Understanding and Preventing Inappropriate Osteogenic Differentiation

One of the major issues affecting individuals who have recieved some sort of traumatic injury, is heterotopic ossification. This impacts 22% of spinal injury cases, 11% of traumatic brain injury cases, and 65% of service personnel injured by explosive blasts. Heterotopic ossification is the growth of bone tissue, in a non-bone structure, such as the muscle. The development of bone in a soft tissue location is regarded as inappropriate differentiation. It can result in decreased movement, and debilitating pain for the patient. One of the problems in developing treatments for this condition is that the biological mechanisms, and even the cells that are turning into bone, remain unknown.

In this research proposal, we propose to utilise a cell type found in the hair follicle, which under the correct stimulating conditions can turn into bone. Like heterotopic ossification seen in individuals after injury, this development of bone from hair follicle cells is regarded as inappropriate differentiation. We will use these hair follicle cells to try and uncover the biological mechanisms that are activated by injury resulting in heterotopic ossification. In our first objective, we will exploit the availability of a modified shock tube that can replicate blast injury in a lab setting, to simulate injury in cells isolated from the skin and hair follicle. We will see if mechanical injury itself can 'stimulate' hair follicle cells, and turn them into bone cells. This will help us to understand if mechanical injury stimulates the cells that are to become bone, or whether they stimulate other cells in the body that then send signals to cells around them, turning them into bone. Our second objective is to establish a genetic profile of hair follicle cells before and after mechanical injury, and bone development, to understand why hair follicle cells have this interesting property, enabling them to turn into bone. This may help to identify therapeutic targets for preventing bone formation. Lastly, the third objective is to screen a number of small molecules, and drugs, to see if any of them can prevent hair follicle cells turning into bone. The concept of this third objective is to identify potential small molecules that could be used clinically to prevent or reduce the heterotopic ossification observed in patients after traumatic injury.

This entire proposal utilises human cells, isolated from scalp biopsies. We are therefore assessing pathways and genes involved in inappropriate bone formation in a human cell model, making it ultimately translational for clinical application. Our overall goal is to identify new targets for therapeutic intervention, to prevent or reverse heterotopic ossification.

Heterotopic ossification (HO) is frequently observed in individuals after traumatic injury. The cell types that inappropriately differentiate into osteoblasts, in addition to the molecular mechanisms underlying HO remain elusive. Current research approaches often utilise bone marrow stem cells, and evaluate how they differentiate into osteoblasts. In this project, we rationalised that since HO is regarded as inappropriate differentiation, we should use a cell type that can, but shouldn't turn into bone in normal circumstances. We settled on using hair follicle dermal papilla cells, which in the correct culture conditions can inappropriately differentiate into bone.

In Objective 1 we will utilse a modified shock tube to mechanically injure dermal papilla cell cultures. We aim to establish the conditions whereby mechanical injury can drive osteogenesis, assessing whether the cells themselves provide all the signals necessary to drive differentiation.

In Objective 2, we will use ChIP-Seq to identify the location of Ets1 and Runx2 transcription factor (TF) binding sites. Ets1 and Runx2 are two TFs known to bind DNA and drive osteogenesis. We will determine whether these binding sites are in active, poised, or repressed states, comparing fibroblasts, and papilla cells, both before and after they have been injured using the shock tube. This will resolve if papilla cells have TF binding sites that are always open, or whether injury elevates these sites into an active state, resulting in gene expression.

In Objective 3 we establish a reporter screening assay that will express GFP when papilla cells are pushed to an osteoblast fate. We will utilise this assay to detect small molecules and proteins that can inhibit inappropriate osteoblast differentiation in dermal papilla cells.

Completion of this work will provide new insights into inappropriate osteoblast differentiation, which is a fundamental requirement if we aim to prevent HO.

Who will benefit?

Several groups will benefit from this research. Academics, including PI's, postdocs and students will all benefit, as will Academic clinicians and clinical students. Other beneficiaries include those in the commercial private sector, military personnel, patients and the NHS, the wider public, and the staff working on this project.

How will they benefit?

-Academics (PI's, postdocs, students)
Academics will benefit though an increased understanding of the mechanisms of stem cell differentiation, and inappropriate osteogenic differentiation.

-Academics (Clinicians and clinical students)
Medical clinicians and clinical students interested in traumatic injury will gain an increased understanding of the impact that injury can have on the body, and its cellular processes.

-Commercial private sector
Radiation therapy, NSAIDS, and further surgical procedures are currently used to alleviate the chronic pain caused by heterotopic ossification. However, there is no drug that can effectively prevent, or reverse long standing heterotopic ossification. The market potential for pharmacological agents to treat heterotopic ossification is huge, and we believe that an improvement in the understanding of inappropriate ossification will directly impact the commercial private sector. We hope that our research will help to identify new therapeutic targets for prevention of heterotopic ossification. This is important as it will lead to new drug development for treatment of heterotopic ossification.

-Military personnel
Up to 65% of military personnel injured in high impact blasts develop heterotopic ossification. Understanding the mechanisms of inappropriate differentiation may provide insight into therapeutic targets to alleviate, or prevent heterotopic ossification. It may also impact the schedule of treatment, or the cells that are targeted by said treatment, and enhance quality of life for affected individuals.

-Patients and the NHS
Heterotopic ossification complicates the extremities in 22% of spinal injury cases, and 11% of traumatic brain injury cases. It is also often a complication of elective surgeries for conditions such as hip arthritis, and is seen in the congential disorder Fibrodysplasia Ossificans Progressiva. Ectopic bone can push against blood vessels and nerves resulting in chronic pain and reduced movement. While there is no conclusive treatment, radiation therapy, NSAIDS, and further surgeries are all proposed to be beneficial. We hope that this research will lead to the development of drugs that prevent or mitigate the impact of heterotopic ossification, resulting in improved human health.

-The general public
Heterotopic ossification has been known about for 1000 years. However, the recent military conflicts in Iraq and Afghanistan have resulting in an increased incidence, with 65% of military personnel with traumatic injuries developing heterotopic ossification. This sudden increased incidence means that the general public is gradually becoming aware, and more interested in heterotopic ossification.
Cellular reprogramming is also a very 'hot' topic at the moment, and mechanisms that result in adult cells differentiating into another cell type are of public interest.

-Staff working on this project
The postdoctoral researcher who is employed to work on this project will gain an in-depth experience of a broad variety of techniques, including microdissection, in vitro cell culture, shock tube manipulation, ChIP-Seq, bioinformatics and molecular biology, drug screening, presentation and writing skills. All these skills are highly transferable.