Bone regeneration using ultrasound beat frequencies

在英国，骨折是骨科服务面临的重大医疗挑战。英国一项为期10年的研究(2004-2014)显示，有250万人因骨折入院，其中髋部、半径、脚踝和手部是最常见的骨折部位。据国际骨质疏松症基金会估计，英国脆性骨折的数量在2019年至2034年期间增长了26%，相当于每年665,000例脆性骨折。标准骨折的修复通常依赖于手术复位和固定，根据严重程度需要等待几个月。出于这个原因，骨骼刺激剂的市场已经出现了增长。这一市场估计价值21亿美元(10.1%的复合年增长率)，包括超声波和电刺激设备，旨在改善各种临床场景中的骨骼生长，包括手术后。相比之下，在骨刺激方面，使用khz频率的声刺激的研究相对较少。我们的研究团队此前已经证明，当应用于骨髓间充质干细胞(MSCs)的体外培养时，振幅为30 nm的1 kHz振动(称为纳米振动)是成骨的。这一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作为一种可穿戴设备应用，类似于触觉技术。虽然血液中骨形成的标记物升高(数据未发表)，但在大鼠模型中没有看到骨形态的变化。挑战的一部分涉及选择正确的活体模型，但也要确保振动通过软组织和进入骨骼。长期以来，LIPUS形式的超声波可以利用相对较高的载波频率和重复脉冲产生类似的治疗效果，这种重复脉冲可以复制被发现在纳米点击中促进治疗益处的1 kHz振动。这种装置(英国的NICE和美国的FDA)被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有缺陷的实验程序而没有得到优化。优化将扩大患者的利益以及可以用这种非侵入性的门诊技术有效治疗的条件的范围，包括普通骨折和骨质疏松症。在这个项目中，我们建议测试一种替代方法来提供1 khz的骨刺激，利用超声参数阵列在受控的焦点区域产生1 khz的拍频。我们的假设是，与以前的标准纳米振动设备相比，这将允许信号更好地穿透到骨骼核心，具有更大的潜在频率范围。可以使用两个超声换能器产生拍频，每个换能器的频率略有不同。产生的波前变得越来越非线性，由此声速对密度的二次依赖导致和场和差场的产生，或等于两个原始产生的波前之间的差的拍频。这种参数阵列将提供对将纳米振动传递到目标组织区域的新程度的控制，从而允许对治疗效果进行真正详细的表征，并旨在更有效地将纳米振动传递到目标。在这个PHD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拍频与我们现有的大宗压电致动器振动设备的应用。测试将包括细胞的2D和3D培养。对于3D培养，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构建物的组织学进行评估。然后，我们将基于这些数据，寻求开发一种适用于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.