Proximal digit tip amputation initiates simultaneous blastema and transient fibrosis formation and results in partial regeneration

Abstract

Complete extremity regeneration in mammals is restricted to distal amputations of the digit tip, the terminal phalanx (P3). In mice, P3 regeneration is mediated via the formation of a blastema, a transient population of progenitor cells that form from the blending of periosteal and endosteal/marrow compartmentalized cells that undergo differentiation to restore the amputated structures. Compartmentalized blastema cells are formed independently, and periosteal compartment‐derived cells are required for restoration of amputated skeletal length. P3 regenerative capacity is progressively attenuated at increasingly more proximal amputation levels, eventually resulting in regenerative failure. The continuum of regenerative capacity within the P3 wound milieu is a unique model to investigate mammalian blastema formation in response to distal amputation, as well as the healing response associated with regenerative failure at proximal amputation levels. We report that P3 proximal amputation healing, previously reported to result in regenerative failure, is not an example of complete regenerative failure, but instead is characterized by a limited bone regeneration response restricted to the endosteal/marrow compartment. The regeneration response is mediated by blastema formation within the endosteal/marrow compartment, and blastemal osteogenesis progresses through intramembranous ossification in a polarized proximal to distal sequence. Unlike bone regeneration following distal P3 amputation, osteogenesis within the periosteal compartment is not observed in response to proximal P3 amputation. We provide evidence that proximal P3 amputation initiates the formation of fibrotic tissue that isolates the endosteal/marrow compartment from the periosteal compartment and wound epidermis. While the fibrotic response is transient and later resolved, these studies demonstrate that blastema formation and fibrosis can occur in close proximity, with the regenerative response dominating the final outcome. Moreover, the results suggest that the attenuated proximal P3 regeneration response is associated with the absence of periosteal‐compartment participation in blastema formation and bone regeneration.

The Potential of Nail Mini-Organ Stem Cells in Skin, Nail and Digit Tips Regeneration

“…The digit tips of rodents and primates are recognized for their endogenous regeneration capability, including the digit's nail plate, epidermis, nerves and bone. Nevertheless, this capacity was observed to be limited to the region adjacent to the nail organ area [38,39]. The distal amputation, which ensures consequent complete regeneration, removes around 23% of the distal phalanx length and 15% of the bone volume but leaves the bone marrow (BM), proximal nail matrix and footpad intact [40].…”

“…At this point, as the blood vessels are opened, the soft tissue of the injured site swells and a fibrin clot forms over the cut area [25]. During the following phase, called histolysis, the extracellular matrix, including the bone's surface, is enzymatically degraded and erodes into two segments, ultimately exposing the bone marrow [39]. Accountable for this erosion response are the osteoclasts, which were observed to increase their volume directly after the amputation and during the histolysis process.…”

“…In the early healing stages, the bone-forming regions are located alongside the periosteal surface of the remaining stump and are rich in actively proliferating cells that express markers for osteoblast commitment: Runx2, Sp7 and Osterix (OSX) [15,25,39,40]. The essential role of the periosteum in a successful digit tip regeneration was recognized by Dawson et al [40], who showed that the tissue's mechanical removal leads to a substantial reduction of the regenerated bone's length and volume.…”

“…At this point, the bone re-differentiation occurs, which is perceived as an ossification occurring distally to the stump [40]. Cells that participate in the process organize themselves through the extracellular matrix production, rich in Collagen III, which is subsequently degraded and replaced as the newly regenerated structures develop [39,66]. The process of ECM degradation and remodeling is promoted by matrix metalloproteinases (MMPs), activated by mechanical and chemical stimuli, triggered by the injury [67].…”

Epidermal-Derived Hedgehog Signaling Drives Mesenchymal Proliferation during Digit Tip Regeneration

“…The mouse model of digit tip injuries provides a promising avenue to study the key cellular and molecular mediators of limb and digit regeneration [ 9 , 17 ]. The regenerative capacity of murine digits is dependent upon the level of amputation: successful regeneration occurs when at least 60% of the distal phalanx remains after amputation and does not occur if less than 60% of the distal phalanx remains [ 1 , 10 , 18 ], although a recent study demonstrated that there is still a limited endosteal blastema formation even in a more proximal distal phalanx amputation [ 19 ]. A comparison of the transcriptional changes in the cells that populate the pro-regenerative versus pro-fibrotic wound beds after digit amputations may identify key molecular signaling pathways involved in regenerative healing.…”