EARLY DIAGNOSIS AND INTERVENTION STRATEGIES FOR POST-TRAUMATIC HETEROTOPIC OSSIFICATION IN SEVERELY INJURED EXTREMITIES

Heterotopic sssification (HO) is the growth of bone in abnormal places, for example, in soft tissue. HO occurs frequently (>60%) after traumatic amputations, such as those experienced by warfighters in Iraq and Afghanistan. In a study of 300 military amputees, HO emerged as the single most important barrier to meaningful functional mobility, independence, and return to military service. This leads to an enormous economic cost to both the military health care system and the military community in general. HO also affects civilian patients affected by severe trauma and burns, broadening the impact of the current proposal. Currently, a diagnosis of HO can only be made after it is too late for drug or minimally invasive treatment strategies, and more drastic surgical procedures must be performed. These facts highlight the pressing needs that will be addressed in this proposal. Our research objectives are to improve the ability of medical science to identify not only which patients will develop HO, but also where it will develop, and to expand and improve treatment options so that traumatic injury patients enjoy improved recoveries.

In Aim 1, we will define the biological environment in the tissue near an amputation (where HO might develop), using a variety of techniques to identify the tissue, cellular, and gene expression features that are predictive of the future development of HO. In this aim, we will use histology (microscopic examination of stained sections of tissue that identifies different kinds of cells and whether bone is forming); Raman spectroscopy (a method that provides information about the chemical composition of tissues, a kind of chemical 'fingerprint'); and high-resolution ultrasound, a non-invasive approach that uses sound waves to see inside the body. We will measure how many stem or progenitor cells are present, how fast they divide, and how likely they are to become bone-forming cells. These cells are called 'connective tissue progenitors,' or CTPs, because they can differentiate into many different types of cells that make up connective tissue. In this sense, bone is a 'connective' tissue, as is muscle and cartilage and fat. We will also determine the gene expression profile of the bone-forming tissue during development, which will provide an enormous amount of data concerning the metabolic state of these cells.

In Aim 2, we will use advanced statistical methods (Bayesian models) to test whether the data that we collect in Aim 1 can predict the onset of HO. We have reason to believe that we will be successful because (1) members of the team have already published papers demonstrating that there are more bone-forming cells in affected tissue in injured patients that develop HO compared to affected tissue in injured patients that do not develop HO; and (2) members of the team have shown that Raman spectroscopy can identify different chemical fingerprints in injured muscle tissue versus pre-HO muscle tissue versus mature HO tissue.

In Aim 3, we will attempt to redirect the differentiation of stem cells from a bone-forming (osteogenic) pathway to an fat-forming (adipogenic) pathway. The occurrence of HO requires bone formation, and bone formation requires a population of CTPs to become activated to proliferate in the wounded tissue, giving rise to daughter cells that go on to differentiate locally to form bone. We will do this by treating stem cells isolated from the patient's tissue near the traumatic injury with biochemicals that have been reported to affect the osteogenic/adipogenic differentiation ratio. These biochemicals range from simple glucose (sugar) to drugs that affect how cells react to chemical signals around them. The most promising treatments can be selected for testing in animal models and eventual clinical trials in humans.

The ultimate applicability of this research is to improve our understanding of the factors and events that can predict in which patients and at which anatomic locations within wounds where HO will form before it cannot be stopped and to combine that knowledge with treatment(s) that can be targeted to specific patients and anatomic locations on an individual basis. A clinically relevant outcome may occur within the 2 years of the proposed research and would be in the form of either a method to predict the onset of HO or a treatment to advance to animal models.