(WHS-P32) Matrix Mechanics Dictate Odontoblast Responsiveness to Photobiomodulation Treatments
Friday, May 17, 2024
7:30 AM – 5:00 PM East Coast USA Time
Objectives: Regenerative dentistry promotes the directed differentiation of stem cells. While the predominant focus of these efforts has been genetic manipulations, the critical epigenetic role of extracellular matrix (ECM) mechanics has been poorly investigated. This project aims to dissect the cellular responses of odontoblasts to matrix mechanics by replicating a wound-like environment.
Methods: Polydimethylsiloxane (SYLGARD 184) matrices, composed of a 10:1 ratio (base: curing agent) were poured into 12-well polystyrene plates. The polydimethylsiloxane (PDMS) plates were degassed for 5 cycles and cured for 48 hours in a 95°C oven. Mechanical stiffness was then assessed using a Shore A Durometer (Insize). Following sterilization with 70% ethanol, UV treatments, and serum coating, odontoblasts (MDPC-23) were seeded (100,000 cells/well) in hypoxic (1µg/mL CoCl2 + DMEM 10% FBS) and serum-deficient (0.2% FBS DMEM) to simulate a wound-like environment. Alamar blue assay was performed at 24 hours to assess cell viability, and fluorescence was assessed with a Spectrophotometer (i3Max, Molecular Devices). An adhesion assay was performed to observe cell-substrate interaction 5 hours after cell seeding. Photobiomodulation (PBM) treatments were performed with a near-infrared (810 nm) laser at 10 mW/cm2, 5 min, 3 J/cm2, 4.5 p.J/cm2 or 1 Einstein. Signaling pathways for TGF- β1 and FAK were investigated using small molecule inhibitors and recombinant protein.
Results: The shore stiffness assessment noted the ratio 10:1 had a stiffness of 1.14 ± 1.65 MPa, 2:1 of 1.16 ± 0.48 MPa, and 40:1 of 0.57 ± 0.27 MPa. These stiffnesses were significantly (n = 3, p < 0.05) lower compared to the polystyrene culture dish control of 5.41 ± 0.96 MPa. No differences were observed in cell adhesion assays in any of the conditions. Statistically significant (n = 3, p < 0.05) changes were observed in cell proliferation with varying matrix stiffness. TGF-β1 and FAK1 signaling was noted to modulate these responses. Ongoing studies are examining the role of PBM activation of these discrete pathways in mediating odontoblast adhesion and survival. Further, direct activation of these molecular pathways is being assessed with Western Blotting for pSmad2/3, pFAK, pAKT, and pPI3K.
Conclusions: The results from this study suggest that precision engineering of biomaterial mechanical properties and PBM treatments can synergistically promote directed differentiation for optimal dentin regeneration in a simulated wound environment. These findings will next be extended to in vivo animal models for ultimate human clinical translation as a novel Endodontic Regenerative Therapy.