Using Stages to Assess the Relationship Between Osteoclasts and Cellular Proliferation During Regeneration of the Terminal Phalanx in CD1 Mice Following Amputation

Abstract

Compared to amphibians like the Axolotl, mammals display limited endogenous regenerative ability. However, the digit tips of mice, humans, and other primates possess some regenerative capabilities, providing hope that enhanced mammalian regeneration might be possible. Mouse digit tip regeneration progresses through a series of well-characterized and discrete events: histolysis, blastema formation and redifferentiation. However, the onset of these events varies between digits as well as individual mice. This variation has the potential to dilute meaningful results and hinder the development of future therapies which could improve the lives of many. To address this issue, we used micro-computed tomography (μCT) to develop a novel method for staging digits during regeneration, focusing on the histolytic phase. Features easily identifiable from a μCT scan, i.e. bone pitting and secondary amputation, were used to categorize regenerating digits. When comparing bone volumes, we found that the bone volumes of digits within the same stage were more similar than digits on the same day post amputation. We then used this method of categorization to look at the relationship between osteoclasts, a cell known for its catabolic role in bone, and cellular proliferation, an anabolic event. Although these two, catabolism and anabolism, seem to be opposing events, the role of bone degeneration and bone formation in regeneration is not known. To explore these unknowns, the digit tips of adult CD1 female mice were amputated. The digits were scanned using the μCT and were immunostained for osteoclasts (Cathepsin K) and an indicator of cellular proliferation (EdU). These scans were used to categorize digits based on stage within the degradation event. Osteoclasts and cellular proliferation were quantified by manual cell counting and compared between and across stages. We found that osteoclasts were associated with cellular proliferation, suggesting that degeneration may be important to the regenerative process. This study provides a method for staging digits and a step towards understanding the role of histolysis during regeneration.