Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro.

The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3-E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density-dependent. MC3T3-E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number-response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 10(5) per cm2 once cultures reached steady state, (2) most cells entered the G0/G1 phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density-dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density-dependent growth control in cultures of fibroblasts rather than epithelial cells.

Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro.

Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l-lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l-lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation.

Serial passage of MC3T3-E1 cells down-regulates proliferation during osteogenesis in vitro.

Generally, fibroblast-like cells and other types of human cells have been used to demonstrate the principles of replicative senescence in vitro and in vivo. These cells go through three stages of proliferation, including vigorous proliferation, declining proliferation and quiescence or no proliferation. Any variation of this process occurring in osteoprogenitor cells may offer insight into the mechanism of age-related osteopaenia that predisposes individuals to osteoporosis and bone fractures. We selected MC3T3-E1 cells derived from mouse calvaria to study the mechanism of replicative senescence of pre-osteogenic cells because: (i) these cells constitute a well-known model for studying osteogenesis in vitro; (ii) they undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture; and (iii) they show signs of replicative senescence. These cells were aged by multiple passaging before their use for studying growth kinetics and the effects of population density, effect of extracellular matrix (ECM), size and phases of the cell cycle. Our results show that (i) MC3T3-E1 cells go through the first two stages of proliferation in a manner similar to human cells, but escape the quiescent phase; (ii) the rate of proliferation is similar for low passage (LP) and high passage (HP) cells, but is decreased in very high passage cells (VHP); (iii) growth inhibition is observed using HP cells seeded at high density; (iv) HP ECM stimulates proliferation of both LP and HP cells; (v) a small increase in cell size is observed in HP cells, but no change is seen in the distribution analysis of their cell cycle; (vi) distribution analysis of the cell cycle of VHP cells reveals a decreased and an increased frequency of cells in S and G2 + M phases of their cell cycle, respectively. These results suggest that the mouse MC3T3-E1 cell line exhibits many of the cellular and molecular markers associated with replicative senescence in culture as defined by human cells, such as fibroblast-like cells. Alteration in the sensitivity of MC3T3-E1 cells to intercellular contact and increase in cell size are the primary factors contributing to decreased proliferation of HP cells.

The late effects of selected immunosuppressants on immunocompetence, disease incidence, and mean life-span. III. Disease incidence and life expectancy.

The effect of various immunosuppressive treatments on mean life-span and disease incidence have been studied. Significant life shortening was seen only in mice which recieved X-irradiation early in life and can be ascribed primarily to an increased incidence of certain malignancies. Marginal life shortening was seen in cyclophosphamide-treated animals, however, survival patterns between those and control animals did not differ until 30 months of age and the magnitude of life-shortening never approached that seen in X-irradiated animals. Thymectomy, splenectomy or cortisone treatment did not alter survival. All immunosuppressive treatments enhanced mortality due to non-neoplastic diseases, however, only a small percentage of animals die with these disease entities. With the exception of cortisone all immunosuppressive treatments increased the incidence of neoplastic disease. However, their effects on various neoplastic processes were variable and unpredictable. Four primary patterns in terms of relative immune competence, disease incidence and life expectancy were seen. Thus, immunodepression may of may not correlate with increased disease incidence, which in turn may or may not have a life-shortening effect. These findings are discussed in terms of the marked reduction of both humoral and cell-mediated immunity normally seen in aged mice and the significance of postulated immune surveillance mechanisms to survival.

The late effects of selected immunosuppressants on immunocompetence, disease incidence, and mean life-span. II. Cell-mediated immune activity.

The late effects of various immunosuppressive insults on cell-mediated immunity in mice were studied in an attempt to assess the role of immune surveillance in the aging process. Results were obtained using susceptibility to allogeneic tumor cell challenge, graft-versus-host reaction (GVHR), blastogenic response to PHA, a thymus derived T cell-specific plant mitogen, and cytolytic activity against allogeneic tumor cells as measures of immunologic activity. In vivo studies late in life show that resistance to allogeneic tumor cells is significantly decreased in thymectomized mice, whereas those treated with cortisone, cyclophosphamide and sublethal X-ray remain unchanged. Spleen cells from only the thymectomized and the sublethally irradiated mice show reduced activity in the GVHR. No difference is seen in the activity of bone marrow cells. Results consistent with these findings were obtained in in vitro studies. Thus spleen cells from thymectomized or sublethally irradiated mice show decreased activity is response to PHA, whereas no change is seen in spleen cells from other treated groups. Hence, surgical and physical insults are more likely to induce long-lasting immunosuppression in those immunocompetent tissues whose activity normally diminishes with advancing age. Furthermore, the degree of immunosuppression seen in this study is not of the order of magnitude that one could reasonably predict a significant decrease in mean life-span.

The late effects of selected immunosuppressants on immunocompetence, disease incidence, and mean life-span. I. Humoral immune activity.

The effect of different immunosuppressive treatments during young adulthood or humoral immune competence late in life was determined. It was found that the marked reduction in humoral immune competence in aged mice is further compromised when severe insults are administered early in life. Thus, thymectomy, splenectomy, and sublethal X-irradiation produced lasting immunodepression as measured (1) in situ and (2) by the hemolysin, direct and indirect plaque forming cell responses of adoptively transferred spleen cells. In contrast, treatment with cyclophosphamide and cortisone acetate were without effect, indicating that drug-damaged cells of the immune system were replaced by competent cells during the course of time. Decrease in immune competence of aged thymectomized animals could not be correlated with a decrease in numbers of theta-bearing T or immunoglobulin receptor-bearing B lymphocytes. The significance of the observed unequal effects of these immunosuppressants on immune competence, as they relate to disease incidence and life expectancy, are dealt with in the third paper in this series.

A method to assess the proliferative activity of small numbers of murine peripheral blood mononuclear cells.

An economical, reproducible method was developed to assess the mitogenic response of small numbers of murine peripheral blood mononuclear cells (PBMC, 10(3)/ml) to phytohemagglutinin (PHA) stimulation in the presence of feeder layer cells (FLC) and interleukin-2 (IL-2). A linear cell dose-mitogenic response relationship was then demonstrated between the activity of PBMC and spleen cells of individual mice (slope = 1.054 +/- 0.037; r = 0.989 +/- 0.003). This would suggest that mice can be used for longitudinal studies because small blood volumes can be obtained frequently over a period of time from individual mice and the PBMC can be assessed for their T cell proliferative activity.

Skeletal muscle-smooth muscle interaction: an unusual myoelastic system.

The serratus superficialis metapatagialis (SSM) of pigeons is a skeletal muscle with unusual properties. It lies between the ribs and the trailing edge of the wing, where it is attached to the skin by a system of smooth muscles having elastic tendons. Wing movements during flight induce marked changes in this muscle's length. The SSM inserts onto the deep fascia, and at its termination the skeletal muscle contains large numbers of microtubules. Many myofibrils attach to leptomeric organelles, which then attach to the terminal end of the skeletal muscle fiber. The deep fascia next connects to the dermis of the skin by bundles of smooth muscles that have elastic tendons at both ends. This system allows large movements of the muscle while preventing its fibers from overstretching. The movements and presumed forces acting at this muscle make the presence of sensory receptors such as muscle spindles unlikely. Spindles are absent in this muscle.

The Anchorage Skid Row.

A survey of residents of the Anchorage Skid Row identified four subgroups: highly mobile workers, working residents, semi-employed residents and the homeless unemployed.

The possible importance of wintering yards in the transmission of Parelaphostrongylus tenuis to white-tailed deer and moose.

Terrestrial gastropods were collected, 15 June to 25 November 1994, from beneath cardboard sheets on deer range in northeastern Minnesota (USA) and examined individually for larvae of Parelaphostrongylus tenuis, the meningeal worm of white-tailed deer (Odocoileus virginianus). Overall, 10 (0.08%) of 12,096 snails and slugs were infected with a mean (+/- SD) of 3.2 +/- 2.5 P. tenuis larvae. The prevalence of infection in gastropods was greater in a traditional deer wintering yard (seven of 4,401, 0.16%), where deer aggregated for almost 5 months at a density of 50/km2, than on summer range (three of 7,695, 0.04%) where they occurred at 4/km2. Despite relatively low densities of infected gastropods, their ingestion purely by chance remains a tenable explanation for the high prevalence of P. tenuis infection observed in white-tailed deer.

Infrapopulation dynamics of Parelaphostrongylus tenuis in white-tailed deer.

The prevalence and intensity of Parelaphostrongylus tenuis was determined by examining the head and a fecal sample from each of 379 white-tailed deer (Odocoileus virginianus) of known age that had been killed by vehicles in northeastern Minnesota (USA), November 1991 to May 1993. Small numbers of adult worms (mean +/- SD, 3.2 +/- 2.2; maximum, 13) were found in the cranium of 311 (82%); but over a third (118 of 311) of the infected deer were not passing larvae in their feces. Most occult infections were sterile because only one sex of the parasite was present. Adult P. tenuis were not found in the vertebral canal of deer. Prevalence of adult worms and larvae was lower in fawns (68% and 35%, respectively) than in older age classes of deer (89% and 63%, respectively). Forty-three of 45 deer between 7 and 15 yr old were infected. Mean (+/- SD) intensity of adult worms was lower in fawns (2.7 +/- 1.8) and yearlings (3.0 +/- 2.1) than in deer 7 to 15 yr (4.1 +/- 2.5). Conversely, the mean (+/- SD) number of larvae in feces was higher in fawns (103 +/- 119 larvae/g) than in adults 2 to 6 yr old (36.2 +/- 46 larvae/g) and 7 to 15 yr old (35.6 +/- 60 larvae/g). Mean (+/- SD) fecundity of female worms was greatest in fawns (51.6 +/- 64.8 larvae/g of feces/female worm). Deer of all ages passed more lavae in the spring. Deer from an area where year-round density was 30 deer/km2 had a mean (+/- SD) of 3.5 (+/- 1.8) adult worms; deer from the study area, with a summer density 2 deer/km2, had 3.2 (+/- 2.2) worms; however, deer at the greater density passed a greater mean number of larvae (93.8 and 57.1 larvae/g, respectively). Based on our results we propose that P. tenuis is a long-lived parasite and that most deer become infected in their first or second summer of life, and acquire few additional worms thereafter.

A T cell model for age-related loss of homeostasis: identification of the cell type responsible for age-related delay in recovery of the PHA-induced mitogenic response from radiation injury.

Immunologic recovery from radiation injury was used to assess age-related loss of immunologic homeostasis. Mice were exposed to 500 rad to determine whether macrophages, T helper cells and/or T responder cells were responsible for the age-related delay in recovery of PHA-induced mitogenesis. Recovery of macrophages was determined by facilitation of macrophage-deficient cultures, recovery of T helper cells by interleukin 2 production, and recovery of T responder cells by incorporation of tritiated thymidine. The results showed that in old mice macrophages are resistant to radiation and aging, T helper cells recover completely from radiation, and recovery is incomplete for T responder cells.

Differential effects of age on mitotically active and inactive bone marrow stem cells and splenic T cells in mice.

Young and old BALB/c mice and thymectomized young mice were subjected to continuous exposure of 6-thioguanine (6-TG), and the numbers of their bone marrow spleen colony-forming units (CFU-S) and in vitro culture colony-forming units (CFU-C) and the number and mitogen-induced proliferative activity of their splenic T cells were determined at various time intervals. The results indicated that (a) old mice have seven times more 6-TG-resistant (6-TGr) CFU-S than young mice, (b) the mitogen-induced proliferative activity of 6-TGr T cells is comparable to that of 6-TG-sensitive (6-TGs) T cells of both young and old mice, and (c) 6-TGr CFU-S and T cells are resistant to 6-TG because they are mitotically inactive and not because they are drug-resistant mutants.

Comparison of the radiosensitivity of interleukin-2 production between species, between tissues, and between young and old individuals.

The radiosensitivity of interleukin-2 (IL-2) production was assessed of (a) peripheral blood mononuclear cells (PBMC) of young humans, dogs, and mice (C57BL/6); (b) PBMC and splenic cells of young mice; and (c) PBMC of young and old humans and the splenic cells of young and old mice. The results indicate that (a) large differences in radiosensitivity exist between the PBMC of humans, dogs, and mice (e.g., the radiation doses which resulted in 37% remaining IL-2 activity (D37) of human, dog, and mouse PBMC were 3771, greater than 10,000, and 1398 rads, respectively); (b) only a small difference exists between the PBMC and splenic cells of mice; and (c) no difference exists between the PBMC of young and old humans and between splenic cells of young and old mice. Topological abnormalities, as judged by scanning electron microscopic analysis, could not be detected in dog PBMC after their exposure to 1800 rads, but could be detected in mouse PBMC after their exposure to 400 rads.

Immune potentiation after fractionated exposure to very low doses of ionizing radiation and/or caloric restriction in autoimmune-prone and normal C57Bl/6 mice.

Very low doses of ionizing radiation can enhance immune responsiveness and extend life span in normal mice. Total lymphoid irradiation at relatively high doses of radiation can retard autoimmune disease in genetically susceptible mice, but may impair immune function. In order to determine whether fractionated low dose exposure would enhance immune response and retard lymphadenopathy in autoimmune-prone mice, groups of C57B1/6 lpr/lpr mice were sham irradiated, exposed 5 days/week for 4 weeks to 0.04 Gy/day (0.8 Gy cumulative dose), or to 0.1 Gy/day (2.0 Gy cumulative dose). After the radiation protocol, the mice were evaluated for splenic T cell proliferative capacity, T cell subset distribution, and total spleen cell numbers. The independent and additive effect of caloric restriction was additionally assessed since this intervention has been shown to increase immune responsiveness and retard disease progression in autoimmune-prone mice. The congenic C57B1/6 +/+ immunologically normal strain was evaluated in parallel as congenic control. The results indicated that mitogen-stimulated proliferation was up-regulated in both strains of mice after exposure to 0.04 Gy/day. The proliferative capacity was additively enhanced when radiation at this dose level was combined with caloric restriction. Exposure to 0.1 Gy/day resulted in further augmentation of proliferative response in the lpr/lpr mice, but was depressive in the +/+ mice. Although the proportions of the various T cell subpopulations were altered in both strains after exposure to LDR, the specific subset alterations were different within each strain. Additional experiments were subsequently performed to assess whether the thymus is required for LDR-induced immune potentiation. Thymectomy completely abrogated the LDR effect in the +/+ mice, suggesting that thymic processing and/or trafficking is adaptively altered with LDR in this strain. In contrast, augmentation in proliferative activity after LDR in the lpr/lpr mice was maintained, although attenuated, in thymectomized mice. Taken together, these results indicate that fractionated exposure to LDR augments the proliferative response of spleen cells in both autoimmune-prone and immunologically normal mice; however, within each strain, the mechanisms appear to be different.

Role of T cells and adherent cells in age-related decline in murine interleukin 2 production.

Spleen and lymph node cells from young (3 to 4 months) and old (24 to 36 months) mice of two inbred lines and one hybrid stock were assessed for the effect of age on IL 2 production. The results revealed that the production of IL 2 by old spleen and lymph node cells is reduced to 42% that of young cells. Cell counting, IL 2 inhibition and absorption, and cell mixture studies were then performed to determine the basis for the reduced IL 2 production. Cell counting studies revealed that the reduced IL 2 production by old cells cannot be caused by a decrease with age in the total number of Lyt 1+ T helper cells or by an increase with age in Lyt 2+ T suppressor cells. IL 2 inhibition, IL 2 absorption, and young-old spleen cells mixture studies revealed that the decrease in the level of IL 2 activity with age is caused neither by the formation of excessive amounts of soluble IL 2 inhibitors, by the generation of excessive numbers of IL 2 responsive blast cells, nor by an increase in suppressor cells. Further cell mixture studies revealed a decrease in IL 2 production by young T cells in presence of old adherent cells, which did not approach that of the old T cell-old adherent cell mixture, and an increase in IL 2 production by old T cells in the presence of young adherent cells, which did not approach that of the young T cell-young adherent cell mixture. These results would indicate that the decline in activity of both the T cells and adherent cells involved in IL 2 production are responsible for the reduction. Finally, it was found that the reduced proliferative activity of Con A-stimulated old spleen cells can be enhanced to the same extent as that of young spleen cells by exposing them to exogenous IL 2.

Transforming growth factor-beta, osteogenin, and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells.

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues-processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-beta) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-beta and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-beta for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by 3H-thymidine (3H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of 3H-TdR at doses of 50-800 ng/ml. Similarly, TGF-beta inhibited uptake of 3H-TdR at doses of 2-32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 microgram/ml) or higher dose of TGF-beta (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-beta on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-beta (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-beta on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-beta at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC50) calculated for BMP-2 and TGF-beta at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-beta. The observation may suggest that TGF-beta may have effects upon cytoskeletal elements in osseous tissues.