IntroductionNo matter the context, losing a limb is a devastating experience with tremendous impact on an individual’s quality of life. In the US alone, there are nearly two million people affected by limb loss,1 and the only current treatment is replacement of the limb with prostheses. Although prosthetic limb technology has been around for a significant portion of recorded human history,2 evolving from iron hand replacements during the Roman era to modern day microprocessor-controlled prosthetic knees,3 it is still an imperfect solution to a major quality of life issue.4 In pursuit of a better treatment, scientists have been inspired by certain specialized vertebrates, such as salamanders, which are able to fully regrow amputated limbs. Studying these vertebrates will allow us to identify mechanisms that could possibly be translated to induce limb regeneration in humans. Similar to salamander limbs, the mouse digit tip is also capable of epimorphic regeneration, or the near perfect replacement of a limb or other complex body structure following injury. However, this regenerative capability is only present at the distal digit tip, or the digit tip furthest from the core of the body. If an amputation injury occurs closer to the body rather than around the middle or end of the distal digit tip bone, the limb will not regenerate and instead undergo a non-regenerative healing response like forming nonfunctional scar tissue.5 Studying the mechanisms of these regenerative and non-regenerative wound healing processes has led to the discovery of a “regeneration window,” or a time period during healing where wounds that normally would not regenerate can undergo partial regeneration in response to treatment with certain morphogenic agents, or proteins that regulate cell development.6 For example, it has been recently discovered that treatment with morphogenic agents such as bone morphogenic protein 2 (BMP2) and bone morphogenic protein 9 (BMP9) during the regeneration window will stimulate bone and joint regeneration in normally non-regenerative amputation injuries in mice.7 With such advancements in mouse regeneration biology, application of these concepts to human biology may not be far behind. According to Dr. Ken Muneoka, a regeneration biologist in Texas A&M University's College of Veterinary Medicine & Biomedical Sciences, “with adequate funding, human-finger regeneration in children will be possible within 20 years.”8 Looking to the future, human regenerative therapies must be effective for all age groups, especially considering that adult and elderly demographics have the largest need for regenerative treatments. With advanced age comes a litany of new variables to account for such as changes in blood pressure, respiratory cycles, vision, and most importantly, overall loss of regenerative capacity. Therefore, exploring the effects of aging on regeneration of the distal (regenerative) part of the mouse digit may give clues for inducing regeneration specifically optimized for older patients. To study this age-related decline of regenerative power, the digit tips of mice modified to have progeria, a disease of accelerated aging, were analyzed at different time points of their regenerative processes. |
STUDYING THESE VERTEBRATES WILL ALLOW US TO IDENTIFY MECHANISMS THAT COULD POSSIBLY BE TRANSLATED TO INDUCE LIMB REGENERATION IN HUMANS. |
Methods
Two groups of lab mice were raised to two months of age, one group with progeria (the mutant/ experimental group) and one group without (the wild type/control group). A two-month timeframe was selected from mouse work previously published by the lab in order to keep a consistent procedure.9 Digit tips on each paw of each mouse were scanned with computer-assisted microtomography analysis (a three-dimensional x-ray scanning technology) to set a normal starting bone volume. The mice were then given inhalation anesthetic and 20-30% of each rear digit tip was amputated to analyze differences in bone formation during the healing process. After amputation, the same digit tips were scanned at 3–7-day intervals to track the progress of bone regeneration, until 28 days post amputation when the regeneration process had finished.
In addition to computer-assisted microtomography analysis, immunohistochemical (IHC) analysis was conducted. In IHC analysis, antibodies are used to mark cells of interest because they can attach to specific cell types within selected tissue samples. This is possible since specific cell types have unique protein markers which only certain antibodies can attach to. For this project, select mice were euthanized to collect samples of their full digits at various time points during the healing process. These samples were immunohistochemically stained to measure the quantities of osteoclasts (bone resorbing cells, or cells that eat and degrade bone) and osteoblasts (bone forming cells). Collected digit samples were fixed in zinc-buffered formalin, decalcified, mounted in paraffin, and serially sectioned to a thickness of 4µm per section. These sections were attached to microscope slides and immunohistochemically stained for osteoclasts and osteoblasts using their respective protein markers, cathepsin K and osterix. These were detected through specific primary antibodies binding onto the markers and fluorescently labeled secondary antibodies binding onto the primary antibodies.10 Then, an automated microscope took images of the sections to detect the fluorescent label attached to the osteoclasts and osteoblasts.
Acknowledgments
I would like to thank my research advisors, Dr. Regina Brunauer, and Dr. Ken Muneoka for their guidance and support throughout the course of this research.
Ian Guang Xia '21Ian Guang Xia '21 is a biomedical engineering major with a minor in electrical engineering from San Antonio, Texas. Ian started conducting academic research at Claudia Taylor Johnson High School for his AP chemistry teacher during senior year, and carried that interest in academic research into his undergraduate studies. Ian plans to work in the software industry and possibly conduct research on current questions and issues he encounters there. Vertical Divider
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