By Ian Guang Xia '21

Introduction

No 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.

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Results

Data generated through computer-assisted microtomography of the regenerating digits found that both bone resorption and bone formation were delayed in the digit tip bone of mice with progeria when compared to mice without it. This data is displayed in Figure 1. The point of lowest bone volume occurs at 7–10 days post amputation (DPA) for mice without progeria while occurring at 10–21 DPA for mice with progeria. Before these specified time points, bone resorption or degradation is occurring as osteoclasts eat away at damaged bone structures near the wound site to clear away debris for bone formation to begin. Following bone resorption, new bone is formed, and regeneration is completed at 28 DPA in healthy digits. As can be seen in Figure 1C, digits from progeria mice exhibit a much lower bone volume, which may indicate that progeria inhibits regeneration.

Amputation of the distal half (farthest away from the body) of the terminal digit bone induces a regeneration response, starting with bone resorption driven by osteoclasts (“bone-eating cells”). Bone resorption is typically completed by 7–10 DPA and is followed by bone formation driven by osteoblasts (“bone forming cells”). Regeneration is typically completed by 28 DPA. Minimal bone volume in progeria digits is achieved later (between 10–21 DPA) and is significantly lower than minimal bone volume in wild type digits, suggesting higher total osteoclast activity but slower osteoclast activation. Bone volume in progeria digits when compared to wild type digits is significantly lower, suggesting that aging inhibits osteoblast activity and, ultimately, digit regeneration.

Figure 1. This figure shows a general schematic of (A) the distinct steps which occur in the process of digit bone regeneration in mice illustrated by how the bone looks during each phase, and (B) differences in minimal bone volume (smallest bone volume caused by bone resorption) and (C) final bone volume (bone volume at 28 DPA), relative to preamputation volume, between progeria mice and healthy control mice (WT). The dashed line indicates 50% of the preamputation bone volume in B, and 100% in C. Each data point represents one digit, while bars indicate the mean ± 95% confidence interval. BV means bone volume; DPA, days post amputation; WT, wild type.

Since progeria seemed to affect digit regeneration by affecting both bone resorption and bone formation, it was anticipated that osteoclast and osteoblast behavior must be altered by accelerated aging. Immunohistochemical staining using a primary antibody for the protein cathepsin K was employed to study osteoclast behavior. Figure 2 shows the results of the histology on representative images (images which can be used to portray data from each group) for each time point, suggesting that osteoclast quantity sharply decreases in wildtype mice around 10 DPA while no such decrease exists for the progeria mice at any time point accounted for in this investigation. This result indicates that progeria extends the timeframe of osteoclast activity and explains the observed reduced bone volume by the end of the resorption phase.

Furthermore, immunohistochemical staining using a primary antibody for the protein osterix was employed to assess the osteoblast prevalence and temporal changes thereof in the regenerative process. Figure 3 shows the results of the histology on representative images for each time point, suggesting that osteoblast quantity sharply increases in wildtype mice around 14 DPA while no such increase exists for the progeria mice at 14 DPA. This suggests that the recruitment of osteoblasts to the wound site is impaired by progeria, which explains the lack of regeneration observed in Figure 1.

Conclusion

This project found that progeria significantly alters the timing and magnitude of both bone degradation and bone formation during the digit tip regeneration process. For progeria mice, bone degradation occurred later and with a greater magnitude than for healthy control mice. While bone formation also occurred later for mice with progeria, the magnitude of bone formation was significantly reduced when compared to mice without progeria. These results suggest that the regeneration window is postponed in older tissues. Since mice share around 97.5% of DNA with humans and are one of the most popular mammalian models for medical research, it can be argued based on the findings in this research that regenerative medicine therapies tailored to older patients should be administered a few days later compared to therapies for younger patients.

Possible future directions for this work include inducing regeneration in typically non-regenerative wounds of progeria or aged mice by adapting existing strategies, and the establishment of a preclinical large animal model such as sheep, bovine, or pig models which more accurately approximate human skeletal physiology. Once further work has been conducted, the novel techniques and theories may be translated to human patients, where induced regeneration may replace prostheses in the treatment of limb loss.

Figure 2. These images show sections of digit tips stained with an antibody marking the protein cathepsin K (in green) and DAPI, which marks cell nuclei (in grey). The white scale bar in the top left-hand section has been set to 500 µm, and the digit tip bone has been outlined in a white dotted line in each section image. The “Mutant” label refers to image display sections from the experimental group of mice with progeria.

Figure 3. These images show sections of digit tips stained with an antibody marking the protein osterix (in green), and DAPI, which marks cell nuclei (in grey). The white scale bar in the top left-hand section has been set to 500 µm, and the digit tip bone has been outlined in a white dotted line in each section image.

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.

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Ian Guang Xia '21

Ian 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.

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