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1.
J Vis Exp ; (124)2017 06 28.
Article in English | MEDLINE | ID: mdl-28715367

ABSTRACT

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.


Subject(s)
Flow Cytometry/methods , Microfluidics/methods , Microscopy/methods , Optical Imaging/methods , Humans
2.
J Tissue Eng Regen Med ; 10(6): 475-85, 2016 06.
Article in English | MEDLINE | ID: mdl-23950016

ABSTRACT

Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd.


Subject(s)
Cell Differentiation , Dental Pulp/metabolism , Liver/metabolism , Stem Cells/metabolism , Cells, Cultured , Dental Pulp/cytology , Humans , Liver/cytology , Stem Cells/cytology
3.
Stem Cell Reports ; 3(4): 585-93, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25358787

ABSTRACT

Huntington's disease (HD) is a dominant neurodegenerative disorder caused by the expansion of glutamine residues in the N-terminal region of the huntingtin (HTT) protein. The disease results in progressive neuronal loss, leading to motor, cognitive, and psychiatric impairment. Here, we report the establishment of neural progenitor cell (NPC) lines derived from induced pluripotent stem cells (iPSCs) of transgenic HD monkeys. Upon differentiation to neurons, HD neural cells develop cellular features of HD, including the formation of nuclear inclusions and oligomeric mutant HTT (mHTT) aggregates, as well as increased apoptosis. These phenotypes are rescued by genetic suppression of HTT and pharmacological treatment, demonstrating the ability of our HD cell model to respond to therapeutic treatment. The development and reversal of HD-associated phenotypes in neural cells from HD monkeys provides a unique nonhuman primate (NHP) model for exploring HD pathogenesis and evaluating therapeutics that could be assessed further in HD monkeys.


Subject(s)
GABAergic Neurons/cytology , Huntington Disease/pathology , Induced Pluripotent Stem Cells/cytology , Neural Stem Cells/cytology , Phenotype , Animals , Antiparkinson Agents/pharmacology , Apoptosis , Cells, Cultured , GABAergic Neurons/drug effects , GABAergic Neurons/metabolism , Haplorhini , Huntingtin Protein , Huntington Disease/genetics , Induced Pluripotent Stem Cells/metabolism , Memantine/pharmacology , Mutation , Nerve Tissue Proteins/genetics , Neural Stem Cells/metabolism , Neurogenesis
4.
BMC Cell Biol ; 12: 39, 2011 Sep 12.
Article in English | MEDLINE | ID: mdl-21910887

ABSTRACT

BACKGROUND: Dental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntington's disease (HD), have not been fully investigated. RESULTS: To examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile. CONCLUSIONS: Our results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.


Subject(s)
Adult Stem Cells/metabolism , Huntington Disease/therapy , Serotonin Plasma Membrane Transport Proteins/metabolism , Stem Cell Transplantation , Stromal Cells/metabolism , Adult Stem Cells/pathology , Animals , Animals, Genetically Modified , Cell Survival/genetics , Cells, Cultured , Dental Pulp/pathology , Disease Models, Animal , Haplorhini , Humans , Huntington Disease/genetics , Huntington Disease/pathology , Mutation/genetics , Serotonin Plasma Membrane Transport Proteins/genetics , Stromal Cells/pathology , Transgenes/genetics
5.
Histol Histopathol ; 26(3): 377-94, 2011 03.
Article in English | MEDLINE | ID: mdl-21210351

ABSTRACT

Osteoarthritis (OA) is the most common form of joint disease. Histopathologically, OA is characterized by a progressive loss of articular cartilage, osteophyte formation, thickening of subchondral bone, and subchondral cyst formation. All current therapies are aimed at symptomatic control and have limited impacts on impeding or reversing the histopathologic progression to advanced OA. Previous studies have shown that overexpression of matrix-degrading proteinases and proinflammatory cytokines is associated with osteoarthritic cartilage degradation. However, clinical trials applying an inhibitor of proteinases or proinflammatory cytokines have been unsuccessful. A more sophisticated understanding of the regulatory mechanisms that control the function of articular chondrocytes is paramount to developing effective treatments. Since multiple catabolic factors and pathological chondrocyte hypertrophy are involved in the development of OA, it is important to identify which upstream factors regulate the expression of catabolic molecules and/or chondrocyte hypertrophy in articular cartilage. This review summarizes the current studies on the molecular regulation, with a main focus on transcriptional regulation, of the function of adult articular chondrocytes and its significance in the pathogenesis and treatment of OA. Recent studies have discovered that transcription factor Nfat1 may play an important role in maintaining the physiological function of adult articular chondrocytes. Nfat1-deficient mice exhibit normal skeletal development but display most of the features of human OA as adults, including chondrocyte hypertrophy with overexpression of specific matrix-degrading proteinases and proinflammatory cytokines in adult articular cartilage. ß-catenin transcriptional signaling in articular chondrocytes may also be involved in the pathogenesis of OA. Activation of ß-catenin leads to OA-like phenotypes with overexpression of specific matrix-degrading proteinases in articular cartilage of adult mice. These and other regulatory mechanisms described in this review may provide new insights into the pathogenesis of OA and the development of novel therapeutic targets for the treatment of OA.


Subject(s)
Cartilage, Articular/pathology , Chondrocytes/pathology , Osteoarthritis/pathology , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthroscopy , Cartilage, Articular/metabolism , Cartilage, Articular/physiology , Cartilage, Articular/surgery , Cell Differentiation/physiology , Chondrocytes/drug effects , Chondrocytes/metabolism , Humans , Injections, Intra-Articular , Joints/growth & development , Joints/pathology , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Osteoarthritis/surgery
6.
Connect Tissue Res ; 44 Suppl 1: 312-7, 2003.
Article in English | MEDLINE | ID: mdl-12952215

ABSTRACT

The development of new bone formation strategies offers tremendous therapeutic implications in a variety of musculoskeletal diseases. One approach involves harnessing the regenerative capacity of osteoprogenitor bone cells in combination with biomimetic scaffolds generated from appropriate scaffold matrices and osteoinductive factors. The aims of our study were to test the efficacy of two innovative osteoinductive agents: the osteoblast stimulating factor-1 (osf-1), an extracellular matrix-associated protein, and osteoinductive extracts of Saos-2 cells on human osteoprogenitor cells. Saos-2 extracted osteoinductive factors significantly stimulated alkaline phosphatase specific activity in basal and osteogenic conditions. Osf-1 significantly stimulated chemotaxis, total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase specific activity at concentrations as low as 10 pg/ml compared with control cultures. Osteoinductive factors present in Saos-2 cell extracts and osf-1 promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on 3-D scaffolds. The successful generation of 3-D biomimetic structures incorporating osf-1 or osteoinductive factors from Saos-2 cells indicates their potential for de novo bone formation that exploits cell-matrix interactions.


Subject(s)
Biocompatible Materials , Carrier Proteins/pharmacology , Culture Media, Conditioned/pharmacology , Cytokines/pharmacology , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Aged , Aged, 80 and over , Alkaline Phosphatase/metabolism , Bone Marrow Cells , Calcification, Physiologic/drug effects , Calcification, Physiologic/physiology , Cell Division/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Female , Humans , Immunohistochemistry , Lactic Acid , Male , Mesenchymal Stem Cells/enzymology , Middle Aged , Osteogenesis/physiology , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , Polymers , Tissue Engineering
7.
J Histochem Cytochem ; 48(11): 1493-502, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11036092

ABSTRACT

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Cartilage, Articular/metabolism , Diaphyses/metabolism , Epiphyses/metabolism , Growth Plate/metabolism , Animals , Cartilage, Articular/embryology , Cartilage, Articular/growth & development , Chondrocytes/metabolism , Diaphyses/embryology , Diaphyses/growth & development , Embryo, Mammalian , Epiphyses/embryology , Epiphyses/growth & development , Growth Plate/embryology , Growth Plate/growth & development , Humans , Immunohistochemistry , Organ Specificity , Osteoblasts/metabolism , Osteoclasts/metabolism , Rats , Rats, Sprague-Dawley , Stromal Cells/metabolism
8.
Biochim Biophys Acta ; 1416(1-2): 320-32, 1999 Jan 12.
Article in English | MEDLINE | ID: mdl-9889389

ABSTRACT

Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage.


Subject(s)
Adenosine Triphosphate , Calcium Pyrophosphate/analysis , Cartilage/drug effects , Detergents/pharmacology , Extracellular Matrix/drug effects , Skull/drug effects , Animals , Calcinosis/etiology , Cartilage/metabolism , Cartilage Diseases/etiology , Extracellular Matrix/metabolism , Octoxynol/pharmacology , Pyrophosphatases/analysis , Rats , Rickets/complications , Skull/metabolism , Spectroscopy, Fourier Transform Infrared , Tetramisole/pharmacology
9.
IEEE Trans Neural Netw ; 9(6): 1529-31; author reply 1531-2, 1998.
Article in English | MEDLINE | ID: mdl-18255831

ABSTRACT

The above paper claims that under a set of minor restrictions radial basis function networks and fuzzy inference systems are functionally equivalent. The purpose of this letter is to show that this set of restrictions is incomplete and that, when it is completed, the said functional equivalence applies only to a small range of fuzzy inference systems. In addition, a modified set of restrictions is proposed which is applicable for a much wider range of fuzzy inference systems.

10.
Am J Pathol ; 151(6): 1555-61, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9403706

ABSTRACT

Hypophosphatasia, a heritable disease characterized by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), results in rickets and osteomalacia. Although identification of TNSALP gene defects in hypophosphatasia establishes a role of ALP in skeletal mineralization, the precise function remains unclear. The initial site of mineralization (primary mineralization) normally occurs within the lumen of TNSALP-rich matrix vesicles (MVs) of growth cartilage, bone, and dentin. We investigated whether defective calcification in hypophosphatasia is due to a paucity and/or a functional failure of MVs secondary to TNSALP deficiency. Nondecalcified autopsy bone and growth plate cartilage from five patients with perinatal (lethal) hypophosphatasia were studied by nondecalcified light and electron microscopy to assess MV numbers, size, shape, and ultrastructure and whether hypophosphatasia MVs contain apatite-like mineral, as would be the case if these MVs retained their ability to concentrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in approximately normal numbers and distribution and that they are capable of initiating internal mineralization. There is retarded extravesicular crystal propagation. Thus, in hypophosphatasia the failure of bones to calcify appears to involve a block of the vectorial spread of mineral from initial nuclei within MVs, outwards, into the matrix. We conclude that hypophosphatasia MVs can concentrate calcium and phosphate internally despite a deficiency of TNSALP activity.


Subject(s)
Apatites/metabolism , Bone Matrix/metabolism , Hypophosphatasia/metabolism , Osteomalacia/metabolism , Bone Matrix/ultrastructure , Bone and Bones/metabolism , Bone and Bones/ultrastructure , Cartilage/metabolism , Cartilage/ultrastructure , Crystallization , Female , Growth Plate/metabolism , Growth Plate/ultrastructure , Humans , Hypophosphatasia/pathology , Infant, Newborn , Male , Osteomalacia/pathology , Pregnancy
12.
J Biol Chem ; 271(42): 26383-8, 1996 Oct 18.
Article in English | MEDLINE | ID: mdl-8824294

ABSTRACT

Accumulating evidence indicates that calcification by isolated mammalian matrix vesicles (MVs) can be initiated by ATP. Since ATP can be hydrolyzed by either a specific ATPase or by nonspecific alkaline phosphatase (ALP), it remains to be established whether ATPase or ALP mediates ATP-initiated Ca and Pi deposition. To support the hypothesis that specific ATPase is responsible for ATP-initiated calcification by MVs isolated from mammalian cartilage and bone, the effects of ATP analogs, ALP substrates, and specific inhibitors on ATP hydrolysis and ATP-initiated calcification were compared between intact MVs and monoclonal antibody affinity-purified MV ALP. ATP analogs such as ADP and AMP exerted marked inhibitory effects on both [gamma-32P]ATP hydrolysis and ATP-initiated calcification by intact MVs, whereas phosphomonoesters such as beta-glycerophosphate or phosphoethanolamine had no effect. In contrast to intact MVs, purified MV ALP failed to calcify, and its [gamma-32P]ATP hydrolytic activity was readily inhibited by phosphomonoesters. Additionally, [gamma-32P]ATP hydrolysis by purified ALP in contrast to that by intact vesicles was completely inhibited by l-tetramisole, a specific inhibitor of ALP, suggesting a loss of specific ATPase during purification. Vanadate inhibition of ATP hydrolysis by purified ALP can be decreased by increasing ATP concentrations. On the contrary, ATP concentrations did not affect vanadate inhibition of ATP hydrolysis by intact MVs if ALP activity was blocked by l-tetramisole. These observations, therefore, suggest that: 1) a portion of [gamma-32P]ATP hydrolysis by MVs is attributable to a specific ATPase, whereas the remaining activity is due to ALP; and 2) a specific ATPase, but not ALP, is responsible for ATP-dependent Ca- and Pi-depositing activity of MVs isolated from bone or cartilage.


Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Bone and Bones/metabolism , Calcification, Physiologic , Cartilage/metabolism , Adenosine Monophosphate/pharmacology , Alkaline Phosphatase/metabolism , Animals , Bone and Bones/ultrastructure , Calcium/metabolism , Cartilage/ultrastructure , Egtazic Acid/pharmacology , Ethanolamines/metabolism , Glycerophosphates/metabolism , Microscopy, Electron , Phosphates/metabolism , Rats , Tetramisole/pharmacology
13.
J Dent Res ; 75(7): 1518-23, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8876605

ABSTRACT

Freeze-dried Saos-2, human osteosarcoma cells, and extracts of Saos-2 cells contain all components necessary to induce ectopic new bone and marrow when implanted into athymic Nu/Nu nuce. On the other hand, human osteosarcoma cells of the U-2 OS strain failed to induce bone formation under the same experimental conditions. Our aim was to compare the relative expressions of known osteoinductive factors including bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGF-beta) in these two cell lines in an attempt to explain the unique bone-inducing ability of the Saos-2 cells. Saos-2 cells expressed mRNA for BMP-1, -2, -3, -4,-6, and TGF-beta 1. The non-osteoinductive U-2 OS cells expressed BMP-2, -4, -5, -6, and -7 as well as TGF-beta 1 mRNA, while levels of BMP-1 and BMP-3 mRNA were either not detectable or detectable at a very low level in U-2 OS cells. The presence of BMP-1 and -4 protein was confirmed in Saos-2 cells by immunofluorescence, and TGF beta protein was demonstrated by bioassay in both cell types. These findings suggest that Saos-2 cells are endowed qualitatively and quantitatively with sufficient amounts of many bone morphogenetic proteins-especially BMP-1, -3, and -4-to confer osteoinductivity upon these cells. However, the absence of osteoinductivity in U-2 OS cells, despite significant mRNA expression levels of several bone morphogenetic proteins, suggests that, even though expression of one or more bone morphogenetic proteins may be present, it may not necessarily be sufficient to confer osteoinductivity upon U-2 OS cells. U-2 OS cells may be non-osteoinductive because (1) they contain inhibitors to the BMPs or secrete inhibitory binding proteins, (2) they do not process BMPs correctly, or (3) the BMPs are inappropriately localized and sequestered within the U-2 OS cells. Saos 2 cells may be osteoinductive because (1) they uniquely express BMP-1, (2) they express an appropriate combination of interactive BMPs at appropriate levels, and/or (3) the Saos-2 cells elaborate as-yet-unidentified osteoinductive factor(s).


Subject(s)
Bone Morphogenetic Proteins/biosynthesis , Bone Neoplasms/metabolism , Osteogenesis/genetics , Osteosarcoma/metabolism , Animals , Biological Assay , Blotting, Northern , Blotting, Western , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Fluorescent Antibody Technique , Gene Expression Regulation , Humans , Mice , Mice, Nude , Ossification, Heterotopic/metabolism , Osteosarcoma/genetics , Osteosarcoma/pathology , RNA, Messenger/analysis , Transforming Growth Factor beta/biosynthesis , Tumor Cells, Cultured/metabolism
14.
J Orthop Res ; 14(4): 605-10, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8764870

ABSTRACT

Implants of defatted, freeze-dried Saos-2 human osteosarcoma cells grown to confluency induce de novo bone formation in athymic mice. These cells are also richly endowed with bone morphogenetic proteins and express mRNA for bone morphogenetic proteins 1, 2, 3, 4 and 6, as well as for transforming growth factor-beta 1. Our aim was to study whether the ability to induce bone formation is related to the level of expression of bone morphogenetic protein. We studied the osteoinductive abilities and levels of expression of bone morphogenetic protein of Saos-2 cells both during the growth phase and after confluency was reached. Subconfluent cells were at least 70% less effective in their osteoinductive ability than confluent cells. Comparison of bone morphogenetic protein mRNA expression in confluent and subconfluent cells revealed that the latter had lower expression of all the mRNAs studied. The expression of bone morphogenetic protein-1, bone morphogenetic protein-2, and bone morphogenetic protein-6 mRNAs was 2, 3, and 6 to 10-fold lower, respectively, in subconfluent cells. These results suggest that the ability of Saos-2 cells to induce de novo bone formation may be correlated with the relative expression of these proteins; the expression of bone morphogenetic proteins in Saos-2 cells also may be dependent on the cell cycle.


Subject(s)
Bone and Bones/cytology , Proteins/genetics , Animals , Biological Assay , Blotting, Northern , Bone Morphogenetic Proteins , Cell Count , Cell Cycle/physiology , Gene Expression/physiology , Humans , Mice , Mice, Nude , Osteosarcoma , RNA, Messenger/analysis , Transforming Growth Factor beta/genetics , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/physiology
15.
J Bone Joint Surg Am ; 78(1): 41-8, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8550678

ABSTRACT

Devitalized extracts from cultured human osteosarcoma cells (Saos-2) can induce ectopic bone formation. The ability of an extract from Saos-2 cells to stimulate healing of an operatively created four-millimeter defect in the femoral diaphyses of rats was compared with that of collagen and that of autogenous bone graft. Forty adult rats were randomized into four groups of ten each. In Group 1 (controls), no material was placed in the defect; in Group 2, the defect was filled with pure bovine collagen; in Group 3, it was filled with autogenous graft obtained by morseling of the resected segment of the femur; and in Group 4, it was filled with ten milligrams of extract from Saos-2 cells that was mixed with an equal amount of bovine collagen. Five rats from each group were killed at four weeks and the remaining five, at eight weeks. Each femoral defect was analyzed radiographically and histologically for osseous healing. There was no evidence of healing at either four or eight weeks in Groups 1 and 2. Although there was some new-bone formation in Group 3, none of the defects had united at eight weeks. There was early, almost complete union in all five four-week specimens in Group 4 and complete healing of the defect in four of the five rats assessed at eight weeks. The Saos-2 cell extract was found to be the most effective agent, promoting union by mature lamellar bone within eight weeks.


Subject(s)
Bone Remodeling/drug effects , Cell Extracts/pharmacology , Osteosarcoma/physiopathology , Wound Healing/drug effects , Animals , Bone Remodeling/physiology , Bone Transplantation , Cell Extracts/therapeutic use , Collagen/pharmacology , Femur/diagnostic imaging , Femur/injuries , Femur/pathology , Femur/physiopathology , Humans , Male , Radiography , Random Allocation , Rats , Tumor Cells, Cultured , Wound Healing/physiology
17.
Bone ; 17(5): 473-7, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8579959

ABSTRACT

Elsewhere it has been shown that zinc is highly concentrated in the hypertrophic zone of epiphyseal cartilage. It has also been shown that zinc deficiency can result in abnormal bone development, suggesting a direct or indirect role for zinc in calcification. Because matrix vesicles have been implicated in the initiation of calcification, we tested the effect of zinc and its chelators, such as EGTA and phenanthroline, on ATP-dependent Ca uptake by rat matrix vesicles. EGTA pretreatment of matrix vesicles inhibited ATP-dependent Ca uptake by 50%. To see if zinc depletion by EGTA pretreatment is responsible for decreased levels of ATP-dependent Ca uptake, ZnCl2 concentrations, ranging from 5 to 100 mumol/L, were tested for their ability to restore Ca deposition. Zinc exerted a striking enhancing effect on ATP-dependent Ca uptake of both untreated and EGTA-pretreated matrix vesicles in a dose-dependent manner. A 50% activation occurs at about 16 mumol/L Zn2+. At 63 mumol/L Zn2+, there was a fourfold increase in Ca-depositing activity. Addition of an excess amount of phenanthroline relative to Zn2+ concentration to the reaction mixture failed to abolish activation of Ca uptake by Zn2+, indicating that the putative chelator-Zn2+ complex formation did not prevent activation. The observed partial inhibition of ATPase and the activation of ATP-dependent Ca uptake of Zn2+ suggest that, in addition to ATPase, some other Ca and/or Pi uptake activators responsive to Zn2+ treatment are present in mammalian matrix vesicles.


Subject(s)
Adenosine Triphosphate/pharmacokinetics , Bone Matrix/drug effects , Calcium/metabolism , Chelating Agents/pharmacology , Egtazic Acid/pharmacology , Phenanthrolines/pharmacology , Rickets/physiopathology , Zinc/pharmacology , Adenosine Triphosphatases/antagonists & inhibitors , Animals , Bone Matrix/metabolism , Bone Matrix/ultrastructure , Disease Models, Animal , Dose-Response Relationship, Drug , Growth Plate/drug effects , Growth Plate/metabolism , Hydrolysis , Microscopy, Electron , Rats , Rickets/metabolism
18.
Clin Orthop Relat Res ; (314): 266-80, 1995 May.
Article in English | MEDLINE | ID: mdl-7634645

ABSTRACT

Matrix vesicles are extracellular 100-nanometer-diameter membrane-invested particles selectively located within the matrix of bone, cartilage, and predentin. They serve as the initial site of calcification in all skeletal tissues. Matrix vesicle biogenesis occurs by polarized budding and pinching off of vesicles from specific regions of the outer plasma membrane of chondrocytes, osteoblasts, and odontoblasts. Seeding of selected areas of matrix with matrix vesicles explains the localized distribution of subsequent zones of mineralization. Matrix vesicle biogenesis in the growth plate is linked to the chondrocyte cell cycle and reflects a stage in programmed cell death (apoptosis). Generation of initial hydroxyapatite mineral crystals occurs within the matrix vesicle membrane during Phase 1 of biologic mineralization. Phase 1 is controlled by phosphatases (including alkaline phosphatase) and Ca-binding molecules with which the matrix vesicles are well endowed. Phase 2 of biologic mineralization begins with breakdown of matrix vesicle membranes, exposing preformed hydroxyapatite to the extracellular fluid after which mineral crystal proliferation is governed by extracellular conditions. Phase 1 and Phase 2 of mineralization are under cellular control. Phase 1 is initiated by cells generating calcifiable matrix vesicles and releasing them into sites of intended calcification. Phase 2 is controlled by cells regulating extracellular ionic conditions and matrix composition.


Subject(s)
Bone Matrix/physiology , Calcification, Physiologic , Alkaline Phosphatase/metabolism , Animals , Apoptosis/physiology , Bone Matrix/chemistry , Bone Matrix/ultrastructure , Cells, Cultured , Collagen/physiology , Growth Plate/physiology , Humans , Microscopy, Electron , Molecular Biology/methods
19.
Clin Orthop Relat Res ; (313): 129-34, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7641470

ABSTRACT

Saos-2 cultured human osteosarcoma cells contain an extractable bone inducing agent that can induce heterotopic bone in the muscle of Nu/Nu mice. A semipurified GuHCl extract of Saos-2 cells also can promote healing and complete bony union in otherwise non-healing surgically induced defects of rat femur. Northern blot analyses indicate expression of mRNAs for bone morphogenetic proteins (BMP)-1, 2, 3, 4, 6 and transforming growth factor beta (TGF beta) in Saos-2 cells, and BMP-2, 3, 4, 5, 7 and TGF beta in nonosteoinductive U20S human osteosarcoma cells. Saos-2 cells exceeded U20S cells in expression levels of BMP-1, 3, 4 and TGF beta, whereas U20S cells expressed higher levels of BMP-2, 6 and also expressed trace amounts of BMP-5 and 7 not seen in Saos-2 cells. The authors hypothesize that Saos-2 cells contain an optimal admixture of known bone growth factors plus possible other unknown components that, acting alone or in combination with bone morphogenetic protein and/or TGF beta, can induce bone. Although bone inducing agent-induced heterotopic bones have half lives of only a few weeks, the reparative bone induced by bone inducing agent in femoral defects gives every indication of being permanent and self-sustaining. This suggests a fundamental difference between heterotopic and orthotopic osteoprogenitor cells with those involved in orthotopic bone repair more closely resembling the committed or determined osteoprogenitor cells of marrow as described by Friedenstein.


Subject(s)
Bone Development , Bone Neoplasms/chemistry , Bone Regeneration , Osteosarcoma/chemistry , Animals , Blotting, Northern , Bone Morphogenetic Proteins , Bone Neoplasms/pathology , Gene Expression , Growth Substances/biosynthesis , Humans , Mice , Mice, Nude , Osteosarcoma/pathology , Protein Biosynthesis , RNA, Messenger/genetics , Transforming Growth Factor beta/biosynthesis , Tumor Cells, Cultured
20.
Bone ; 16(1): 91-6, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7742091

ABSTRACT

A monoclonal antibody that reacts with murine and human bone morphogenetic protein-4 (BMP-4) has been developed using recombinant BMP-4 as an immunogen. The antibody that bound most tightly to recombinant murine (rm)BMP-4 was selected, subcloned, and characterized. The specificity of the antibody was confirmed using Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The antibody reacts with murine and human BMP-4 in both the reduced and nonreduced condition; however, this antibody shows cross-reactivity with neither human BMP-2 nor TGF-beta 1. Thus, the produced antibody could recognize the disulfide-linked dimeric structure of bioactive BMP-4, regardless of the species. Immunocytochemical study using this antibody successfully shows the cytosolic localization of BMP-4 in osteoinductive cells; i.e., BFO and Saos-2 in which the level of mRNA for BMP-4 was proved to be constitutively high by Northern blot analysis. In addition, the antibody could demonstrate the presence of BMP-4 in developmental bone formation in the alveolar bone of rat embryo by immunohistochemistry. The antibody could be used for a more sensitive approach for quantitative analysis of BMP-4.


Subject(s)
Antibodies, Monoclonal , Neoplasm Proteins/analysis , Proteins/analysis , Animals , Bone Morphogenetic Proteins , Cell Line , Embryonic and Fetal Development/physiology , Escherichia coli , Fetal Proteins/analysis , Humans , Mice , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/biosynthesis , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis
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