Open questions for Mechanobiologics

A · Acoustic dose definition

1. critical Is the quoted pressure (4 kPa in Aging Cell; 4–8 kPa on the 2026 slide; 6–7 kPa in the patent; "4–10 Pa" in NCT06562374) peak, peak-to-peak or RMS? Is "Pa" in the trial record a typo for kPa?
2. critical Where exactly is it measured: on the beam axis at the target plane, a spatial average over the target zone, or the maximum in the tank? At what distance from the transducer face?
3. critical What is the spatial uniformity of the field at the target (a map, or min/max over the zone)? How much do standing waves vary with water level and with the subject's position?
4. critical Is a frequency sweep / jitter or beam steering used during treatment (as the patent describes)? If so, what is the sweep range (e.g. 32.2 ± x kHz), rate and waveform?
5. important Which parameter matters biologically: peak pressure, time-averaged intensity, particle displacement or velocity, or acoustic radiation force / streaming? Was a dose–response for pressure (the 1×, 1.3×, 2× power groups) measured in kPa?
6. important Was cavitation (stable or inertial) monitored with the MCT-2000? What cavitation levels were present in the cell, mouse and human setups? Is sub-cavitation a requirement?
7. important Why 32.2 kHz specifically? Was it the transducer's resonance, or a biologically optimal value found by scanning? What was the frequency–response curve (33 vs 39 kHz, anything below 30 kHz)?
8. important Duty cycle: 1.5 s or 1.6 s on/off? Rise/fall time (hard gating or ramped)? Is there a continuous-wave within the "on" period, or sub-pulsing?
9. nice Waveform purity: harmonic content and generator output waveform. The patent explicitly contemplates a rectangular drive and notes its strong 2×–7× harmonics — do the inventors believe the harmonic content is part of the active agent?
10. critical The Onda MCT-2000 is a cavitation meter, not a needle hydrophone. Was the "self-calibration to an absolute reference" performed, so that the kPa scale is real? Over what averaging time was the reading taken? Does "4 kPa" mean the meter's time-averaged total pressure, including harmonics?
11. critical Figure S1B maps the field in millivolts with a 9× spread across the sample plane. Where in that distribution does "4 kPa" sit: the centre, the mean, or the peak? What was the sample's position within the map?
12. critical Measured on your own equipment, with one convention: how far apart are the rejuvenation dose and the tumour-apoptosis dose of Tijore et al.? The published numbers imply only 12–18 dB, which is inside most replication error.

B · Hardware

1. critical Transducer manufacturer and model (Langevin / bolt-clamped? radiating face diameter, horn/plate?), resonant frequency, impedance at resonance, static capacitance, rated power.
2. critical Generator / amplifier: make and model, or a custom design? What does "varying power input 135–275 V" refer to: RMS drive voltage at the transducer, peak voltage, or supply voltage? What is the corresponding electrical power?
3. important Matching network (series/parallel inductor, transformer)? Is there resonance tracking (PLL) or a fixed frequency?
4. important Mouse and cell tank: exact vessel (4 L glass beaker, wall thickness), water height, transducer mounting (bonded under the glass or immersed?), position of the mesh, and any acoustic absorbers.
5. important Human tub: how many transducers (our count of ~10–12 on articulated arms is only our reading of a conference photo, and is corroborated by nothing published), their placement and orientation, per-channel amplitude/phase control, sequencing, tub material and dimensions, water volume.
6. important "Generators encased in epoxy within the baths" (OA trial): which epoxy/potting, electrical insulation design, and cable routing? What electrical safety standard was applied (IEC 60601-1, leakage currents, isolation transformer, RCD)?
7. nice Hydrophone calibration chain: MCT-2000 probe model and calibration certificate (frequency range, sensitivity at 33 kHz, uncertainty); how often it was recalibrated.
8. nice Would they share CAD drawings, schematics or the field maps of the mouse device, the older non-patented set-ups, or at least a reference measurement protocol?
9. important Is the senescence rig the same family as the cancer rig described in the sister papers — PZT4 rings with an aluminium cone, epoxy and silicone potted — or a Langevin transducer like the APC 90-4050 / PiezoDrive PDU 210 set-up used by the Bangalore group?
10. important "Varying power input 135–275 V" on Figure S1A: is that peak-to-peak or RMS at the transducer terminals, and what electrical power does it correspond to?

C · Medium & environment

1. important Degassing method and target dissolved-oxygen level (mg/L or % saturation). How long does degassed water stay usable?
2. important Temperature control: heater type, tolerance (32–35 °C), and whether sonication itself changed the temperature.
3. nice Water type (tap, deionised, additives, disinfectant such as chlorine), and whether the conductivity matters for safety or coupling.
4. nice Hygiene protocol between human sessions (filtration, disinfection) and whether it affects the dissolved gas content.

D · Exposure protocol

1. critical Human exposure: which body parts are immersed, what the subject's posture and distance to the transducers are, and whether the head is immersed. How is the dose at tissue estimated, given body attenuation and reflections?
2. important Why 45 min × 3/week in the RCT vs 30 min every 2–3 days in mice? Is there a known minimum effective dose, or an upper limit / saturation? What was the rationale for the mouse D3 (every 3rd day) schedule outperforming daily?
3. important Acute effects: how long after a session do cellular changes (Ca²⁺, autophagy, SASP) peak and fade? When should blood be drawn relative to a session?
4. important Does effectiveness depend on body temperature (warm immersion itself is a stimulus, e.g. heat-shock and sauna-like effects)? How was the sham matched thermally?
5. nice Are there contraindications identified so far (implants, pregnancy, cancer, open wounds, epilepsy, pacemakers)?

E · Biology & safety

1. critical Oncogenic safety: has LFU been tested on oncogene-induced (e.g. RAS) or DNA-damage-induced senescent cells? Do those cells re-enter the cell cycle? Did the γH2AX/53BP1 foci or karyotype of re-proliferating cells normalise, or do damaged cells divide?
2. critical Why was the UTMB osteoarthritis trial suspended by the IRB (March 2026)? Any adverse events in humans so far?
3. important Which blood biomarkers respond in humans, and in what time frame: SASP proteins, p16 in T cells, epigenetic clocks? What exactly is "senescence in blood via IHC/biochemistry/RNA-seq" in NCT07168525?
4. important Do the effects persist after stopping, and for how long (mice: rest periods of 2 weeks)? Is continuous treatment needed?
5. important Cancer-model data: the same group reports ultrasound killing tumour cells (mechanotherapy). What are the parameter differences between "rejuvenating" and "tumour-killing" regimes, and how close are they?
6. nice Raw data availability (RNA-seq accession, survival data, activity data) for independent re-analysis.
7. critical Auditory effects: were audiograms or tinnitus reports collected in the human pilots? The exposure sits above the ACGIH waterborne limit, whose evidence base is long-term tinnitus.
8. important How do they reconcile the largely negative diabetes paper (no change in glucose control or physical function in aged mice) with the strong performance effects in the lifespan study?
9. critical Has anyone run a single dose series on one cell population, from the rejuvenation regime up through the apoptosis regime? And at the rejuvenation dose, are transformed or pre-malignant cells killed, unaffected, or primed (as the 2025 capillary-constriction work on PIEZO1 would suggest)?
10. important In the mouse tumour study the highest power produced more tumour growth than untreated controls, and chick-embryo survival fell to 60% after the second exposure. How do you interpret those two results, and what upper bound do they place on human exposure?
11. important Has anyone tested whether the sharp 32.2 kHz optimum survives a change of vessel? If it is a mode of the 15.2 cm beaker rather than a property of cells, it will not transfer to a bathtub.

F · IP & collaboration

1. important Would Mechanobiologics tolerate or support a non-commercial, open-science replication (e.g. a research covenant not to sue, or an explicit statement that it is permitted)?
2. nice Interest in receiving independent calibration and biomarker data from DIY builders (a data-sharing agreement)?
3. nice Which of the "three other pending" applications cover device design versus methods?

Questions derived from the evidence review, the hardware analysis and the biomarker plan. Suggestions are welcome via GitHub issues.