Bone regeneration using ultrasound beat frequencies

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Bone fractures are a significant healthcare challenge for orthopaedic services in the UKk. A 10-year study in England (2004-2014) showed 2.5 million fracture admissions with hip, radius, ankle and hand being the most prevalent fracture locations. The number of fragility fractures in the UK is estimated by the International Osteoporosis Foundation to grow by 26% between 2019 and 2034, amounting to 665,000 fragility fractures per year. Repair of standard fractures often relies on surgical reduction and fixation, with a wait of several months depending on the severity. For this reason, a growing market of bone stimulators has emerged. Worth an estimated $2.1 billion (10.1% CAGR), this market includes ultrasound and electrical stimulation devices aimed at improving bone growth in a variety of clinical scenarios, including post surgery. In comparison, the use of acoustic stimulation, at kHz frequencies is relatively underexplored in terms of bone stimulation. Our research team has previously demonstrated that 1 kHz vibration, with an amplitude of 30 nm (dubbed 'nanokicking'), is osteogenic when applied to in vitro cultures of mesenchymal stem cells, MSCs (bone forming cells). The process utilises cellular sensitivity to mechanical forces, exploiting it for phenotypic control. We recently applied this stimulation as a wearable device, similar to haptic technologies. No changes to bone morphology were seen in a rat model, although blood borne markers of bone formation were elevated (data unpublished). Part of the challenge involves selecting the correct in vivo model, but also ensuring delivery of the vibration through soft tissue and into the bone. It has been long established that ultrasound in the form of LIPUS can generate similar therapeutic effects utilising a comparatively higher carrier frequency and a pulse repetition that replicates the 1 kHz vibration found to promote therapeutic benefit in nanokicking. Such devices are approved (by NICE in the UK and the FDA in the USA) to treat non-union fractures but are not optimised due to an absence of clear mechanistic understanding and in some cases flawed experimental procedures. Optimisation would expand patient benefit as well as the range of conditions that could be effectively treated with this non-invasive, outpatient technique, to include general fractures and osteoporosis as examples.In this project, we propose to test an alternative method to deliver 1 kHz bone stimulation, utilising an ultrasound parametric array to generate a 1 kHz beat frequency over a controlled focal region. Our hypothesis is that this will allow better penetration of the signal into the skeletal core over prior standard nanovibration devices, with a larger potential range of frequencies able to be applied. A beat frequency can be generated using two ultrasound transducers, each driven at slightly differing frequencies. The wave fronts generated become increasingly non-linear, whereby the quadratic dependence of sound speed on density leads to the generation of sum and difference fields, or a beat frequency equal to the difference between the two originally generated wave fronts. This parametric array will provide a new degree of control over the delivery of nanovibration to a targeted region of tissue permitting truly detailed characterisation of the therapeutic effects and the aim to more effectively deliver nanovibration to the target. In this PhD project, we will focus on initial in vitro testing with osteogenic cells (MG-63s, MSCs), comparing application of a 1 kHz beat frequency to our existing bulk piezo actuator vibration devices. Testing will include 2D and 3D culture of cells. For 3D culture, a key assessment will be spatial resolution of cellular osteogenesis, e.g. assessed through immunofluorescent staining or histology of the constructs. We will then seek to develop a prototype wearable device, suitable for patient stimulation based on this data.