Biophysical characterization of low-frequency ultrasound interaction with dental pulp stem cells

BACKGROUND Low-intensity ultrasound is considered an effective non-invasive therapy to stimulate hard tissue repair, in particular to accelerate delayed non-union bone fracture healing. More recently, ultrasound has been proposed as a therapeutic tool to repair and regenerate dental tissues. Our recent work suggested that low-frequency kilohertz-range ultrasound is able to interact with dental pulp cells which could have potential to stimulate dentine reparative processes and hence promote the viability and longevity of teeth. METHODS In this study, the biophysical characteristics of low-frequency ultrasound transmission through teeth towards the dental pulp were explored. We conducted cell culture studies using an odontoblast-like/dental pulp cell line, MDPC-23. Half of the samples underwent ultrasound exposure while the other half underwent 'sham treatment' where the transducer was submerged into the medium but no ultrasound was generated. Ultrasound was applied directly to the cell cultures using a therapeutic ultrasound device at a frequency of 45 kHz with intensity settings of 10, 25 and 75 mW/cm(2) for 5 min. Following ultrasound treatment, the odontoblast-like cells were detached from the culture using a 0.25% Trypsin/EDTA solution, and viable cell numbers were counted. Two-dimensional tooth models based on μ-CT 2D images of the teeth were analyzed using COMSOL as the finite element analysis platform. This was used to confirm experimental results and to demonstrate the potential theory that with the correct combination of frequency and intensity, a tooth can be repaired using small doses of ultrasound. Frequencies in the 30 kHz-1 MHz range were analyzed. For each frequency, pressure/intensity plots provided information on how the intensity changes at each point throughout the propagation path. Spatial peak temporal average (SPTA) intensity was calculated and related to existing optimal spatial average temporal average (SATA) intensity deemed effective for cell proliferation during tooth repair. RESULTS The results demonstrate that odontoblast MDPC-23 cell numbers were significantly increased following three consecutive ultrasound treatments over a 7-day culture period as compared with sham controls underscoring the anabolic effects of ultrasound on these cells. Data show a distinct increase in cell number compared to the sham data after ultrasound treatment for intensities of 10 and 25 mW/cm(2) (p < 0.05 and p < 0.01, respectively). Using finite element analysis, we demonstrated that ultrasound does indeed propagate through the mineralized layers of the teeth and into the pulp chamber where it forms a 'therapeutic' force field to interact with the living dental pulp cells. This allowed us to observe the pressure/intensity of the wave as it propagates throughout the tooth. A selection of time-dependent snapshots of the pressure/intensity reveal that the lower frequency waves propagate to the pulp and remain within the chamber for a while, which is ideal for cell excitation. Input frequencies and pressures of 30 kHz (70 Pa) and 45 kHz (31 kPa), respectively, with an average SPTA of up to 120 mW/cm(2) in the pulp seem to be optimal and agree with the SATA intensities reported experimentally. CONCLUSIONS Our data suggest that ultrasound can be harnessed to propagate to the dental pulp region where it can interact with the living cells to promote dentine repair. Further research is required to analyze the precise physical and biological interactions of low-frequency ultrasound with the dental pulp to develop a novel non-invasive tool for dental tissue regeneration.