Osteogenesis after bone and bone marrow transplantation. Studies of cellular behaviour using combined myelo-osseous grafts in the subscorbutic guinea pig

In order to study factors influencing osteogenesis after bone and bone marrow transplantation, we have caused guinea pigs to become scorbutic, and looked at the cell morphology at sites of bone formation. We had previously studied normal guinea pigs and found that autologous marrow in intermuscular implants was associated with bone production by the ninth day, regardless of the type of stored allogeneic bone transplanted with it. In subscorbutic guinea pigs, using identical implants, bone did not appear within the first 13 days, and the cell population around the implants was different. These experiments support the dominant role of bone marrow cells in osteogenesis and cast further doubt on the primary role of devitalised bone as an inducer of bone formation. Interference with cell function by deprivation of a single essential molecule, Vitamin C, produces great change in the ability of cells to synthesise bone, or pre-osseous matrix.     

Jaundice following bone marrow transplantation. The problem of diagnosis

Jaundice presented a major diagnostic and therapeutic problem in 6 out of 20 patients undergoing allogeneic bone marrow transplantation for severe acute aplastic anaemia or leukaemia in relapse. In the first 2 cases histological features of graft-versus-host disease were demonstrable in the skin but absent in the liver. In the 3rd case B-virus hepatitis was the most likely diagnosis, in the 4th cumulative cytotoxic chemotherapy was incriminated, and in the last 2 cases the jaundice was obstructive. These 6 cases illustrate the varied causation of jaundice in patients undergoing bone marrow transplantation, and emphasize that correct diagnosis is essential for rational management.     

The fate of cancellous and cortical bone after transplantation of fresh and frozen tissue-antigen-matched and mismatched osteochondral allografts in dogs

After implantation, a massive osteochondral allograft cannot be completely protected from the stresses that are produced by weight-bearing, and it is susceptible to collapse during incorporation, revascularization, and substitution. How these processes are affected by disparities between the tissue antigens of the host and the graft remain unclear. To clarify the role of histocompatibility antigen-matching in the incorporation of cancellous and cortical bone, we orthotopically implanted both fresh and cryopreserved dog leukocyte-antigen-matched and mismatched proximal osteochondral radial allografts in beagles. Four groups of beagle dogs were used; they received (1) a dog leukocyte-antigen-mismatched frozen allograft, (2) a dog leukocyte-antigen-mismatched fresh allograft, (3) a dog leukocyte-antigen-matched fresh allograft, or (4) a dog leukocyte-antigen-matched frozen allograft. In twelve dogs, a sham operation was done in the contralateral limb (the first living donor had a sham operation), and in the remaining ten dogs, the proximal part of the contralateral radius was removed and then replaced as an autogenous (control) graft. The animals were given fluorochromes periodically, and they were killed eleven months after the operation. The osseous portion of the grafts was evaluated radiographically, biomechanically, and histomorphometrically. No dog had grossly obvious clinical abnormalities, all host-graft interfaces healed, and no joints dislocated. Radiographic examination of the allografts frequently showed deformation of the radial head and variable peripheral resorption. No significant difference in the modulus of elasticity at the host-graft interface was found among the groups. The repair process of the cortical bone was similar for all grafted segments. New periosteal and endosteal bone formed, and the cortical bone became porotic as vessels penetrated it. The uptake of fluorochrome was the most active in the autogenous grafts and the least active in the fresh antigen-mismatched grafts. The volume of cancellous bone was significantly greater and the trabeculae were thicker in all allografts compared with the bones on which a sham operation had been done and compared with the autogenous grafts. The volume of intertrabecular fibrous connective tissue was directly proportional to the immunogenicity of the allografts, and the percentage of the surface on which bone was forming tended to be inversely proportional to the immunogenicity of the allografts. The grafts were revascularized by the ingrowth of vessels into the intertrabecular spaces; necrotic trabeculae were not penetrated by vessels. This pattern was particularly pronounced in the antigen-mismatched grafts, regardless of whether they were fresh or frozen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Engraftment following T-cell-depleted bone marrow transplantation. II. Stability of mixed chimerism in semiallogeneic recipients after total-body irradiation

Chronic, stable mixed chimerism of both lymphocytes and erythrocytes was observed in semiallogeneic murine recipients of T-cell-depleted bone marrow transplants that had been conditioned with supralethal total-body irradiation (1100 cGy). Mixed chimerism was extensive, with a wide range of donor engraftment persisting for at least one year after transplant. In both erythrocyte and lymphocyte lineages, decreasing donor engraftment correlated with decreasing marrow dose; however, complete red cell engraftment was more easily achieved than complete lymphocyte engraftment. There were no late graft failures, even among animals exhibiting a substantial host component of hematopoiesis. The extent of mixed hematopoietic chimerism therefore appears to be much greater than had been expected in recipients of T-cell-depleted bone marrow transplants.     

The short-term changes of bone mineral metabolism following bone marrow transplantation

Organ transplantation is now the treatment of choice for many patients with life-threatening chronic diseases. A new set of side effects unique to these groups of patients has become recognized, and bone disease is one of these complications. However, little is known about the effects of myeloablative treatment followed by bone marrow transplantation (BMT) on bone mineral metabolism. We have prospectively investigated 31 patients undergoing BMT for hematologic diseases. Serum concentrations of calcium, phosphorus, creatinine, gonadotropins, sex hormones, and the biochemical markers of bone turnover were measured. The samples were collected before BMT and 1, 2, 3, 4, and 12 weeks, 6 months, and 1 year after BMT. Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry before BMT and 1 year after BMT. The serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 weeks after BMT. Thereafter, it began to decrease and reached basal values after 1 year. Serum osteocalcin decreased progressively until 3 weeks after BMT. After that, it increased and reached basal values after 3 months. No distinct differences were observed in the serum biochemical turnover markers between males and females, or between patients who received total body irradiation and those who did not. One year after BMT, lumbar spine BMD had decreased by 2.2%, and total proximal femoral BMD had decreased by 6.2%. Eighty-six percent of the women (12/14) went into a menopausal state immediately after BMT. This was caused by high gonadotropin levels and low estradiol levels. In contrast, gonadotropin levels and testosterone levels did not change significantly in the male patients after BMT. In conclusion, the rapid impairment of bone formation and the increase in bone resorption, as shown by the biochemical markers in this study, might play a role in post-BMT bone loss.     

Early events in natural resistance to bone marrow transplantation. Use of radiolabeled bone marrow cells

Natural resistance against the proliferation of splenic colony-forming units (CFU-S) is seen in certain combinations of bone marrow donors and irradiated hosts. In order to examine the early events following bone marrow transplantation and to determine whether genetically determined CFU-S repression is due to elimination of the transplanted cells from the spleen or to inhibition of their proliferation, we labeled proliferating cells in freshly isolated bone marrow or long-term bone marrow cultures (LTBMC) with radioactive I-iododeoxyuridine prior to injection. A heterogeneous mixture of cells was labeled in both freshly isolated marrow and LTBMC; CFU-S precursors were among the cells labeled as indicated by reduction of CFU-S numbers in the radiolabeled cell population. Radiolabeled cells from C57BL/6J, DBA/2J, and B6D2F1 mice were injected into syngeneic, semisyngeneic, and allogeneic irradiated mice that were killed at various times after injection to determine the amount of radioactivity remaining and the organ distribution of the labeled cells; additional mice were killed 7-8 days later to count CFU-S. Retention of label in the spleen was predictive of subsequent CFU-S numbers, suggesting that killing or elimination of the transplanted cells from the spleen is the cause of CFU-S depression in resistant animals. Further genetic analysis of the survival of the radiolabeled cells in the spleen indicated that mismatching of H-2 homozygous donor cells with the host at H-2D was a prerequisite for resistance and that H-2 heterozygous cells were not resisted in spite of H-2 mismatching. Although natural killer (NK) cell-deficient beige mice were able to resist H-2-nonidentical bone marrow cells, pretreatment of the host with anti-asialo GM-1 antibodies completely abrogated natural resistance as assessed by splenic survival or radiolabeled cells. The findings that splenic survival or radiolabeled bone marrow cells reflects the immunogenetic specificity of CFU-S repression and that abrogation of CFU-S repression increases the splenic survival of the labeled cells strengthen the postulate that repression of CFU-S proliferation involves events occurring within the first 24 hr after injection.     

Repetitive DNA polymorphisms in following chimerism after allogeneic bone marrow transplantation

Information about the chimeric status of patients is of great importance in comparison of different conditioning and prophylactic regimens as well as for the post-bone marrow transplantation (BMT) therapies. In some cases, mixed chimerism (MC) can also be predictive of relapse. Analysis of the short tandem repeats (STR) loci by polymerase chain reaction (PCR) is a choice method for this purpose. In this study, we monitored 15 patients after BMT. Twelve of them underwent classical-conditioning regimen while the remaining three patients were subjected to non-myeloablative conditioning (minitransplantation). Evaluation of chimerism was performed using five STR and one variable number of tandem repeats (VNTR) locus. Four additional loci were PCR-amplified in cases of minitransplantation. Samples were analyzed by electrophoresis in an ALFexpress sequencer. MC was detected in seven cases of which it was predictive of relapse for two patients, who suffered from acute lymphocytic leukemia (ALL). The PCR-STR method proved to be a fast and relatively simple method, while the tested STR loci showed a high level of informativeness.     

自体骨髓联合脱钙牙基质治疗骨折不愈合

2006年3月～2008年12月,我科采用自体骨髓移植联合脱钙人牙基质材料(骨又生,decalcified dentin matrix,DDM)治疗骨折延迟愈合或不愈合患者19例,取得了良好的疗效.     

Kidney transplantation following treatment with cyclophosphamide and bone marrow grafting.

Canine cyclophosphamide (CY) chimeras permanently accept kidney and skin grafts as do radiation chimeras. Three of five dogs with reversion of chimerism rejected their kidney grafts within 11-16 days, while two of them retained their kidney grafts permanently. These results suggest that the reversion of chimerism in CY chimeras may be due to different mechanisms, either immunologic rejection or a nonimmunologic substitution of the grafted marrow by the host's own hemopoiesis.     

The effect of decalcified bone matrix on the osteogenic potential of bone marrow

Rabbit bone marrow cells were cultured in diffusion chambers with or without decalcified bone matrix. The chambers were assayed after 28 days for alkaline phosphatase activity, deoxyribonucleic acid (DNA), calcium, and phosphorus contents. Morphologically, marrow cells incubated with or without matrix differentiated to form bone and cartilage. With bone matrix, the calcium and phosphorus contents of chambers were significantly higher than control chambers. Alkaline phosphatase activity and DNA content were not influenced by inclusion of bone matrix. These results indicate that bone matrix constituents exert a stimulatory effect on bone formation from marrow cells. This osteogenic stimulation could be due to the influence of an osteoinductive factor and/or to stimulation of osteoprogenitor cells known to be present in the marrow.     

骨髓输注和抗淋巴细胞球蛋白诱导对同种异体移植肾特异性免疫无反应性的临床研究

国外使用抗淋巴细胞球蛋白（ＡＬＧ）和输入供体特有骨髓（ＢＭ）诱导同种移植物的耐受力，并建立了动物模型。我们将这研究用于诱导特异性免疫无反应性病例２０例（骨髓组），并随机抽样２２例尸体肾移植患者作为对照组。骨髓组接受常规三联免疫抑制治疗，在１４天从ＡＬＧ诱导期后，停用１周，于第７天输入冷藏骨髓，随访５～４１个月。结果：１～６个月发生排斥反应２例（２／２０），均用大剂量激素逆转；另１例合并败血性肺炎死亡。对照组采用三联常规免疫抑制治疗剂量较骨髓组大，但不输入骨髓，结果同期发生排斥反应６例（６／２２），后期排斥反应２例，其中４例因排斥反应未能逆转而移植肾失功，有１例因合并心衰死亡。从两组结果分析，人的一年成活率两组无明显差别。移植肾一年成活率则骨髓组高于对照组（Ｐ＜０．０１），排斥反应发生率骨髓组明显低于对照组（Ｐ＜０．０５），且骨髓组常规免疫抑制剂用量较对照组小。     

Lung function changes after allogenic bone marrow transplantation.

The lung function of 21 patients with leukaemia (11 with acute myeloid leukaemia, six with acute lymphatic leukaemia, four with chronic myeloid leukaemia) and of five with severe aplastic anaemia was tested before and after allogenic bone marrow transplantation. Vital capacity (VC) was lowered in patients with leukaemia before transplantation. VC and FEV1 fell significantly after transplantation. Residual volume (RV) and RV as a percentage of total lung capacity (RV % TLC) were already increased and rose significantly after transplantation. Patients with severe aplastic anaemia had noticeably increased RV and RV % TLC, values that did not change after transplantation. In contrast to the patients with aplastic anaemia, the patients with leukaemia had significantly reduced VC, RV, RV % TLC, and FEV1 before and after transplantation. The specific airway resistance (sRaw) was raised significantly before and after transplantation in the leukaemic patients. In addition, transfer coefficient (Kco) fell significantly more after transplantation in the patients with leukaemia than in those with severe aplastic anaemia. In three patients with histologically established obstructive bronchiolitis in conjunction with chronic graft versus host disease after transplantation, VC, FEV1 and FEV1 % VC fell, while RV, RV % TLC, and sRaw rose; Kco was far below normal. On the basis of these findings it is concluded that in patients with leukaemia obstructive disorders of ventilation develop or, if they are already present, worsen. In patients with severe aplastic anaemia lung function was not impaired in the early phase after transplantation. These differences are probably due to the more intensive immunosuppressive and cytotoxic preparatory regimen before transplantation in the leukaemic patients. Obstructive bronchiolitis, a complication of graft versus host disease, first manifests itself in a typical rise in specific airway resistance and must be treated early.     

Mixed chimerism of cardiomyocytes and vessels after allogeneic bone marrow and stem-cell transplantation in comparison with cardiac allografts

Controversy persists about mixed chimerism (mCh) occurring in the hearts of patients after orthotopic cardiac transplantation in comparison with allogeneic bone marrow (BM) and peripheral blood stem-cell (PBSC) transplants. Cadaver hearts were examined after sex-mismatched transplantation by immunophenotyping combined with dual color fluorescence in situ hybridization (X and Y chromosome-specific probes). A striking disparity in the extent of mCh depending on the different transplantation procedures was recognizable. After allografting with PBSCs, 1.7% chimeric cardiomyocytes were detectable contrasting 5.4% of donor cells after full BM transplantation. In cardiac transplants, host-type endothelial cells (16.2%) and myocytes (14.3%) of the vessel walls were more often encountered than after BM and PBSC allografting. A sprouting of vascular structures into the donor heart after orthotopic cardiac transplantation has to be assumed, as does a pivotal role of the mesenchymal stem cells of the BM in the development of mCh.