Research Summary Research topic:
Studying the role of major cell signaling pathways, such as bone morphogenetic protein (BMP), sonic hedgehog (Shh), and Wnt in directing the growth, development, differentiation, and maintenance of pre- and postnatal intervertebral disks Research results:
Understanding the biological mechanisms that preserve the integrity of intervertebral disks and ensure proper function Patient care application of results:
Repairing degenerative and injured intervertebral disks at the molecular and cellular level, restoring tissue health and functionality
Simplified patient care application: Regenerating the shock-absorbing power of aging, injured, or degenerataed intervertebral disks to eliminate back pain yet retain spinal flexibility
Disk Regeneration: A Dream Within Reach?
OREF grant recipient is discovering clues for disk regeneration
Jay D. Lenn
The Catch 22 of managing back pain due to disk degeneration is that while surgical treatment usually eliminates pain, it often reduces flexibility of the spine.
The precise biologic mechanisms that preserve disk integrity and thus ensure proper function are still a mystery. Among research studies to date, there is no consensus on treatment methods that will best alleviate pain and preserve flexibility With support from a 2011 Orthopaedic Research and Education Foundation (OREF) Career Development Grant, Chitra L. Dahia, PhD, assistant professor, Division of Orthopaedics at Cincinnati Children’s Hospital Medical Center, is determined to change that. She is investigating cell signaling pathways that induces intervertebral disk growth, development, and differentiation in an in vitro mouse model.
Finding pathways to disk generation
An intervertebral disk has a flexible outer wall called the annulus fibrosus and a jelly-like center core called the nucleus pulposus. The nucleus pulposus produces large amounts of proteins (proteoglycans) with properties that enable it to retain water, which is key to its role as a shock absorber between vertebrae. Degenerative disk disease involves the loss of nucleus pulposus cells, changes in water and protein content, and loss of cushioning function.
Each disk is a self-contained, avascular unit. “It is a microenvironment,” said Dr. Dahia. “Our central hypothesis is that the nucleus pulposus of each disk is a signaling center that controls the proliferation and differentiation of cells to maintain the components of that particular disk.”
Dr. Dahia and her colleagues have studied cultured mouse disks, which resemble the structure and function of human disks. Because development of the disk is not complete at birth in mice, the postnatal mouse disk provides a window into the cellular and molecular processes for creating and maintaining disk components.
In previous research with their mouse model, Dr. Dahia and her team observed a growth spurt during the first nine weeks after birth. While the basic structure of the disk is present at birth, cell proliferation and differentiation continue after birth to produce the mature properties of the nucleus pulposus and the surrounding annulus fibrosus, as well as the mineralized endplates that cover the surfaces of vertebrae where they come into contact with disks.
Previous research also enabled Dr. Dahia and her colleagues to demonstrate that certain signaling proteins, known to be critical players in embryonic development, continue to be expressed in postnatal nucleus pulposus cells. Signaling proteins essentially function as switches on molecular circuits. They direct events inside the cell that can lead to such outcomes as cell proliferation, differentiation, or apoptosis.
Dr. Dahia explained, “We are asking a number of questions: ‘What are the signaling pathways? Which cells respond to each pathway? And what is the particular response to each signal?’ ”
Two signaling proteins of particular interest to the team are sonic hedgehog (Shh) and bone morphogenetic protein (BMP), both of which are expressed in the postnatal nucleus pulposus.When the researchers inhibited Shh in their mouse model, they observed a decline in cell proliferation, induction of apoptosis, loss of a protein essential in maintaining the extracellular matrix, and an increase in response to BMP.
Following pathways to regeneration
In the OREF-funded investigation, Dr. Dahia and her colleagues are teasing out the details of BMP’s role. They want to learn if BMP is a step leading to one or more of the other outcomes of Shh inhibition. For example, does inhibiting Shh lead directly to apoptosis, or is BMP upregulation a necessary next step for inducing cell death?
Dr. Dahia and her research team are employing an antagonist that will specifically target and inhibit BMP in cultured neonatal mouse disks. They will assess the effects by assaying molecular markers of cell proliferation, cell apoptosis, and extracellular matrix synthesis in the nucleus pulposus, annulus fibrosus, and end plates.
They will then restore signaling by reintroducing BMP molecules to determine the specificity of the signaling pathway. Dr. Dahia noted, “We look at the same molecular markers. If the role of BMP signaling is very specific, then rescuing the signaling pathway should reverse the outcome. If a molecular marker was gone, it should come back. If a molecular marker went up, then it should go down again.”
Another aim of the research is to determine how BMP interacts with other signaling pathways. Therefore, using the same type of BMP inhibition and rescue in their model, they are assessing changes in other signaling proteins expressed in the nucleus pulposus.
Developing new treatments
The long-term goal of Dr. Dahia’s work is the development of new treatments. BMP molecules are already being used in experimental interventions, but more information about its function is critical in developing successful treatments. She said, “Once we identify the basic cellular mechanisms, we can work with tissue engineering groups on methods to regenerate damaged disks. This work provides the platform for understanding what signaling pathways we should activate.”
Dr. Dahia added, “Because of the OREF grant, I will be able to generate and publish important preliminary data that will be necessary to qualify for National Institutes of Health grants. I think if we can expand the basic understanding of the disk—if we understand the normal signaling pathway—we may eventually be able to regenerate a disk in the same way it was originally formed.”