Understanding the Role of Mechanical Load in Endogenous and Induced Mammalian Digit Regeneration - Project Summary This new RO1 application entitled “Understanding the Role of Mechanical Load in Endogenous and Induced Mammalian Digit Regeneration” is focused on determining how mechanical load regulates various aspects of mammalian appendage regeneration. In the US, one in 190 people is living with limb loss, and that number is projected to double by the year 2050. Present treatments for limb amputation include replantation of amputated parts, revision amputations to accommodate prosthesis fitting, transferring tissue from other locations (e.g. toe-to-thumb transfer), or hand transplantation. These treatments are not without risks and the restoration of normal function is rarely realized. An alternative approach is to utilize the mouse digit, which possesses amputation-level-dependent regeneration capabilities, to inform the development of targeted therapies for inducing multi-tissue regenerative responses at otherwise non-regenerative digit amputation wounds, with the ultimate goal of scaling those therapeutic strategies up to the amputated limb. This approach has led to the discovery that BMP2 induces patterned bone regeneration at otherwise non-regenerative digit amputation wounds, and remarkably, BMP2 induces patterned tibia and fibula regeneration at limb amputation wounds. These findings are a proof-of-concept that induced-regeneration outcomes in the digit can be scaled up to the amputated limb. We have recently reported that mouse digit tip regeneration is mechanical load dependent, and that hindlimb unloading mice during digit tip regeneration inhibits regeneration at the earliest stages. In our preliminary studies, we have identified the mechanosensitive ion channel, Piezo1, is expressed throughout the regenerating digit. Moreover, injecting a Piezo1-agonist rescues the catabolic stage of digit tip regeneration when mice are hindlimb unloaded. Studies in Aim 1 will determine if the Piezo1-agonist is capable of rescuing the anabolic component of digit tip regeneration, as well as determine if Piezo1 expression in monocytes and osteoprogenitor cells is required for successful regeneration. In Aim 2, we will determine the mechanical properties of the unamputated and regenerating digit tip. These studies will use compression based microcomputed tomography scanning and atomic force microscopy combined with fluorescent imaging various to quantify the mechanical properties of the regenerating bone. These data will subsequently be incorporated into a finite element model which we will use to predict various components of digit tip regeneration. In Aim 3, we will determine if mechanical load is required for BMP2-induced regeneration following non-regenerative digit amputation, as well as determine if increased mechanical load, either with treadmill running exercise or a Piezo1-agonist, can enhance BMP2-induced regeneration. The successful completion of these studies will determine the specific role of Piezo1 during endogenous digit tip regeneration, as well as elucidate if mechanical load is required for non-regenerative appendage injuries.
