Bone marrow cells of swine: collection and separation

Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy.     

Automatic recognition of myeloma cells in microscopic images using bottleneck algorithm,modified watershed and SVM classifier

Plasma cells are developed from B lymphocytes, a type of white blood cells that is generated in the bone marrow. The plasma cells produce antibodies to fight with bacteria and viruses and stop infection and disease. Multiple myeloma is a cancer of plasma cells that collections of abnormal plasma cells (myeloma cells) accumulate in the bone marrow. The definitive diagnosis of multiple myeloma is done by searching for myeloma cells in the bone marrow slides through a microscope. Diagnosis of myeloma cells from bone marrow smears is a subjective and time‐consuming task for pathologists. Also, because of depending on final decision on human eye and opinion, error risk in decision may occur. Sometimes, existence of infection in body causes plasma cell's increment which could be diagnosed wrongly as multiple myeloma. The computer diagnostic process will reduce the diagnostic time and also can be worked as a second opinion for pathologists. This study presents a computer‐aided diagnostic method for myeloma cells diagnosis from bone marrow smears. At first, white blood cells consist of plasma cells and other marrow cells are separated from the red blood cells and background. Then, plasma cells are detected from other marrow cells by feature extraction and series of decision rules. Finally, normal plasma cells and myeloma cells could be classified easily by a classifier. This algorithm is applied on 50 digital images that are provided from bone marrow aspiration smears. These images contain 678 cells: 132 normal plasma cells, 256 myeloma cells and 290 other types of marrow cells. Applying the computer‐aided diagnostic method for identifying myeloma cells on provided database showed a sensitivity of 96.52%; specificity of 93.04% and precision of 95.28%.     

Behavior of graft and host cells in underlying subchondral bone after transplantation of osteochondral autograft

Transplantation of osteochondral autograft is widely used as a therapeutic strategy for the defect of articular cartilage. In the repair process, although underlying subchondral bone becomes necrotic and then is followed by bone reconstruction, the fate of graft and host cells during remodeling of underlying subchondral bone has not been elucidated. The objectives of this study were to establish a method to follow graft and host cells after transplantation of osteochondral autograft, and to elucidate the fate of both graft and host cells during remodeling of underlying subchondral bone. For these purposes, autologous transplantation models employing transgenic rats and wild-type rats, which were genetically identical to each other except for transgenes, were used. Two transplantation models were designed so that either the graft or the host cells had transgenes. Model I: transgenic rats were the donor, and wild-type rats were the recipient; model II: conversely, wild-type rats were the donor, and transgenic rats were the recipient. The grafted bone marrow cells and osteocytes in the trabeculae survived in the graft at 3 weeks after transplantation. Invasion of the host bone marrow cells into the graft was also found. Thus, bone marrow cells in the host as well as both bone marrow cells and osteocytes in the graft could potentially participate in the remodeling of underlying subchondral bone. Furthermore, the interface between graft and host was consisted with both graft and host derived cells. Since new bone formation was found in this space, both graft and host cells could have the potential to contribute to remodeling of underlying subchondral bone. The two models of the transplantations using the transgenic rats were found to be beneficial in following graft cells as well as host cells and in understanding their function on healing after autologous transplantation.     

Young's modulus repeatability assessment using cycling compression loading on cancellous bone

For various applications, precision of the Young's modulus of cancellous bone specimens is needed. However, measurement variability is rarely given. The aim of this study was to assess the Young's modulus repeatability using a uniaxial cyclic compression protocol on embedded specimens of human cancellous bone. Twelve specimens from 12 human calcanei were considered. The specimens were first defatted and then 1 or 2 mm at the ends were embedded in an epoxy resin. The compression experiment consists in applying 20 compressive cycles between 0.2 per cent and 0.6 per cent strain with a 2 Hz loading frequency. The coefficient of variation of the current protocol was found to be 1.2 percent. This protocol showed variability similar to the end-cap technique (considered as a reference). It can be applied on porous specimen (especially human bone) and requires minimal bone length to limit end-artifact variability. The current method could be applied in association with noninvasive measurements (such as ultrasound) with full compatibility. This possibility opens the way for bone damage follow-up based on Young's modulus monitoring.     

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry,PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis. Microsc. Res. Tech. 73:1067–1071, 2010. © 2010 Wiley‐Liss, Inc.     

Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells

An intense network of nerve fibers can be demonstrated in skeletal tissues, not only in the periosteum but also within cortical bone, growth plate, and bone marrow. This neuro-osteogenic network expresses a restricted number of signalling molecules, including neuropeptides, neurotransmitters, and neurotrophins. Several lines of evidence indicate that receptors for these molecules are present on bone cells and that activation of these receptors leads to changes in bone cell activities. In addition, deletion of signalling molecules has been shown to alter bone metabolism. In the present review, these studies are summarized with a focus on distribution and effects of vasoactive intestinal peptide.     

Hematopoietic progenitor constituents and adherent cell progenitor morphology isolated from black-rumped agouti (Dasyprocta prymnolopha, Wagler 1831) bone marrow

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black‐rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10⁷ cells (ACP). Thirteen 25‐cm² culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10⁶ cells per milliliter, and placed in liquid nitrogen at ?196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation

A potential strategy to enhance ligament healing by transplantation of mesenchymal stem cells (MSCs), which are demonstrated to differentiate into fibroblast-like cells in vitro, is presented. The objective of this study was to follow transplanted nucleated cells from bone marrow, which contain MSCs, in the healing medial collateral ligament (MCL) over time, and to examine their phenotype and survivability. It was hypothesized that MSCs in nucleated cells from bone marrow would differentiate into fibroblast-like cells in the healing ligament following adaptation to the environment. The transplantation model employed in this study eliminates the immune response to a donor by the recipient using a transgenic rat (donor), which does not produce foreign protein from transgenes, and its wild-type rat (recipient) in order to simulate autologous transplantation. The MCL of the wild-type rat was ruptured, where 1 x 10(6) nucleated cells of bone marrow from the transgenic rat were injected. The transgenes in transplanted nucleated cells were detected throughout the healing MCL for 28 days by in situ hybridization. At 3 days, many donor cells were evident in the injury site and fascial pocket, and some were found in the midsubstance. Morphologically, transplanted cells with elongated nuclei were found at the ruptured edge of the midsubstance and surface of the unruptured site after 3 days. At 28 days, these cells continued to survive in the healing MCL. Their shapes were similar to those of surrounding recipient MCL fibroblasts. Thus, transplanted cells might differentiate into fibroblasts. Therefore, it was demonstrated that there is a potential for nucleated cells from bone marrow to serve as a vehicle for therapeutic molecules as well as to be a source in enhancing healing of ligaments.     

Morphometric studies on mouse bone using a computer-based image-analysis system

The morphological structure of the ilium, femur, third lumbar vetrebra and a central caudal vertebra of the female CBA mouse has been studied using 5 μm thick, plastic embedded, transverse and longitudinal sections. The sections were analysed on a Quantimet 720, system 30, image analyser connected on-line to a PDP11 computer. Separate endosteal and periosteal surface to volume ratios were calculated for each position of sampling in each bone. For this calculation the anisotropy of the bone was estimated from measurements of mean chord lengths in longitudinal sections of the bone using a new analytical technique. Chord length distributions in transverse sections of bone were also measured and the relevance of such measurements to the study of morphological changes in the bone and its included marrow are briefly discussed.     

Alkaline hydrolysis/methylation-acetylation: a new technique for ultrastructural DNA cytochemistry

A new technique for the visualization of DNA-containing structures in electron microscopy is described. Samples of glutaraldehyde-fixed bone marrow from rats were subjected to alkaline hydrolysis to remove RNA and the phosphate of phospho-proteins, followed by a combined blockage of protein carboxyl and amino groups through methylation-acetylation. After uranyl acetate staining of epoxy-embedded ultrathin sections, chromatin from all cell types showed a highly selective and intense electron opacity. Staining methods for DNA were also positive in semithin sections. This simple procedure could be very useful in ultrastructural cytochemistry of DNA and chromatin.     

Rheology of bovine bone marrow

The viscosity of bovine bone marrow was measured using samples taken from proximal and distal sites of five radii. The viscosity was found to be independent of shear rate and temperature above 37 degrees C (distal samples) and 42 degrees C (proximal samples). The viscosity of all samples fell to a lower limit of 0.04 Pas above these temperatures, irrespective of the treatments used. Below them the measured viscosity of the proximal marrow was some ten times that of the distal marrow at 35 degrees C and some 15 times that of the distal marrow at 30 degrees C. Below 30 degrees C the proximal marrow solidified. Distal marrow remained liquid to below 20 degrees C.     

Simultaneous 3D visualization and quantification of murine bone and bone vasculature using micro-computed tomography and vascular replica

Recent evidence suggests a close functional relationship between osteogenesis and angiogenesis as well as between bone remodeling and bone vascularization. Consequently, there is a need for visual inspection and quantitative analysis of the bone vasculature. We therefore adapted and implemented two different vascular corrosion casting (VCC) protocols using a polyurethane-based casting resin in mice for a true three-dimensional (3D), direct, and simultaneous measurement of bone tissue and vascular morphology by micro-computed tomography (μCT). For assessment of vascular replicas at the level of capillaries, a vascular contrast perfusion (VCP) protocol was devised using a contrast modality based on a barium sulfate suspension in conjunction with synchrotron radiation (SR) μCT. The vascular morphology quantified using the VCP protocol was compared quantitatively with the results of a previously established method, where the vascular network of cortical bone was derived indirectly from cortical porosity. The presented VCC and VCP protocols have the potential of serving as a valuable method for concomitant 3D quantitative morphometry of the bone tissue and its vasculature. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat

We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.     

Microwave exposure increases bone demineralization rate independent of temperature

There is a long‐standing controversy regarding an effect of microwaves, independent of increasing temperature, on the rate of bone demineralization. In this study, we exposed standardized samples of gerbil femur to constant microwave exposure while maintaining the demineralizing solution (ethylenediamine tetraacetic acid, EDTA) at 20 °C. Random samples were selected at 3 h intervals, embedded in plastic and sectioned for histological evaluation to determine the extent of demineralization. The time to complete demineralization was significantly faster with microwave exposure (33 h) compared to non‐exposure on a tissue rotator (45 h) in a limited amount (5 mL/24 h) of EDTA. The presence of bone marrow was a significant barrier to the rate of demineralization and resulted in an asymmetrical pattern of mineral extraction. Samples without bone marrow were completely demineralized after 21 h of exposure to microwaves and EDTA. Additional comparisons were made between samples exposed to an effectively infinite supply of demineralizing agent (bone marrow intact). There was a significant increase in rate with unlimited demineralizing agent with (24 h) or without (30 h) microwaves when compared to tissue demineralized on the rotator. Our results establish a positive effect of microwaves on the rate of bone demineralization which is independent of temperature.     

Cytogenetic profiling by FISH microscopy and comparison with light microscopy and complete blood count findings in myelodysplastic syndrome

Karyotype, bone marrow blast percentage and cytopenia influence the prognosis of myelodysplastic syndrome. We studied the abnormalities detected by fluorescence in situ hybridization (FISH) in myelodysplastic syndrome and associated haematological profile with abnormalities detected by FISH. Complete blood counts, peripheral blood and bone marrow of patients were evaluated for cytopenia, dysplasia and blasts. FISH probes were used to detect del(5q), gain of chromosome 8, de (7q/‐7) and del(20 q). Multiple regression analysis was used to study the association of FISH abnormalities, age and sex with haematological profile. Mc Nemar's test studied the relationship between FISH abnormalities and dysplastic features in bone marrow. Cytogenetic abnormalities were detected by FISH in 25.7% of patients. Del(20 q) was seen in 14.2% of patients. FISH was able to predict changes in peripheral blood blast count by 80% (p ? 0.0001). Cytogenetic abnormalities were not seen in 74.2% of patients. Groups with FISH abnormalities have a different haematological profile, and these abnormalities have a significant effect on blast percentage.     

Computer aided detection and classification of acute lymphoblastic leukemia cell subtypes based on microscopic image analysis

Acute lymphoblastic leukemia (ALL) is a cancer that starts from the early version of white blood cells called lymphocytes in the bone marrow. It can spread to different parts of the body rapidly and if not treated, would probably be deadly within a couple of months. Leukemia cells are categorized into three types of L1, L2, and L3. The cancer is detected through screening of blood and bone marrow smears by pathologists. But manual examination of blood samples is a time‐consuming and boring procedure as well as limited by human error risks. So to overcome these limitations a computer‐aided detection system, capable of discriminating cancer from noncancer cases and identifying the cancerous cell subtypes, seems to be necessary. In this article an automatic detection method is proposed; first cell nucleus is segmented by fuzzy c‐means clustering algorithm. Then a rich set of features including geometric, first‐ and second‐order statistical features are obtained from the nucleus. A principal component analysis is used to reduce feature matrix dimensionality. Finally, an ensemble of SVM classifiers with different kernels and parameters is applied to classify cells into four groups, that is noncancerous, L1, L2, and L3. Results show that the proposed method can be used as an assistive diagnostic tool in laboratories.     

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.     

Effects of filgrastim on granulopoietic cells of mice pretreated with methotrexate.

We have evaluated the effect of filgrastim on proliferation and differentiation activity of granulopoietic cells in mice pretreated with methotrexate. Filgrastim was injected daily, from day 8 to 28 after cytotoxic agent administration. The granulopoiesis changes were measured by assessment of GM-CFU cells content, marrow and spleen granuloid cells pool as well as circulating neutrophils. In MTX pretreated mice, bone marrow GM-CFU oscillating values were higher than normal values, but these changes were not followed by high proliferative activity in granuloid precursor cell compartment. After MTX treatment, filgrastim administration was unable to stimulate marrow granulopoiesis as observed in normal mice. In the spleen, MTX led to dramatic changes in the proliferative activity of GM-CFU cells, but did not result in spleen granuloid cell changes. However, filgrastim treatment induced a spleen granuloid amplification, similar to the changes observed in circulating neutrophils values. We suggest that these findings can be explained by inhibition of differentiation of marrow GM-CFU cells into the more mature granulopoietic cells and/or by an inhibited proliferative activity of marrow granuloid cells. They can be also explained in terms of an unfavorable marrow microenvironment for granulopoiesis, contrary to a supportive spleen microenvironment.     

Three-dimensional metacarpophalangeal joint kinematics using two markers on the phalanx

A protocol for analysing three-dimensional metacarpophalangeal (MCP) joint motion in vivo using two markers on the proximal phalanx is described. The analysis uses an assumption that the rotation of the phalanx about its own long axis is zero. In an experimental study 24 volunteers had surface markers applied to the dorsal surfaces of their hands and index and long finger proximal phalanges, with three-dimensional marker positions recorded in two hand and finger postures in an incomplete box design using a test-retest protocol. Kinematic parameters from the optoelectronic system were compared with those obtained from three-dimensional reconstruction of bone landmarks and of the marker positions identified on stereoradiographs. Pronation/supination angles obtained from bone landmarks showed high test-retest variability, reflecting the difficulty in obtaining reliable pronation/supination data in small bones without the use of implanted markers. Changes in MCP joint extension and deviation angles determined using two surface markers agree with those obtained from bone landmarks. The results indicate a reproducible protocol for tracking MCP joint motion using only two phalangeal markers, suggesting that the 'no-rotation assumption' can be applied without affecting measures of extension and deviation motion in the normal joint.