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The Journal of Bone and Joint Surgery (American) 84:1420-1429 (2002)
© 2002 The Journal of Bone and Joint Surgery, Inc.

Scientific Article

Cryopreservation of Osteochondral Allografts: Dimethyl Sulfoxide Promotes Angiogenesis and Immune Tolerance in Mice

Carsten Wingenfeld, MD, Rainer J. Egli, MD, Axel Hempfing, MD, Reinhold Ganz, MD and Michael Leunig, MD

Investigation performed at the Department of Orthopedic Surgery, University of Berne, Inselspital, Berne, Switzerland

Carsten Wingenfeld, MD
Rainer J. Egli, MD
Axel Hempfing, MD
Reinhold Ganz, MD
Michael Leunig, MD
Department of Orthopedic Surgery, University of Berne, Inselspital, CH-3010 Berne, Switzerland. E-mail address for M. Leunig: michael.leunig{at}insel.ch

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from the Swiss National Science Foundation, the Sandoz Foundation, and the Maurice E. Müller Foundation. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Background: 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 microscopy.

Methods: 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.

Results: 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 fresh allografts.

Conclusion: 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.

Clinical Relevance: 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.


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