Two-Photon-Excited FLIM of NAD(P)H and FAD-Metabolic Activity of Fibroblasts for the Diagnostics of Osteoimplant Survival.

Bioinert materials such as the zirconium dioxide and aluminum oxide are widely used in surgery and dentistry due to the absence of cytotoxicity of the materials in relation to the surrounding cells of the body. However, little attention has been paid to the study of metabolic processes occurring at the implant-cell interface. The metabolic activity of mouse 3T3 fibroblasts incubated on yttrium-stabilized zirconium ceramics cured with aluminum oxide (ATZ) and stabilized zirconium ceramics (Y-TZP) was analyzed based on the ratio of the free/bound forms of cofactors NAD(P)H and FAD obtained using two-photon microscopy. The results show that fibroblasts incubated on ceramics demonstrate a shift towards the free form of NAD(P)H, which is observed during the glycolysis process, which, according to our assumptions, is related to the porosity of the surface of ceramic structures. Consequently, despite the high viability and good proliferation of fibroblasts assessed using an MTT test and a scanning electron microscope, the cells are in a state of hypoxia during incubation on ceramic structures. The FLIM results obtained in this work can be used as additional information for scientists who are interested in manufacturing osteoimplants.

In Vivo Quantification of the Effectiveness of Topical Low-Dose Photodynamic Therapy in Wound Healing Using Two-Photon Microscopy.

The effect of low-dose photodynamic therapy on in vivo wound healing with topical application of 5-aminolevulinic acid and methylene blue was investigated using an animal model for two laser radiation doses (1 and 4 J/cm2). A second-harmonic-generation-to-auto-fluorescence aging index of the dermis (SAAID) was analyzed by two-photon microscopy. SAAID measured at 60-80 µm depths was shown to be a suitable quantitative parameter to monitor wound healing. A comparison of SAAID in healthy and wound tissues during phototherapy showed that both light doses were effective for wound healing; however, healing was better at a dose of 4 J/cm2.