Title: A Microsurgical Approach to Bone Marrow Transplantation Using an Extraperitoneal Isolated Vascularized Bone/bone Marrow Composite Transplant
Chau Y. Tai, MD, Louise F. Strande, Riva Eydelman, Xiaoli Sheng, MD, Jean-Luc van Tran, Martha S. Matthews, MD, and Charles W. Hewitt, PhD
An isolated vascularized bone marrow transplant model (iVBMT) was previously developed in the rat in order to study the contribution of the bone marrow and its environment in a composite tissue allograft (CTA).1 It was an intraperitoneal model that required a long laparotomy time. This study aimed to develop an extraperitoneal model of iVBMT and test its functionality in reconstituting an irradiated animal.
Methods: For evaluating technical feasibility, Sprague-Dawley rats (n=5) were used and sacrificed at 24 hrs. For the long-term study, Lewis-Brown-Norway (LBN) rats (n=5) were used and sacrificed at various time points from 2 to 8 weeks. To evaluate the hematopoietic repopulation of irradiated animals, four LBN rats received 900cGy of total body irradiation prior to transplant, and three irradiated LBN animals served as controls.
The iVBMT model consisted of a left donor femur that was harvested with its nutrient vessels as previously described.1 The dissected graft was then left in situ, perfused by the anesthesized donor, while the recipient’s right femoral vessels were prepared. The vessels were anastomosed in an end-to-end fashion using interrupted 10-0 nylon sutures. After ensuring perfusion and hemostasis, a subcutaneous pocket was bluntly created in the ipsilateral abdominal wall and the vascularized graft gently situated without tension or torsion on the anastomoses(Fig. 1).
At sacrifice, the graft bones were fixed in Histochoice, decalcified in 5% formic acid overnight, stored in 70% cold alcohol, then paraffin embedded. Four micrometer sections were H&E stained and examined at 200x.
Results: Technique group – Mean ischemic time was 103 minutes. Four out of five vessels were patent at 24 hrs.
Long term group – All animals appeared well, and the position of the graft did not interfere with normal movements. Mean ischemic time was 80 minutes. The right limb functioned normally postoperatively. At sacrifice, five out of five vessels were grossly patent. Histology of the grafts in the long term animals showed a normal appearing bone marrow (Fig. 2).
Irradiated group – All three irradiated control animals died between 15 to 17 days postoperatively. All four irradiated and transplanted animals gained weight and remained well-appearing 30 days after the procedure.
Discussion: An extraperitoneal iVBMT model was possible based on the recipient’s femoral vessels and a subcutaneous placement of the graft. One thrombosed graft vessel in the technical feasibility study was due to excess manipulation at time of anastomosis. Abundant collateral vessels in the rat’s limb permitted ligation of the femoral vessels without morbidity.
We tested the functionality of the vascularized bone marrow graft by transplanting it to an irradiated animal. Although without complete cell count data, clinical recovery of the transplanted recipients compared with the controls indicated hematopoietic reconstitution. The surgical vascularized bone marrow transplant may have an advantage over conventional cellular bone marrow transplant since the stem cells were transferred within their stromal environment, eliminating the engraftment period prior to proliferation. Direct comparisons between cellular and vascularized bone marrow transplant will help answer this question in the future.
With the surgical model established, studies using the iVBMT across semiallogeneic and allogeneic barriers with and without immunosuppression will help to define the role of the bone marrow compartment in a CTA and for potentially inducing immune tolerance.
Fig. 1 Placement of the extraperitoneal isolated vascularized bone marrow transplant graft.
Fig. 2 Normal histologic appearance of the graft marrow 55 days post-transplant (H&E 200x).
Reference
1. Suzuki, H., Patel, N., Matthews, M.S., et al. Vascularized Bone Marrow Transplantation: A New Surgical Approach Using Isolated Femoral bone/bone marrow. J Surg Res, 80: 176-183,2000.