Mechanical behavior of a new biphasic calcium phosphate bone graft

The aim of this study was to create a new porous calcium phosphate implant for use as a synthetic bone graft substitute. Porous bioceramic was fabricated using a foam-casting method. By using polyurethane foam and a slurry containing hydroxyapatite-dicalcium phosphate powder, water, and additives, a highly porous structure (66 ± 5%) was created. The porous specimens possess an elastic modulus of 330 ± 32 MPa and a compressive strength of 10.3 ± 1.7 MPa. The X-ray diffraction patterns show hydroxyapatite and beta-pyrophosphate phases after sintering. A rabbit model was developed to evaluate the compressive strength and elastic modulus of cancellous bone defects treated with these porous synthetic implants. The compressive mechanical properties became weaker until the second month post implantation. After the second month, these properties increased slightly and remained higher than control values. New bone formed on the outside surface and on the macropore walls of the specimens, as osteoids and osteoclasts were evident two months postoperatively. Considering these properties, these synthetic porous calcium phosphate implants could be applicable as cancellous bone substitutes.     

A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.

A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

The frequency of bone marrow cells that bind erythropoietin

The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was, in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.     

The construction of an in vitro three-dimensional hematopoietic microenvironment for mouse bone marrow cells employing porous carriers

Spatial development of mouse bone marrow cellsemploying porous carriers was investigated in order todesign a bioreactor with a three-dimensionalhematopoietic microenvironment. Three types of porouscarriers were used for examining the spatialdevelopment of anchorage-dependent primary stromalcells as feeder cells. Stromal cells were found tospread well at a high density on a polyester nonwovendisc carrier (Fibra cel (FC)) under a scanningelectron microscope, while cells on porous cellulosebeads (Microcube (MC), 500 m pore diameter)spread at a low density; cells on another type ofcellulose porous beads (CPB, 100 m pore diameter)were globular. Mouse bone marrow cells wereinoculated to dishes containing three types of porouscarriers which shared more than 30% of the bottomsurface in a dish. The concentration of stromal cellsin the well containing FC was lower than that on theother two carriers. However, the weekly output oftotal hematopoietic cell (suspension cells) increasedbetween day 21 and 28 in the culture using FC while itdecreased monotonously in the cultures by use of theother two carriers. The proportion of progenitorcells (BFU-E, CFU-GM) in the total hematopoietic cellpopulation, after showing an initial decrease,increased after 1 week in the culture using FC whilethe proportion decreased monotonously to zero in thecultures using MC and CPB.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

Combined computational study of mechanical behaviour and drug delivery from a porous, hydroxyapatite-based bone graft

This paper presents a numerical model of a porous, hydroxyapatite-based bone graft also suitable as a drug delivery device. The graft was positioned in different sites and with different porosities inside a human femur model. The structural analyses were carried out to verify the graft mechanical strength, using the Tsai–Wu criterion, and the maximum porosity at which static failure does not occur. A local stress shielding risk was also calculated as the ratio between the bone stress in the intact condition and the stress after implantation of the graft. Drug release kinetics was calculated by means of the finite element method. High porosity grafts were found to fail in all implantation sites. Lower porosity grafts showed to have adequate strength if implanted in some positions, while provided insufficient resistance for other implantation sites. Drug release kinetics was found to be strongly dependent both on the porosity of the graft and the bone density near the bone-graft interface.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Modulation of one of three murine bone marrow stromal cell lines to adipose cells by serum and insulin

Adipose cells have been recognized as an integral component of the bone marrow hematopoietic microenvironment in vivo and as an essential cell type required for in vitro maintenance of stem cells. Four stromal cell lines obtained from the adherent cell population of murine bone marrow cultures have been enriched and purified by multiple trypsinizations. We noted that these cell lines exhibited an accumulation of vacuoles of lipid, the extent of which varied be-tween cell lines in response to a change from medium containing 10% fetal calf serum to medium containing 20% horse serum. The lipid was lost when the cell lines were transferred back into the medium supplemented with fetal calf serum. In light of the reported lipogenic and antilipolytic effects of insulin on fibroblasts and adipocytes, we investigated the ability of insulin to induce adipocyte transformation of these bone marrow stromal cell populations. Three cell lines were exposed to bovine insulin at concentrations ranging from 10^?9 to 10^?6 M. All three cell lines responded to the insulin by accumulating lipid, but the extent of accumulation and the insulin concentration at which maximum lipid content was attained were population specific. One cell line (MC₁) responded fully at physiological levels of insulin (10^?9 M), whereas the other two showed lipid accumulation only at pharmacological concentrations. The initial growth of MC₁ was inhibited in the presence of 10^?9 M insulin which is compatible with the observed differentiation to adipocytes. The growth of MC₃ was unaltered in the presence of physiological concentrations of insulin, whereas that of MC₄ was accelerated. Grafts of organ cultures of the cell lines under the kidney capsule of syngeneic mice developed specific characteristics rep-resentative of the different cell lines. In particular, the majority of the grafts of MC₁ consisted primarily of fat cells which were not observed in the grafts of MC₃ and MC₄. These data strongly suggest that these cell lines comprise cells with different potentialities and that the MC₁ line represents a preadipocyte stromal cell of bone marrow.     

Differentiation of embryonic stem cells in adult bone marrow

Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as "seed" cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

NK cells in allogeneic bone marrow transplantation

NK cells, until recently an ignored subset of lymphocytes, have begun to emerge as important cytotoxic effectors. It is now accepted that NK cells together with T cells constitute major actors in graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Over the last several years the mechanisms regulating the activation of NK cells have been the subject of intense investigations encouraged by the clinical implications that these studies will have. This article provides a general overview of NK-cells biology and regulation pertinent to their function in allogeneic BMT, followed by a review of the in vivo preclinical and clinical evidence for the beneficial effect of NK cells in the adoptive immunotherapy of leukemia.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Selective intra-lysosomal concentration of niobium in kidney and bone marrow cells: a microanalytical study

Niobium is used as an alloy in the industrial and biomedical fields. The concentration of the toxic element in organs of a number of animal species has been defined by using radioactive niobium (⁹⁵Nb). However, tissue lesions induced by niobium have only been studied at the light microscopy level. In this study, we used an electron probe X-ray analyzer equipped with a transmission electron microscope to define the localization of this element in kidney and bone marrow cells. Results demonstrated that niobium is located in the lysosome and that this element coprecipitates with phosphate. In kidney, lysosomes and precipitates are eliminated in the tubular lumen. In contrast, precipitates appear to be eliminated more slowly from the lysosomes of bone marrow macrophages. These processes therefore correspond to one of the mechanisms by which lysosomes eliminate certain toxic mineral elements and thus play a role in the more general process of the body's defenses.     

Unbiased selection of bone marrow derived cells as carriers for cancer gene therapy

BACKGROUND: There is currently great interest in development of cell-based carriers for delivery of viral vectors to metastatic tumors. To date, several cell carriers have been tested based largely upon their predicted tumor-localizing properties. However, cell types may exist which can be mobilized from the circulation by a tumor which have not yet been identified. Here we use an unbiased screen of bone marrow (BM) cells to identify cells which localize to tumors and which might serve as effective candidate cell carriers without any prior prediction or selection. METHODS: Unsorted BM cells from green fluorescent protein (GFP)-transgenic donor mice were adoptively transferred into C57Bl/6 mice bearing pre-established subcutaneous B16 melanoma tumors. Forty-eight hours and eight days later, tumors, organs and blood were analyzed for GFP-expressing cells by flow cytometry. The phenotype of GFP cells in organs was determined by co-staining with specific cell surface markers. RESULTS: CD45(+) hematopoietic cells were readily detected in tumor, spleen, bone marrow, blood and lung at both time points. Within these CD45(+) cell populations, preferential accumulation in the tumor was observed of cells expressing Sca-1, c-kit, NK1.1, Thy1.2, CD14, Mac-3 and/or CD11c. Lymphodepletion increased homing to spleen and bone marrow, but not to tumors. CONCLUSIONS: We have used an in vivo screen to identify populations of BM-derived donor cells which accumulate within tumors. These studies will direct rational selection of specific cell types which can be tested in standardized assays of cell carrier efficiency for the treatment of metastatic tumors.     

Segmental bone replacement via patient‐specific,three‐dimensional printed bioresorbable graft substitutes and their use as templates for the culture of mesenchymal stem cells under mechanical stimulation at various frequencies

The treatment of large segmental bone defects remains a challenge as infection, delayed union, and nonunion are common postoperative complications. A three‐dimensional printed bioresorbable and physiologically load‐sustaining graft substitute was developed to mimic native bone tissue for segmental bone repair. Fabricated from polylactic acid, this graft substitute is novel as it is readily customizable to accommodate the particular size and location of the segmental bone of the patient to be replaced. Inspired by the structure of the native bone tissue, the graft substitute exhibits a gradient in porosity and pore size in the radial direction and exhibit mechanical properties similar to those of the native bone tissue. The graft substitute can serve as a template for tissue constructs via seeding with stem cells. The biocompatibility of such templates was tested under in vitro conditions using a dynamic culture of human mesenchymal stem cells. The effects of the mechanical loading of cell‐seeded templates under in vitro conditions were assessed via subjecting the tissue constructs to 28 days of daily mechanical stimulation. The frequency of loading was found to have a significant effect on the rate of mineralization, as the alkaline phosphatase activity and calcium deposition were determined to be particularly high at the typical walking frequency of 2 Hz, suggesting that mechanical stimulation plays a significant role in facilitating the healing process of bone defects. Utilization of such patient‐specific and biocompatible graft substitutes, coupled with patient’s bone marrow cells seeded and exposed to mechanical stimulation of 2 Hz have the potential of reducing significant volumes of cadaveric tissue required, improving long‐term graft stability and incorporation, and alleviating financial burdens associated with delayed or failed fusions of long bone defects.     

Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow.

Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.     

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free‐floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP‐expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin⁺/ GFP⁺ cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen‐1 and smooth muscle α‐actin. This was observed in the injured area as well as in the surrounding not‐injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte‐derived cells, closely related with the tissue‐repairing cells known as ‘fibrocytes’ and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free‐floating coelomic cells.     

骨髓基质细胞的特征及其在细胞和基因治疗中的应用

骨髓基质细胞是一类独特的间质干细胞,可分化为多种非造血系的组织。骨髓基质细胞具有贴壁生长的特性,因而易于在体外分离和扩增;另外骨髓基质细胞可在体内外表达多种治疗性的外湖目的基因。因此,骨髓基质细胞被认为是一种理想的治疗性细胞的基因治疗中的靶细胞。本文对骨髓基质细胞的研究进展及其在细胞和基因治疗中的应用作一综述。     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.