Partial graft cell survival and enhanced graft revascularization have suggested fast freezing using the cryoprotective substance dimethyl sulfoxide (DMSO) as a promising means to improve the biologic function and immune tolerance of allograft bone. This study determines the presence of osteoblasts (cola(1)(I) mRNA), osteoclasts (TRAP), and cytotoxic T cells (CTLs; GrA mRNA) within pretreated bone grafts 12 days after transplantation. The grafts were transplanted either as isografts, allografts, or allografts in presensitized recipients. In fresh isografts, serving as control, well-formed blood vessels and the highest numbers of viable osteoblasts and osteoclasts were found. In fresh allografts, blood vessels were observed within the marrow cavity and the bone was partially covered by osteoblasts and osteoclasts accompanied by CTLs. In DMSO-pretreated frozen allografts, blood vessels together with osteoblasts were observed in three of five, but in none of five grafts frozen without DMSO. However, infiltration with CTLs was higher in DMSO-pretreated frozen allografts when compared to grafts frozen without DMSO. In presensitized allograft recipients, independent of the pretreatment, in none of the grafts were either blood vessels or osteoblasts found. Thus, fast cryopreservation of bone using DMSO improves vascularization and expression of cola(1)(I) mRNA (osteoblasts) after allografting when compared to cryopreservation alone, potentially improving graft incorporation. As these grafts were still invaded by CTLs, the long-term effect of DMSO pretreatment needs to be defined.
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