Background: Vascularized composite allotransplantation has emerged as a viable treatment option for patients with devastating tissue loss not amenable to traditional reconstructive techniques. Different from solid organs, these grafts often include and intact, vascularized bone marrow niche (BMN.) Here we examined the role of this vascularized BMN in allograft survival and the induction and maintenance of mixed chimerism.

Methods: Grafts containing a BMN (murine orthotopic hind limb, OHLTx) were compared to a non-BMN containing graft (full thickness skin, FTSTx) infused with an equivalent amount of donor BM cells. All transplants were performed across a full MHC mismatch (Balb/C into C57BL6.) Transplant recipients received 250cGy non-myeloablative total body irradiation on POD -1, CTLA4-Ig and anti-CD154 mAb on POD 0, 2, 4, and 6. In addition, FTSTx recipients were infused on POD 0 with 10^7 unfractionated donor BM cells. This quantity represents the average amount of BM cells contained within the tibia of a transplanted Balb/C hind limb. Graft survival and post transplant peripheral mixed chimerism of PBMCs was analyzed using flow cytometry.

Results: OHLTx recipients showed increased graft survival compared to FTSTx recipients. 8/8 OHLTx showed rejection-free graft survival >200 days; 3/8 FTSTx were rejected with a mean survival time of 31.7, while the remaining five have vital ongoing grafts on POD 50. Moreover, mixed chimerism induced by donor derived bone marrow components was detected in the OHLTx group at significantly higher levels than FTSTx recipients on POD 40 within the B220+ (p<0.05,) CD3+ (p<0.01,) and CD11b+ (p<0.001) lineages. Analysis of the proportion of T cells expressing T cell receptor vβ chains normally expressed in C57BL6 but not in Balb/C showed significantly (p<0.005) decreased levels of circulating alloreactive T cells in animals receiving a BMN compared to FTSTx recipients, suggesting actuation of central tolerance only in the former group.

Conclusions: The presence of a BMN significantly extended allograft survival and sustained higher chimerism levels compared to non-BMN containing transplants. Our preliminary data suggests that an intact BMN plays a more important role in allograft survival than the simple administration of BM cells. This is likely due to the BMN's role in hematopoietic cell proliferation, differentiation, and trafficking.

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