Bone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and beta-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3-4 and 10-11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 micrograms/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 micrograms/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day-21 mineralization and only the high dose, 10.0 micrograms/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 microgram/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.
The act of chondrocyte preparation for primary, enchondral, mineralization is associated with a decline in mitochondrial respiration toward the end of the proliferative zone and the hypertrophic zone in the growth plate. Dexamethasone (Dex)-stimulated cultures of rat marrow stroma constitute a differentiation model simulating, in its energy metabolism, chondrocyte mineralization. In this model, early inhibition of succinate dehydrogenase (SDH) enriches the culture with mineralizing cells, whereas levamisole inhibits mineralization. Dex also increases mitochondrial membrane potential in stromal cells, especially on days 7-8 of stimulation. In the present study, suicide inhibition of SDH, by nitropropionic acid (NPA), in Dex-stimulated cells showed a dose-dependent increase in day 21 mineralization; the maximal effect was induced on days 2-4 of stimulation. Mineralization under 2-day-long exposure to NPA showed a similar trend to the previously studied effect of continuous exposure to malonate applied between days 3-11. Unlike malonate, the effect of NPA required its presence in the cultures for only 2 days and resulted in higher mineralization than that seen under 8 days of malonate. NPA delineated a period, days 2/4 to 7/9, in which inhibition of succinate oxidation is necessary to augment mineralization. During this period, NPA also exhibited OPC selection capacity. Early application of levamisole, under conditions previously shown to decrease day 21 mineralization, maintained mitochondrial membrane potential at the beginning of Dex stimulation but decreased or had little effect on it during days 5-10. By contrast, malonate previously found to increase day 21 mineralization decreased the membrane potential at the beginning of Dex stimulation but increased it later on day 7, or during days 5-10. These results indicate that during osteoprogenitor differentiation, before the mineralization stage, a surge in mitochondrial inner membrane potential during late matrix maturation may be a marker that heralds the extracellular matrix mineralization.
Bone marrow stromal cells give rise to osteoprogenitor cell (OPC) colonies, with characteristic mineralized bone nodules in vitro. During differentiation, OPCs in the culture are surrounded by heterogeneous populations of various cell lineages and by different OPC differentiation stages. In the present study, attempts were made to increase the homogeneity of OPCs in culture. The reliance on energy metabolism restricted to glycolysis, which is specific to the premineralizing skeletal cells, was tested as a selectable marker for cells in this stage. Day 12 alkaline phosphatase (ALP) and day 20-21 calcium precipitates were used as early and late OPC differentiation markers. Malonate, a competitive inhibitor of succinate dehydrogenase, was added to the OPC stimulation medium, to interfere with the Krebs cycle-dependent energy metabolism operating in most of the stromal cells. OPCs that entered the stage of energy metabolism restricted to glycolysis were expected to become malonate resistant. Malonate showed dose and time dependence, 10 mM malonate added on day 3, decreased day 12 ALP activity/well to the lowest level. Variations in time and length of exposure to malonate used during the first 12 days of differentiation showed an inverse correlation between specific ALP activity and cell yield. Malonate-treated variations of specific ALP and of cell yield indices were up to 30- to 40-fold larger than variations within day 21 calcium precipitates. Thus, calcifying cells were almost unchanged relatively to noncalcifying cells. These results indicate that malonate-resistant cells are mostly selected, rather than induced, to differentiate by malonate. The results also show that stromal derived OPCs undergo a similar biochemical stage as in chondrocytes.
The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole-sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole-induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture.(ABSTRACT TRUNCATED AT 250 WORDS)
von Recklinghausen neurofibromatosis (NF1) is an autosomal dominant genetic disorder associated with congenital pseudoarthrosis and with short stature. To examine whether the NF1 phenotype includes functional osteogenic defects, embryonic bone-derived cells affected with NF1 were tested in culture for specific alkaline phosphatase (ALP) activity and cell-mediated mineralization and compared with other embryonic bone derived cells. NF1 showed a relatively higher specific ALP activity, which has further increased in response to dexamethasone + beta-glycerophosphate (beta GP) (Dex medium) coordinately with a decrease in cell proliferation. In In the control group, two samples showed increased ALP activity, one showed decreased activity and the forth one did not show any change in ALP. NF1 cells were distinguished from other cells regarding day 21 mineralization, they did not mineralize when cultured with ascorbate alone in the absence of Dex medium, whereas control cells did mineralize. Adding beta GP resulted in mineralization by NF1 cells but less than in other cells. In addition, NF1 cells responded to dexamethasone by increasing the beta GP-induced mineralization, as opposed to cells from other embryonic bones, which either did not respond or have even decreased mineralization under dexamethasone. Upon cis-hydroxyproline exposure, Dex medium has also distinguished NF1 cell ALP activity from that of other cell origins. Inhibition of respiratory complex II by malonate showed that most embryonic bone-derived cells of 12 weeks gestation are malonate resistant; thus, malonate selection was ineffective. This is in contrast to rat marrow stromal cells previously shown to undergo mineralizing cell enrichment in response to malonate. Exposure to levamisole, of Dex-treated cells, at days 0-11 has inhibited day 21 mineralization in all tested cultures in spite of the increase in day 11-specific ALP activity. Both malonate and levamisole did not distinguish NF1 cells from the osteogenic phenotype of other cells. Essentially embryonic bone-derived cells from 12 weeks gestation, cultured in the absence of beta GP, retained their mineralization capacity, which does not increase under dexamethasone, as distinguished from NF1 cells which require beta GP for mineralization and positively respond to dexamethasone. Therefore, bone-derived NF1 cells may be useful for studying the regulation of the mineralization process.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.