AAHS ASPN ASRM - 2013 Annual Meeting - American Association ...

Rapamycin-Treated Alloantigen-Pulsed Host Dendritic Cells for the Induction of Hind-Limb Allograft Survival
Institution where the work was prepared: University of Pittsburgh Medical Center, Pittsburgh, PA, USA
Justin Michael Sacks, MD; Ryosuke Ikeguchi; Jignesh Unadkat; Elaine Horibe; Linda Lu; W.P. Andrew Lee; Maryam Feili-Hariri; University of Pittsburgh Medical Center
Introduction:
Risks of chronic immunosuppression hinder composite tissue allograft (CTA) transplantation such as face and hand. Chronic immunosuppressive therapy can
ultimately lead to side effects such as malignancy, opportunistic infections and organ toxicity. Thus, novel approaches to induce immunological tolerance for
the treatment of graft rejection holds considerable promise. We have assessed whether rapamycin (Rapa)-generated GM-CSF dendritic cells (DC) can induce
long-term (>100 day) survival in a CTA animal model.
Methods:
DC were derived from bone-marrow cells cultured in GM-CSF with rapamycin (Rapa-DC) or in GM-CSF (GM-DC). DC phenotype and function were examined.
Orthotopic hind-limb transplantations were performed (day 0) from Wistar-Furth to Lewis rats. Controls included (n=6/group): untreated, cyclosporine A (CsA
10mg/kg, day 0-20), and anti-lymphocyte serum (ALS, day -4,+1 with CsA). Experimental groups included (n=6/group) CsA+ALS combined with GM-DC or
Rapa-DC pulsed with or without donor antigen (Ag) (5 x 106 cells, day +7, +14). Epidermolysis/desquamation of donor skin defined rejection. Recipient's
peripheral blood mononuclear cells were examined for production of pro- and anti-inflammatory cytokines on day 30, 50, and 100 upon Ag restimulation.
Biopsies were performed on day 21 and at rejection.
Results:
Donor alloAg-pulsed GM-Rapa-DC significantly prolonged median survival time (95.5 days) compared to GM-Rapa-DC (46.0 days) and controls (p100 d was observed (3/6 rats) in alloAg-pulsed GM-Rapa-DC group. Rapa inhibited DC maturation. Pulsing DC with donor cell antigens did
not change DC phenotype and function. T cells from donor Ag-pulsed Rapa-DC-treated group produced more IL-10 and less IFN-gamma following donor and
third-party antigenic challenge or via TCR activation in vitro.
Conclusions:
Our data suggest that donor Ag-pulsed host Rapa-DC combined with transient immunosuppression induce CTA survival across a full MHC barrier. This represents
a basis for a clinically applicable strategy to achieve CTA survival with reduced systemic immunosuppression.
Size Limits in Autologous Cell-based Ectopic Prefabrication of Engineered Bone Flaps in Rabbits
Institution where the work was prepared: University Hospital Basel, Basel, Switzerland
Oliver Scheufler, MD1; Dirk J. Schaefer, MD1; Claude Jaquiery, MD1; Alessandra Braccini, PhD1; David J. Wendt, PhD1; Juerg A. Gasser, PhD2; Peter Ingold, PhD2; Gerhard
Pierer, MD, PhD1; Michael Heberer, MD, PhD1; Ivan Martin, PhD1; (1)University Hospital Basel, (2)Novartis Institutes for Biomedical Research
Introduction:
Autologous bone flaps are the gold standard in reconstruction of large bone defects but limited by availability and donor site morbidity. We generated large
ectopic bone flaps in rabbits combining flap prefabrication and bone tissue engineering concepts. We then aimed at determining size limits regarding the depth
of tissue ingrowth and bone tissue formation within the flaps.
Methods:
Porous hydroxyapatite scaffolds (80±3% porosity, Fin-Ceramica, Faenza, Italy) were fabricated in the shape (i) of small disks (4mm height, 8mm diameter), used
to validate the osteogenic capacity of rabbit BMSC in vivo, and (ii) of tapered cylinders (30mm height, 20mm upper base diameter, 10mm lower base diameter),
used to determine tissue ingrowth and bone tissue formation at different construct diameters. Expanded bone marrow stromal cells (BMSC) from 12 NZW
rabbits were uniformly seeded into disks and tapered cylinders at a density of 10 x 106 cells per cm3 of scaffold by continuous perfusion in alternate directions
through the scaffold pores at a velocity of 1.2 ml/min. for 24 hours in a perfusion bioreactor. In each animal, a disk loaded with autologous BMSC (group
1) and a cell-free disk (group 2) were implanted subcutaneously. Two tapered cylinders loaded with autologous BMSC, wrapped in a panniculus carnosus flap
and covered by a semipermeable membrane (vascularized condition; group 3) or covered by a semipermeable membrane and inserted under the panniculus
carnosus (non-vascularized condition; group 4) were implanted on opposite sides. Constructs were explanted after 12 weeks and assessed by MRI, µCT and
histology.
Results:
Uniform bone formation was observed in cell-seeded disks (group 1), whereas no bone formed in cell-free disks (group 2). In tapered cylinders, constructs were
filled by connective tissue in the outer 4.2±0.3 mm and contained bone tissue in the outer 2.5±0.3 mm in vascularized conditions (group 3), whereas no connective
or bone tissue formed under non-vascularized conditions (group 4), resulting in significant differences in all assessed histomorphometric parameters.
Conclusions:
A panniculus carnosus flap supported ectopic prefabrication of large engineered bone flaps in rabbits. The finding that bone tissue was restricted to the outer
region of the flaps could be explained by insufficient vascularization of the central core of the constructs upon implantation and prompts for the development
of strategies to improve vessel ingrowth from the flap.
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