Isomer-Specific Effects of Conjugated Linoleic Acid on Mineralized Bone Nodule Formation from Human Osteoblast-LikeCells

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Ilana Platt*,
Leticia G. Rao and
Ahmed El-Sohemy*1

^* Department of Nutritional Sciences, University of Toronto, Toronto, Ontario M5S 3E2, Canada; Calcium Research Laboratory, Division of Endocrinology and Metabolism, St. Michael’s Hospital and Department of Medicine, University of Toronto, Toronto, Ontario M5B 1A6, Canada

¹ Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Fitzgerald Building, 150 College Street, Toronto, Ontario, Canada, M5S 3E2. E-mail: a.el.sohemy{at}utoronto.ca

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 µM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formationof mineralized bone nodules using bone cells of human origin,and provide evidence for isomer-specific effects of CLA on bonehealth.

Keywords: conjugated linoleic acid, osteoporosis, SaOS-2 cells, bone, alkaline phosphatase

Osteoporosis is a major cause of morbidity and mortality; it is characterized by decreased bone mass, increased bone fragility, and increased susceptibility to bone fractures (1). Attaining maximal peak bone mass through proper nutrition and lifestyle choices is an important strategy for preventing osteoporosis later in life (2, 3). Consumption of dairy products is associated with improved bone health in a variety of different populations (4). Although calcium and vitamin D are major nutrients in dairy products that promote bone health, other constituents, such as conjugated linoleic acid (CLA), may also be important (5–14). CLA refers to a group of positional and geometric isomers of linoleic acid that are produced by the bacterial biohydrogenation of linoleic acid via an enzymatic isomerase reaction (15). CLA has diverse physiological effects (16, 17) and is found naturally in foods from ruminant animals, predominantly as the 9cis,11trans isomer (18). Synthetic CLA preparations are abundant in both the 9cis,11trans and 10trans,12cis isomers. These isomers have both been shown to be biologically active (19) and are known to have different physiological effects. Several studies have examined the effects of CLA on the development of chronic diseases such as cancer, diabetes, and cardiovascular disease (20–24), and there is growing evidence that CLA may also affect bone health (5–14, 25–32).

Several studies using experimental animals have shown equivocal effects of CLA on bone formation (5–14, 25, 26, 31, 32). Rodent studies have shown that dietary CLA supplementation increases body ash (10, 11, 31, 32), suggesting a favorable effect of CLA on bone formation. However, other animal studies showed no effect of CLA on bone mineral content (7, 25). CLA has also been shown to increase (8, 9) or decrease bone formation rates (12) and to have variable effects on markers of bone formation and resorption (13, 14). In rodent calvarial cells, CLA alters protein levels of the osteoblast-specific transcription factor core binding factor alpha 1 (Cbfa1) and increases osteoblast differentiation (26).

Only a few studies have examined the effects of CLA on human bone physiology (27–30). Intake of dietary CLA has been shown to be positively associated with bone mineral density in postmenopausal women (27), while supplemental CLA has been shown to have no effect on bone mineral density in men (28, 29). Using bone cells of human origin, individual and mixed isomers of CLA have been recently shown to have variable stimulatory effects on alkaline phosphatase (ALP) activity (30), a marker of osteoblast differentiation. Osteoblasts are mononucleated bone-forming cells that actively synthesize and secrete an organic bone matrix which undergoes rapid mineralization (33). ALP regulates bone matrix mineralization by hydrolyzing phosphate esters to increase the local phosphate concentration necessary for bone formation (34, 35). Although CLA has been shown to variablyincrease ALP activity, the effects of individual and mixed CLAisomers on mineralized bone nodule formation using bone cellsof human origin are not known. The objective of the presentstudy is to determine the direct effects of the individual andmixed isomers of CLA on osteoblastic bone formation and differentiationusing the human osteoblast-like SaOS-2 cell line.

Materials.

SaOS-2 cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). The 9cis,11trans and 10trans,12cis (>98% pure) isomers of CLA were purchased from Matreya (Pleasant Gap, PA). Mixed CLA isomers (~41% 9cis,11trans/ 9trans,11cis; ~44% 10trans,12cis; ~10% 10cis,12trans; ~5% 9trans,11trans, 10trans,12trans and 9cis,11cis) were purchased from Nu-Chek Prep (Elysian, MN).The Bio-Rad Protein Assay reagent was purchased from Bio-Rad(Mississauga, Canada). Fetal bovine serum (FBS) was purchasedfrom Cansera (Etobicoke, Canada). Antibiotic-antimycotic waspurchased from GIBCO (Burlington, Canada). Ham’s F12 andPBS were purchased from Central Technical Services, Universityof Toronto. Alkaline phosphatase assay reagent was purchasedfrom Teco Diagnostics (Anaheim, CA). β-Glycerophosphatewas purchased from ICN Pharmaceuticals (Costa Mesa, CA). Allother chemicals were purchased from Sigma Chemical Co. (St.Louis, MO).

Cell Culture.

Cells were grown in 24-well dishes at a density of 5 x 10³ cells per well in Ham’s F12 medium, containing 10% FBS, 28 mM HEPES buffer (pH 7.35), 1.4 mM CaCl₂, 2 mM glutamine, 1% antibiotic-antimycotic solution, and 50 µg/ml ascorbic acid. The medium was changed every 2–3 days. On the eighth day and at every medium change thereafter, the medium was replaced with medium containing 10 mM β-glycerophosphate as well as varying concentrations (25, 50, and 100 µM) of mixed, 9cis,11trans, or 10trans,12cisCLA or vehicle (0.1% ethanol). CLA was added to the medium byfirst dissolving it in ethanol, which was then added to FBSsupplemented with 1 g/l of fatty acid–free bovine serumalbumin (BSA). The final concentration of ethanol and BSA ineach well was 0.1% and 1 g/l, respectively.

Determination and Quantification of Mineralized Bone Nodules.

After 21 days in culture, the cells were washed twice with PBS, fixed overnight with 4% p-formaldehyde, and stained in situ using the standard von Kossa technique (36, 37). Briefly, thenodules were stained with 5% silver nitrate under UV light for30 mins, background color was removed with 5% sodium thiosulfate,and the cells were maintained in 50% glycerol. The mineralizednodule areas and numbers were quantified using a FluorChem imagingsystem.

Alkaline Phosphatase (ALP) Activity.

After 2, 4, and 10 days of treatment, the cells were washed twice with 50 mM Tris-HCl (pH 7.35). Cells were lysed in buffer containing 0.05% Triton-X-100 in 50 mM Tris-HCl (pH 7.35) following one freeze-thaw cycle. ALP activity was determined according to the method of Lowry (38) in cell sonicates using a commercially available kit from Teco Diagnostics. ALP activity was determined as the amount of p-nitrophenol produced from p-nitrophenyl phosphate over time at 37°C, standardized for protein concentration. The absorbance of p-nitrophenol was measured at 405 nm using a Fusion plate reader every 2 mins for 15 readings. Protein concentration of the cell sonicates was determined using the Bio-Rad Protein Assay reagent. The ALP activity of each sample was normalized to its protein concentration, calculated as units/g protein, and expressed as percent of control. One unit of ALP activity is defined as the amount of enzyme that catalyzes the conversion of one micromole of p-nitrophenyl phosphate to p-nitrophenoland phosphate per minute.

Statistical Analyses.

Results are expressed as mean ± SEM with at least three replicates in each group. Differences were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. The effects of each CLA treatment were assessed on separate plates, with each plate having its own controls. The effects of CLA on mineralized bone nodule formation and ALP activity were analyzed relative to the controls on the corresponding plates. P values <0.05 were considered significant. All datawere analyzed using GraphPad Prism Software, Version 4.00.

The Effects of CLA on the Formation of Mineralized Bone Nodules.

Mineralized bone nodules formed from SaOS-2 cells were first observed after 10 days of treatment (Day 18). The nodules appeared three-dimensional in appearance under a phase contrast microscope and grew in size until the end of the culture period (Day 21). After von Kossa staining, the mineralized bone nodules could be observed with the naked eye as dark spots. Mixed and 9cis,11trans CLA increased the formation of mineralized bone nodules from these cells (Fig. 1). Mixed CLA isomers at concentrations of 50 and 100 µM increased both the number and size of mineralized bone nodules formed, as quantified using a FluorChem imaging system. Compared to control cells, 50–100 µM mixed CLA increased the area of nodules by approximately 3- to 4.5-fold (Fig. 2A), while 100 µM increased the number of nodules by approximately 5-fold (Fig. 2B). The 9cis,11trans isomer of CLA increased both the number and size of mineralized bone nodules formed. Compared to control cells, 25–100 µM 9cis,11trans CLA increased the area of nodules formed by approximately 2- to 5-fold (Fig. 2C), while 25–100 µM of the 9cis,11trans isomer of CLA increased the number of nodules by approximately 4- to 11-fold (Fig. 2D). In contrast, 10trans,12cis CLA didnot appear to increase the formation of mineralized bone nodulesat any of the concentrations tested (Fig. 2E and F).

Effects of CLA on ALP Activity.

The effect of increasing concentrations of mixed, 9cis,11trans, and 10trans,12cis CLA on ALP activity in SaOS-2 cells is shown in Figure 3. As shown in Figure 3A, 25 µM mixed CLA increased ALP activity after 2 days of treatment by approximately 40%, while 50 and 100 µM mixed CLA increased ALP activity after 4 days of treatment by approximately 110% and 90%, respectively. As shown in Figure 3B, 100 µM 9cis,11trans CLA increased ALP activity at all time points by approximately 40%. After 4 days of treatment, 25 µM increased ALP activity by approximately 40%, while 50 µM increased ALP activity by approximately 100%. As shown in Figure 3C, 10trans,12cis CLA increased ALP activity at a later time in culture beginning at 4 days of treatment. At this time point, 50 µM 10trans,12cis CLA increased ALP activity by approximately 50%, while 100 µM increased ALP activity approximately 65%, compared to control cells. After 10 days of treatment, 100 µM increased ALP activity byapproximately 40%.

In the present study we have shown that 9cis,11trans CLA, the most abundant isomer found in food products from ruminant animals, increases the formation of mineralized bone nodules from human osteoblast-like cells. This effect was accompanied by an increase in osteoblast differentiation as measured by ALP activity. Although mixed CLA isomers also increased both the formation of mineralized bone nodules and ALP activity, these effects were likely due to the 9cis,11trans isomer, because the effects were intermediate between those of the 9cis,11trans and 10trans,12cis isomers. These results suggest a potential beneficial effect of the 9cis,11trans isomer of CLA on bone health in humans. To our knowledge, this is the first study to examine the effect of CLA on mineralized bone nodule formation in vitro, although a few studies have examined its effects on ALP activity (8, 26, 30). Our results are consistent with the findings of another study which demonstrated that individual and mixed CLA isomers variably increase ALP activity in human osteoblast-like cells (30). Similarly, CLA has also been shown to increase ALP activity in murine calvarial cells; however, this effect was observed with the 9cis,11trans but not the 10trans,12cis isomer (26). In murine calvarial cells, treatment with 9cis,11trans CLA for 7 days increased, while 14-day treatment decreased, the expression of Cbfa1 in murine calvarial cells (26). Cbfa1 expression is required for early osteoblast differentiation but negatively regulates late-stage osteoblast differentiation (39), suggesting that 9cis,11trans CLA may regulate bone formation through Cbfa1 expression. This hypothesis is consistent with our results showing that 9cis,11transCLA increases early osteoblast differentiation as assessed byan increase in ALP activity.

There are several possible explanations for the isomer-specific effects of CLA that we observed on mineralized bone nodule formation. One rodent study demonstrated that 9cis,11trans and 10trans,12cis CLA are preferentially retained by different organs and tissues. Compared to 10trans,12cis CLA, the 9cis,11trans isomer is preferentially retained by bone tissues, and of the tissues analyzed, bone appears to retain the highest levels of CLA (40). This suggests that 9cis,11trans CLA may affect bone formation through direct effects on bone physiology. The stimulatory effect of 9cis,11trans CLA on mineralized bone nodule formation could also result from favourable alterations in prostaglandin E₂ (PGE₂) biosynthesis in these cells. PGE₂ is a major regulator of bone metabolism (41, 42). At high concentrations (10^–6 M) in vitro PGE₂ inhibits bone formation (41) and stimulates bone resorption (42), while at low concentrations (10^–10–10^–8 M) PGE₂ stimulates bone formation (41). It was recently shown that relative to vehicle, mixed and 10trans,12cis CLA decrease PGE₂ biosynthesis in SaOS-2 cells and human osteoblastic MG-63 cells to a greater extent than 9cis,11trans CLA (30). Thus, 10trans,12cis CLA may reduce PGE₂ levels below that which is required for bone formation, whereas 9cis,11trans CLA may reduce PGE₂ levels to within the range that stimulates bone formation. It is also possible that the 9cis,11trans CLA increases mineralized bone nodule formation by decreasing 3-hydroxy-3-glutaryl CoA (HMG-CoA) reductase. Statins, which inhibit HMG-CoA reductase, have been shown to increase bone formation in vitro and in vivo by stimulating BMP-2 expression (43).

Several studies using experimental animals have shown equivocal effects of mixed CLA isomers on bone formation. Mice fed 5 g/kg dietary CLA had higher levels of whole-body ash (11), and chickens had higher tibial bone ash (10), suggesting that CLA may enhance bone formation. Chicks fed 5.2 g/kg butterfat, a rich source of CLA, were found to have 60% higher bone formation rates (9). Furthermore, a diet containing 5 g/kg CLA increased the tibial bone formation rate in rats (8). The studies showing a null or negative effect of dietary CLA on bone physiology provided CLA as 10 g/kg of the diet (7, 12, 13). At this concentration, CLA did not alter markers of bone formation (osteocalcin) or resorption (urinary pyridinium cross-links) in Wistar rats (13), but it reduced the tibial mineral apposition and bone formation rates of weanling Sprague-Dawley rats without affecting bone mineral content (12). In weanling rats with polycystic kidney disease, which is characterized by elevated tissue levels of arachidonic acid, elevated parathyroid hormone, and reduced bone mass, 10 g/kg CLA had no effect on bone mineral density (7).

All of the above studies were conducted using young, growing male animals. Although there may be important sex differences in lipid metabolism and bone physiology, few studies have examined the effect of CLA on bone in aging female animals. Because the rate of bone loss is accelerated after menopause, ovariectomized animals are useful models for studying the effect of CLA on bone resorption. For example, ovariectomized Fisher rats fed 5–10 g/kg CLA displayed less bone resorption compared to control rats (14). In nonovariectomized female pigs, however, 0.7–5.5 g/kg dietary CLA did not alter bone mineral content (25). Inconsistencies between animal studies may be due to a number of differences between studies, including the sex, species, or strain of the animal model used or differences in the dose or duration of CLA treatment. In addition, commercially prepared diets may contain varying concentrations of 9cis,11trans and10-trans,12cis,as well as other isomers of CLA.

To our knowledge, the present study is the first to examine the direct effects of CLA on mineralized bone nodule formation using bone cells of human origin. We demonstrate that 9cis,11trans CLA greatly increases the formation of mineralized bone nodules in long-term cultures of human SaOS-2 cells. Because bone formation is tightly coupled with resorption in healthy bone, ongoing studies aim to determine whether CLA affects bone resorption by modulating osteoclast formation and function in vitro. Although isolated bone cell cultures are useful models to directly test the effects of purified CLA on bone formation and resorption, they do not account for differences in CLA metabolism or bone physiology, which are both affected by a number of factors, including age, sex, diet, and genetic variability. Findings from this study warrant further investigation of the effects of the naturally occurring 9cis,11trans isomer of CLA on boneformation in humans.

This research was supported by the Dairy Farmers of Canada andthe Natural Sciences and Engineering Research Council of Canada.

Received for publication March 25, 2006.

Accepted for publication July 3, 2006.

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This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Platt, I. Articles by El-Sohemy, A. Search for Related Content Social Bookmarking What’s this?