High-resolution ultrasonic monitoring of cellular differentiation in an ex vivo produced oral mucosal equivalent (EVPOME)

Background, Motivation and Objective This study examines the use of high-resolution ultrasound to monitor an ex vivo produced oral mucosal equivalent (EVPOME) as it develops from oral keratinocytes being seeded on a dermal cadaveric scaffold, with surface variations, into a stratified uniform cellular layer. Ultrasonic profilometry should be able to detect filling and smoothing of surface irregularities as seeded cells proliferate. As these tissue-engineered structures develop, seeded cells stratify due to their differentiation in which they produce a keratinized protective upper layer. These cells change in shape and composition, lose water content, and accumulate proteins (keratins) transformations which could alter ultrasonic backscatter. If non-invasive ultrasonic monitoring could be developed then tissue cultivation could be adjusted in-process to account for variations in the development and manufacture of the stratified cellular layer. Statement of Contribution/Methods To create an EVPOME specimen, oral mucosa keratinocytes were dissociated from human oral tissue samples and then seeded onto a scaffold of acellular cadaveric dermis. EVPOME's were cultured submerged for 4 days to form a continuous epithelial monolayer and then raised to an air-liquid interface for another 7-10 days. At specific intervals (1, 2, 4, and 7 days) specimens were imaged with an ultrasound acoustic microscope that consists of a single-element transducer (61 MHz center frequency, 32 MHz bandwidth, 1.52 f#) with a three-axis stepper-motor-controlled positioning system. Lateral step size was 15 ?m, about half of the 37 ?m resolution. Ultrasonic images were created using confocal image reconstruction. Tissue surfaces were determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise. Roughness was measured with root-mean-squared (rms) surface height. Echogenicity was measured using apparent integrated backscatter (AIBS) with respect to a fused silica flat. AIBS was calculated over bandwidth after gating the signal with a window centered at the specimen surface. Results There was no considerable change in measured roughness (20 to 22 ?m) from unseeded scaffold through day 4 after seeding. Between days 4 and 7 rms heights decreased from 22 ?m to 13 ?m. AIBS first changed between days 1 and 2 when it dropped from -30 dB to -41 dB. Echogenicity increased again from days 4 to 7 from -40 dB to -29 dB. Discussion and Conclusions Ultrasonic profilometry did not detect seeded cells filling in scaffold irregularities until cells had sufficiently stratified and differentiated by day 7. The initial drop in AIBS on day 2 may be due to undifferentiated seeded cells attenuating but not scattering ultrasound at that layer. More maturely differentiated cells, present at day 7, show brighter echogenicity possibly due to cellular changes. These initial results show that ultrasonic characterization may have potential to monitor EVPOME development during its manufacturing process.