Effect of Cryopreservation on the Immunogenicity of Osteoblasts

Tissue-engineered bone is the most perspective and ideal prosthesis for bone defects. But the immunological reaction in allograft is unavoidable. To date there has not been an analysis of the effect of cryopreservation on osteoblast immunogenicity. In this study, we investigated the effects of fluid nitrogen cryopreservation on immunogenicity of osteoblasts.The osteoblasts were harvested and cultured from the tibial periosteum of New Zealand rabbits, examined by alkaline phosphatase (ALP) and alizarin red S stains, and then cryopreserved for 90 days. Cryopreserved (group C) and fresh (group F) osteoblasts were labeled by the immunofluorescene technique, and the MHC class I antigen was examined by flow cytometric (FC) assay. At the same time, by establishing an osteoblast and lymphocyte mixed culture model (OLMC), the immunogenicity of osteoblasts was examined.The positive rate of MHC class I antigen in group F was 46.36%+/-3.15%, while in group C it was 26.43%+/-2.57%. The MHC I antigen expression was significantly stronger in fresh cells than that in cryopreserved ones (P<.01). In the OLMC assay, the stimulation index (SI) of group F was 3.55+/-0.78, which was significantly higher than that of group C (1.83+/-0.41; P<.01).The FC assay and OLMC experiments showed that osteoblasts surface antigen expression and allostimulatory function were greatly reduced by cryopreservation. Fluid nitrogen cryopreservation is an ideal method to reduce the immunogenicity in allograft.