Clinical and immunopathological significance of chimerism in bone marrow and organ transplantations

Chimerism is an exceptional immunogenetic state, characterized by the survival and collaboration of cell populations originated from two different individuals. The prerequisites to induce chimerism are immunosuppression, myeloablation or severe immunodeficiency of the recipients on one side and donor originated immuno-hematopoietic cells in the graft on the other. Special immunogenetic conditions to establish chimerism are combined with bone marrow transplantation, transfusion and various kinds of solid organ grafting. There are various methods to detect the type of chimera state depending on the immunogenetic differences between the donor and recipient. The chimera state seems to be one of the leading factors to influence the course of the post-transplant period, the frequency and severity of graft-versus-host disease (GVHD), and the rate of relapse. However, the most important contribution of the chimeric state is the development of graft versus leukemia (GVL) effect. A new conditioning protocol (DBM/Ara-C/Cy) for allogeneic BMT in CML patients and its consequence on chimera state and GVL effect is demonstrated.     

Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration

Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.     

Effect of irradiation on autogenous bone transplantation in rat parietal bone

To determine the appropriate time for bone reconstruction after irradiation, the healing process after autogenous iliac bone transplantation in the irradiated parietal bone was examined by scanning electron microscopy and light microscopy. Bone transplantation was carried out at the second and the fourth weeks after Cobalt-sixty (60Co) irradiation with calculated dose and fractionation. Animals without irradiation were used as control. The results show the appearance of mesenchymal cells and blood vessels around the transplantation to be extremely few one week after transplantation which was carried out at the second week after irradiation. These inhibitions were still seen two weeks after transplantation. Four weeks after transplantation, there were no differences in the bone formation among the experimental groups. Bone formation in the transplantation at the fourth week after irradiation was similar to that of the control group. Microvascularization in the transplantation at the second week after irradiation was inhibited one week after transplantation. The delay in bone healing was responsible for the retardation of revascularization and caused microcirculatory failures as well as the damage of osteogenic cells. It is quite clear that damaged cells and tissues recovered by the elapse of time under the irradiation procedure employed in this study and also that bone formation was carried out in the physiological process. We think that bone transplantation after irradiation should be done after recovery from the radiation damage to the periosteal cells and the blood vessels.     

Overview of signal transduction between LL37 and bone marrow-derived MSCs

Journal of Molecular Histology - As a key signaling molecule, cationic antimicrobial peptide LL37 helps mediate intracellular and extracellular signal transduction. It interacts with various cells...     

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.     

The value of cell cultures in leukemias, aplastic anemias and bone marrow transplantations

In a survey different methods of culture are represented for the purpose of identifying and quantifying haemopoietic stem cells (CFU-GEMM, CFU-GM, CFU-E/BFU-E) in human bone-marrow or peripheral blood respectively. On the basis of findings from international medical literature their validity is explained in the diagnostics and prognosis of some haematological diseases, such as acute and chronic myeloic leukemia, aplastic anemia, preleukemia. Special attention is given to their significance within bone-marrow transplantation. Their importance in evaluating transplantations after their preceding in-vitro manipulation as to the separation of rest tumour and T-cells is particularly referred to.     

The comparative analysis of homologous fresh frozen bone and autogenous bone graft,associated or not with autogenous bone marrow,in rabbit calvaria: a clinical and histomorphometric study

The aim of this study was to evaluate the potential of fresh frozen homologous and autogenous grafts, associated or not with autogenous bone marrow, to form bone. Sixty titanium cylinders were used, and were fixed to the skulls of 30 rabbits. These cylinders were filled with (A) autogenous bone (AM) autogenous bone associated with the bone marrow (H) fresh frozen homologous bone (HM) fresh frozen homologous bone associated with the bone marrow (M) pure autogenous bone marrow and (C) blood clot. The animals were sacrificed after 02 and 03 months. After clinical evaluation, the samples were stained with hematoxylin, eosin and Mallory Trichrome dyes for optical microscopy analysis and histomorphometric analysis. Experimental groups that received mineralized materials (A, AM, H, HM) showed the best bone formation results, presenting no statistical difference between them (P > 0.05). Groups that did not receive mineralized materials (M and C) showed the worst results (P < 0.05), but the M group showed better results than the C group. Most of the autogenous and homologous bone particles were resorbed and there was a larger amount of residual particles in the homologous graft (H, HM) when compared with the autogenous graft (A, AM; P < 0.05). These findings suggest that fresh frozen homologous grafts produced similar amounts of new bone when compared with the autogenous grafts. However, the amount of residual bone particles was larger in the homogenous groups, which may indicate a slower remodeling process. The homologous fresh frozen bone seems to be a good osteoconductive material. The use of only autogenous bone marrow showed better results when compared to the bood clot. However, this research indicates that association with mineralized materials is required.     

A biomechanical comparison of vascularized and conventional autogenous bone grafts

Vascularized and conventional autogenous rib grafts were used to reconstruct 6-cm ulnar defects in the forelegs of the nine dogs. Each dog served as its own control. Biomechanical torsional testing of the grafted ulnas showed that vascularized grafts were 234 percent stronger than the conventional grafts. Bone toughness (energy absorbed) was 483 percent greater in the vascularized grafts, and elastic modulus and proportional limits were 263 and 246 percent greater, respectively. We conclude that vascularized bone grafts are significantly stronger than conventional autogenous bone grafts after 3 months of healing in the dog ulna model.     

Ciprofloxacin concentrations in serum, bone and bone marrow of rabbits

Ciprofloxacin concentrations were determined in serum, bone and bone marrow of rabbits. Four experimental groups of animals were examined: group A (n = 6) received a dosage of 60 mg/kg/day intramuscularly for 4 weeks, groups B (n = 6), C (n = 15) and D (n = 15) received dosages of 120 mg/kg/day subcutaneously for 2 days, 2 weeks, and 4 weeks, respectively. In the kinetic portion of the study, peak serum concentrations of ciprofloxacin measured at the 15 min sampling time were: 2.61 +/- 0.27 micrograms/ml in the 60 mg/kg/day group (group A) and 3.24 +/- 0.78 micrograms/ml in the 120 mg/kg/day group (group B). At necropsy, rabbits in group A had mean ciprofloxacin concentrations of 3.60 +/- 2.27 micrograms/ml in serum, 2.24 +/- 1.19 micrograms/g in marrow and 1.19 +/- 0.44 micrograms/g in bone. Rabbits in group B achieved mean levels of 4.02 +/- 1.23 micrograms/ml in serum, 2.48 +/- 0.79 micrograms/g in marrow, and 1.35 +/- 0.40 micrograms/g in bone. Rabbits in group C achieved mean levels of 5.65 +/- 2.16 micrograms/ml in serum, 3.74 +/- 1.33 micrograms/g in marrow and 1.92 +/- 0.94 micrograms/g in bone. Rabbits in group D achieved mean levels of 7.24 +/- 2.50 micrograms/ml in serum, 4.48 +/- 1.68 micrograms/g in marrow, and 1.93 +/- 0.54 micrograms/g in bone. Differences between mean values for the four experimental groups were not statistically significant.     

硫酸钙作为微小颗粒骨载体的实验研究

目的：研究硫酸钙作为微小颗粒骨载体,解决微小颗粒骨的自身缺点的实际效果,为其临床应用提供依据。方法：将49只日本大耳白兔随机分成4组并通过手术造成双侧桡骨中段1．5cm骨缺损,以植入硫酸钙为载体的自体微小颗粒骨为实验组,同时设立单纯植入自体微小颗粒骨,单纯植入硫酸钙和不植入任何物质的空白对照组。术后4周和8周分别行大体观察。X线摄片,组织学观察,骨生物力学测定。结果：以硫酸钙为栽体的自体微小颗粒骨组比单纯自体微小颗粒骨组及单纯硫酸钙组能更有效地修复骨缺损,单纯颗粒骨组成骨效果优于单纯硫酸钙组。空白组无骨愈合迹象;组织学观察示以硫酸钙为载体的自体微小颗粒骨实验组的成骨效果最好,单纯自体微小颗粒骨组次之;生物力学测定证明以硫酸钙为载体的自体微小颗粒骨实验组的力学强度优于单纯自体微小颗粒骨组及单纯硫酸钙组。结论：硫酸钙是微小颗粒骨的优良载体,以硫酸钙为载体的自体微小颗粒骨成骨速度快,成骨量多,质量高,骨的机械强度高,修复骨缺损的能力较单纯应用微小颗粒骨和硫酸钙强;二者结合可充分发辉各自的优势。     

Allogeneic hepatic cell suspension in the treatment of liver insufficiency

A study was made of a possibility of using isolated hepatocytes for the treatment of diseases of the liver in animals. The optimal dose of the cell suspension in intravascular, intraperitoneal, intrapleural and subcutaneous administration was determined; a reaction of the experimental animals to this biological substrate was studied. The efficacy of the isolated hepatocytes in the treatment of hepatic insufficiency is demonstrated; also a comparative assessment of the mentioned methods of the cell suspension administration is given.     

Engineering microdent structures of bone implant surfaces to enhance osteogenic activity in MSCs

Problems persist with the integration of hip and dental implants with host bone tissues, which may result in long-term implant failure. Previous studies have found that implants bearing irregular surfaces can facilitate osseointegration. An improvement to this approach would use implant surfaces harboring a well-defined surface microstructure to decrease variability in implant surfaces. In this study, we tested whether well-defined surfaces with arrays of microdents (each with depth approximately 3 µm) significantly affected the morphology, proliferation, and osteogenic activity of mesenchymal stem cells (MSCs). Arrays of microdents tested had diameters of 9 µm, 12 µm, and 18 µm, while spacing between arrays ranged from 8 µm to 34 µm. Effects on MSC morphology (cell spreading area) and proliferation were also quantified, with both significantly decreasing on micropatterned surfaces (p<0.05) on smaller and denser microdents. In contrast, MSCs were found to deposit more calcified matrix on smaller and denser arrays of microdents. MSCs on a pattern with arrays of microdents with a diameter of 9 µm and a spacing 8 µm deposited 3–4 times more calcified matrix than on a smooth surface (p<0.05). These findings show that well-defined surface microtopographies promote osteogenic activity, which can be used on implant surfaces to improve integration with the host bone tissue.     

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke.