Rapid Bone Graft Synthesis Through Dual Piezoelectric/Nanomechaniocal Stimulation

Bone graft is regularly used in surgery (plastics, maxillofacial surgery and orthopaedics); bone is actually the second most grafted tissue after blood. Ideally the surgeon wishes to take bone from one area (donor site) to another area (recipient site) to support the operation they are performing or to replace damaged, infected or cancerous tissue . However, a patient's own donor bone is in short supply and its removal can lead to complications at the donor site. This means the surgeon will often recourse to allograft - decellularised (and thus biologically inferior) - bone from cadavers or other people. A third, and growing, option is synthetic graft. Synthetic graft can be made from biologically active materials, but is not viable at the time of implantation and thus not yet as good as living bone, as cells must be made to infiltrate the material prior to regeneration.
It has been known for many decades that bone tissue responds to mechanical loading and commonly, mechanical loading via a bioreactor device is employed to enhanced bone formation in vitro. Recently, it has been also discovered that cells respond to nanoscale mechanical stimulation, and in particular, that nanoscale vibrations induce rapid bone formation from cells cultured in vitro. Interestingly, bone tissue produces minute electrical fields when loaded, a phenomenon which is hypothesised to help with the bone remodelling process during regeneration. However, if this electrical stimulation is important for the rapid generation of bone graft is not yet known and is a focus of this project.
Our bioreactor, that supplies nanoscale 'kicks' to cells in culture can be used to convert mesenchymal stem cells (the stem cells of the bone, simple to isolate from a patient's iliac crest or fat tissue) to bone forming osteoblasts. We will optimise this by designing new 3D architectures for the cells to grow in that work in synergy with the bioreactor. The scaffolds will emit physiological electrical signals that the body uses as cues to heal bone. Further, we will look at the biological building blocks, metabolites, that the cells use to form bone and incorporate these into delivery systems that also work with the bioreactor. The electricity produced by the scaffolds will trigger release of the metabolites, further driving bone formation.
This will ultimately allow us to improve the quality of living bone graft derived from a patient's own cells while reducing the lab time we need to make it.

We have developed a new bioreactor, the Nanokick, that supplies nanoscale high frequency-low amplitude mechanical cues to MSCs in 2D and 3D to induce osteogenesis. We aim to develop consumables that partner the Nanokick and further stimulate the MSCs. Our goal is to induce large numbers of MSCs to enter osteogenesis as quickly as possible within a 3D environment to provide tissue engineered bone graft and to study this process as a function of electromechanical stimulation.
To achieve this will make full use of the mechanical cues provided by the Nanokick and use piezoactive materials to provide electrical stimulation to the cells in order to potentiate osteogenesis. These will be both in disposable (coverslip that sits in the bottom of the well plate and can be discarded after use) and scaffold format (to enable future tissue engineering as in vivo toxicology is addressed). Cells will be seeded over the coverslips or scaffolds in synthetic hydrogels. We will tailor the hydrogels with adhesive and degradable motifs to optimise the 3D environment; the move to synthetic materials is important as it will facilitate xeno-free protocols as we develop the graft further.
Further, we will use our metabolomics workflows to identify bioactive metabolites that interact with biochemical pathways that we will identify. The metabolites will be incorporated into electrostimulated drug delivery vehicles and incorporated into the piezoactive coverslips or scaffolds. This will enable drug stability and delivery only as the nanokicking process is commenced.
Finally, we will investigate mechanotransductive pathways with particular focus on transmembrane mechanoresponsive ion channels such as piezo 1 and 2.
The work is important primarily as tissue engineered bone graft is required to support the aging population. However, it will also deliver mechanoresponsive-cell stimulating scaffolds an drug delivery systems containing novel metabolite drugs with known biochemical pathway.

Scientists and Healthcare Professionals.
We will use open access publishing to disseminate as broadly as possible. This will be of direct interest to the biomaterials, tissue engineering, stem cells, pharma and medical communities.

Laboratories.
There is a range of bioreactors available to academic laboratories including mechanical (eg BOSE, EBERS), perfusion (eg BOSE, Minucell) and rotary (eg Synthecon). All have drawbacks for bone production ranging from affordability, sterility, usability and functional outcome. The advantage of our system is that it uses standard cell culture consumables and thus standard protocols. This will clearly open up 3D bone research to many more labs.

Industry.
In the first instance our work will be of interest to industrialists working in the mechanotransduction (bioreactor) and material science (synthetic bioactive graft) areas. We will develop a bioreactor, consumables/scaffolds for it and try to move to use synthetic environments for our 3D bone graft. Companies working in this area include those with interest in bioreactors (e.g. BOSE, EBERS, Minuth, Synthecon etc) and providers of innovative tissue engineering scaffolds (e.g. BiogelX, Stem Cell Technologies, Baxter Healthcare, Medtronic etc). These companies would generate economic benefit from co-developing, licensing and exploiting our bioreactors and consumables for them. Secondly, we will appeal to Pharma with our novel mass spectrometry approaches to drug design (e.g. GSK). Thirdly, we will appeal to companies with an interest in advanced technologies, such as Medtronic, Genzyme and Advanced Tissue Solutions, who will be interested in tissue engineering approaches to bone graft.

Patients.
Bone graft is the second most transplanted tissue after blood. Autograft harvest from e.g. the iliac crest has a range of associated complications including herniation, instability, nerve damage, infection, deep hematoma requiring surgical intervention and pain [1]. Synthetic bone graft is increasing in use but is biologically inferior and tends to be used with BMP (e.g. Medtronic Infuse)[2]. BMP has to be used in high doses [3] and this has led to serious respiratory, neurological and inflammatory complications and the issuing of a public health warning by the FDA [4]. Subsequently, reports of the carcinogenic potential of BMP have also been noted. The development of "clean" tissue engineered bone graft with good volume (cms3) would be a major advantage. All surgeons we have surveyed strongly agree there is urgent unmet need.

Health Service Providers.
- Delivery of tissue engineered bone graft would reduce the need for two surgeries. This would represent a saving on surgical time and could be competitively placed compared to e.g. BMP treatment, which can cost up to £3k per patient in non-union fixation [4].
- Delivery of novel drugs and delivery systems. We will identify bioactive metabolites to enhance graft engineering. Our model could also be used to test 3rd party drugs and to advise personal therapies.
Public
Our work will raise interest in science, increasing awareness of the scientific challenges of supporting an aging population. We will engage with the next generation of scientists to inspire them to overcome the burden and limitations of bone grafting and bone regeneration.

1. Arrington 1996. Clin Orthop Relat Res. 329, 300-9.
2. Epstein 2013. Surg Neurol. 4, S343-S352.
3. Garrison et al 2007. NIHR Health Technology Assessment programme.
4. FDA Public Health Notification.
5. Dahabreh 2007. JBJ Editorial.