Neural pain pathways found to play crucial role in bone fracture healing

When a house catches on fire, we assume that a smoke alarm inside will serve one purpose and one purpose only: warn the occupants of danger. But imagine if the device could transform into something that could fight the fire as well. 

In a new study in today's issue of Science, a multi-institutional team lead by researchers at Johns Hopkins Medicine has shown in mice that the body's "pain alarms" - sensory neurons - actually have such a dual function. In the event of a bone fracture, these nerves not only report the trauma, but they also morph into "reconstruction commanders" that actively direct the cellular workforce to rebuild the skeleton. 

The mostly federally funded study reports on and details for the first time, a network among peripheral afferent neurons - nerves that send signals from all areas of the body to the central nervous system (brain and spinal column) - through which the nerves communicate directly with bone-building cells after a skeletal injury, using specific protein signals to stimulate the generation, growth and spread of new, healing bone. 

For the first time, we have mapped the circuitry of this neural network, defined which specific sensory neurons innervate [supply nerves to] bone, determined how these neurons change after an injury and identified which signals they produce that are necessary to promote bone formation and repair."

Zhao Li, M.D., Ph.D., study co-first author, senior research specialist in the James Laboratory, Johns Hopkins University School of Medicine Department of Pathology

To map the neural network by which signaling for bone repair occurs, Li and his colleagues used a laboratory-engineered adeno-associated virus with peripheral nerve tropism - a strong attraction toward peripheral nerves that innervate bone - to label which dorsal root ganglion (DRG) neurons - nerves along the spinal cord that are critical in transmitting signals from peripheral nerves to the central nervous system - actually do the job. 

"The technique, known as retrograde tracing, is akin to following a single electrical wire from a light bulb back through the walls to find where the circuit breaker lies," says James Laboratory leader and study corresponding author Aaron James, M.D., Ph.D., professor of pathology at the Johns Hopkins University School of Medicine. 

"We combined retrograde tracing with a second technique, single-cell RNA sequencing isolation, to study individual innervating DRG nerve cells in mice - before and after bone fractures - and then isolated them to determine the proteins each one produces," says James. "Putting the data for all of the profiled cells together, we created the first comprehensive single-cell atlas of bone-innervating sensory neurons, a map of the neural network and signals necessary for bone repair." 

In a 2019 study in the Journal of Clinical Investigation, James Laboratory-led researchers showed in mice that at a fracture point, two proteins - tropomyosin receptor kinase-A (TrkA) and nerve growth factor (NGF) - bind together to promote innervation and a process leading to the production of new bone. 

"When we blocked the response of TrkA+ neurons, either genetically or chemically, we saw a dramatic reduction in not only innervation but also in the three follow-up activities critical to successful recovery from a fracture: blood vessel formation, production of bone-synthesizing cells and mineralization of new bone," says James. "This finding indicated that fracture repair is truly dependent on the neural signaling directed by TrkA-expressing nerve fibers, yet the downstream molecular underpinnings were still unknown." 

First discovered in the 1950s, NGF is now known to direct the growth, maintenance, proliferation and preservation of neurons throughout the body, It also helps nerve cells alert the brain to which tissues, including bones, are experiencing pain from injury or disease. 

In their new study, the researchers dove even deeper into the bone healing process, looking for what transforms bone-specific DRG neurons from nerve cells that report inflammation or injury to the central nervous system into ones that trigger local bone repair. 

"What we discovered is that there are dynamic changes associated with sensory neuron response to bone injury and that these changes reflect how bone repair is done in phases," says Li. "Just after injury, DRG neurons are nociceptors, nerves that produce signals focused on pain perception and inflammatory responses, but then at later timepoints, they enter a different phase, a pro-regenerative state, where they produce and release proteins that promote the generation of new neurons, blood vessels, and of course, bone and cartilage." 

Li says that the team found three nerve-derived morphogens (signaling proteins that guide the formation and organization of tissues and organs, including bone) - transforming growth factor beta 1 (TGFB1), fibroblast growth factor 9 (FGF9) and sonic hedgehog (SHH) - of particular interest because they were expressed (produced) by genes in neurons during the first phases of bone repair. The researchers then surgically removed or genetically blocked the neurons responsible for these proteins, a process called denervation, in fractured bones for a group of mice. 

"We saw in the denervated mice that there was defective skeletal cell proliferation, less differentiation of stem cells into bone and cartilage cells, and overall poor bone repair," Li explains. "And with more extensive analysis, we were able to single out one protein, neuron-derived FGF9, as an essential paracrine [cell-to-cell] signal for communicating the steps needed to repair injured bone." 

Li says that the researchers confirmed FGF9's key role in the bone repair process using a method in which the neurons secreting the protein were ablated in mice. 

The work done in this study, James says, shows that "by bridging neuroscience, skeletal biology and regenerative medicine, we now know that nociceptive DRG nerve cells - specialized for transmitting severe pain after bone injury - simultaneously drive bone regeneration." 

"Resolving the paradox of how nociceptors play this dual role, and establishing FGF9 signaling as a key signal for making bone repair happen, gives us a potential target for drugs that might one day enhance bone healing, especially in people dealing with compromising situations, such as aging, diabetes or neuropathy," says James. 

Along with Li and James, the members of the research team from Johns Hopkins Medicine are study co-first author Mingxin Xu and co-authors Mary Archer, Patrick Cahan, Masnsen Cherief, Mario Gomez-Salazar, Yun Guan, Chunbao Rao, Sowmya Ramesh, Neelima Thottappillil, Xin Xing and Manyu Zhu. Study co-authors from other institutions include Thomas Clemens, University of Maryland School of Medicine; Xue-Wei Wang and Chi Zhang (previously a postdoctoral student at Johns Hopkins Medicine), University of South Florida; Juliet Mwirigi, Theodore Price, Ishwarya Samkaranarayanan and Diana Tavares-Ferreira, University of Texas at Dallas; and Robert Tower, University of Texas Southwestern Medical Center. 

Federal funding for the study includes two grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases at the National Institutes of Health (NIH): R01AR079171 and R21AR078919; two grants from the National Institute of Dental and Craniofacial Research at NIH: R01 DE031488 and R01 DE031028; and a grant from the Department of Defense: USAMRAA HT9425-24-1-0051. 

Non-federal support for research by the James Laboratory includes grants from the Alex's Lemonade Stand Foundation, the American Cancer Society and the Maryland Stem Cell Research Foundation.