Development and regeneration of the neonatal digit tip in mice

Abstract

The digit tips of children and rodents are known to regenerate following amputation. The skeletal structure that regenerates is the distal region of the terminal phalangeal bone that is associated with the nail organ. The terminal phalanx forms late in gestation by endochondral ossification and continues to elongate until sexual maturity (8 weeks of age). Postnatal elongation at its distal end occurs by appositional ossification, i.e. direct ossification on the surface of the terminal phalanx, whereas proximal elongation results from an endochondral growth plate. Amputation through the middle of the terminal phalanx regenerates whereas regenerative failure is observed following amputation to remove the distal 2/3 of the bone. Regeneration is characterized by the formation of a blastema of proliferating cells that appear undifferentiated and express Bmp4. Using chondrogenic and osteogenic markers we show that redifferentiation does not occur by endochondral ossification but by the direct ossification of blastema cells that form the rudiment of the digit tip. Once formed the rudiment elongates by appositional ossification in parallel with unamputated control digits. Regenerated digits are consistently shorter than unamputated control digits. Finally, we present a case study of a child who suffered an amputation injury at a proximal level of the terminal phalanx, but failed to regenerate despite conservative treatment and the presence of the nail organ. These clinical and experimental findings expand on previously published observations and initiate a molecular assessment of a mammalian regeneration model.

Figure 1

A case of regenerative failure after proximal amputation injury of the fingertip (*) of a 2-years old child that was conservatively treated. A: Radiograph at the time of injury indicated about 70% of the terminal phalangeal bone was lost. B–E. Fingertip 10 months after injury. B: Dorsal view, C: Ventral view and D: Lateral view. The wound healed to form a small bump with normal contour and sensibility (B–D), but there was no elongation of the terminal phalangeal bone (E).

Figure 2

Regeneration of the terminal phalanx of digit tips in neonatal mice. Amputations were carried out at a distal level through bone, (A) and a proximal level through cartilage (B) at postnatal day 3 (PN3). Control unamputated digits were used for comparison (C, F). After 6 weeks, digits were analyzed using whole mount bone stain with Alizarin Red S (C–E) and histological analysis with Mallory’s triple stain (F–H). Proximal amputations show no signs of regeneration (E, H). Distal amputations regenerate anatomically normal digit tips (D, G), however the length of the terminal phalanx of these digit tips never reach that of unamputated control digits (I). Scale bars: A,B − 200µm; C–E − 300µm; F–H − 400µm.

Figure 3

Differentiation and elongation of the terminal phalangeal bone. A–D: Whole mount differential bone staining of developing hindlimb digits with Alizarin Red S and Alcian Blue. Ossification of the terminal phalangeal bone is initiated at the distal tip between E17.5 and E18.5 (A, B). Ossification progresses in a proximal direction after birth (C), and the entire bone is Alizaring Red S positive by PN14 (D). E, F: Mallory’s triple stained sections of neonatal digits. At PN7 an endochondral growth plate is clearly evident in the proximal region of the terminal phalangeal bone (arrowheads in E), however this growth plate is absent by PN21 (F). Scale bars: A,B − 150µm; C–F − 300µm.

Figure 4

Expression of cartilage-specific (A–F) and bone-specific (I–L) transcripts in the E17.5 and E18.5 terminal phalanx at the onset of ossification. Type II collagen (Col II; A, B) is a marker for proliferating chondrocytes, Ihh (C, D) is a prehypertrophic chondrocyte marker, and Type X collagen (Col X; E, F) is a hypertrophic chondrocyte marker. G, H: Mallory’s triple stained section showing the deposition of collagen along the dorsal tip of the terminal phalangeal bone. Collagen staining in G and H correlate with the expression of Type I collagen transcripts (Col I; I and J, respectively). The osteoblast-specific marker, Osteocalcin (K, L) identifies a distal ossification center during the maturation of the terminal phalangeal bone. Scale bars: 100µm.

Figure 5

Ossification and elongation of the terminal phalangeal bone. A–D and I–K: Calcein incorporation into the terminal phalangeal bone identifies new bone deposition. E–G and L: Calcein incorporation into the terminal phalangeal bone is used as a vital marker to mark existing bone so subsequent bone deposition (unlabeled) can be identified. During postnatal digit elongation new bone is deposited throughout the terminal phalangeal bone at 1 week of age (A). At 3 weeks new bone deposition is restricted to the proximal (*) and dorsal-distal (arrow) regions of the bone (B). At 5 weeks new bone deposition is only observed at the dorsal-distal (arrow) region of the bone (C). At 7 weeks new bone deposition is not localized but occurs throughout the bone (D). E–G: The terminal phalangeal bone was labeled with calcein at PN1 (E) to characterize regions of new bone formation during digit elongation. Unlabeled bone distal to the calcein label (double arrow lines) identifies progressive deposition of new bone at the digit tip in mice 4 weeks (F) and 7 weeks (G) of age. H: BrdU, introduced at birth, labels bone cells at the distal tip of the terminal phalanx 3 weeks later (arrow). I–L. Calcein labeling during digit tip regeneration. Calcein was injected 1 day before collecting samples of regenerating digits 1 week (I), 2 weeks (J), and 3 weeks (K) post-amputation. Note that the general morphology of the digit tip is regenerated by 2 weeks post-amputation (J), and that by 3 weeks post-amputation the normal dorsal-distal bone deposition pattern (arrow) is observed in the regenerating digit tip (K). To demonstrate that the bulk of the regeneration response occurs between 1 and 2 weeks post-amputation, calcein incorporation at 1 week post-amputation labeled the stump bone and digits collected at 2 weeks post-amputation were collected and stained with Alizarin Red S to label all bone (L). The double arrow line in L shows the extent of regenerated new bone during this 1 week period. Scale bars: 250µm.

Figure 6

Histological and in situ hybridization analyses of digit tip regeneration. A–D: Histological sections of regenerating digit tips stained with Mallory’s triple stain at the time of amputation (A), 4 days post-amputation (B), 6 days post-amputation (C), and 8 days post-amputation (D). A blastema-like structure is evident at PA6 (C) and collagen deposition (c) is evident between the bone stump and the blastema-like structure at PA8 (D). E–H: BrdU incorporation was used to identify regions of cell proliferation in the PN7 (E) and PN10 unamputated control digit (G), and in the regenerating digit at PA4 (F; correspondent to PN7), and PA7 (H; correspondent to PN10). Regions of proliferation in the unamputated digits (E and G) include the epidermis (arrowheads) and the bone marrow (*). At PA4 (F) epidermal proliferation appears more widespread, extending distalward in association with epidermal closure, but not including the leading edge (*). In addition, a region of enhanced proliferation is found at the interface between the forming blastema-like structure and the bone marrow (arrow). At PA7 (H) many cells in the connective tissue between the nail bed and the bone are proliferating (*), as are the cells at the interface of the bone marrow and the blastema-like structure. I–L: In situ hybridization analyses documenting the expression of developmental genes during regeneration. Transcripts for Msx1 (I) are absent from the blastema-like structure, but are localized in the dorsal connective tissue between the nail bed and the terminal phalangeal bone. This pattern is similar to that observed in the unamputated digit (not shown). Msx2 expression at PA4 (J) indicates a transient up-regulation in the connective tissue cells associated with the amputation wound (arrow), however Msx2 expression is absent in the blastema-like structure at PA8 (K). At PA8 Bmp4 transcripts (L) are present throughout the blastema-like structure (*) and also in connective tissue cells just proximal to the regenerate (arrow). Scale bars: 100µm.

Figure 7

Cartilage-specific and bone-specific gene expression during regeneration. A, C, E, G: 4 days after distal amputation, B, D, F, H: 8 days after distal amputation. The cartilage-specific marker, Type II Collagen (ColII), is expressed in the proximal region of the terminal phalanx but not in the regenerating distal region (A, B). The pre-hypertrophic cartilage-specific marker, Ihh, (C,D) and the hypertrophic cartilage-specific marker, Type X Collagen (ColX), (E,F) are expressed in the proximal bone stump but not distally. The osteoblast-specific marker, Osteoclacin, is expressed proximally at PA4 (G), and both proximally and at the interface between the bone stump and the blastema-like structure at PA8 (H, arrows). Scale bars: 100µm.