Impact of MHC mismatch and freezing on bone graft incorporation: An experimental study in rats

Abstract

Cortical bone graft failure develops for poorly defined reasons, and the effects of the immune responses on the incorporation of an allograft are less clear. In a rat model of tibial allotransplantation, we have studied biometric and histological changes of the graft and the humoral immune response against it. We have also compared fresh with prefrozen grafts to study putative effects of freezing on the healing of the graft and the immune response against it. Fresh and frozen cortical bone grafts matched or mismatched for major histocompatibility complex antigens (syngeneic and allogeneic grafts) were implanted in an 8-mm segmental defect in the tibia. The construct was stabilized with intramedullary nailing. Incorporation of the graft was assessed with use of conventional radiography, micro computed tomography (CT), biomechanical testing and histological examination. The immune response was evaluated by monitoring distribution of leukocytes in the blood and by measuring antibodies in a tailor-made fluorescence activating cell scanning (FACS) analysis. We found that the fresh syngeneic grafts were well integrated after 8 weeks with intact bone cells. In the fresh allogeneic grafts, all cells were dead with radiological signs of resorption, and mechanical testing indicated failure of incorporation. The frozen grafts showed poorer overall reconstruction than the fresh syngeneic grafts, but the incorporation was better than the fresh allogeneic grafts. A measurable alloantibody response was only detected after fresh allografting. The combined results suggest that freezing of bone allograft impedes the antibody response against major histocompatibility complex (MHC) antigens and improves incorporation, but frozen allografts still perform poorer than do frozen syngeneic grafts. ß

Impact of freezing on immunology and incorporation of bone allograft

“…We found that long term freezing of the allograft did not induce any detectable antibody response nor impeded healing of the graft. Previously we have shown that the antibody response we have measured reflects immune responses in general, 7 and our present results indicate that deep freezing of bone allograft for 1 year has been sufficient to destroy immunogenicity.…”

“…PVG rats promptly reject organ grafts from PVG.1U rats on the basis of differences in the MHC region, and we and others have shown strong immunological reactions after MHC mismatched transplantation of fresh bone. 6,7 However, allogeneic bone for clinical application is usually stored deep frozen for longer periods of time, and in the present experiments we studied aspects of immunity and outcome of bone bank grafts. We employed rats of a similar body weight that were genetically identical except for the major histocompatibility complex region.…”

“…Accordingly, in a previous study we found that during a shorter follow up of 2 months, a fresh syngeneic graft healed faster that a frozen. 7 However, with time bone grafts are penetrated by vessels and substituted with host bone and ultimately remodeled. This creeping process is dependent on osteoconductive properties of the graft, and our study indicates that long term freezing of bone grafts does not significantly influence its structure for osteoconduction, and ultimately deep frozen and fresh bone grafts incorporate with the same biomechanical competence.…”

Effect of Soft-Tissue Attachment on Tibial Fracture Healing in Rats

“…32,33 Histology at 8 weeks after grafting of a non-vascularised graft showed normal osteocyte density with signs of revascularisation and remodelling; the grafts were well-integrated, with a continuous bridge of hard tissue between the implant and the cortical bone of the host. 34 Direct contact of soft tissues to bone is not a prerequisite for osteogenesis, which may be achieved by soluble factors. [11][12][13][14] It is unclear whether vascularity is essential for osseous integration of reinserted cortical lids.…”

Review of Shino et al (1984) on anterior cruciate ligament reconstruction using allograft in the dog

“…When allografts were first being used, fresh allografts, rather than fresh frozen were used and incited a marked inflammatory and rejection response which was characterised by a perivascular high density of infiltrating cells and lymphocyte invasion 15. An experimental study in rats suggested that the fresh-frozen technique, which is currently a major allograft processing method as is the freeze-dried process, impeded the antibody response to major histocompatibility complex antigens in bone allografts and improved incorporation compared with fresh allografts 70. A comparative experiment between fresh-frozen and freeze-dried allografts concluded that incorporation and replacement of fresh-frozen bone allografts were delayed compared with the freeze-dried bone allografts 71.…”