Title: Composite Face/Scalp Allograft Transplantation in Rat Model
Authors: Maria Siemionow, MD, PhD, Betul G. Ulusal, MD, Ali E. Ulusal, MD,
Selahattin Ozmen, MD, Dariusz Izycki, MD, PhD, Yavuz Demir, MD, James E. Zins, MD
Introduction Advances in transplantation immunology opened the discussion on routine clinical
applicability of the composite tissue allografts (CTAs).1-3 Composite tissue allografts differ from solid
organ allografts as they contain different type of tissues. Due to the higher antigenicity of its skin
component, transplantation of composite tissue cannot be routinely used in clinical practice.
Solid organ transplantations have been used successfully for many years with established protocols.
Composite tissue allograft transplantations could also be performed in recent years, with the advance in
immunosuppressive drugs. Especially successful hand allotransplantations were reported from different
centers. Repair of extensive facial defects with optimal functional and aesthetic results remains a major
challenge in reconstructive surgery. The most challenging, but most rewarding, would be restoration of
the composite facial and/or scalp component. Here we introduced the feasible and applicable face/scalp
allograft transplant model across MHC barrier in rat.
Method: Anatomic studies were performed in 6 rats to find out the vascular territories of the face/scalp
transplant flap. Anatomic studies on facial/scalp harvesting technique and pilot isograft transplants
(n=14) in Lewis (LEW;RT1l) rats revealed that the composite face/scalp flap could be successfully
transplanted. Nonvascular isograft (LEWLEW) and allograft rejection controls (LBN(F1);
RT1l+nLEW) (n=4) were studied in order to show whether face/scalp composite tissue transplant
could survive as a non-vascular graft. Vascularized isograft (LEWLEW; n=4) and vascularized
allograft transplantations (LBNLEW;n=7) across MHC barrier were studied. Allograft recipients were
treated with Cyclosporine-A (CsA) tapered from 16 mg/kg/day to 2 mg/kg/day in four weeks and
maintained at this level as chronic treatment protocol. Surgical Technique: In the donor, the face
transplant flap was marked. The flap was raised based on the bilateral common carotid arteries and
jugular veins. The flap included skin facial mimic muscles and both external ears. In the recipient, both
ears were removed along with the skin and the external carotid artery and anterior facial veins were used
for anastomosis. At postoperative follow-up, the animals were evaluated clinically and histologically for
the presence of rejection. In order to test chimerism and efficacy of the immunosuppressive therapy,
flow cytometric analysis was performed. Mixed lymphocyte reaction (MLR) assay was performed to test
donor specific tolerance in the recipient.
Results: Mean total operation time was 5 hours and 30 minutes and mean warm ischemia time was 2
hours. Non-vascularized grafts resulted in facial flap necrosis within 5 to7 days post-transplantation,
indicating that composite face/scalp transplant cannot survive as a non-vascular graft. Vascularized face
allotransplants survived between 90 and 370 days, which are still alive and under evaluation (Fig.1).
Flow cytometric assessment of chimerism at day 120 post-transplant showed 1.2 % of CD8+/RT1n+
positive cells in the peripheral blood of recipients (Fig. 2). MLR assay at day 120 post-transplant reveled
hyporesponsiveness to the host, but increased reactivity to the donor and third-party (ACI; RT1a)
alloantigens (Fig. 3). The immunostaining of frozen skin sections with FITC-conjugated mouse-anti rat
CD25 monoclonal antibodies revealed CD25+ positive cells in skin obtained from the recipients of face
allograft transplants (Fig. 4).
Fig 1: At >140 and >180 days, the face transplant recipient showed no clinical signs of rejection and is
in good health condition under low dose of the CsA (2 mg/kg/day) mono-therapy.
RT1nFITC/CD8PE
IgG2a-PE
CD8-PE
ISOTYPI C
CONTROL
RT1n FITC
IgG1- FITC
Fig 2: Two-color flow cytometry analysis performed at day 120 post-transplant demonstrated RT1n
antigen expression of the donor origin on the surface of CD8+ T cell subpopulation (1.2%) (dashed
circle).
3H thymidine uptake
25000
MIXED LYMPHOCYTE REACTION ASSAY
SI=16.6
20000
15000
SI=13.6
SI=10.9
SI=9.5
10000
5000
0
NAÏVE LEWIS
LEWIS
FACE TRANSPLANT
BROWN NORWAY
ACI
Fig 3: At day 120 post-transplant MLR assay revealed absence of donor specific tolerance in vitro. At
the same time immunocompetence of the face transplant recipient was confirmed by strong response to
the third-party alloantigens (ACI).
(10X)
Fig 4: The immunostaining evaluation of the infiltrates of the frozen skin sections, isolated from the
face allograft and isograft transplant recipients and stained with FITC -conjugated mouse anti rat
CD25 monoclonal antibody revealed CD25+ positive cells in skin obtained from allotransplant
recipients.
Conclusion: To the best of our knowledge this is the first report on composite face/scalp transplantation
in animal model. The composite face allotrasnplants achieved indefinite survivals over 370 days with
low dose immunosuppressive treatment.
This model will allow studying the pattern of acute and chronic rejection and tolerance including
strategies in the face/scalp allograft transplants. Once tolerance is induced application of this procedure
in the clinical scenario will be more justified in humans.
References
1. Siemionow M, Oke R, Ozer K, Izycki D, Prajapati R. Induction of donor-specific tolerance in rat hindlimb allografts under antilymphocyte serum and cyclosporine A protocol. J Hand Surg [Am].
2002;27:1095.
2. Jones JW, Gruber SA, Barker JH, Breidenbach WC. Successful hand transplantation. One-year follow-up.
Louisville Hand Transplant Team. N Engl J Med. 2000;343:468.
3. Strome M. et al. Laryngeal transplantation and 40-month follow-up. N Engl J Med. 2001;344:1676.