Tolerogenic Property Of Vascularized Bone Marrow Transplantation Combined With Intravenous Bone Marrow Transplantation In Vascularized Composite Allotransplantation

BackgroundComparing with the traditional autologous tissue transfer,vascularized composite allotransplantation（VCA）was preferred for the restoration of mass soft tissue or limb defect caused by serious burn or trauma because of its abundant tissue source,satisfactory aesthetical and functional reconstructive effect.However,the clinical application of VCA has been hindered by the current life-long immunesuppression after transplantation and inevitable acute or chronic rejection toward the allograft.To solve these problems,the induction of donor specific immune tolerance is necessary.Donor-recipient mixed chimerism as well as immune tolerance could be induced by combined donor-derived intravenous bone marrow transplantation（i.v.BMT）.However,myeloblative conditioning regime is often required before transplantation,and the resulting serious complications has limited the application of i.v.BMT in clinical VCA.Vascularized bone marrow（VBM）consisted of various stem cells and their intact microenvironment exists in some VCA allografts such as limb or face.Therefore,vascularized bone marrow transplantation（VBMT）was simultaneously conducted while the transplantation of these allografts.Numerous animal studies have shown that VBMT is superior to i.v.BMT in the induction process of mixed chimerism and immune tolerance.Additionally,as the microenvironment of bone marrow cells remains intact in VBM,it is unnecessary to conduct myeloblative conditioning regimen.Therefore,comparing with i.v.BMT,VBMT might be more suitable for usage in clinical VCA.However,it is also shown that donor-derived bone marrow cells in VBM allograft could be gradually replaced by recipient-derived cells after transplantation,eventually leading to the rejection of VCA allografts.By the combination of VBMT and i.v.BMT,their disadvantages might be overcomed while their advantages being adopted.Therefore,a nonmyeloblative immune tolerance induction protocol might be created for the broader clinical application of VCA.ObjectivesFirst,developing two simple and reliable VCA models in miniature swine.The VCA allograft in one model contains VBM component,while the other one does not.Second,building an effective method to obtain a large number of bone marrow mononuclear cells（BM-MNCs）from pig.Finally,investigating the impact of VBMT,i.v.BMT or their combination on the survival of VCA allografts under a nonmyeloblative condition regimen as well as its possible mechanism.Methods1.The development of modified VCA model in miniature swine.Tibial osteomyocutaneous flap was transplanted between Bama miniature swines to build a simple and reliable VCA model in large animals.There were two kinds of VCA allograft,one containing VBM component（allograft 1）while the other（allograft 2）not.The vascular pedicles of the allograft were anastomosed to the recipient femoral vessels under microscopic magnification.Then the allograft was placed into the dorsolateral abdominal wall of the recipient.The anatomic parameters of the allografts were observed and recorded,so was the time for each operational process.Allografts were observed grossly and pathologically 1 hour after the transplantation.These observation was used to test the applicability of this model in the following experiments.2.The method to obtain large amount of porcine BM-MNCs.All the thoracic and lumbar vertebrae of the donor pig were harvested and crushed.Then the small bone particles were flushed to obtain suspension of whole bone marrow cells（BMCs）.A sedimentation method using hydroxyethyl starch（control group）and a closed centrifugation method（experimental group）were used to isolate the BM-MNCs form the suspension of whole bone marrow cells.The morphology of BM-MNCs from these two groups were observed using inverted microscope and scanning electron microscope.The isolated BM-MNCs in the two groups was counted manually to obtain the general cell amount and cell harvest rate of the two methods.Cell vitality and proportion of subtype cells were investigated with flow cytometry analysis.3.The influence of VBMT,i.v.BMT or their combination on the survival of VCA allograft under nonmyeloblative conditioning regimen and its possible mechanism.Male Landrace pigs and female Bama pigs were used as donors and recipients,respectively.All recipients received glucocorticoids,thymus irradiation and tapered tacrolimus monotherapy as nonmyeloblative therapy until 90 days post transplantation.The donors were divided into 4 groups based on the transplantation method of donor bone marrow.Group 1（VCA,n=4）only received VCA allografts without VBM component.Group 2（VCA+VBMT,n=4）received VCA allografts containing VBM component.Group 3（VCA+i.v.BMT,n=4）received VCA allografts without VBM component,then receiving i.v.BMT from the donor at 7 days post-transplant.Group 4（VCA+VBMT+i.v.BMT,n=6）reveived VCA allografts containing VBM component,then receiving i.v.BMT from the donor at 7 days post-transplant.Recipients and their allografts were observed daily after the operation to record the survival time of allografts.Regular skin biopsy was carried out to assess the rejection degree of the allograft.Peripheral chimerism level of the recipient in each group was measured with q PCR analysis.At the end of the observation,VBM component in the allografts of group 2 and 4 was taken out to be observed pathologically.Bone marrow cells in the allografts of group 2 and 4 were isolated and counted.Percentage of donor-derived cells in bone marrow cells isolated from the tibial allograft was measured using q PCR analysis,too.Results1.Anatomic parameters of harvested tibial osteomyocutaneous flap were shown as following: arterial pedicle（length: 10.3±1.4 cm;diameter: 2.9±0.4 mm）,venous pedicle（length: 11.8±1.0 cm;diameter: 3.2±0.4 mm）,area of myocutaneous flap（skin: 65.3±18.0 cm²;muscle: 61.7±14.0 cm²）,bone（length: 5.1±0.5 cm）.The mean harvest time of graft 1 and 2 were 57.2±3.6 and 52.5±4.2 min,respectively.The mean warm ischemia time of the allograft was 65.3±3.0 min.No transplant failure caused by surgical procedure was observed.Mean survival time of allograft 1 and 2 were 7.8±1.3 d and 7.5±0.9 d,respectively.Difference between the mean survival time of these two allografts was not statistically significant（P>0.05）.Skin biopsy at 1 h post transplantation showed well blood supply.A large number of bone marrow cells was observed in the tibial metaphysis but not the diaphysis of allograft 1.In allograft 2,there was only cortical bone.2.The mean weight of the vertebrae harvested from the donor was 415.3±21.2 g.Morphological observation showed that most of the isolated cells were BM-MNCs.The morphology of BM-MNCs in the control group was better than that in the experimental group.In control group,the mean general cell amount was（1.53±0.32）×10¹⁰.The mean cell harvest rate was（3.82±0.44）×107/g.In experimental group,the mean general cell amount was（2.36±0.24）×10¹⁰.The mean cell harvest rate was（5.73±0.56）×107/g.The difference between these corresponding indexs of the two groups was statistically significant（P<0.05）.The viable cell rate of the experimental group was less than that of the control group（98.43±0.70% vs 93.1±0.75%,P<0.05）.However,the viable cell amount of experimental group(（2.17±0.34）×10¹⁰)was greater than that of control group(（1.49±0.21）×10¹⁰,P<0.05).Difference between the cell subtype ratios of the two groups was not statistically significant（P>0.05）.3.During the initial 21 days after transplantation,the nonmyeloblative conditioning regimen could effectively decreased the cell amount of peripheral lymphocyte in recipients,which was necessary for VCA and implantation of donor BM-MNCs.The mean survival time of VCA allografts in group 1 and 3 was 10.3±1.3 d and 12±2.2 d,respectively.The mean survival time of VCA allografts in group 2（40±10.7 d）was significantly longer than that of group 1 or 3.Compared with group 1,2 or 3,the mean survival time of VCA allografts in group 4（110±10.2 d）was significantly longer（P<0.05）.In group 1,peripheral chimerism was never detected during the observation.In group 2,peripheral chimerism was detected at 3 days post transplantation（2.2±0.3%）,then it was detected to be persistently decreasing.At 60 days post-transplant,the peripheral chimerism level of group 2 was undetectable.Transient peripheral chimerism was detected in group 3（1.2±0.1%）immediately after i.v.BMT at 7 days post-transplant.Then it faded away.In group 4,peripheral chimerism was detected at 3 days post-transplant（2.2±0.3%）.Then the chimerism level increased to 3.2±0.2% at day 7 post-transplant and sustained to be among 3% during the subsequent period of immunosuppressive regimen.The cells amount of BMCs and percentage of donor-derived cells in allografts of group 4 at 90 days post-transplant were significantly higher than that of group 2 and 4 at the time when their allografts were rejected.The pathological observation also confirmed the above-mentioned results.Conclusions1.The two miniature swine VCA models developed in this research were simple and reliable for the study of VCA as well as the tolerogenic property of VBMT.2.In comparision with the traditional method of hydroxyethyl starch sedimentation,the method of closed centrifugation method developed in this research could isolate more viable BM-MNCs with higher efficiency and safety.Therefore,the modified isolation method was more appreciable for the massive isolation of BM-MNCs.3.Under nonmyeloblative immunosuppressive regimen,the combination of VBMT and i.v.BMT could increase the quantity and percentage of donor-derived cells in the VBM component of allograft.Thus,the survival time of VCA allograft could be significantly increased due to stable peripheral chimerism level in the recipient.