Living bone allotransplants survive by surgical angiogenesis alone: development of a novel method of composite tissue allotransplantation

Abstract

Background: Segmental bone defects pose reconstructive challenges. Composite tissue allotransplantation offers a potential solution but requires long-term immunosuppression with attendant health risks. This study demonstrates a novel method of composite-tissue allotransplantation, permitting long-term drug-free survival, with use of therapeutic angiogenesis of autogenous vessels to maintain circulation.

Methods: Ninety-three rats underwent femoral allotransplantation, isotransplantation, or allografting. Group-1 femora were transplanted across a major histocompatibility complex barrier, with microsurgical pedicle anastomoses. The contralateral saphenous artery and vein (termed the AV bundle) of the recipient animal were implanted within the medullary canal to allow development of an autogenous circulation. In Group 2, allotransplantation was also performed, but with AV bundle ligation. Group 3 bones were frozen allografts rather than composite-tissue allotransplantation femora, and Group 4 bones were isotransplants. Paired comparison allowed evaluation of AV bundle effect, bone allogenicity (isogeneic or allogeneic), and initial circulation and viability (allotransplant versus allograft). Two weeks of immunosuppression therapy maintained blood flow initially, during development of a neoangiogenic autogenous blood supply from the AV bundle in patent groups. At eighteen weeks, skin grafts from donor, recipient, and third-party rats were tested for immunocompetence and donor-specific tolerance. At twenty-one weeks, bone circulation was quantified and new bone formation was measured.

Results: Final circulatory status depended on both the initial viability of the graft and the successful development of neoangiogenic circulation. Median cortical blood flow was highest in Group 1 (4.6 mL/min/100 g), intermediate in Group 4 isotransplants (0.4 mL/min/100 g), and absent in others. Capillary proliferation and new bone formation were generally highest in allotransplants (15.0%, 6.4 μm³/μm²/yr) and isotransplants with patent AV bundles (16.6%, 50.3 μm³/μm²/yr) and less in allotransplants with ligated AV bundles (4.4%, 0.0 μm³/μm²/yr) or allografts (8.1%, 24.1 μm³/μm²/yr). Donor and third-party-type skin grafts were rejected, indicating immunocompetence without donor-specific tolerance.

Conclusions: In the rat model, microvascular allogeneic bone transplantation in combination with short-term immunosuppression and AV bundle implantation creates an autogenous neoangiogenic circulation, permitting long-term allotransplant survival with measurable blood flow.

Fig. 1

Diagram showing the surgical technique. A: Donor procedure: resection of the femoral diaphysis on the basis of the nutrient vessels to the bone. B: Microvascular anastomosis in the recipient; the slight size mismatch in donor and recipient animals permitted matching of the donor common iliac vessels to the recipient femoral vessels. C: AV bundle implantation; with the transplanted bone in the abdominal pocket, the recipient saphenous vessels were carefully pulled through the medullary canal and fixed to the abdominal wall.

Fig. 2

Photograph of the AV bundle dissection in the left hind limb of a recipient animal. A 3 × 3-mm distal fascial flap, including the saphenous artery and venae comitantes, is elevated to the bifurcation in the groin. This permitted enough vascularity and length to ensure proper placement and survival inside the bone.

Fig. 3

Bone blood flow (mL/min/100 g) per group. The median value for each group is denoted by a plus sign (+).

Fig. 4-A Fig. 4-B

Figs. 4-A and 4-B Hematoxylin-eosin sections (original magnification, ×400). Fig. 4-A Representative sample from Group 1, in which more than 50% of the lacunae are filled with normal osteocytes and the sample exhibits normal bone morphology. Fig. 4-B Representative sample from Group 2, showing very few osteocyte-holding lacunae and grossly altered bone morphology with cortical necrosis and absent peritrabecular lining.

Fig. 4-A Fig. 4-B

Figs. 4-A and 4-B Hematoxylin-eosin sections (original magnification, ×400). Fig. 4-A Representative sample from Group 1, in which more than 50% of the lacunae are filled with normal osteocytes and the sample exhibits normal bone morphology. Fig. 4-B Representative sample from Group 2, showing very few osteocyte-holding lacunae and grossly altered bone morphology with cortical necrosis and absent peritrabecular lining.

Fig. 5

Bone viability per group, as measured by histologic grading of necrosis. The median value for each group is denoted by a plus sign (+).

Fig. 6

Capillary density (%) per group. The median value for each group is denoted by a plus sign (+).

Fig. 7

Cropped images showing a representative decalcified and cleared allotransplant from the Group-1 (top) and Group-2 (bottom) AV bundle groups. Polymerized Microfil infusion permits visualization of neovascularization (which is extensive in the Group-1 sample) from the implanted host-derived AV bundle.

Fig. 8

Bone formation rate per bone surface area (μm³/μm²/yr) per group. The median value for each group is denoted by a plus sign (+).

Fig. 9-A Fig. 9-B

Figs. 9-A and 9-B Histomorphometric analysis of calcein and tetracycline-labeled, methylmethacrylate-embedded 5-μm sections (original magnification, ×200). Both samples are from Group 1. Double fluorescence was discernible both on the perimeter of the transplant (Fig. 9-A) and within the cortical boundaries (Fig. 9-B).

Fig. 9-A Fig. 9-B

Figs. 9-A and 9-B Histomorphometric analysis of calcein and tetracycline-labeled, methylmethacrylate-embedded 5-μm sections (original magnification, ×200). Both samples are from Group 1. Double fluorescence was discernible both on the perimeter of the transplant (Fig. 9-A) and within the cortical boundaries (Fig. 9-B).

Fig. 10

Inflammation rate in the medullary canal per group. The median value for each group is denoted by a plus sign (+).