Bone regeneration using ultrasound beat frequencies

骨折是一个重大的医疗保健挑战骨科服务在英国。在英国进行的一项为期10年的研究（2004-2014）显示，250万例骨折入院，髋部、桡骨、踝关节和手部是最常见的骨折部位。据国际骨质疏松基金会估计，2019年至2034年间，英国的脆弱性骨折数量将增长26%，每年发生66.5万例脆弱性骨折。标准骨折的修复通常依靠手术复位和固定，根据严重程度需要等待几个月。由于这个原因，骨刺激器的市场正在增长。这个市场估计价值21亿美元（复合年增长率10.1%），包括超声和电刺激设备，旨在改善各种临床情况下的骨骼生长，包括手术后。相比之下，使用声波刺激，在千赫频率在骨刺激方面的探索相对不足。我们的研究小组先前已经证明，1 kHz振幅为30 nm的振动（称为“纳米效应”）在体外培养间充质干细胞（MSCs）时具有成骨作用。该过程利用细胞对机械力的敏感性，利用它进行表型控制。我们最近将这种刺激应用于可穿戴设备，类似于触觉技术。在大鼠模型中没有观察到骨形态的变化，尽管骨形成的血源性标志物升高（数据未发表）。部分挑战包括选择正确的体内模型，但也要确保通过软组织传递振动并进入骨骼。长期以来，LIPUS形式的超声波可以产生类似的治疗效果，利用相对较高的载波频率和重复的脉冲，复制1khz的振动，以促进纳米化的治疗效果。此类器械已被批准（由英国NICE和美国FDA批准）用于治疗骨不连骨折，但由于缺乏明确的机制理解和在某些情况下存在缺陷的实验程序而未得到优化。优化将扩大患者的受益范围，以及可以通过这种非侵入性门诊技术有效治疗的疾病范围，包括一般骨折和骨质疏松症。在这个项目中，我们建议测试一种提供1khz骨刺激的替代方法，利用超声参数阵列在受控焦点区域产生1khz的拍频。我们的假设是，与之前的标准纳米振动设备相比，这将使信号更好地渗透到骨骼核心中，并且能够应用更大的潜在频率范围。使用两个超声波换能器可以产生一个节拍频率，每个换能器的驱动频率略有不同。产生的波前变得越来越非线性，声速对密度的二次依赖导致和场和差场的产生，或者一个拍频等于最初产生的两个波前之差。这种参数阵列将提供对纳米振动传递到目标组织区域的新程度的控制，允许真正详细地描述治疗效果，并旨在更有效地将纳米振动传递到目标。在这个博士项目中，我们将专注于成骨细胞（MG-63s, MSCs）的初步体外测试，比较1 kHz拍频与我们现有的块压电致动器振动装置的应用。测试将包括细胞的2D和3D培养。对于3D培养，一个关键的评估将是细胞成骨的空间分辨率，例如通过免疫荧光染色或结构的组织学进行评估。然后，我们将根据这些数据寻求开发一种适合患者刺激的可穿戴设备原型。

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.