In vitro osteogenesis of rat adipose-derived stem cells: comparison with bone marrow stem cells

Introduction
Various methods have been proposed for the repair of bone defects in the field of plastic and orthopaedic surgery. A previous approach to this problem focused on the development of various artificial materials that might be used instead of autogenous bone [1]. Current studies, however, rely mainly on the use of fresh autograft bone, various bone graft substitutes [2] and tissue-engineering techniques that incorporate appropriate implants seeded with cells having osteogenic potential [3]. Mesenchymal stem cells have recently received widespread attention because of their potential use in tissue-engineering applications [4]. Mesenchymal stem cells (MSCs), also known as marrow stromal cells or mesenchymal progenitor cells, are defined as self-renewable, multipotent progenitor cells with the capacity to differentiate into several distinct mesenchymal lineages [5]. To date, MSCs of various adult vertebrate species have been demonstrated to differentiate into lineage-specific cells that form the bone, cartilage, fat, tendon, and muscle tissue [6-8]. Bone marrow derived mesenchymal stem cells have been shown to be multipotential in that they differentiate in culture [8] or after implantation in vivo into osteoblasts [9]. Although bone marrow provides the most universal source of MSCs, other tissues such as periosteum [10], muscle [11], synovial membrane [12] and adipose tissue also appear to possess MSCs [13]. Adipose tissue is particularly attractive because of its easy accessibility and abundance [14-16]. Adipose tissue-derived mesen-chymal stem cells (ADSCs) obtained from lipoaspirates have been shown to have the multi-lineage potential to differentiate into adipogenic, chondrogenic, myogenic and osteogenic cells [13, 14]. Adipose-derived stem cells mineralized their extracellular matrix (ECM), and increased the expression of osteocalcin and alkaline phosphatase (ALP) [16]. However, it is not well established whether ADSCs have the same potential for osteogenesis and chondrogenesis as bone marrow-derived mesenchymal stem cells (BMSCs). Thus, the purpose of this study is to investigate whether ADSCs are equal to BMSCs with regard to their osteogenesis potential.

Material and methods
Isolation and culture of mesenchymal stem cells
About 6- to 8-week-old male Wistar rats of the albino strain were killed using diethyl ether and the bones were collected under sterile conditions, then all the bones were cut at both ends. The bone marrow from each bone was collected by flushing the bone with Dulbecco’s Modified Eagle’s Medium (Sigma) containing 1000 U/ml Penicillin G. The cells after filtering were centrifuged at 3,000 rpm for 5 min. The purified cells were finally dispersed in DMEM with 15% fetal bovine serum (Sigma) containing 100 U/ml penicillin, 100 mg/ml streptomycin [17].
Primary adipose-derived stem cells were harvested from the scrotal fat pad of the same age rats. Epididymal adipose tissue was excised, placed on a sterile glass surface, and finely minced. The minced tissue was placed in a 50 ml conical tube (Greiner, Germany) containing 0.05% tissue culture grade collagenase type 1 (Sigma) and 5% bovine serum albumin (Sigma). The tube was incubated at 37°C for 1 h, being shaken every 5 min. The tube contents, after filtering through a sterile 250 mm nylon mesh, were centrifuged at 250 γ for 5 min. The cell pellet was resuspended in adipose-derived stem cell medium: DMEM/F12 (Sigma), 10% fetal bovine serum (Gibco), and 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma). Cell count was determined with a haemacytometer [18].
Cell culture and expansion
The isolated stem cells were plated in T75 tissue culture flasks, containing appropriate stem cell medium at a density of 10 × 105 cells per flask. The flasks were maintained in a tissue culture incubator at 37°C and 5% carbon dioxide. The medium was replaced every third day afterwards. Cell viability was confirmed by continued cell division and the cells were subcultured using 3 ml of trypsin/EDTA (Sigma) when the flasks reached 90% confluence.
Osteogenic differentiation
Both types of cells were used to assess melatonin’s effect on osteogenic differentiation. Cells (passage 3) were then seeded at an initial density of 10 × 105 cells per flask (25 cm²) in osteogenic medium (OS) containing 0.05 mM ascorbate, 1 mM dexamethasone and 10 mM b-glycerophosphate for 4 weeks [18].
Confirmation of osteogenic differentiation
Confirmation of osteogenesis was confirmed by means of Von Kossa and Alizarin Red S staining (highlights extracellular matrix calcification) and assessment of alkaline phosphatase activity and expression of osteocalcin gene.
Von Kossa Staining
Cells in flasks (25 cm²) were rinsed with phosphate-buffered saline and were fixed in 4% paraformaldehyde for 20 min. The cells were incubated in 5% silver nitrate in the dark, then the flasks were exposed to ultraviolet light for 1 h. Secretion of calcified extracellular matrix was observed as black nodules with von Kossa staining [1].
Alizarin Red S Staining
Cells in flasks (25 cm²) were washed with phosphate-buffered saline (PBS) and fixed in 10% (v/v) formaldehyde (Sigma-Aldrich). After 15 min ARS 2% (pH = 4.1) was added to each flask. The flasks were incubated at room temperature for 20 min. Then the flasks were washed four times with dH₂O while being shaken for 5 min [19].
Quantification of mineralization
The analysis of the amount of calcium deposition in osteogenic media was modified from a previous report [19]. In brief, 2 ml 10% (v/v) acetic acid was added to each flask. After 30 min the monolayer was scraped off the plate with a cell scraper and transferred with 10% (v/v) acetic acid to a 15 ml microcentrifuge tube. After vortexing for 30 s, the slurry was overlaid with 1.25 ml mineral oil (Sigma-Aldrich), heated to exactly 85°C for 10 min, and transferred to ice for 5 min. The slurry was then centrifuged at 20,000 × γ for 15 min and 500 ml of the supernatant was removed to a new 1.5 ml microcentrifuge tube. Then, 200 ml of 10% (v/v) ammonium hydroxide was added to neutralize the acid. Aliquots (150 ml) of the supernatant were read in triplicate at 405 nm in 96-well format using opaque-walled, transparent-bottomed plates.
ALP activity
The cells were lysed by sonication for three cycles, and then protein solutions were centrifuged at 2000 × γ for 15 min at 4°C. The total protein content of each sample was determined according to Bradford [20]. ALP was performed using an ALP kit (Ziest Chem, Tehran, Iran) and following the manufacturer’s instructions. The levels of activity were neutralized with an amount of protein in cell lysate solution (units/mg protein).
RNA extraction and reverse transcription-polymerase chain reaction analysis of gene expression
After extraction of total RNA, reverse trans-criptase polymerase chain reaction assays were performed as described [21].
Flow cytometry
DNA fragmentation, as a late feature of apoptosis, was evaluated using a flow cytometer. PI staining was performed as previously described [22]. Sample acquisition was performed by a FACScan flow cytometer equipped with Cell Quest software.
Cell viability assay
The MTT (Sigma) test measures the mito-chondrial (metabolic) activity in the cell culture, which reflects the number of viable cells [23]. In brief, the cultures (5 × 104 were seeded to a 96-well plate) were washed in PBS, and 200 ml of MTT reagent were added. Following incubation for 3 h in the incubator (in 5% CO2 at 37°C) the absorption of the medium was measured in an ELISA Reader (Anthos 2020) at 440 nm.
Statistical analysis
The results are listed as the mean ± SD. The statistical difference was analyzed by one-way ANOVA followed by Dennett’s test. The p value < 0.05 was considered to be significant. All assays were performed in triplicate.

Results
Cultivation and passaging of human mesenchymal stem cell culture
No adherent cells were removed from the dish during medium changes and the subsequent passaging. Typically 80-90% of confluence was reached by day 10 in BMSC cultures and by day 5 for ADSC cultures. Cells at third passage were used for the experiments.
Rat bone marrow stem cells and adipose-derived stem cells grown in culture appeared spindle-shaped. Cells cultured in osteogenic media demonstrated a dramatic change in cell morphology from day 5 induction, with the cells changing morphology from an elongated fibroblastic appearance to polygonal, more cuboidal shape (Figure 1).
The cells of the two cell populations were cultured for 28 days in osteogenic media. After this period, samples were taken for analyses.
Quantitative estimation of ALP and mineralization
As a marker for BMSCs and ADSCs diffe-rentiation into osteoblasts, ALP levels were measured in both groups. In our study, ALP activity was measured in BMSCs and ADSCs in osteogenic medium after 14 and 28 days (Figure 2). The data showed that BMSCs underwent a much higher increase in ALP activity compared with ADSCs.
The cells were stained positively for extracellular mineralization after 2 and 4 weeks of culture in osteogenic media, as confirmed by Von Kossa and Alizarin Red S staining (Figure 3). Calcium level quantification was measured in both groups after 2 and 4 weeks after osteogenic induction in ADSCs and BMSCs (Figure 4).
The calcium measurement indicated more calcium for the BMSCs. The ADSC groups revealed a low level.
Osteocalcin gene expression
To determine gene expression of osteocalcin mRNA in both MSCs, RT-PCR was carried out with primer specific for osteocalcin. Figure 5 shows the results. The presence of the osteocalcin mRNA band indicated osteogenesis.
Apoptosis and cell viability
In individual experiments, the incidence of apoptotic cells among ADSCs was higher than BMSCs (Figure 6). The flow cytometry proves that cell growth reduction is due to a decrease of cells entering the S phase of the cell cycle. These data were demonstrated by MTT assay (Figure 7). These data indicated that viable cells among ADSCs were lower than BMSCs in the control groups.

Discussion
In the present study, we confirm that both BMSCs and ADSCs have the potential to differentiate into an osteogenic lineage. However, BMSCs have a greater osteogenesis potential as evidenced by greater matrix production when compared with ADSCs.
Over recent years, stem cells have generated great interest given their potential therapeutic use. Recent reports have provided clear evidence that multipotent adult stem cells exist in many more organs and tissues than previously expected. Mesenchymal cells capable of differentiation into a variety of specific cell phenotypes have been isolated from tissues such as bone marrow, muscle, fat, periosteum and synovial membrane both from rodents and humans [8, 12, 13, 24, 25].
Although bone marrow provides a universal source of MSCs, adipose tissue also possesses abundant and easily accessible MSCs. Recent advances in cosmetic surgery add to its advantage with a huge amount of available fatty tissue. It may have a further advantage when the morbidity associated with large volume bone marrow harvests is taken into consideration [26, 27].
Regarding comparison between BMSCs and ADSCs there are similar findings [28-30] but the overall results of the present study do not corroborate the results of the previous studies that suggested equal or comparable capacity of ADSCs for osteogenesis [13, 31, 32]. There are several possible reasons for such a difference. The reason suggested by Gun et al. [28] was that ADSC isolates may represent a fairly heterogeneous population of cell types with only a small number of progenitor cells capable of osteogenic differentiation. This would be consistent with the results of some studies which indicated differences in cell surface antigen expression between cellular preparations of adipose-derived and bone marrow-derived stromal cells [33-35]. Another explanation is that ADSCs may represent distinctly different cell populations that are at different stages of lineage-specific commitment from BMSCs [36]. Generally, studies have shown that BMSCs and ADSCs are not a homogeneous population of multilineage progenitors; rather, they are made up of a heterogeneous population of pluripotent stem cells and tripotent, bipotent, and unipotent progenitors [13, 37, 38].
Therefore, the differences between BMSCs and ADSCs observed here may not be due to the inherent difference between multipotent BMSCs and multipotent ADSCs. Rather, it could be due to the fact that BMSC cultures may be dominated by osteogenic and chondrogenic progenitors, whereas ADSCs have mainly adipogenic progenitors [38].
There are limitations of this study that preclude definite conclusions. First, we had different rats for BMSCs and ADSCs. Second, the gene expression profiles were not thoroughly investigated using a quantitative PCR technique. This will be pursued in the following study.
In conclusion, the results of this study show that ADSCs differ from BMSCs in their osteogenic potential. When equal amounts of bioactive factors are given, ADSCs have inferior capacity to differentiate into bone, suggesting the limited utility of ADSCs as a source of cells needed for tissue engineering of bone. As a further step forward, a search for the culture conditions that would induce successful osteogenesis from ADSCs is warranted.

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