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A tissue construct model for investigating the therapeutic effects of ultrasound on cartilage

15 February 2008

Clinical trials show that intensity pulsed ultrasound accelerates the healing of bone fractures by 30-40%.

The American Food and Drug Administration have approved the use of an ultrasonic osteogenic healing device, to augment normal fracture healing and treat bone non-union by daily application of a twenty minute localised ultrasound treatment.

Unfortunately, without a recognised human-based in vitro test model, human clinical trials are delayed whilst animal tests are conducted – and inevitably, these prove to be contradictory.

A new Lord Dowding Fund grant to researchers at the Eastman Dental Institute in London aims to result in a novel biological model of cartilage, free from animal-derived products, which can provide high quality tissue regeneration data for the establishment of clinical trial protocols.

Importantly, this model will be able to assess therapeutic response within a human-derived biological system, thereby avoiding species differences.
This could prevent animal suffering and benefit over 2,000,000 people in the UK alone.

Background

Cartilage degeneration and damage results in over two million annual visits to the local doctor in the UK, and is the leading cause of joint replacement surgery.

The expense and donor tissue limitations of surgical treatment mean that a non-invasive, ultrasound-based technique that enhances cartilage self-regeneration could be hugely important.

An increasing body of literature suggests that, like bone cells, chondrocytes, (the cells that make up cartilage), respond beneficially to ultrasound. This results in up-regulation of key extracellular matrix-associated proteins and genes in vitro. Thus, ultrasound treatment has been suggested as a potential clinical tool for the repair of damaged cartilage tissue.

Unfortunately, studies to date have revealed variations in ultrasonic effects on chondrocyte proliferation, depending on the studies and cell species.
The establishment of an experimental method of chondrogenic cells within an animal derivative-free matrix, will create a biological model from which histological, cell proliferation and other data can be gathered without animal dissection.

The model will also provide a high degree of control over ultrasound dose delivery. This avoids some of the problems with previous animal studies, such as variation in tissue thickness, as well as potential for poor dose delivery from misalignment of ultrasound.

Additionally, the use of human-derived biological specimens, rather than animal tissues, avoids justifiable concern regarding the extrapolation of animal findings to human subjects.

Constructs can be manufactured with reproducible physical, chemical and mechanical properties to match human tissue properties such as viscosity, density and elasticity. These properties, which may differ in animal tissues, fundamentally define the acoustic properties of tissue.

Project Aims

The LDF’s policy is to encourage the replacement of cell culture media originating from animals – for example foetal calf serum. This study will therefore use an animal-free alginate culture system, hitherto unused in ultrasound studies but successfully applied in other physical therapy studies.
The alginate matrices can be manufactured with specific mechanical properties and cell populations dispersed, cultured over several weeks and sectioned or digested for investigation by a variety of methods.

There are five stages to the project, encompassing different areas of expertise.
In the first six months an hMSC-alginate model of cartilage tissue is to be established. Using human patient cells, initial experiments will confirm the progress of each cell population down a chondrogenic pathway, following pelleting by centrifugation and application of specific chemical reagents. The protein and gene expression of macromolecules constituting the cartilage matrix will be determined over culture time. Gels of varying weights and volumes will be manufactured and their mechanical properties investigated against alginate content. Gel matrix concentrations will be tailored, as much as possible, to match the properties of cartilage tissue.

Simultaneously, a dosimetric evaluation of new ultrasound exposure system is planned with the construction of an ultrasound exposure device at the Open University. The ultrasound fields will be characterised with a needle hydrophone to ensure no reflections or standing waves are formed within the system. Measurements will be taken across each of the six piezoelectric ultrasound transducers and the dose distribution and mean dose will be established. Following this, hydrophone measurements will be made at the entrance, exit and at intermediate path lengths through the alginate gel to establish the mean ultrasound dose per cell within the cartilage model.
This will be followed by extensive examination of the effects of standard SAFHS treatment on developed cartilage model at the Eastman Dental Institute. The bulk of the work will examine the effects of standard SAFHS exposure parameters, which are currently used in clinical treatment of bone fracture, on the cartilage model. Twenty minute exposures will be applied, which is what occurs in the clinical protocol.

This study will represent the first SAFHS investigation of human cells within a tissue substrate.

Studies will initially address effects on cytotoxicity and cell growth, by using an established staining technique and also by measuring the incorporation of radiolabeled DNA precursors into the newly synthesised genetic material. DNA content will then be analysed to determine the proportion of cells at various stages of multiplication providing crucial data about whether, and which parameters of the ultrasound influence the cell cycle.

Thereafter the expression of key cartilage proteins and generic growth factors will be investigated for SAFHS exposure using flow cytometry. Gel sectioning and staining of notable changes over extended time periods will follow. If sufficient progress is made, the effects of ultrasound on apoptosis (programmed cell death will be examined due to its importance in regenerative processes.

Work at the Eastman will also examine the ultrasound intensity which initiates beneficial effects in cartilage. This will be performed by insertion of a thin urethane disc within the exposure assembly to provide controlled attenuation of ultrasound from the prototype exposure unit.

This work will determine the maximum attenuation (thus calculated tissue depth dependant on anatomical site) that can be applied to SAFHS therapy in order to induce a positive effect on cartilage repair. This will generate new information as to whether cartilage, located at deep sites, is a legitimate choice for treatment by ultrasound, information useful within the medical community. Keeping all human radiation exposure “As Low As Reasonably Achievable” is a key aspect of Medical Devices legislation (the ALARA principle). This may permit future therapy protocols where exposure time can be increased if the device output is lowered.

To optimise the parameters of the therapy, a new prototype unit will be supplied by Smith and Nephew by the later stages of the study. This unit will emit, provisionally, a choice of three ultrasound frequencies and a range of pulsing characteristics. Pulsing is particularly useful for cartilage tissues where blood supply is poor and so there is potential to thermally damage tissue. Studies will focus on determining if pulsing regimes can use greater beam-off time, to reduce thermal damage, without diminishing the benefits of ultrasound exposure. This will be measured by gene and cartilage matrix production.

At the Open University, the final part of the study will examine the acoustic mechanisms that may elicit such biological effects within the alginate gels. The extent of the three main acoustic mechanisms, which will induce heating, cavitation and radiation force, will each be examined by experimental techniques to assess their likelihood and impact on biological response within the ultrasound exposure system.

The magnitude and presence of these three acoustic mechanisms will be examined using previously described methods. These are infra-red thermometry for heating, spectral analysis to determine sub-harmonic emission for cavitation presence and Doppler laser or Doppler ultrasound measurement for bulk streaming effects.

By approaching this study from a biological angle and a mechanistic one too, it is hoped that this will greatly increase the multi-disciplinary interest and impact of the published work.

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