Impact of Freezing on Immunology and Incorporation of Bone Allograft Olav Reikera˚s,1 Ulf W. Sigurdsen,1 Hamid Shegarfi2 1 Faculty Division Rikshospitalet, Department of Orthopaedics, University of Oslo, N-0027 Oslo, Norway, 2Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
Received 1 October 2009; accepted 20 January 2010 Published online 1 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.21121
ABSTRACT: With an increasing clinical use of deep frozen allograft for bone reconstruction, it is important to understand the immunological and biological events of allograft incorporation. In this study, we have investigated the impact of deep freezing on immunology and biopotency for incorporation of bone allografts. Deep frozen bone grafts matched or mismatched for major histoscompatibilty complex (MHC) were implanted in an 8-mm segmental defect in the tibia in rats. The construct was stabilized with intramedullary nailing. The immune response was evaluated by determination of serum antibody against the grafts MHC molecules at day 1 and after 2 and 4 months. Incorporation of the graft was compared with fresh syngeneic grafts and assessed with the use of conventional radiography, biomechanical testing and measurement of bone mineral content and density after 4 months. The analyses revealed no antibody responses in the rats that received grafts from donors differing at histocompatibility loci, and at 4 months the frozen grafts showed an overall reconstruction that was not significantly different from the fresh grafts. This study indicates that in the long run there are no significant consequences; either immunological or biomechanical, of the use of deep frozen allogenous bone as compared to fresh autogenous bone grafts in this animal model. ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1215–1219, 2010 Keywords: bone graft; immune response; incorporation; MHC mismatch; transplantation;
Bone grafting in orthopedic surgery has increased dramatically in recent years, and in clinical practice bone is the second most transplanted tissue after blood transfusion. Autogenous grafting is the gold standard as there is no risk of infectious disease transmission and there is no deleterious immune response after transplantation. However, complications and donor site morbidity may develop,1 and also the shape and size of autogenous bone often do not conform to the defect in the recipient site. Therefore allografting has emerged as a suitable option to autograft procedures. However, upon implantation of allogenous bone the host is expected to experience an intricate immune response.2,3 Deep freezing at 808C in sterile tubes may reduce the immunity, but freezing may also alter the properties of the graft. The treatment of allografts and the degree of histocompatibility, then, are two principal determinants of importance for healing. With an increasing application of grafting for massive bone reconstruction, it is important to understand the immunological and biological events of graft incorporation. In this study, we have investigated the impact of long term deep freezing of allogenous cortical bone on immunity and biopotency for graft incorporation.
METHODS Animals Prior to the experiments 5 PVG.1U (RT1u) and 5 PVG (RT1c) 3 months old male rats (Harlan UK Limited, Shaw’s Farm, Blackthorn, Bicester, England) were sacrificed. On both sides two osteotomies at the shaft of the tibial bone were made 8 and 16 mm from the knee joint with a fine-toothed circular saw blade mounted on an electric drill. The bone segments were elevated from soft tissues and totally emptied of cancellous bone and bone marrow. Thereafter they were deep frozen at 808C for a year in the bone bank. The PVG.1U and PVG rats Correspondence to: Olav Reikera˚s (T: þ47 23076013; F: þ47 23076010; E-mail:
[email protected]) ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
are identical except for the major histoscompatibilty complex (MHC), where PVG.1U rats have their MHC (RT1) complex derived from the AO strain. An isolated effect of MHC mismatches (class I, class II, and other polymorphic antigens within the MHC) on graft survival and other biological parameters can therefore be monitored. Bone Transplantation The experiment conformed to the Norwegian Council of Animal Research Code for the Care and Use for Experimental Purposes. In general anaesthesia (Hypnorm/Dormicum VetaPharma, Sheburn, Leeds, England) 30 male PVG rats 3 months old were operated. The proximal part of either the right or left tibia was exposed through an incision anteriorly, and the muscles were carefully elevated. Two osteotomies at the shaft of the bone were made 8 and 16 mm from the knee joint with a fine-toothed circular saw blade mounted on an electric drill, and the bone segment was removed. By random, 10 rats were transplanted with a fresh syngeneic segment which was emptied of cancellous bone and bone marrow; 10 rats were transplanted with a deep frozen syngeneic segment and 10 rats were transplanted with a deep frozen allogeneic segment from the bone bank. A 0.8 mm steel pin (cannula) was used for intramedullary stabilization. Fixation was achieved without radiographic control, but proper pin placement was confirmed later by radiographs taken at the end of the experiment. The rats were bled from the tail vein immediately after grafting, at 2 months and at termination of the experiments at 4 months, and blood serum were examined for changes in antibodies against MHC antigens in the graft. Graft Incorporation The strength of the composite (transplanted) bone was tested for torsion strength (MTS 858, MTS Systems Corporation, Eden Prairie, MN). The tibial ends were fixed with a clamp and the instrument was run at a constant rate of 0.08 rad/s. The load values were recorded and transformed to a load deformation curve (Origin 7.5, Northhampton, MA). The strength was calculated as the torsion moment necessary to produce fracture of the composite bone. The torsion rigidity was determined from the slope of the linear elastic part of the curve. The energy was defined as the energy absorbed during JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2010
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loading to fracture. Bone mineral content (BMC) and density (BMD) were examined by dual energy X-ray absorptiometry (DEXA). DEXA was performed on a GE Lunar Piximus (Lunar Corporation, Madison, WI) with a tube current of 400 mA. The frequency of the scanning unit was 50/60 Hz, and the X-ray tube had a focal spot size of 0.25 mm 0.25 mm with a coefficient of variation of 1%. After scanning of the whole tibia, a region of interest was centralized at the graft. Antibody Measurements The antibody response in individual rats was measured as previously described.4 In short, rat B lymphoma cells, YB2/0, expressing RT1u antigens were incubated with serum dilutions from the transplanted rats at 48C for 30 min, washed and then incubated with a FITC-labeled secondary anti rat Ig (Jackson ImmunoResearch Laboratories, West Grove, PA) for another 30 min and washed. The fluorescence intensity was measured in a FACScalibur (Becton Dickinson, San Jose, CA), as previously described,4 with gates set to exclude dead cells. Cells labeled with secondary anti rat IgG were uniformly negative. Experiments included the secondary mAb alone (negative control) and a standard 68 mAb against MHC class I of the u haplotype (positive control). Statistics Data are presented as mean and standard deviation of the mean. To test statistical differences, ANOVA was used followed by LSD test when appropriate (SPSS, Inc., Chicago, IL). The level of significance was set at 0.05.
RESULTS The rats tolerated the operation well and resumed full weight-bearing after a few days. There were no failures.
The animals were weighed at operation and at sacrifice, and there were no differences in weight gain between the groups. Healing of the grafted segment occurred in all transplanted bones, either fresh or frozen as evaluated by radiography at 4 months (Fig. 1). There were no significant differences in torsion strength between the three groups, nor were there any significant differences in either rigidity or fracture energy (Table 1). For comparison, intact bone had torsion strength of 14.8 3.02 (Nm 102) which was significantly higher than the transplanted bones (P ¼ 0.019), rigidity of 3.08 0.60 (Nm/degree 103); also significantly higher than the transplanted bones (P ¼ 0.032), and energy to fracture of 73.2 26.3 (Nm degree 101) (P ¼ 0.043). There were no significant differences in either bone mineral content or bone mineral density between the three groups (Table 2). For comparison, BMC of intact bone was 35 3 (103 g) and BMD was 171 7 (103 g/ cm2); which was significantly lower than in the transplanted segments (P ¼ 0.009 and 0.004, respectively). The alloantibody response to frozen allogeneic versus frozen syngeneic grafts is shown in Figure 2 using YB2/0 cells of the RT1u haplotype as test cells. Individual rats of both groups showed no responses, at titers of 1/4 and 1/8. The antibody response was consistent between the different rats and remained negative up to termination of the experiment after 4 months.
Figure 1. Radiographs of a fully incorporated fresh syngeneic graft (a), frozen syngeneic graft (b) and a frozen allogeneic graft (c). JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2010
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Table 1. Torsion Moment (Nm 102), Rigidity (Nm/degree 103) and Energy (Nm degree 101) of the Composite Bone (Graft and Tibia) 4 months following Syngeneic/Allogeneic Grafting of a Fresh or Frozen Segment. (Mean Standard Deviation of the Mean)
Moment Rigidity Energy
Fresh Syngeneic
Frozen Syngeneic
Frozen Allogeneic
ANOVA
10.0 3.13 2.22 0.87 39.5 13.8
10.2 3.75 1.98 0.76 55.0 34.1
11.0 2.86 2.32 0.67 46.6 13.5
0.794 0.668 0.401
DISCUSSION MHC in rats is termed RT1, a gene complex encoding MHC antigens located on chromosome 20. It is analogous to the human leukocyte antigen (HLA) with a general overall similarity in construction of the class II and class III regions, but with varying numbers of MHC class I antigens.5 Like in humans, the gene region encodes MHC class I and II molecules that provoke strong alloimmune responses to organ transplantation, leading to their rejection. 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. We found that differences in genes within the MHC region did not influence incorporation of the graft when the bone grafts were frozen over a long time period. For example, during a 4-month period deep frozen bone incorporated to the same degree as fresh bone. The most powerful immunogenic response in bone allografting is generated by donor bone marrow cells in the graft.8 These cells are recognized as foreign by host T lymphocytes, which respond with a cell-mediated immune response. Further, ostocytes display class-I and class-II histocompatibility antigens. Furthermore, Type I collagen, proteoglycans, and other components of extracellular matrix stimulate both a cell-mediated and humoral immune response. Altogether, alloantigens from a graft are presented to the recipient’s immune system in two ways. Direct recognition occurs when recipient T lymphocytes respond to donor antigens on cells in the allograft. Indirect recognition occurs when
recipient antigen presenting cells ingest donor MHC antigens and present peptides of donor MHC in the groove of the recipient’s own MHC molecules. This latter scenario may be predominant when donor tissue is damaged by freezing. 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. Different animal models have been developed to study bone graft incorporation,9–11 and different conclusions have be drawn from these studies. Bone graft incorporation is a complicated process with multiple variables influencing rate, pattern, and completeness. The healing process at the graft and host bone interface can be divided into different stages.12 Stage 1, or the inflammatory stage, is marked by the arrival of various inflammatory cells to the bone graft site, attracted via (the common mechanisms of) chemotaxis. Stage 2 is marked by the differentiation of host mesenchymal cells into osteoblasts (osteogenesis), while stage 3, or osteoinduction, involves the functioning of both osteoblasts and osteoclasts. Stage 4 is osteoconduction, in which new bone forms over an existing scaffold. Stage 5 is remodeling, and this final stage continues for years after graft implantation. The net biological activity of a graft is the sum of the interactions between the cells of the inflammatory wound healing response in the host and the biological activity of the graft, its capacity to induce surrounding host tissues to produce relevant biological activity (osteoinduction mediated by bioactive factors within the matrix), and its ability to support the ingrowths of osteogenic host tissue (osteoconduction).13 Autogenous bone possesses all these properties, while a deep frozen necrotic allograft lacks osteogenetic and
Table 2. Bone Mineral Content (BMC, 103 g) and Bone Mineral Density (BMD, 103 g/cm2) of the Composite Bone (Graft and Tibia) 4 months following Syngeneic/ Allogeneic Grafting of a Fresh or Frozen Segment. (Mean Standard Deviation of the Mean)
BMC BMD
Fresh Syngeneic
Frozen Syngeneic
Frozen Allogeneic
ANOVA
46 12 198 32
45 11 197 21
48 9 210 31
0.816 0.595
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Figure 2. FACS plots showing no antibody production in PVG rats transplanted with either frozen bone from PVG (syngeneic) or PVG.1U (allogeneic) rats. (A) Monoclonal antibody 68 against MHC class I of the u haplotype used as a positive control to YB2/0 cells is shown. Experiments included the secondary mAb alone (negative control, not shown). (B) Comparison between syngeneic (left) and allogeneic (right) grafts at day 1, and after 2 and 4 months. Final serum dilution was 1/8. Plots from three representative rats of each group are shown.
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osteoinductive properties and only serves as a scaffold for bone ingrowths. The incorporation of an allograft, then, will only take place from the host bone site, and the initial healing processes will be slower than for a fresh autograft. 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. Successful incorporation of a bone graft should result in a composite that can bear physiological loads and adapts its structure in response to load changes. In our animal model the graft was placed in a weight-bearing environment so that it was subjected to in vivo mechanical stresses. Stability is needed for optimum graft healing and incorporation. Motion at the interface between grafted and host bone interferes with revascularization (and) affects new-bone formation. However, no massive allograft is implanted under ideal conditions. In our model stability was ensured by the intact fibula, and we consider it as relevant for clinical practice. DEXA scanning determines the mineral mass in the skeletal region with acceptable accuracy and precision and can describe its changes under different treatments.14,15 The vast majority of whole body calcium and phosphate is stored as the mineral hydroxylapatite in the skeleton, and bone tissue is essential in the physiological mechanisms governing calcium and phosphate transport. DEXA investigations thereby describe altered regulations and changes in bone minerals under different treatments and conditions. In an avascular bone graft there is no dynamic shift in mineral mass until the segment is vascularized. In our study this was indicated by significantly higher mineral mass as evaluated by BMC and BMD in the grafts as compared to intact tibial bone. The fact this was observed after 4 months reflects that even at this time the vascular incorporation of the segments was not completed. This is in agreement with the fact that the transplanted bones only had reached strength of 80% of intact bone.
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In conclusion, our results indicate that long term deep freezing of allogenous bone destroy immunogenicity, and in the long run deep frozen bone incorporates like fresh autogenous bone.
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