Long-term cultures of chicken bone marrow cells

We report an adaptation to cultures of chicken bone marrow cells of the Dexter culture technique for obtaining long-term hemopoiesis in vitro. Cells were seeded in DMEM supplemented with fetal calf serum (20%) and hydrocortisone (10(-6) M) with or without chicken serum (1%). Cultures were incubated at 37 degrees C and fed every 2 weeks. An adherent cell layer composed of macrophages, fibroblasts, and adipocytes became established, over which hemopoietic cells formed foci and were released into the supernatant. Granulocytes and monocytes-macrophages differentiated in a constant proportion until Week 6, whereafter differentiation became progressively restricted to the monocytic lineage. As demonstrated by the generation of colony-forming cells, hemopoiesis was maintained for either 12 or 28 weeks.     

Fine structure of the bone marrow of the chicken and pigeon

The structure of bone marrow from chickens and pigeons was studied with light and electron microscopy. Erythropoiesis occurs in the lumen of the medullary sinuses. Immature erythroid cells appear to adhere to the sinus wall and may thus be prevented from entering the peripheral circulation. The wall of the medullary sinuses is formed by elongated lining cells, lacking a basement membrane, which are continous except at sites where blood cells are passing through them. When viewed with the electron microscope, developing heterophil myelocytes, which occur only in the extravascular spaces, possess two populations of granules; one type is globular in content, the other is fibrillar in content. The globular type predominates during all stages of development and appears to be the specific granule. Specific granules originate from material which is formed in the Golgi complex, pinches off, and accumulates in expanded vesicles. The origin of the material in the fibrillar granules was not determined. Like the globular granules of heterophil leucocytes, granules of eosinophil leucocytes arise from material which is formed in the Golgi complex.     

Lipid composition of human bone marrow

1. A modified method for the analysis of phospholipid mixtures by selective hydrolysis is described. 2. The phospholipid compositions of normal human bone marrow and of the bone marrows of patients who died with anaemia or various forms of leukaemia were investigated. 3. Phospholipids from normal bone marrow comprised about 44% of lecithin, 4% of choline plasmalogen, 7% of glyceryl ether phospholipid (choline base), 10% of sphingomyelin, 22% of phosphatidylethanolamine plus phosphatidylserine, 8% of ethanolamine plasmalogen and 5% of glyceryl ether phospholipid (ethanolamine base). 4. The proportion of kephalin (i.e. phosphatidylethanolamine plus phosphatidylserine) in the pathological bone marrows tended to be lower than normal. No other consistent differences were observed between the normal and pathological samples. 4. A ceramide dihexoside was isolated from normal bone marrow.     

Cellular isolation, culture and characterization of the marrow sac cells in human tubular bone

The goal of this study is to characterize the epithelioid-like human marrow sac cells that separate the myeloid and osteoblast populations in situ and to determine if they express osteoblast cytoplasmic markers. Tubular segments of femoral diaphyseal bone were obtained from healthy young (4-8 yr) male and female patients undergoing femoral shortening surgeries. The interface between bone and marrow was examined by scanning (SEM) and transmission electron microscopy (TEM). The marrow sac cells were isolated and cultured in a-MEM medium with and without dexamethasone, glycerophosphate, and ascorbic acid [DGPA]. Alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2) and osteocalcin were evaluated. In the SEM, the marrow sac presented a distinctive pattern of large overlapping cells. TEM studies showed that marrow sac was one or two cells thick, which were attenuated with elongated nuclei, few cellular organelles, and appeared to display intercellular gap junctions. In culture, the marrow sac cells stained positively for ALP and BMP-2, and their expression was enhanced two- to three- fold when the cells were grown in DGPA. DGPA did not enhance osteocalcin expression. The cells of the human marrow sac reside proximate to endosteal osteoblasts and express osteoblastic markers. It is possible that these stromal cells constitute an osteoprogenitor pool from which replacement osteoblasts are recruited, and that they are involved in normal bone formation and in bone diseases (e.g., osteoporosis and osteopenia).     

Post-translational processing of chicken bone phosphoproteins. Identification of the bone phosphoproteins of embryonic tibia.

After periodate oxidation and incubation with a dihydrazide, cross-linking of the two heavy chains of immunoglobulins G from several species proceeds specifically through their oligosaccharides. We have used malonic acid dihydrazide, adipic acid dihydrazide and dithiodipropionic acid dihydrazide. The last compound is introduced in this work as a cleavable-carbohydrate-specific cross-linker. It was found that in rabbit and human immunoglobulins the degree of cross-linking was strongly dependent on the oxidation conditions but only very weakly dependent on the concentration and size of the dihydrazides. Papain cleavage of the cross-linked rabbit IgG indicated that the cross-linking occurred predominantly, if not exclusively, in the Fc region, probably through the two glycans linked to Asn-297 in the CH2 domain of each of the two heavy chains. The immunoglobulins from sheep, pig, goat and guinea pig show a comparable cross-linking pattern, indicating that the sugar chains from these immunoglobulins have a spatial structure closely related to that of rabbit and human IgG. When dithiodipropionic acid dihydrazide was used as the cross-linker, the cross-link could be cleaved by mercaptoethanol.     

Curvature, length, and cross-sectional geometry of the femur and humerus in anthropoid primates

The aims of this study were to describe the curvature of anthropoid limb bones quantitatively, to determine how limb bone curvature scales with body mass, and to discuss how bone curvature influences static measures of bone strength. Femora and humeri in six anthropoid genera of Old World monkeys, New World monkeys, and gibbons were used. Bone length, curvature, and cross-sectional properties were incorporated into the analysis. These variables were obtained by a new method using three-dimensional morphological data reconstructed from consecutive CT images. This method revealed the patterns of curvature of anthropoid limb bones. Log-transformed scaling analyses of the characters revealed that bone length and especially bone curvature strongly reflected taxonomic/locomotor differences. As compared with Old World monkeys, New World monkeys and gibbons in particular have a proportionally long and less curved femur and humerus relative to body mass. It is also revealed that the section modulus relative to body mass varies less between taxonomic/locomotor groups in anthropoids. Calculation of theoretical bending strengths implied that Old World monkeys achieve near-constant bending strength in accordance with the tendency observed in general terrestrial mammals. Relatively shorter bone length and larger A-P curvature of Old World monkeys largely contribute to this uniformity. Bending strengths in New World monkeys and gibbons were, however, a little lower under lateral loading and extremely stronger and more variable under axial loading as compared with Old World monkeys, due to their relative elongated and weakly curved femora and humeri. These results suggest that arboreal locomotion, including quadrupedalism and suspension, requires functional demands quite dissimilar to those required in terrestrial quadrupedalism.     

Altered architecture and cell populations affect bone marrow mechanobiology in the osteoporotic human femur

Age-related increases in trabecular bone porosity, as seen in osteoporosis, not only affect the strength and stiffness, but also potentially the mechanobiological response of bone. The mechanical interaction between trabecular bone and bone marrow is one source of mechanobiological signaling, as many cell populations in marrow are mechanosensitive. However, measuring the mechanics of this interaction is difficult, due to the length scales and geometric complexity of trabecular bone. In this study, a multi-scale computational scheme incorporating high-resolution, tissue-level, fluid–structure interaction simulations with discrete cell-level models was applied to characterize the potential effects of trabecular porosity and marrow composition on marrow mechanobiology in human femoral bone. First, four tissue-level models with different volume fractions (BV/TV) were subjected to cyclic compression to determine the continuum level shear stress in the marrow. The calculated stress was applied to three detailed models incorporating individual cells and having differing adipocyte fractions. At the tissue level, compression of the bone along its principal mechanical axis induced shear stress in the marrow ranging from 2.0 to 5.6 Pa, which increased with bone volume fraction and strain rate. The shear stress was amplified at the cell level, with over 90% of non-adipocyte cells experiencing higher shear stress than the applied tissue-level stress. The maximum shear stress decreased by 20% when the adipocyte volume fraction (AVF) increased from 30%, as seen in young healthy marrow, to 45 or 60% AVF typically found in osteoporotic patients. The results suggest that increasing AVF has similar effects on the mechanobiological signaling in bone marrow as decreased volume fraction.     

Cellular and molecular mechanisms underlying bone marrow and liver fibrosis: a review

Chronic fibroproliferative diseases are an important cause of morbidity and mortality in the world. Fibrotic diseases occur in a large variety of vital organs, and the process of fibrosis seems common to all tissues. In all of fibrotic reactions, the underlying cellular and molecular mechanisms involve leukocyte infiltration, the persistence of inflammation in the tissue, and the proliferation of cells with a myofibroblast phenotype. The different cell types participating to this process sustain production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements that progressively destroy and remodel normal tissue architecture. This review focuses on the comparison of two, major, chronic fibroproliferative diseases: the myelofibrosis which develops in bone marrow, a "fluid" tissue producing circulating haematopoietic cells, and liver fibrosis, which demonstrates all the features of solid tissue damage. We discuss the etiology and histological quantification of each type of fibrosis, the implication of cell partners, cytokines and growth factors, animal models developed to study fibrosis, and antifibrotic therapies for each of these two fibroproliferative disease models.     

Immunoglobulin-positive cells from the gland of harder and bone marrow of the chicken

Cells were collected from the gland of Harder (GH) and bone marrow (BM) of 14-, 21- and 32-week-old birds and were incubated with an ¹²⁵I-labeled rabbit anti-chicken Ig (IgG and IgM) serum. At 14 weeks of age the percentage of Ig⁺ small lymphocytes (SL) in the GH and BM was similar. However, by 21 weeks of age Ig⁺ SL in BM had increased to approximately 19% of total lymphocytes while the Ig⁺ SL in the GH represented less than 1.7% of the lymphocyte pool. A marked drop in the number of Ig⁺ SL in BM occurred by 32 weeks of age. These data suggest that either the BM may be dependent on the bursa for maintenance of its Ig⁺ SL or it is unable to produce in situ or maintain Ig⁺ SL with age. In the GH the predominant cell was the plasma cell (PC). Labeled PC (> 20 grains) exceeded 80% of the total PC pool in the GH. These data contrast with the apparent deficiency of Ig receptors on murine PC. The maintenance of a large number of PC in the GH without the presence of Ig⁺ SL illustrates the uniqueness of this gland.     

Structural allometry of the femur and tibia in Hominoidea and Macaca

Allometric scaling relationships between body weight, bone length, and cross-sectional dimensions of the lower limb bones which measure structural strength and rigidity (area, second moments of area) are investigated in Homo, Gorilla, Pan, Pongo, and Macaca. Cross-sectional dimensions are slightly positively allometric and highly correlated with body weight; within-bone proportional differences are largely a result of differences in relative bone length to body weight. Orangutans show the greatest deviation from general scaling relationships between lower limb bone structural strength and weight, probably due to habitual upper limb suspension. Formulas for the prediction of weight from cross-sectional dimensions are presented.     

Isolation and characterization of mesenchymal stem cells from chicken bone marrow

The bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells, which can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit and other mammals, but rarely in poultry. So, in this study, Thirty- to sixty-day old chicken was chosen as experimental animal, to isolate and characterize BMSCs from them. To investigate the biological characteristics of chicken BMSCs, immunofluorescence and RT-PCR were used to detect the characteristic surface markers of BMSCs. Growth curves were drawn in accordance with cell numbers. To assess the differentiation capacity of the BMSCs, cells were induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The surface markers of BMSCs, CD29, CD44, CD31, CD34, CD71 and CD73, were detected by immunofluorescence and RT-PCR assays. The growth curves of different passages were all typically sigmoidal. Karyotype analysis showed that these in vitro cultured cells were genetically stable. In addition, BMSCs were successfully induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The results suggest that the BMSCs isolated from chicken possess similar biological characteristics with those separated from other species, and their multi-lineage differentiation potentiality herald a probable application for cellular transplant therapy in tissue engineering.     

Familial comparison of bone mineral density at the proximal femur and lumbar spine

Familial resemblance of bone mineral density (BMD) was studied in the lumbar spine and three regions of the proximal femur in 41 biological mother-daughter (M-D), 42 mother-son (M-S), 24 mother-grandmother (M-G) pairs and 18 mother-grandmother-daughter (M-G-D) triads. Children were placed into three maturity categories based on an assessment of secondary sex characteristics and growth velocities. Two sets of standardized BMD Z-scores were derived for the children based on either their chronological age or their maturational status. These scores were compared with maternal Z-scores derived from age-specific norms. Similar comparisons were made between the Z-scores of the mothers and grandmothers. For all three regions of the proximal femur and for the total AP lumbar spine the correlations between Z-score values were similar and significant (P < 0.05) between the M-G and M-D pairs ranging from 0.41 to 0.57. In general, the familial correlations improved when maturity-status based Z-scores were used for comparison. The absolute BMD values measured in the grandmothers and the three maturity groups of the children — expressed as a percentage of the BMD of the mothers — showed that at the neck and the trochanteric regions of the proximal femur the late-pubescent girls and boys had a significantly (P < 0.05) greater bone density than their mothers (115–123%), whereas at the AP spine these groups averaged only 88% of their mothers BMD. This site differential was not apparent when comparing the post-menopausal grandmothers with the pre-menopausal mothers (80% at both sites). Three generation comparisons demonstrated a strong familial resemblance in bone mineral density. The value of incorporating maturity-based versus chronological-based parameters for comparison with adult measures in studies that involve growing children at different stages of development was also demonstrated.     

Isolation of a cDNA clone encoding the RNase-superfamily-related gene highly expressed in chicken bone marrow cells.

The RNase gene superfamily combines functionally divergent proteins which share statistically significant sequence similarity. Known members assigned to this family include secretory and nonsecretory RNases; angiogenin; eosinophil cationic protein; eosinophil-derived neurotoxin; sialic-acid binding lectin and anti-tumor protein P-30. We report the cDNA cloning of the chicken RNase Super Family Related (RSFR) gene that is specifically overexpressed in normal bone marrow cells and bone marrow-derived AMV transformed monoblasts. It codes for a 139 amino acid protein with a putative signal peptide and remarkable conservation of active-site residues, other residues known to be important for substrate binding and catalytic activity and half-cystine residues common for all RNase family members. Phylogenetic tree analysis shows that RSFR defines a new group of genes within the family. We also conclude that an amino acid sequence block CKXXNTF(X) 11C is a "shortest RNase superfamily signature" which is both necessary and sufficient to identify all previously recognized family members as well as chicken RSFR.     

Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.