Figure 1.
Schematic diagram of swine heterotopic hind limb transplant.
Osteomyocutaneous flap consisting of femur, tibia, fibula, knee joint, overlying muscles and skin is transplanted to a subcutaneous pocket in recipient with externalized skin component.
Figure 2.
Immunohistochemical staining at Day 75 and 100.
(A) Lack of PGP9.5 staining at Day 75 demonstrating complete denervation. (B) Axonal sprouts visualized in a 3 mm punch biopsy 0.5 cm from skin margin at Day 100. (C) Native skin adjacent to flap demonstrating dense intra-epidermal axons.
Figure 3.
Polynomial regression model demonstrating centripetal distance of collateral axonal sprouting from adjacent native skin as a factor of time since transplant with estimated rate of regeneration 0.06 mm per day.
Figure 4.
Schwann cell staining (p75) and confocal microscopy triple staining with PGP9.5, GAP43 and DraQ5 at the junction between native skin and alloflap.
Skin sections immunostained with PGP9.5 (A), nerve growth factor receptor, p75 (C, D and E) and confocal microscopy montage triple-stained with the axonal marker PGP9.5 (red), GAP 43 (green) indicating co-localization as yellow and the nuclear marker DraQ 5 (blue).
(A) At the junction between native and grafted skin, a thick deep dermal nerve bundle ( arrow) surrounded by dense collagen extending out collateral sprouting fibers ( slashed arrow) towards the grafted skin.
(B) These dermal bundles at the grafted skin exhibited newly regenerating fibers (yellow, arrow).
(C) Native skin served as control showing thick epidermal layers and vertical Schwann cell bands (arrows, Sch) entering from the deeper dermis into the papillary dermis.
(D) On day 240, Schwann cells in the center of grafted flap had completely degenerated (lack of p75 staining) indicating that collateral sprouting only could serve as the major pathway for re-innervation. The epidermis is thin at this site.
(E) Schwann cell tubes at the papillary dermis (arrows, Sch) at the junction between native and the donor flap indicate the Schwann cell support for the collateral sprout guidance. The border shows dense aggregation of inflammatory cells (broken arrow). Scale bar: A=100µm, B=10µm, C and D=50 µm.
Figure 5.
Sequential sections of skin component of allograft (Day 240 post-transplant).
PGP9.5 (pan-axonal marker) demonstrates dense epidermal fibers (solid arrow) in native skin 0.5 cm away from the graft (A). Intra-epidermal fibers also visualized in skin component of VCA at 1cm (B) and 1.5 cm (C) away from the junction with native skin. No epidermal fibers seen in the center of alloflap (D).
Figure 6.
Schematic diagram of a denervated ostemyocutaneous flap.
Blood vessels are anastomosed without apposition of nerve endings allowing the regenerative pathway to undergo atrophy (Dotted line). Re-innervation of the denervated VCA epidermis is achieved through collateral sprouting of uninjured axons outside the transplant region (shown in black).