Experimental study of changes in osteoblastic shape induced by calcitonin and parathyroid extract in an organ culture system

Summary Neonate rat endocranial osteoblasts were cultured on their bone surfaces in control medium (CC) or medium to which either parathyroid extract (PTE) or calcitonin (CT) had been added for 2, 4, 8 or 24 h. Some were cultured for 24 h in CC, then for 2, 4, 8 or 24 h in either CT or PTE medium; or for 24 h in PTE, then for 2, 4, 8 or 24 h in either CC or CT; or 24 h in CT and 2, 4, 8 or 24 h in CC. The dorsal ruffling of the cells in CC was found to be suppressed by later culturing with PTE and the disoriented cells reorganized to form arrays of parallel cells. The effects of PTE were also reversed by CC or CT: the osteoblasts in the second culture (CC) lost elongation and order, and proceeded through a proliferative phase before exhibiting the ruffling form similar to a single CC 24 h culture. PTE-cultured osteoblasts showed an increase in cell overlap and contact so that a more competent barrier was formed separating the bone from the medium. In control or CT medium, however, intercellular gaps were greater than in vivo.We are grateful for the expert technical assistance of Elaine Bailey, for laboratory facilities kindly provided by Dr. Martin Evans, and for financial support from the Medical Research Council     

Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca²⁺ release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single‐cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain‐derived neurotrophic factor (BDNF) in the culture medium. The ryanodine‐induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 467–480, 2014     

Osteoblasts and collagen orientation

Summary Bone was removed from the calvaria of anaesthetized 70 g rats or freshly killed young monkeys and the fibrous periosteum dissected off the inner, formative surface under 0.15 M cacodylate buffer. The bone and undisturbed osteoblasts were fixed in 3% glutaraldehyde in the same buffer for 24 to 48 hours, critical point dried and coated with evaporated carbon and gold for scanning electron microscopy (SEM). Fields of osteoblasts were photographed and chosen cells dissected off the osteoid using a tungsten needle. The control of the dissection was made possible by the use of a system of real-time stereo TV-speed SEM. The fields were rephotographed and the orientations of the osteoblasts were compared with that of the underlying collagen fibres. 62% of all osteoblasts lay with their long axes within 15° of the collagen fibre orientation below and 80% within 30°. Montages of large areas of osteoblasts were also made, and then compared with ones of the same area after the cells had been stripped off on adhesive tape. In general, the orientation of the collagen tended to be the same as the cell that formed it. Collagen fibres below cells at the periphery of a domain sometimes had the orientation of the cells in the adjacent patch. It is not possible to determine whether the cells controlled the orientation of the collagen, or vice versa, from this experiment, but other SEM evidence suggests that the collagen orientation in hard tissue matrices depends on the freedom of cells to move with respect to the matrix surface. Acknowledgements. This work has been supported by generous grants from the Medical Research Council and the Science Research Council. We are grateful to Elaine Bailey and Mr. P. Reynolds for technical assistance.     

Human chorion cells respond to growth factors but lose steroidogenic capacity in primary monolayer cell culture

Summary This study has defined a method for preparation and monolayer culture of cells fromchorion laeve. Cell number and cell protein content are table over 7 d in culture. The cells will divide in response to epidermal growth factor in the presence of a supplemented, enriched medium and a collagen matrix, but they lose steroidogenic activity over time in culture. This culture system can be used as the starting point for the development of a chemically defined hormone-supplemented, serum-free culture system for studies of chorion cell differentiation and fetal membrane cell interactions. This work was supported by the Medical Research Council of Canada through Grant MT-7867 (to A. K. T.), Grant MA-7731 (to B. F. M.), and a Summer Studentship (to K. A. F.).     

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

Summary Thrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of⁵¹Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the⁵¹Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells. This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council for Cardiovascular Diseases. We express our gratitude for their grants.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

Physical and nutritional factors in gel culture of mammalian cells

Summary The growth of human glioma cells, cultured as spherical colonies in agarose gel, stopped after about 10 days for both large and small colonies apparently due to an increased osmolality in the gel. When osmolality was kept under control by addition of distilled water, growth continued. However, a continuous increase in the population-doubling period, similar for both large and small colonies, then was observed. The increase persisted although excess amounts of nutrition were added. When the cells were cultured in liquid suspension above a thin layer of agarose gel and most of the medium was repeatedly changed, the colonies continued to grow beyond the limits in gel culture. HeLa and hamster embryonic lung cell colonies showed a growth pattern in agarose gel similar to the glioma cells. The results imply that the osmolality must be kept under precise control to prevent growth inhibition. However, it seems difficult to ascertain optimal growth in gel culture for more than about 2 weeks probably because of the accumulation of toxic products. The work was supported financially by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

Some morphological observations on osteoclasts

Summary Osteoclasts of the peripheral portions of the endocranial aspect of young rat parietal and frontal bones were studied by scanning electron microscopy of glutaraldehyde fixed, critical point dried specimens. These studies show Osteoclasts to have a much more complicated form than has previously been realised. Extensively branching, elongated, smooth-surfaced cells, which are for the most part elevated above the level of the surrounding bone matrix surface and sometimes above portions of osteoblasts or other osteoclasts, were identified as motile non-resorbing cells. Portions of the former and other entire cells may be embowered in Howship's lacunae, have microvilli on their dorsal surface, and are surrounded by a serrated border of microprojections which have an apparently firm attachment to the matrix surface. Osteoclasts in short term culture show additional free surface ruffles which are not encountered in specimens taken fresh from the animal. No evidence of recruitment of osteoblasts or osteocytes into osteoclasts was found. Disinterred osteocytes retained an ability to migrate from their lacunae on to surrounding bone matrix surface.We would like to thank Elaine Maconnachie for expert technical assistance and Dr. Martin J. Evans for the use of his tissue culture laboratory. These studies have been supported in part by a grant from the Medical Research Council     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

Isolation of fetal mouse motor neurons on discontinuous percoll density gradients

Summary The spinal cords of fetal NIH∶CR mice, gestational age Day 12 to 14, were dissected free of meninges and dorsal root ganglia, chemically dissociated, and layered onto discontinuous Percoll gradients at densities 1.040, 1.050, and 1.060 g/ml. After centrifugation (800 Xg for 15 min at 4° C), three morphologically, biochemically, and immunohistologically distinct cell populations were collected from the gradient interfaces. The first interface, located at a density of 1.040 g/ml, was choline acetyltransferase enriched (0.86±0.08) compared to the second and third fractions (0.42±0.01 and 0 pmol acetylcholine synthesized/μg protein, respectively). When simultaneously cultured with fetal mouse cardiac muscle on a gelatin-polylysine-laminin substrate in serum-free medium, these cells developed the characteristics of motor neurons. Dr. Strong is supported in part by a Research Fellowship from the Medical Research Council of Canada.     

The growth and phenotypic expression of human osteoblasts

The care of patients with a skeletal deficiency currently involves the use of bone graft or a non-biologic material such as a metal or polymer. There are alternate possibilities in development which involve the growth of bone cells (osteoblasts) on degradable polymer scaffolds. These tissue engineering strategies require production of the polymeric scaffold, cellular harvest followed by either ex vivo or in vivo growth of the cells on the scaffold, and exploration of the interaction between the cell and scaffold. Research into these strategies utilizes cells from a variety of species, but clinical applications will likely require human osteoblasts. This study explores the process whereby human osteoblasts are harvested under sterile conditions during joint replacement surgery from normally discarded cancellous bone, transported from the operating room to the lab, and grown in culture. This process is feasible, and the cells express their phenotype via the production of alkaline phosphatase and collagen in culture.     

Indirect osteoblast differentiation by liposomal clodronate

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow‐derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real‐time RT‐PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow‐derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre‐treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate‐injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK‐containing exosomes in response to clodronates.     

Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases

Mesenchymal stem cells (MSC) are a group of cells present in bone-marrow stroma and the stroma of various organs with the capacity for mesoderm-like cell differentiation into, for example, osteoblasts, adipocytes, and chondrocytes. MSC are being introduced in the clinic for the treatment of a variety of clinical conditions. The aim of this review is to provide an update regarding the biology of MSC, their identification and culture, and mechanisms controlling their proliferation and differentiation. We also review the current status of their clinical use. Areas in which research is needed to enhance the clinical use of MSC are emphasized. This work was supported by grants from the Danish Medical Research Council, Danish Center for Stem Cell Research, and Novo Nordisk Foundation.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Characterization of human osteoblastic cells: Influence of the culture conditions

Summary Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)₂ vitamin D₃. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D₃. 10 mM βGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.     

The effects of growth conditions on the esterases of Paramecium aurelia

The esterase isozymes of Paramecium aurelia grown in the presence and absence of bacteria were examined by starch gel electrophoresis. Where present, bacteria make a positive contribution to the esterase zymograms. Different bacteria contribute different esterases. Paramecia growing in axenic medium possess a different esterase which is not produced by the same parmecia growing in the presence of bacteria. This axenic esterase disappears when bacteria are added back to the medium.Supported by a research grant from the Science Research Council.     

Cytological studies on HeLa,a strain of human cervical carcinoma

Summary Cells of the HeLa strain show considerable activity of nuclear movements in the fashion of complete rotation, incomplete rotation and rocking. The activity decreases when the culture becomes older but changing fresh medium restores it. Approximately two-thirds of the cells entering division show premitotic movements, mostly of the rocking type.Supported in part by a grant-in aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council and in part by a contract from the Office of Naval Research N6onr-266 T.O. 5.     

Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa

Summary Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca²⁺ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments, the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg?rden, Stockholm, Sweden.     

Establishment and comparison of fibroblast cell lines from the medial collateral and anterior cruciate ligaments of the rabbit

Summary We have developed a procedure to explant fibroblasts from the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) of the rabbit knee, and have optimized conditions for maintaining them in culture. Maximal growth for both ACL and MCL cells was obtained with Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 250 μM ascorbate. ACL and MCL fibroblasts displayed intrinsic differences in their responses to changes in culture parameters. Specifically, they displayed different growth responses when plated at different densities and responded to RPMI 1640 medium in very different ways. There were also biochemical differences between the cell types. Both cell types produced similar amounts of collagen in culture, but the ratio of type I to type III, the major collagen subtypes produced by these cells, were different. ACL fibroblasts produced 86.7% type I and 13.3% type III, and MCL fibroblasts produced 71.1% type I and 28.9% type III. In addition, total protein produced by ACL fibroblasts was higher than that produced by MCL cells. This confirms the suggestions of previous researchers that such differences might exist. This work was funded by a grant-in-aid from Medtronic of Canada, by an R&D Grant from the Alberta Ministry of Technology, Research and Telecommunications, and by the Alberta Heritage Foundation for Medical Research.