Effects of Flaxseed (Linum usitatissimum) Extract on the Osteoblast Differentiation Potential of Stem Cells Derived from Human Exfoliated Deciduous Teeth

Background: Flaxseed promotes bone health and possibly induces bone regeneration. However, the capacity of flaxseed to induce the differentiation of stem cells into osteoblasts remains unreported. Accordingly, this study aimed to determine the effects of flaxseed extract on the osteoblast differentiation potential of stem cells derived from human exfoliated deciduous teeth (SHED). Methods: SHED cultured in osteoblast induction media (OIM) were treated with 4 mg/mL flaxseed extract. RNA was collected and extracted with Total RNA Mini Kit (Geneaid) from cells cultured at days 1, 3, 7, 14, and 21 and subjected to reverse-transcriptase PCR for osteoblast markers (OSX, OCN, and DMP1). Alkaline phosphatase (ALP) activity was determined by ALP assay, and Alizarin Red-S staining was performed to evaluate calcium deposition in SHED. Results: All osteoblast markers were expressed in all samples analyzed. OSX expression was reduced in the SHED treated with flaxseed extract. In addition, the SHED treated with flaxseed extract had lower ALP activity than the control (p < 0.05). Calcium deposition was positive in the SHED cultured in OIM only. Conclusions: Flaxseed can reduce the expression of osteoblast markers, ALP activity, and calcium deposition in SHED. Thus, flaxseed potentially inhibits the osteoblast differentiation of SHED.


I N T R O D U C T I O N
Flaxseed (Linum usitatissimum) contains beneficial compounds recognized for its health benefits. Flaxseed has attracted considerable interest because of its potential benefits associated with its biologically active components, such as approximately 59% a-linoleic acid and lignan secoisolaricirecinol diglycoside (SDG). [1][2][3][4] These compounds may exert protective effects on bone formation and bone metabolism. Consuming food rich in omega-3, such as flaxseed, improves bone health. 5 The consumption of flaxseed also alleviates menopausal effects and osteoporosis. 6 Flaxseed extract promotes bone health, especially in estrogen-deficient individuals, and possibly induces bone regeneration. [6][7][8] Dental pulp tissue is an attractive source of mesenchymal stem cells (MSCs) because of its readily accessible and high yield source. 9 In addition, stem cells from dental pulp possess similar gene expression and comparable regenerative potential to bone marrow MSCs. 10 Previously, we have shown that 4 mg/mL flaxseed extract influences the bioavailability and growth of stem cells from human deciduous teeth (SHED). 11 SHED is a type of MSCs that can differentiate into multiple cell lineages, including osteoblasts. 12 The osteogenic potential of SHED has been demonstrated previously. 13,14 In specific, SHED can differentiate into osteoblast cells when cultured in osteoblast induction media containing ß-glycerophosphate, ascorbic acid, and dexamethasone. 15,16 Moreover, the expression levels of osteoblast-related genes increase in SHED cultured in the presence of growth factors, indicating the application of SHED in tissue engineering because of its osteogenic differentiation potential. 14 Bone regeneration involves the physiological process of bone formation during normal fracture healing and is involved in continuous remodeling throughout adult life. 17 Given their minimal side effects, natural compounds obtained from natural resources give added value in regenerative medicine. Although flaxseed extract affects bone formation, its effect in inducing stem cells to differentiate into osteoblasts has not been reported. Hence, this study aimed to determine the effects of flaxseed extract on the osteoblast differentiation potential of SHED.

M E T H O D S
Culture of SHED SHED (ALLCells, USA) were cultured in complete growth media (alpha-MEM; 10% FBS; 0.5% Pen-Strep) until confluence. SHED at 80%-90% confluence were subjected to osteoblast differentiation. The complete growth medium was replaced with osteoblast induction media (OIM) to initiate differentiation. 14,18 The cells were incubated in 5% CO2 incubator at 37°C for 21 days.
SHED cultured in complete growth media and OIM were treated with or without 4 mg/mL flaxseed extract. The flaxseed extract was prepared, and the optimum concentration of extract was determined as previously described. 11 Four groups were set up for the analysis: SHED cultured in growth media alone (A), SHED cultured in growth media treated with 4 mg/mL flaxseed extract (B), SHED cultured in OIM alone (C), and SHED cultured in OIM treated with 4 mg/mL flaxseed extract (D).
Alkaline phosphatase activity of SHED SHED were seeded in a 12-well plate (1 mL of 5 × 10 4 cell suspension/well) and placed in 5% CO2 incubator until confluence. For background control, 1 mL of media (without cells) was aliquoted into the same 12 well plate. The media were discarded and replaced with media consisting of growth media as negative control, OIM as positive control, growth media with 4 mg/mL flaxseed extract, and OIM with 4 mg/mL extract. Alkaline phosphatase (ALP) activity of SHED was evaluated at days 1, 3, 7, 14, and 21 by ALP assay.
Alizarin red-s staining for the calcium deposition of SHED SHED (1 × 10 5 cells) were seeded in a 6-well plate in OIM and cultured in 5% CO2 incubator until confluence. The media was discarded and replaced with media containing 4 mg/mL flaxseed extract. The cells were maintained in 5% CO2 incubator. The media was replaced every 3 days. Calcium deposition of SHED was analyzed at days 14 and 21 by using Alizarin Red-S staining. The stained mineral deposits were viewed under an inverted phase contrast microscope (Leica, Germany). SHED cultured in OIM without flaxseed extract served as control.

Treatment of SHED for gene expression analysis
SHED (1 × 10 6 cells) were cultured in OIM and treated with 4 mg/mL flaxseed extract. Non-treated SHED cultured in OIM acted as control. The cells were collected at days 1, 3, 7, 14, and 21. RNA was extracted using Total RNA Mini Kit (Geneaid, Korea). Approximately 20 ng of total RNA was used for cDNA synthesis (ReverTra Ace qPCR Master Mix, Toyobo, Japan) and subjected to reverse-transcriptase PCR analysis for osteoblast markers OSX, DMP1, and OCN by using primer sequences as described previously. 19 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal positive control.

Statistical analysis
All data were expressed as mean (±SD) from at least three independent experiments and were subjected ANOVA and t-test by using SPSS software (SPSS version 17). Statistical significance was considered at p < 0.05.

ALP activity
The ALP activity of all groups was detectable after 3 days of incubation. The ALP activity of groups A, B, and D gradually increased until day 21. The ALP activity of group C gradually increased from day 3 until day 14 and then significantly decreased at day 21 (p < 0.05) ( Figure  1). A significant difference in ALP activity was found between groups A and B at day 14 (p < 0.05, t-test) but not at day 21 (p > 0.05). Similarly, the ALP activity of the SHED cultured in OIM (C) was significantly higher than that of the SHED treated with flaxseed (D) at day 14 (p < 0.01, t-test) but not at day 21 (p > 0.05).

Calcium deposition
The calcium deposition of SHED was assessed using Alizarin Red-S staining at days 14 and 21. The SHED cultured in OIM were positively stained at day 14, and the nodules of the mineralized matrix were strongly stained at day 21. A weak staining of Alizarin Red-S was observed in the SHED cultured in OIM treated with 4 mg/mL flaxseed extract at day 14. In addition, the formation of mineralized nodules at day 21 was lesser than that at day 14 (indicated by a weak staining of Alizarin Red-S). By contrast, Alizarin Red-S staining was negative in the other groups, whereas no visible staining of Alizarin Red-S was observed on the SHED cultured in growth media with or without flaxseed extract at days 14 and 21 ( Figure 2).

Expression of osteoblast markers
Analysis of gene expression on osteoblast markers was only performed on samples collected from the SHED cultured in OIM treated with or without 4 mg/mL flaxseed extract. The band intensities were measured using Chemidoc (BioRad, USA) and expressed as average intensity value (Table 1). Figure 3 illustrates the gene expression patterns observed in the analysis.
OSX expression was detectable at days 7 to 21 in the control, whereas day 7 recorded the highest and day 21 recorded the lowest expression. By contrast, OSX expression was detectable at days 1 to 21; it significantly reduced at day 7 in the treatment group but significantly increased at day 21 compared with the control (p < 0.05).

D I S C U S S I O N
Osteogenesis involves the differentiation of mesenchymal cells into pre-osteoblasts and osteoblasts, leading to the synthesis and deposition of bone matrix proteins. 20 Osteoblast differentiation undergoes proliferation, matrix maturation, and mineralization, which are regulated by different transcription factors and signaling proteins. 21 The current study confirmed the potential of SHED to differentiate into osteoblast-like lineage when cultured under specific induction media. 13,14 We demonstrated the differentiation of SHED into osteoblast lineage by ALP and calcium deposition analysis and positive expression of osteoblast-related markers as presented previously. 14 Nonetheless, the addition of flaxseed extract affected the ability of SHED to undergo osteoblast differentiation. ALP is a well-known marker of early osteoblast differentiation stage. ALP is a cell surface protein ubiquitously expressed by several cell types and is used for screening pre-osteoblasts. 22 ALP activity peaked at the second week of culture at about day 14 and then decreased with the onset of mineralization, which is a typical positive indication. 23 In the present study, flaxseed extract reduced the osteoblast differentiation potential of SHED. The ALP activity of the SHED cultured in OIM alone resembled the normal phases of osteoblast differentiation process. In specific, ALP activity started to increase from day 7, peaked at day 14, and ten significantly decreased at day 21. By contrast, the ALP activity of the SHED cultured in OIM treated with flaxseed significantly reduced at day 14 compared with that of the control. In addition, the ALP activity of the SHED treated with flaxseed cultured in normal growth media also reduced. This result might be additional evidence showing that flaxseed reduces the osteoblast differentiation potential of SHED.
Osteoblast differentiation was also analyzed by calcium deposition through Alizarin Red-S staining. Intense Alizarin Red-S staining and mineralized nodules were observed in the SHED cultured in OIM alone at day 21. Conversely, a weaker staining was observed and only few mineralized nodules were detected in the SHED cultured in OIM treated with flaxseed at day 21. This finding further supports that flaxseed extract reduces the osteoblast differentiation of SHED and thus reduces calcium deposition. Mineralization is the final phase of osteoblast differentiation, where the mineral matrix, which predominately contains calcium phosphate in the form of hydroxyapatite, is secreted and deposited by mature osteoblasts. 24 In the present study, SHED cultured in OIM exhibited normal osteoblast differentiation, but the addition of flaxseed extract reduced the differentiation potential of SHED into osteoblast-like lineage.
Gene expression levels of osteoblast markers OSX, DMP1, and OCN were analyzed to evaluate the osteoblast differentiation potential of SHED in the presence of flaxseed extract. Osterix (OSX) is an osteoblast-specific transcription factor important for osteoblast differentiation and bone formation and is also an early osteoblast marker. 25 Although the expression of OSX was only detectable from day 7 onward in control, it was consistent with the normal phases of osteoblast differentiation as demonstrated in the ALP analysis results that ALP activity increased from day 7, peaked at day 14, and reduced at day 21. In the treatment group, OSX was expressed at all days analyzed with highest expression at day 21 compared with the control. This result might be due to the continuous osteoblast differentiation at this stage. ALP results revealed that ALP activity slightly increased at day 21 in the treatment group. In comparison to control, ALP activity reduced at day 21, which could be related to reduction of OSX expression.
Dentin matrix protein 1 (DMP1) expression is localized in the mineralized matrix of bone; it is constitutively expressed during osteoblast differentiation. 26,27 In the present analysis, DMP1 expression reduced from day 14 to day 21 in the flaxseed-treated group compared with the control. Osteocalcin (OCN) is a late-stage marker of osteogenic differentiation. 28 In the present study, OCN was expressed in all days and peaked at day 21 in the control, which can be related to the final phase of mineralization. Intense Alizarin Red-S staining was observed at day 21 in the control, confirming that mineralization is related to the high DMP1 and OCN expression. By contrast, the reduction in DMP1 and OCN expression in the flaxseed-treatment group at day 21 could be related to the reduced ALP activity and weak Alizarin Red-S staining, especially at days 14 and 21.
Overall, our result demonstrated that flaxseed crude extract affected the osteoblast differentiation potential of SHED by reducing the ALP level, calcium deposition, and gene expression of osteoblast-related differentiation markers. Flaxseed extract possibly modulated the activity of ALP, the deposition of calcium, and the gene expression of osteoblast-related markers. Previously, we have shown that flaxseed extract contains high fatty acid contents. 11 Evidence presented over recent years has shown that n-3 polyunsaturated fatty acids (PUFAs) are beneficial for bone health. 29,30 However, PUFAs result in apoptosis at high concentrations and necrosis at even higher concentrations. 31 The inhibitory effects of fatty acids depend on carbon chain length and double bond number. 32 The high contents of fatty acids in flaxseed may inhibit SHED growth and other biological activities, such as ALP activity and osteoblast differentiation. However, further investigation is warranted in the future to clarify this issue.
Most previous studies utilized pure flaxseed oil to study the effects of flaxseed oil on bone health. 8 The present study focused on flaxseed crude extract; the content and ratio of the compound might vary with pure flaxseed oil and thus affect the overall process of osteoblast differentiation. The present study utilized stem cells from dental pulp for in vitro analysis, whereas others used osteoblast primary cell line as a starting material, which could also affect the overall outcome. Nonetheless, the current study provides additional information regarding the utilization of 4 mg/mL flaxseed extract, which can inhibit the osteoblast differentiation potential of SHED.

C O N C L U S I O N S
Flaxseed crude extract reduces ALP activity and calcium deposition in SHED and affects the expression of osteoblast markers. Thus, flaxseed can potentially inhibit the osteoblast differentiation of SHED, suggesting its possible application in the synthesis of natural-based drugs that can control bone development. Nonetheless, the actual mechanism must be elucidated in the future.

C O N F L I C T O F I N T E R E S T
The authors declare that there are no conflicts of interest.

F U N D I N G
The authors would like to acknowledge financial support received from the Ministry of Higher Education, Malaysia (FRGS/1/2015/SKK14/UIAM/02/1; FRGS15-252-0493) and International Islamic University Malaysia.