Title:
Content of Bone Marrow in the Rat Bones and Planning of
Flaps for Experimental Vascularized Bone Marrow
Transplantation
Authors:
Fabio Quatra, MD, Oreste M. Romeo, MD, Dave Lowenberg,
MD, Darrell Brooks, MD, Daniela Perri, MS, Lee Ann
Baxter-Lowe, PhD, Michele R. Colonna, MD, Francesco
Stagno d'Alcontres, MD, and Harry J. Buncke, MD
The search for Tolerance in transplantation is highlighting the
importance of Bone Marrow, which is highly immunogenic, may
contribute to occurrence of rejection, may induce haematological
chimerism (and possibly tolerance) or may be responsible for a
lethal Graft-versus-Host Disease. The aim of this anatomical study
was to evaluate the content of bone marrow in the various bones of
the rat and to identify the corresponding flaps for their possible
use in the study of vascularized bone marrow transplantation,
comparing them to the rat hindlimb, one of the most popular models
for the study of composite tissue transplantation.
Materials and methods: Fifty rats (Sprague Dawley, Simonsen Lab.
Inc.) were used, in a two-part study. In the first part, the
content of bone marrow was evaluated by means of anatomical and
analytical techniques, and the content of stem cells and mature
lymphocytes estimated by flow cytometry. Briefly, bones were
mechanically cleared of the soft tissues, fixed in buffered
formalin for 48 hours, decalcified in a mixture of formic acid and
calcium chelants for further 48 hours, and then were processed in
different ways for bone marrow extraction and weigh
Hematoxylin-Eosin stained sections, blindly performed with
assessment of the ratio of viable bone marrow to fat degenerated
marrow.
Bone marrow was also extracted by freshly harvested bones, flushing
the cavities with RPMI-1641 with the addition 4% Fetal Calf Serum
and HEPES. After centrifugation for 10 minutes at 1,500 rpm, the
pellet was washed twice in Hanff Balanced Salt Solution and red
blood cells removed by a gradient of sedimentation (Lympholyte®,
Cederlane Lab, Ontario). After centrifugation for 20 min at 1500
rpm, the pellet was resuspended, washed, filtered and cells counted
with a fluorescent microscope after staining with acridin orange.
About 500,000 cells were washed in Phosphate Buffered Solution and
double labeled with an anti CD34 monoclonal antibody (FITC
conjugated) as a marker for stem cells and an anti T-cell receptor
antibody (PE conjugated) as a marker for mature T-lymphocytes (both
antibodies from Cederlane Lab., Ontario) and counted on a BecktonDickinson FACSort flow cytometer.
In the second part, we performed a surgical and anatomical study of
the vascular pedicles and corresponding microvascular flaps,
including vascular injection with latex and lead-oxide, x-rays and
experimental in vivo transplantation (anaesthesia: Sodium
Pentobarbital 40 mg/Kg i.p) when indicated.
Results and discussion: Figures for volume and weight slightly
differed between different bones, consistently with a different
density and cellularity. As a general rule, cellularity showed a
centripetal distribution, higher in axial skeleton and very little
bone marrow could be found distally to the wrist or ankle with few
nests of bone marrow sparse in the carpal bones and in the
phalanxes.
The highest cell-yield was from the tibia, followed by femur and
pelvis, then sternum, scapula, humerus. Flow citometry showed a
consistent amount of cells expressing the T-cell receptor. This
cells, mainly mature T-lymphocytes, are often removed before bone
marrow transplantation by means of magnetic beads or incubation
with an anti-TcR antibody and Complement. Some cells expressing the
TcR however, such as CD3+ CD25+ T-lymphocytes, are a possible
candidate as “tolerogenic cell” responsible, or contributing, to
the induction of immune tolerance. Selective removal of only a
subpopulation of TcR+ cells, possibly responsible for the
occurrence of Graft-versus-Host Disease, could be advisable. On the
contrary, putative stem cells expressing the CD34 antigen accounted
for less than 1% of the total content of leukocytes, with lower
content in the sternum than in the long bones (Fig. 1).
A dominant vascular pedicle is present in most bones, with few
exceptions like the humerus (Fig.4). However, harvest of some flaps
such as the pelvis or the acromioclavicular flap may be technically
challenging. On the contrary the femur and tibia flaps rely on
good-sized vessels originating from the common femoral vessels.
We describe the microvascular transplant of a whole hemitorax,
which intercostals system is vascularized both by the internal
mammary vessels and the parietal branches of the thoracic aorta
(see arteriogram in Fig. 2), and can be pedicled on the aorta and
either caval or azygos vein. Possible advantages and disadvantages
of this flaps are listed in Tab.1.
Another new flap described is the cranial vault flap, in which the
scalp is used as a vascular carrier, supplying the bone through a
double row of paramedian perforators. The flap is pedicled on the
common carotid artery and jugular vein. Its main advantage is to
carry virtually no bone marrow.
Conclusions: Only few flaps appear truly useful as experimental
models. For isolated bone marrow transplantation, assuming that the
bone itself is poorly immunogenic, the femur and tibia are the best
combination of ease of dissection, size and length of the pedicle,
and content of bone marrow. The hindlimb, including a modification
developed by us with the removal of most of the bone marrow is
probably still the best flap for the study of composite tissue
transplantation.
Table 1. Advantages and Disadvantages
Advantages
Long pedicle of large calibre
Biggest flap available in the rat
Possibly useful for mouse-to-rat
transplantation
of the Hemitorax Flap
Disadvantages
Tedious dissection
Sacrifice of the donor
Stealth syndrome in ratto-rat transplantation
Fig.1: Putative stem cells expressing the CD34 antigen accounted
for less than 1% of the total content of leukocytes, with lower
content in the sternum than in the long bones
Fig.2: A dominant vascular pedicle is present in most bones, with
few exceptions like the humerus. From the left: humerus, tibia,
femur, forearm.
Fig.3: Arteriogram of the hemitorax flap