Glucocorticoid steroids are commonly used to treat a variety of diseases. It is well recognized that osteonecrosis (ON) and bone loss are the major adverse effects of corticosteroid use. Although corticosteroid-induced ON is suggested to be associated with ischemia of bone, the precise pathogenesis of ON remains unknown.1, 2 Corticosteroid-induced adipogenesis may play a role in the development of ON.3, 4 A bone marrow derived mesenchymal cell line, D1, previously isolated from Balb/c mice, in our laboratory can differentiate into either osteoblasts or adipocytes depending on culture conditions.3, 4 We have previously demonstrated that dexamethasone (DEX) could stimulate D1 cell differentiation into adipocytes while suppressing cell differentiation into osteoblasts.5, 6 These findings support the hypothesis that corticosteroid might alter the differentiating ability of progenitor cells in bone marrow and thus favor induction of ON.
The role of oxidative stress in corticosteroid-induced ON has drawn increasing attention. Oxidative injury was present in the bone shortly after administration of corticosteroid and before development of ON.7 When corticosteroids were administered in a rabbit model, the presence of DNA oxidation injury was confirmed in the early period of corticosteroid treatment.8 The presence of oxidation in bone has been proven by the finding of anti-8-hydroxydeoxyguahosine (anti-8-OHdG) antibody expression, a marker of oxidative stress.9 Moreover, it has also been reported that administration of the pro-oxidant buthionine sulfoximine (BSO) induces the development of ON in rats, together with a deletion of blood reduced glutathione (GSH).10 These findings implicate that oxidative stress is involved in the pathogenesis of ON.11 This is further evidenced by the fact that a well-recognized antioxidant, vitamin E (α-tocopherol) is capable of reducing the incidence of corticosteroid-induced ON in an animal model.12 In another study, the differentiation of stem cells to osteoblasts was enhanced by decreasing the oxidative stress and thus further strengthens the importance of oxidative stress in the regulation of adipogenesis and osteogenesis.13
Due to its unique spherical structure with 30 carbon double bonds, fullerene C60 can react easily with free radicals.14 Therefore it is a very efficient free radical scavenger,15 which labels this molecule as a “radical sponge.” Because pristine C60 is soluble in only a limited number of solvents, such as toluene or dichlorobenzene, water-soluble C60 derivatives have been synthesized and used as free-radical scavengers in biological systems.16, 17 Fullerol (polyhydrolated fullerene, also called fullerenol) is the most extensively studied water soluble fullerene derivative and has been demonstrated to act as a potent antioxidant that protects a variety of tissues and cells from oxidative damage.18–21
The long-term goal of our project is designed to answer two questions. First, is oxidative stress involved in adipogenic differentiation in mesenchymal stem cells in the presence of corticosteroids? Second, if yes, can the antioxidant fullerol decrease adipogenesis and increase osteogenesis? In this study, we examined the effect of fullerol on D1 cell line treated by DEX in vitro. It was found that the treatment of D1 cells with fullerol showed an inhibitory and stimulating effect on adipogenesis and osteogenesis, respectively. Furthermore, fullerol was found to antagonize the overloaded oxidative stress and impairment of gene expression of the antioxidative enzymes, catalase and superoxide dismutase (SOD) caused by DEX.
MATERIALS AND METHODS
Pluripotent mesenchymal cells, D1, which were cloned from Balb/c mouse bone marrow cells, were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, VT), 50 µg/ml sodium ascorbate, and 100 IU/ml penicillin G and 100 µg streptomycin per ml of culture media, in a humidified atmosphere of 5% carbon dioxide at 37°C. For all experiments, cells between passages 4 and 6 were seeded at a density of 5 × 103 cells per cm2 and the experiments were started when the cells reached 80% confluence. The culture medium was replaced every 3 days.
D1 cells in a 12- or 24-well plate were divided into five groups: (1) D1 cells; (2) D1 cells with 10 µM DEX (Sigma Chemical Company, St. Louis, MO); (3) D1 cells with 10 µM DEX and 10 µM GSH (ACROS, ORGANICS, Morris Plains, NJ); (4) D1 cells with 10 µM DEX and 0.1 µM fullerol (formula as C60(OH)22–24(ONa)6–8, MER, Tuscon, AZ); (5) D1 cells with 10 µM DEX and 1.0 µM fullerol. For fullerol preparation, a stock solution of 1 mM was made in pH 10.2 buffer (0.084 g NaHCO3/0.106 g Na2CO3 in 10 ml water). It was then diluted to different concentrations with culture medium and sterilized with a 0.22 µm filter membrane. Cells were treated for up to 21 days.
Oil Red O Staining
Lipid droplets within cells were assessed by staining with Oil Red O (Sigma-Aldrich Company, St. Louis, MO) at day 21. Briefly, cells were washed twice with PBS, fixed with 10% formaldehyde for 30 min at room temperature, and washed twice with distilled water and once with 70% isopropanol. Cells were then stained for 1 h at room temperature with filtered Oil Red O at a ratio of 60% Oil Red O stock solution (0.5% wt/vol in isopropanol) to 40% distilled water. Cells were washed twice with distilled water, twice with PBS, and examined under a light microscope. For quantitative analysis, Oil Red O was extracted with isopropanol, and optical density of each sample was determined at 490 nm on a microplate reader (Eppendorf, Hamburg, Germany).22
Gene Expression Analysis
Total RNA was extracted and purified using an RNeasy kit (QIAGEN Sciences, Valencia, CA) according to the protocol provided by the manufacturer. The RNase-Free DNase is used to digest DNA during RNA purification. The purified RNA was stored at −80°C. The yield of RNA was determined by measuring absorbance at 260 nm.23 Synthesis of cDNA from total RNA and the subsequent real-time PCR was carried out by using the iscript™ cDNA synthesis kit and the iQ™ SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA), respectively. The target genes included adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ) and adipocyte protein 2 (aP2), osteogenic markers runt-related transcription factor 2 (Runx2) and osteocalcin, antioxidative enzymes SOD and catalase. Gene of 18s ribosomal RNA was used as an internal control. The primer sequences are listed in Table 1.
Table 1. The Oligonucleotide Primers Used for Real-Time RT-PCR Analysis of Gene Expression
After removal of culture medium, cells were detached by trypsin and collected by centrifugation. Cells were resuspended with PBS and incubated with 10 µM CM-H2DCFDA (Invitrogen, Eugene, OR) at 37°C for 30 min in the dark to load the dye. Then cells were washed twice with PBS and the fluorescent intensity was measured on a flow cytometer.
Data are expressed as mean ± SD. Statistically significant differences between two groups were determined using two-tailed Student's t-tests. The statistical significance was defined by p < 0.05.
Fullerol Inhibits Adipogenesis in D1 Cells
At day 21, adipogenic changes were not found in D1 cells in the absence of DEX (Fig. 1A). Upon treatment with 10 µM DEX, D1 cells containing lipid vesicles were distinguishable clearly from the surrounding cells under a microscope (Fig. 1B). Treatment of D1 cells with 10 µM GSH or 0.1 µM fullerol or 1.0 µM fullerol, which was added to culture simultaneously with 10 µM DEX, significantly decreased the appearance of fat cells (Fig. 1C–F).
Real-time PCR analysis was also performed to determine the effect of fullerol on expression of two adipogenic markers aP2 and PPARγ mRNA in D1 cells at day 7 (Fig. 2A,B). There were significantly increased levels of aP2 and PPARγ mRNAs after DEX treatment (p < 0.01). Compared with the cells treated with DEX alone, both aP2 and PPARγ mRNA levels decreased remarkably in cells treated with a combination of DEX and GSH (p < 0.05) or DEX and fullerol (p < 0.01). With treatment of 1.0 µM fullerol, PPARγ mRNA returned to the control level, while aP2 mRNA did not. Noticeably, fullerol at 1.0 µM was demonstrated to have greater activity than 10 µM GSH or 0.1 µM fullerol.
Fullerol Inhibits Intracellular ROS Level and Promotes Gene Expression of Antioxidative Enzymes
To elucidate the effect of fullerol on DEX-induced oxidative stress, the level of ROS production was measured by staining the cells at day 14 with a fluorescent probe, CM-H2DCFDA. As shown in Figure 3A–F, the percentage of positively stained cells increased from 91.0% (control, A) to 94.7% in DEX treated cells (B). D1 cells treated with GSH (C) or fullerol (D) in addition to DEX attenuated this trend of change. The lowest percentage of positive cells was found in the presence of 1.0 µM fullerol (E). By performing real-time PCR analysis, we further investigated the effect of fullerol on SOD and catalase mRNA levels in D1 cells at day 7 (Fig. 4A,B). In the presence of 10 µM DEX, both SOD and catalase mRNA levels were lower than the untreated control cells (p < 0.05 for SOD and p < 0.01 for catalase). Combined treatment of DEX with 1.0 µM fullerol caused a significant increase in mRNA level of either SOD or catalase, compared to DEX treatment alone (p < 0.05). However, combined treatment of DEX with 0.1 µM fullerol or 10 µM GSH markedly increased mRNA level of SOD (p < 0.05), but not catalase.
Fullerol Promotes Gene Expression of Osteogenic Markers
We determined gene expression of two specific osteogenic markers Runx2 and osteocalcin in D1 cells at day 7 (Fig. 5A,B). While cells were treated with 10 µM DEX, expression of both genes decreased remarkably, compared to the untreated cells (p < 0.01 for Runx2 and p < 0.05 for osteocalcin). Treatment with either 0.1 or 1.0 µM fullerol efficiently restored the impaired expression of both genes. Treatment of 10 µM GSH was also beneficial, but less effective as compared to fullerol.
In this study, we have assessed the influence of a unique antioxidant, fullerol on the adipogenesis and osteogenesis in the presence of DEX by determining lipid droplet formation, specific gene expression and cellular ROS levels. Experimental data obtained from Oil Red O staining, real-time RT-PCR and flow cytometry demonstrated that DEX induces adipogenesis and suppresses osteogenesis in D1 cells while fullerol can counteract this activity of DEX. Moreover, fullerol is capable of reversing the impaired gene expression of the antioxidative enzymes catalase and SOD by DEX, and restores the abnormal oxidative status within cells.
D1, isolated from bone marrow stroma of a Balb/c mice, is a cloned cell line originally established in our laboratory.4 D1 cells have the capacity of differentiating into adipocyte and osteoblasts in vivo and in vitro, which resembles mesenchymal stem cells that give rise to adipocytes, chondrocytes, and osteoblasts.3, 4, 24 As a matter of fact, D1 cell line has been used as an in vitro system for studies of growth factors and drugs that affect osseous growth, especially relevant to bone ingrowth in total joint replacement, fracture healing, osteonecrosis, and osteoporosis.5, 6, 23–25
It has been reported that the antioxidant capacity of C60 is several hundred-fold higher than that of other antioxidants.15 C60 derivatives have shown some remarkable biologic properties such as promotion of chondrogenesis and protection of skin keratinocytes from ROS-induced cell death after ultraviolet stress.26, 27 Recently, it has been demonstrated that water soluble C60 fullerene can function as a protective agent against catabolic stress-induced degeneration of articular cartilage in vitro and in vivo. Water-soluble C60 significantly reduced articular cartilage degeneration in the osteoarthritis rabbit model.20 These findings suggest that C60 has the potential to protect against free radical-induced pathological features in orthopedic diseases.
There have been reports that glucocorticoids affect osteoblasts by down-regulating type-I collagen and osteocalcin gene expression28 and inducing osteoblast and osteocyte apoptosis.29, 30 Consistent with other reports,31, 32 our previous results demonstrated that DEX down-regulated the expression of Runx2 and osteocalcin while it increased the expression of PPARγ and the adipocytic specific gene aP2 in D1 cells. We suggest that effects of glucocorticoids on bone loss results from down-regulation of osteoblast transcription factors expression with concomitant up-regulation of the adipocyte transcription factors leading to the differentiation of bone marrow stromal cells along the adipocytic lineage. Therefore, drugs that stimulate new bone formation or inhibit corticosteroid-induced adipogenesis would be a significant alternative to prevent bone loss after corticosteroid treatment. Results from the present study indicate that fullerol plays an important role on precursor cells in bone marrow to inhibit adipocyte differentiation, apparently by acting on the expression of fat cell specific genes PPARγ and aP2, and subsequent maturation. Concomitantly, fullerol modulates cell differentiation by enhancing osteoblast differentiation, acting at the level of commitment, through increased expression of the Runx2 and osteocalcin genes. Thus, our data suggest that fullerol promotes uncommitted precursor cells in bone marrow stroma from the adipocytic to the osteoblastic linage and might serve as a valuable drug for preventative treatment of bone loss and ON if administrated simultaneously with a corticosteroid.
To provide further evidence on the role of fullerol, we investigated the cellular ROS level and gene expression of two antioxidative enzymes. We found that DEX caused a higher ROS level and lower mRNA level of either catalase or SOD. In the presence of fullerol, the impaired ROS status and expression of antioxidative enzymes were effectively modulated, indicating that oxidative stress might be a crucial regulator of adipogenesis and osteogenesis. Mody et al.33 showed that oxidative reagents hydrogen peroxide or xanthine/xanthine oxidase or minimally oxidized low-density lipoprotein caused a significant inhibition of osteogenic markers in mouse preosteoblast cell line MC3T3-E1 cells and bone marrow stromal cell line M2-10B4 cells, and these activities could be markedly counteracted by addition of antioxidants. Recently, Barbagallo et al.13 demonstrated that another important antioxidative gene heme oxygenase (HO-1) was able to inhibit adipogenesis and stimulate osteogenesis in human bone marrow mesenchymal stem cells. As such, we conclude that the regulatory role of fullerol in adipogenesis and osteogenesis in the present experiments might be mostly attributed to its antioxidative property.
There are some limitations of the present study. First, although abundant gene expression data by real-time PCR were provided, the respective protein levels were not determined. Second, the ROS data was from one of the two duplicated experiment. Due to limited number of repeats, only a tendency of changes in ROS levels was demonstrated. Lastly, in vivo experiment was not conducted to assess the protective activity of fullerol against corticosteroid-induced bone loss and osteonecrosis. Being aware of these limitations, additional experiments should be carried out in future studies.
So far a number of osteonecrosis models have been established, using various approaches for induction and in different animals including goats, rabbits, rats, mice and chickens.7, 12, 34–39 Particularly, use of a well-recognized corticosteroid, methylprednisolone (MPSL) in rabbits has been reported to evaluate the preventive effects of antioxidants vitamin E and lipoic acid on ONFH.7, 12 However, it is worth mentioning that in these rabbit models, incidence of ON may vary significantly due to different doses of corticosteroid, duration of treatment and means of administration. Motomura et al.34 treated rabbits with 1, 5, 20, and 40 mg/kg of MPSL, resulting in incidence of ON of 0, 42%, 70%, and 96%, respectively. Iwakiri et al.35 described a rabbit model by administering 20 mg/kg MPSL once intramuscularly, leading to an 83% incidence of ON in proximal metaphysis histologically 3 weeks after induction. These aforementioned studies indicate that it is critical to develop a reliable animal model, so that investigations to determine the optimal doses of fullerol and appropriate means of administration, as well as its timing, can be conducted.
The pathogenesis of corticosteroid induced ON remains largely unknown. However, there has been abundant evidence from both in vitro and in vivo experiments implying involvement of over-loaded oxidative stress and enhanced adipogenesis in bone marrow. Reduced blood GSH and increased lipid peroxide levels, as well as presence of advanced glycation end-product expression in the proximal femur were found to occur shortly after corticosteroid administration and before development of osteonecrosis of the femoral head (ONFH) and interestingly, administration of GSH could lower the incidence of ONFH.11 Presence of 8-hydroxy-29-deoxyguanosine, an index of DNA oxidation injury was confirmed in bone marrow hematopoietic cells in the early period after corticosteroid administration.8 Transient extreme oxidative stress by a single injection of a pro-oxidant buthionine sulfoximine could induce ONFH.36 Moreover, two well-recognized antioxidants, vitamin E and lipoic acid were capable of reducing the incidence of corticosteroid-induced ONFH.7, 12 On the other hand, we have previously reported that DEX might promote adipogenesis by D1 cells both in vitro and in vivo.3, 5 Increased adipogenesis has been also demonstrated either in primary marrow stromal cell cultures by DEX or in rabbit or chicken by MPSL.38, 40, 41 Moreover, a lipid-lowering agent, lovastatin can inhibit adipogenesis in D1 cells, as well as adipogenesis and occurrence of corticosteroid-induced osteonecrosis in rabbit.37–39 The results from our current study showed that DEX induced over-loaded ROS and adipogenesis in D1 cells. Taken together, these data indicate that oxidative stress might be a critical mediator for the induction of adipogenesis by the corticosteroid. Consequently, this may lead to the onset of osteonecrosis, especially in consideration of reduction of stem cell numbers to produce osteocytes, and increase in volume of fatty marrow and intraosseous pressure which induces intraosseous venous stasis and decreases arterial perfusion.38–40
In summary, the current study revealed for the first time that fullerol can significantly decrease adipogenesis and enhance osteogenesis by counteracting the effects of DEX in a bone marrow progenitor cell line D1. Fullerol may serve as a putative agent that plays an important role in prevention of corticosteroid induced ON.
The study is supported by Orthopaedic Research and Education Foundation/Zachary B. Friedenberg Clinician Scientist Award. Dr. Cui or an immediate family member has received research or institutional support from Orthopaedic Research and Education Foundation, Musculoskeletal Transplant Foundation, and Arthritis National Research Foundation; received royalties from Elsevier. Dr. Xudong Li or an immediate family member has received research or institutional support from North American Spine Society. Neither of the following authors nor any immediate family member has received anything of value from or owns stock in a commercial company or institution related directly or indirectly to the subject of this article: Dr. HongJian Liu, Dr. Abhijit Dighe, and Dr. Xinlin Yang.