PTH stimulates the proliferation of TE-85 human osteosarcoma cells by a mechanism not involving either increased cAMP or increased secretion of IGF-I,IGF-II or TGFβ

Injections of parathyroid hormone (PTH) result in increased bone formation in several species. Work in our laboratory and others has shown a stimulation of bone cell proliferation and growth factor production by PTH. Our purpose was to study the effects of PTH on a human bone cell line using TE-85 human osteosarcoma cells as a model. After 24 h treatment, PTH caused an increase in cell proliferation as measured by cell counts and [³H]-thymidine incorporation. Proliferation was not inhibited by an anti-transforming growth factor beta (TGFβ) antibody which could abolish stimulation by exogenous TGFβ. PTH did not stimulate cAMP production, alkaline phosphatase activity or production of insulin-like growth factors I or II (IGF-I or IGF-II) in TE-85 cells. Although basal TE-85 proliferation was slowed by incubation with the calcium channel blocking agent verapamil, PTH still caused an increase in growth rate. We conclude that PTH directly stimulates TE-85 proliferation via a mechanism not involving increased adenylate cyclase activity or increased secretion of IGF-I, IGF-II or TGFβ and may stimulate bone formation in vivo by activating some other mitogenic signal to increase bone cell proliferation.     

Cell fate specification during calvarial bone and suture development

In this study we have addressed the fundamental question of what cellular mechanisms control the growth of the calvarial bones and conversely, what is the fate of the sutural mesenchymal cells when calvarial bones approximate to form a suture. There is evidence that the size of the osteoprogenitor cell population determines the rate of calvarial bone growth. In calvarial cultures we reduced osteoprogenitor cell proliferation; however, we did not observe a reduction in the growth of parietal bone to the same degree. This discrepancy prompted us to study whether suture mesenchymal cells participate in the growth of the parietal bones. We found that mesenchymal cells adjacent to the osteogenic fronts of the parietal bones could differentiate towards the osteoblastic lineage and could become incorporated into the growing bone. Conversely, mid-suture mesenchymal cells did not become incorporated into the bone and remained undifferentiated. Thus mesenchymal cells have different fate depending on their position within the suture. In this study we show that continued proliferation of osteoprogenitors in the osteogenic fronts is the main mechanism for calvarial bone growth, but importantly, we show that suture mesenchyme cells can contribute to calvarial bone growth. These findings help us understand the mechanisms of intramembranous ossification in general, which occurs not only during cranial and facial bone development but also in the surface periosteum of most bones during modeling and remodeling.     

A description of chick wing bud development and a model of limb morphogenesis

The location of the prospective cartilage-forming regions in the embryonic chick wing bud was ascertained by implantation of blocks of wing mesenchyme labeled with tritiated thymidine during the early stages of wing development. The position of the implanted cells was determined by autoradiography, and the location of the implanted block in the limb and its relation to the cartilaginous bones was determined by reconstruction of the host limb from serial sections. The areas corresponding to all of the future wing bones, including the digits, were mapped at each stage from stage 18 to stage 24. Growth of the wing and the prospective bone areas was found to be almost exclusively parallel to an axis perpendicular to the base of the limb. The rate of growth in all areas of the wing reflected the rate of cell division, and all changes in the rate of growth corresponded to changes in the number of dividing cells in the wing and each of the prospective bone regions. Differentiative changes and changes in the growth rate are initiated at a constant distance of 0.4-0.5 mm from the apical ectodermal ridge. These results, considered in conjunction with results of earlier studies in this and other laboratories, suggest that the definitive morphogenetic pattern of the limb arises from four component processes; polarized growth, changes in cell proliferation, cell death, and cytodifferentiation.     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Damage and recovery of the bone growth mechanism in young rats following 5-fluorouracil acute chemotherapy

Chemotherapy-induced bone growth arrest and osteoporosis are significant problems in paediatric cancer patients, and yet how chemotherapy affects bone growth remains unclear. This study characterised development and resolution of damage caused by acute chemotherapy with antimetabolite 5-fluorouracil (5-FU) in young rats in the growth plate cartilage and metaphyseal bone, two important tissues responsible for bone lengthening. In metaphysis, 5-FU induced apoptosis among osteoblasts and preosteoblasts on days 1-2. In growth plate, chondrocyte apoptosis appeared on days 5-10. Interestingly, Bax was induced prior to apoptosis and Bcl-2 was upregulated during recovery. 5-FU also suppressed cell proliferation on days 1-2. While proliferation returned to normal by day 3 in metaphysis, it recovered partially on day 3, overshot on days 5-7 and normalised by day 10 in growth plate. Histologically, growth plate heights decreased by days 4-5 and returned normal by day 10. In metaphysis, primary spongiosa height was also reduced, mirroring changes in growth plate thickness. In metaphyseal secondary spongiosa, a reduced bone volume was observed on days 7-10 as there were fewer but more separated trabeculae. Starting from day 4, expression of some cartilage/bone matrix proteins and growth factors (TGF-beta1 and IGF-I) was increased. By day 14, cellular activity, histological structure and gene expression had returned normal in both tissues. Therefore, 5-FU chemotherapy affects bone growth directly by inducing apoptosis and inhibiting proliferation at growth plate cartilage and metaphyseal bone; after the acute damage, bone growth mechanism can recover, which is associated with upregulated expression of matrix proteins and growth factors.     

Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development.     

Modelling the early phases of bone regeneration around an endosseous oral implant

The objective of this study was to see whether a mathematical model of fracture healing was able to mimic bone formation around an unloaded screw-shaped titanium implant as it is well-believed that both processes exhibit many biological similarities. This model describes the spatio-temporal evolution of cellular activities, ranging from mesenchymal stem cell migration, proliferation, differentiation to bone formation, which are initiated and regulated by the growth factors present at the peri-implant site. For the simulations, a finite volume code was used and adequate initial and boundary conditions were applied. Two sets of analyses have been performed, in which either initial and boundary condition or model parameter values were changed with respect to the fracture healing model parameter values. For a number of combinations, the spatio-temporal evolution of bone density was well-predicted. However reducing cell proliferation rate and increasing osteoblast differentiation and osteogenic growth factor synthesis rates, the simulation results were in agreement with the experimental data.     

Effect of cell seeding and mechanical loading on vascularization and tissue formation inside a scaffold: A mechano-biological model using a lattice approach to simulate cell activity

Achieving successful vascularization remains one of the main problems in bone tissue engineering. After scaffold implantation, the growth of capillaries into the porous construct may be too slow to provide adequate nutrients to the cells in the scaffold interior and this inhibits tissue formation in the scaffold core. Often, prior to implantation, a controlled cell culture environment is used to stimulate cell proliferation and, once in place, the mechanical environment acting on the tissue construct is determined by the loading conditions at the implantation site. To what extent do cell seeding conditions and the construct loading environment have an effect on scaffold vascularization and tissue growth? In this study, a mechano-biological model for tissue differentiation and blood vessel growth was used to determine the influence of cell seeding on vascular network development and tissue growth inside a regular-structured bone scaffold under different loading conditions. It is predicted that increasing the number of cells seeded homogeneously reduces the rate of vascularization and the maximum penetration of the vascular network, which in turn reduces bone tissue formation. The seeding of cells in the periphery of the scaffold was predicted to be beneficial for vascularization and therefore for bone growth; however, tissue formation occurred more slowly during the first weeks after implantation compared to homogeneous seeding. Low levels of mechanical loading stimulated bone formation while high levels of loading inhibited bone formation and capillary growth. This study demonstrates the feasibility of computational design approaches for bone tissue engineering.     

Comparative quantitative histology of mammalian growth plates

Variation in the growth rate of long bones is a function of the number of dividing cells in the columns of the proliferation (flat) cell zone of the growth plate, the frequency with which they divide, and the size to which they grow prior to ossification. In a previous study we found that the wide variation in bone growth rates seen among species of birds was largely associated with variation in the numbers of cells in the flat cell zone. Here we have undertaken a similar study of the growth plates of mammals and have examined variation in the morphology and cell kinetics of the tibial growth plates of a variety of species. The bone growth rates tended to he lower than those observed in birds and were particularly low in the anthropoid primates. Although quite marked variation in flat cell numbers is apparent, the results suggest that variation in cell division rate may play a relatively greater role in variation in bone growth rate among mammals than it does in birds. and that the very low hone growth rates seen in the primates are due, in part, to lower rates of cell division than in other species.     

In vitro activity of folic acid on the proliferation dynamics of human bone marrow CFU-GM in propyphenazone-induced granulocytopenia]

The in vitro action of folic acid was tested on the proliferation of bone marrow granulocyte-macrophage progenitor cells from a patient with drug-induced (propyphenazone) neutropenia in remission 20 days after the drug had been suspended. Various bone marrow cultures were prepared with standard stimulant, adding, respectively: folic acid, propyphenazone and both folic acid and propyphenazone together. Growth was tested on day 7, 12 and 19 of incubation. Under baseline culture with standard stimulant, CFU-GM growth was characterized by successive proliferation waves of various entity: the first on day 7 was very high, the second, on day 12 was rather low, and the third, on day 19 was intermediate. This behaviour is different from what is usually observed in normal subjects in steady-state, whose first (AC-A+AC-B) and second (AC-C) proliferation period are of similar entity. The prevalence of the first proliferation period in our case is interpreted as the result of a renewed granulocytopoietic activity after drug-induced bone marrow suppression. This indicates a maintained integrity of the negative-feedback mechanism of homeostatic regulation on granulocytopoietic activity. The sole addition of propyphenazone on the in vitro bone marrow cell cultures of our patient produced a reduction of those CFU-GM that had grown during the first period whereas the growth during the second and third period remained unvaried. Thus the growth peak in cultures treated with propyphenazone occurred on day 19, which seems to correspond with the necessary time for a spontaneous remission from neutropenia, clinically observed to be 20 days after suspension of the propyphenazone-containing drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

The type III transforming growth factor-β receptor inhibits proliferation, migration, and adhesion in human myeloma cells

Transforming growth factor-β (TGF-β) plays an important role in regulating hematopoiesis, inhibiting proliferation while stimulating differentiation when appropriate. We previously demonstrated that the type III TGF-β receptor (TβRIII, or betaglycan) serves as a novel suppressor of cancer progression in epithelial tumors; however, its role in hematologic malignancies is unknown. Here we demonstrate that TβRIII protein expression is decreased or lost in the majority of human multiple myeloma specimens. Functionally, restoring TβRIII expression in myeloma cells significantly inhibited cell growth, proliferation, and motility, largely independent of its ligand presentation role. In a reciprocal fashion, shRNA-mediated silencing of endogenous TβRIII expression enhanced cell growth, proliferation, and motility. Although apoptosis was not affected, TβRIII inhibited proliferation through induction of the cyclin-dependent kinase inhibitors p21 and p27. TβRIII further regulated myeloma cell adhesion, increasing homotypic myeloma cell adhesion while decreasing myeloma heterotropic adhesion to bone marrow stromal cells. Mechanistically, live cell imaging of myeloma and stroma cell cocultures revealed that TβRIII-mediated inhibition of heterotropic adhesion was associated with decreased duration of myeloma/bone marrow stromal cell interaction. These results suggest that loss of TβRIII expression during multiple myeloma progression contributes to disease progression through its functional effects on increased cell growth, proliferation, motility, and adhesion.     

Expression of secreted frizzled related protein 1, a Wnt antagonist, in brain, kidney, and skeleton is dispensable for normal embryonic development

Secreted frizzled related protein-1 (sFRP1), an antagonist of Wnt signaling, regulates cell proliferation, differentiation and apoptosis and negatively regulates bone formation. The spatial and temporal pattern of endogenous sFRP1 expression and loss-of-function were examined in the sFRP1-LacZ knock-in mouse (sFRP1-/-) during embryonic development and post-natal growth. beta-gal activity representing sFRP1 expression is robust in brain, skeleton, kidney, eye, spleen, abdomen, heart and somites in early embryos, but sFRP1 gene inactivation in these tissues did not compromise normal embryonic and post-natal development. Kidney histology revealed increased numbers of glomeruli in KO mice, observed after 5 years of breeding. In the skeleton, we show sFRP1 expression is found in relation to the mineralizing front of bone tissue during skeletal development from E15.5 to birth. Trabecular bone volume and bone mineral density in the sFRP1-/- mouse compared to WT was slightly increased during post-natal growth. Calvarial osteoblasts from newborn sFRP1-/- mice exhibited a 20% increase in cell proliferation and differentiation at the early stages of osteoblast maturation. sFRP1 expression was observed in osteoclasts, but this did not affect osteoclast number or activity. These findings have identified functions for sFRP1 in kidney and bone that are not redundant with other sFRPs. In summary, the absence of major organ abnormalities, the enhanced bone formation and a normal life span with no detection of spontaneous tumors suggests that targeting sFRP1 can be used as a therapeutic strategy for increasing bone mass in metabolic bone disorders or promoting fracture healing by modulating Wnt signaling.     

Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

Vascular endothelial growth factor (VEGF-A) is a crucial stimulator of vascular cell migration and proliferation. Using bone marrow-derived human adult mesenchymal stem cells (MSCs) that did not express VEGF receptors, we provide evidence that VEGF-A can stimulate platelet-derived growth factor receptors (PDGFRs), thereby regulating MSC migration and proliferation. VEGF-A binds to both PDGFRalpha and PDGFRbeta and induces tyrosine phosphorylation that, when inhibited, results in attenuation of VEGF-A-induced MSC migration and proliferation. This mechanism was also shown to mediate human dermal fibroblast (HDF) migration. VEGF-A/PDGFR signaling has the potential to regulate vascular cell recruitment and proliferation during tissue regeneration and disease.     

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2.Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

A mathematical model for describing the metastasis of cancer in bone tissue

Metastasis is the rapid proliferation of cancer cells (secondary tumour) at a specific place, generally leading to death. This occurs at anatomical parts providing the necessary environment for vascularity, oxygen and food to hide their actions and trigger the rapid growth of cancer. Prostate and breast cancers, for example, use bone marrow for their proliferation. Bone-supporting cancer cells thus adapt to the environment, mimicking the behaviour of genetic and molecular bone cells. Evidence of this has been given in Cecchini et al. (2005, EAU Update Ser. 3:214-226), providing arguments such as how cancer cell growth is so active during bone reabsorption. This paper simulates metastasis activation in bone marrow. A mathematical model has been developed involving the activation of molecules from bone tissue cells, which are necessary for cancer to proliferate. Here, we simulate two forms of secondary tumour growth depending on the type of metastasis: osteosclerosis and osteolysis.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.