibronectin was performed to visualize provisional matrix assembly of fibers in tissues 24 hours post-injury. At baseline, NHDFneos healed the wounds in the tissues within 24 hours post-injury, while NHLF tissues failed to close the wounds. On 2D substrates, NHDFneos exhibited lower contractility and smaller adhesions compared to NHLFs. Treatment with manganese (Mn) increased focal adhesion size and contractility of NHDFneos as well as inability to heal full-thickness wounds in tissues. Mn-treated NHDFneo tissues revealed clustering of cellular fibronectin with little to no fibrillogenesis throughout the tissue post-injury, while untreated tissues assembled fibronectin fibers throughout the remodeled tissue post-injury. Conversely, treatment of NHLFs with a low dose FC11, a FAK inhibitor, lowered contractility and the size of focal adhesions to comparable levels as untreated NHDFneos. NHLF tissues treated with FC11 healed within 24 hours post-injury while untreated tissues failed to heal (N = 3, n = 20 tissues per group). Interestingly, NHLFs treated with blebbistatin and Y-27632, a myosin II and ROCK inhibitor respectively, exhibited lower contractility, but continued to fail healing tissues. Together, our data support the hypothesis that the balance between contractility and adhesion levels modulates wound closure in our microtissue system, which may inform new mechanotherapeutic strategies for treating impaired wound healing.