Blastema formation and periosteal ossification in the regenerating adult mouse digit

Abstract

While mammals cannot regenerate amputated limbs, mice and humans have regenerative ability restricted to amputations transecting the digit tip, including the terminal phalanx (P3). In mice, the regeneration process is epimorphic and mediated by the formation of a blastema comprised of undifferentiated proliferating cells that differentiate to regenerate the amputated structures. Blastema formation distinguishes the regenerative response from a scar-forming healing response. The mouse digit tip serves as a preclinical model to investigate mammalian blastema formation and endogenous regenerative capabilities. We report that P3 blastema formation initiates prior to epidermal closure and concurrent with the bone histolytic response. In this early healing response, proliferation and cells entering the early stages of osteogenesis are localized to the periosteal and endosteal bone compartments. After the completion of stump bone histolysis, epidermal closure is completed and cells associated with the periosteal and endosteal compartments blend to form the blastema proper. Osteogenesis associated with the periosteum occurs as a polarized progressive wave of new bone formation that extends from the amputated stump and restores skeletal length. Bone patterning is restored along the proximal-distal and medial digit axes, but is imperfect in the dorsal-ventral axis with the regeneration of excessive new bone that accounts for the enhanced regenerated bone volume noted in previous studies. Periosteum depletion studies show that this compartment is required for the regeneration of new bone distal to the original amputation plane. These studies provide evidence that blastema formation initiates early in the healing response and that the periosteum is an essential tissue for successful epimorphic regeneration in mammals.

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

“…We carried out a longitudinal comparative analysis to investigate changes in bone length, volume, and morphology in digits amputated at constant proximal (n = 12) or distal (n = 15) levels using sequential micro‐computed tomography (μCT) scanning during a regenerative period of 28 days (Figure 1B‐G). While distal amputations displayed a consistent regenerative response as previously described, 7,23,29 the response of proximal amputations ranged from no obvious bone regrowth to the regeneration of a small digit tip (Figure 1B and Supporting Information Figure S1A‐F). The standardized proximal amputation plane 23 removes 75% of the bone length and 60% of the P3 bone volume compared to distal amputation that removes 25% and 15% respectively (Figure 1B‐D).…”

“…The continuum of regenerative competency displayed by the P3 milieu represents a unique model to investigate mammalian blastema formation after amputation. Studies of distal amputation show that the blastema forms concurrent with tissue histolysis and wound closure, and osteoprogenitor cells from the periosteal and endosteal/marrow compartments independently contribute to specific regions of the blastema 29 . Cells associated with the periosteum, the cell‐dense peripheral layer of the bone, 30 proliferate to form the peripheral blastema, while cells within the endosteal/marrow compartment proliferate to form the central blastema core 29 .…”

“…Studies of distal amputation show that the blastema forms concurrent with tissue histolysis and wound closure, and osteoprogenitor cells from the periosteal and endosteal/marrow compartments independently contribute to specific regions of the blastema 29 . Cells associated with the periosteum, the cell‐dense peripheral layer of the bone, 30 proliferate to form the peripheral blastema, while cells within the endosteal/marrow compartment proliferate to form the central blastema core 29 . After bone histolysis is complete, the two compartments merge to form the blastema proper, and osteoprogenitors at the stump/blastema interface differentiate in a proximal to distal sequence via intramembranous ossification to restore the amputated skeletal element 2,7,29 .…”

“…Cells associated with the periosteum, the cell‐dense peripheral layer of the bone, 30 proliferate to form the peripheral blastema, while cells within the endosteal/marrow compartment proliferate to form the central blastema core 29 . After bone histolysis is complete, the two compartments merge to form the blastema proper, and osteoprogenitors at the stump/blastema interface differentiate in a proximal to distal sequence via intramembranous ossification to restore the amputated skeletal element 2,7,29 . The periosteum is required for restoration of amputated skeletal length, but is not essential for endosteal/marrow‐associated blastema formation 29 .…”

“…After bone histolysis is complete, the two compartments merge to form the blastema proper, and osteoprogenitors at the stump/blastema interface differentiate in a proximal to distal sequence via intramembranous ossification to restore the amputated skeletal element 2,7,29 . The periosteum is required for restoration of amputated skeletal length, but is not essential for endosteal/marrow‐associated blastema formation 29 . Thus, the two compartments are established independently and neither dominates the regeneration response, yet both appear to be involved in successful P3 regeneration.…”