Although transplantation of cryopreserved bone allografts has become
a routine procedure in orthopaedic surgery, biological and immunological
impairment remains an unsolved problem that causes clinical failures.
Experimental and clinical evidence has indicated that bone grafts
that are revascularized early remain viable and contribute to union
at the recipient site. Unprotected cryopreservation, used in most bone
banks to reduce graft antigenicity, is associated with complete
loss of graft viability, potentially contributing to graft failure.
The differences in the survival of various cell types during cryopreservation
with use of dimethyl sulfoxide, particularly the increased sensitivity
of leukocytes to fast freezing, has resulted in a new approach to
modulate immunogenicity. On the basis of this concept, it was proposed
that a reduction in the immune response and enhanced revascularization
of osteochondral allografts could be achieved by rapid cryopreservation
with dimethyl sulfoxide. To test this hypothesis, angiogenesis and
immune tolerance were quantified in a murine model with use of intravital
Fresh osteochondral tissue and osteochondral tissue that had been
cryopreserved with and without dimethyl sulfoxide was transplanted
into dorsal skinfold chambers as isografts and as allografts in
presensitized and nonsensitized recipient mice. To quantify angiogenesis,
the onset of hemorrhages in the vicinity of the grafts and the revascularization
of the grafts were determined by means of intravital fluorescence
microscopy. To determine the recipient's intravascular immune response
to the grafts, the leukocyte-endothelium interaction was assessed
on the twelfth day after transplantation.
Nine of nine fresh isografts were revascularized at a mean (and
standard deviation) of 57 ± 33 hours, eight of nine isografts
that had been cryopreserved with dimethyl sulfoxide were revascularized
at 98 ± 50 hours, and zero of nine isografts that had
been cryopreserved without dimethyl sulfoxide were revascularized.
Seven of seven fresh allografts were revascularized at 53 ±
6 hours, and ten of ten allografts that had been cryopreserved with
dimethyl sulfoxide were revascularized at 82 ± 29 hours. However,
signs of revascularization faded in four of the seven fresh allografts
whereas reperfusion was maintained in the majority (seven) of the
ten grafts frozen in the presence of dimethyl sulfoxide. Similar
to the findings associated with unprotected frozen isografts, zero
of ten unprotected frozen allografts were revascularized. None of
the allografts that had been transplanted into presensitized recipients
were revascularized, regardless of whether they had been implanted
fresh (nine grafts) or had been implanted after protected (eight grafts)
or unprotected (nine grafts) freezing. Quantification of the leukocyte-endothelium
interaction revealed a reduction in the intravascular immune response
to frozen allografts (both protected and unprotected) compared with
Osteochondral allografts that had been pretreated by cryopreservation
with dimethyl sulfoxide demonstrated improved angiogenesis induction
and enhanced immune tolerance compared with unprotected frozen grafts.
A selective reduction in donor passenger leukocytes is the proposed
mechanism underlying this phenomenon.
In the absence of presensitization, cryopreservation with dimethyl
sulfoxide appears to reduce the immune response to allografts and
to enhance their revascularization; in the presence of presensitization,
alternatives to allograft transplantation should be considered since
the allografts will be exposed to a deleterious immune response.