7,8-Dihydroxy coumarin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells

Abstract

Objective

To investigate the effects of 7,8-dihydroxy coumarin on the chondrogenic differentiation of rat adipose-derived mesenchymal stem cells (ADMSCs).

Methods

ADMSCs were cultured using a micromass suspension method and incubated with different concentrations of 7,8-dihydroxy coumarin and transforming growth factor (TGF)-β₁ for 3 weeks: group A (negative control, no drug treatment); group B (positive control, 10 ng/ml TGF-β₁); groups C, D and E (incubated with 25, 50 and 100 µg/ml 7,8-dihydroxy coumarin, respectively); groups F, G and H (incubated with 25, 50 and 100 µg/ml 7,8-dihydroxy coumarin, respectively, plus 10 ng/ml TGF-β₁). Markers of chondrogenic differentiation were measured using histology, immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction.

Results

When used alone, 7,8-dihydroxy coumarin only weakly induced the chondrogenic differentiation of ADMSCs. 7,8-Dihydroxy coumarin used in combination with TGF-β₁ strongly induced chondrogenic differentiation of ADMSCs. For some of the markers of chondrogenic differentiation, the extent of the induction was 7,8-dihydroxy coumarin dose-dependent.

Conclusions

7,8-Dihydroxy coumarin appears to work synergistically with TGF-β₁ to strongly induce chondrogenic differentiation of rat-derived ADMSCs.

Introduction

Degeneration of articular cartilage and bone defects caused by trauma and chronic bone joint diseases are common problems. It is difficult to repair damaged articular cartilage because it receives no nourishment from blood vessels and surrounding tissue, and transplanted chondrocytes have very limited proliferative abilities. ¹ Clinical treatments for repairing cartilage damage include arthroscopic lavage debridement, subchondral bone drilling, microfracture technology, periosteum or perichondrium transplantation, and autologous or allogeneic bone and cartilage transplantation. ² These treatments either have a poor prognosis or are unable to restore all of the normal biomechanical characteristics fully, in damaged articular cartilage. ²

Tissue engineering applies biological substitutes in order to repair damaged tissues. ² The autologous chondrocyte is the only cartilage seed cell that has been applied clinically for tissue engineering, ³ but there are a number of limitations associated with its use: the donor source is limited; chondrocyte isolation and in vitro culture are difficult; autologous chondrocytes have a poor proliferative capacity.^4–6 Alternative sources of cells would be embryonic stem cells and induced pluripotent stem cells but, because of ethical and safety issues, their use remains a long way from clinical application.^7,8 Adult stem cells are widely distributed throughout the body, especially adipose-derived mesenchymal stem cells (ADMSCs), which are readily available and easily obtained, making them ideal candidates for use as tissue-engineered seed cells for tissue repair.^9–11

Rapid amplification of a large number of stem cells in vitro and more efficient induction of differentiation towards chondrocytes remain the most important issues to be addressed in this research field.^12,13 Currently, the cytokines and chemokines used for chondrocyte cell culture are very expensive, and simulating the multiple factors that are required to induce chondrocyte differentiation in vitro is difficult. There is a growing interest in the ability of traditional medicines, often derived from plants, to promote osteogenesis.^14–20 Studies have shown that 7,8-dihydroxy coumarin (also known as daphentin), which is obtained from the fruits and seeds of several plants including Daphne mezereum (commonly known as mezereon), can promote bone formation and increase bone mineral density.^21,22 Tang et al. ²¹ reported that coumarin and its derivatives can activate the p38- and extracellular signal-regulated protein-dependent signalling pathways in rat osteoblasts, increase bone morphogenic protein-2 (BMP-2) mRNA levels and promote bone formation. Another study reported that the coumarin-like derivative osthole (also known as 7-methoxy-8-isopentenoxycoumarin), which can be purified from Cnidium monnieri and Angelica pubescens plants, was able to activate the Wnt/β-catenin-BMP-2 signalling pathways in mouse osteoblasts, promote osteoblast differentiation, contribute substantially to mouse skull bone repair and increase bone mineral density. ²²

The present study investigated the ability of 7,8-dihydroxy coumarin to induce the chondrogenic differentiation of rat-derived ADMSCs compared with transforming growth factor (TGF)-β₁, which–as a known inducer of chondrogenic differentiation of ADMSCs–was used a positive control. ¹⁰

Materials and methods

Animals

This study was conducted at the College of Pharmacy of Jilin University, Changchun, Jilin Province, China. Male Sprague–Dawley rats, aged 6–8 weeks with a body weight of 221 ± 30 g, were provided by the Experimental Animal Centre of Jilin University (Changchun, Jilin Province, China). The animals were housed under a 12-h light/12-h dark cycle and allowed free access to food and water.

In the absence of a local Animal Ethics Committee at Jilin University, the study protocol was supported by the College of Pharmacy, Jilin University. All animal feeding, care and experimental testing complied with the guidelines for animal use and care, produced by Jilin University Animal Care and Welfare Committee. Other than the individual reagents specified in detail in the sections below, all other laboratory grade reagents were sourced from within China.

Isolation of Rat-derived ADMSCs and Drug Treatment

A total of 20 rats were sacrificed by neck dislocation, followed by 75% ethanol disinfection. A sample of fat tissue (1 g) was taken from the groin and subrenal area, and washed three times with Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 medium (GIBCO® Cell Culture, Carlsbad, CA, USA) supplemented with 100 IU/ml each of penicillin and streptomycin (North China Pharmaceutical Company, Shijiachuang, China) and 10% fetal bovine serum (FBS; GIBCO® Cell Culture). Visible blood vessels and other tissues were removed from the fat sample, followed by repeated washing with 200 mM phosphate-buffered saline (PBS; pH 7.0) at 37°C. The fat tissues were cut into 1-mm ³ pieces, placed in 0.1% type I collagenase solution (Sigma-Aldrich, St Louis, MO, USA) and incubated at 37°C for 60 min. The digested cells were filtered using a 75 -µm mesh sieve (Xiamen Jingfeng Nylon Netting Company, Xiamen, China), followed by washing with 200 mM PBS (pH 7.0) at 37°C and centrifuged at 260 g to remove the type I collagenase. The cells were cultured in DMEM/F12 medium containing 10% FBS at 37°C with 5% CO₂, in a humidified atmosphere.

Rat ADMSCs were cultured using a micromass suspension method as follows. Rat ADMSCs that were between the third and fifth passage were digested using 0.25% trypsin solution (Sigma-Aldrich), resuspended, counted and adjusted to a final density of 10⁷ cells/ml. Cells (20 µl of cell suspension) were seeded onto the inner wall of the cover of 24-well plates (Costar®, Sigma-Aldrich) and incubated overnight at 37°C with 5% CO₂ in a humidified atmosphere. After 14 h, a cell mass was formed and moved from the wall into the well of the 24-well plate, and 1 ml of DMEM/F12 medium containing 10% FBS was added to the well. At the same time, the experimental drugs were added to induce stem-cell differentiation. The experimental drugs, 7,8-dihydroxy coumarin (Sigma-Aldrich) and TGF-β₁ (Sigma-Aldrich), were dissolved in 200 mM PBS (pH 7.0) prior to use.

The experiments were performed in eight groups, with each group consisting of three wells: group A: negative control, no experimental drug; group B: positive control, 10 ng/ml TGF-β1; groups C, D and E, treated with 25, 50 and 100 µg/ml 7,8-dihydroxy coumarin, respectively; groups F, G and H, treated with 25, 50 and 100 µg/ml 7,8-dihydroxy coumarin, respectively, plus10 ng/ml TGF-β₁. The cell-culture medium was changed every 3 days, accompanied by supplementation of the working concentration of the experimental drug(s). Each time the medium was changed, the cell mass was gently pushed up into suspension from the bottom of the plate. The induction of differentiation phase lasted 3 weeks.

Alcian Blue and Periodic Acid-Schiff Staining

After 3 weeks’ treatment, the cell mass from the cell culture well was fixed in 10% neutral-buffered formalin solution for 24 h, then subjected to ethanol gradient dehydration and embedding in paraffin wax. Sections (5 µm thick) were cut from the specimens, mounted onto glass slides, then dewaxed and washed with deionized water. Sections were treated consecutively with 3% acetic acid for 1 min, 1% alcian blue solution (Wuhan Boster Biological Technology, Wuhan, Hubei, China) for 15 min, deionized water for 3 min, 1% periodic acid (Wuhan Boster Biological Technology) for 10 min, deionized water for 3 min, Schiff Reagent (Wuhan Boster Biological Technology) for 15 min and deionized water for 3 min. For staining with alcian blue alone, sections were treated consecutively with 3% acetic acid for 1 min and 1% alcian blue solution for 15 min. The specimens were subjected to gradient alcohol dehydration and xylene, and were mounted with neutral gum for light microscopy. Alcian blue was used alone to stain acid mucopolysaccharides (stained blue) and the combination of alcian blue and periodic acid-Schiff was used to stain both acid (stained blue) and neutral mucopolysaccharides (stained magenta).

Immunohistochemical Staining to Detect Type II Collagen

Dewaxed slices of cell mass were prepared as described above, then incubated in 3% hydrogen peroxide at room temperature for 10 min and washed three times with 200 mM PBS (pH 7.0). For antigen retrieval, specimens were incubated in 1.0 mM ethylenediaminetetra-acetic acid (EDTA; pH 9.0) in a boiling-water bath for 2 h, and incubated with 10% horse serum (HyClone, Beijing, China) at room temperature for 10 min to block endogenous nonspecific antigens. Mouse antirat type II collagen monoclonal antibody (1 : 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the slides and incubated overnight at 4°C. After washing three times with 200 mM PBS (pH 7.0), biotinylated goat antimouse polyclonal secondary antibody (1 : 5000 dilution; Santa Cruz Biotechnology) was added and incubated at room temperature for 30 min. The specimens were washed three times with 200 mM PBS (pH 7.0), incubated with streptavidin–alkaline phosphatase (1 : 10,000 dilution; Invitrogen, Beijing, China) at room temperature for 30 min, washed three times with 200 mM PBS (pH 7.0), and incubated with 3,3′-diaminobenzidine (DAB) substrate (0.05% diluted in 200 mM PBS, pH 7.0) for 5 min at room temperature. The slides were washed gently using deionized water, which was rinsed slowly down the back of the slide, followed by dehydration through a graded series of alcohol and xylene, and then mounted with neutral gum for light microscopy.

ELISA to Detect GAG Content

Enzyme-linked immunosorbent assay (ELISA) analysis was used to assess the secretion of glycosaminoglycans (GAG) by the cell masses into the cell culture supernatant. After 3 weeks’ treatment, cell-culture supernatants were centrifuged at 314 g for 10 min. An ELILSA kit for GAG (alcian blue method; Wuhan Boster Biological Technology) was used according to the manufacturer’s instructions and the absorbance was measured at 490 nm. The GAG ​​content secreted into the supernatant was determined according to the standard curve for a preparation of standard GAG that was provided with the kit. The minimum detectable concentration of GAG was 10.0 ng/ml. Intra- and interassay coefficients of variation for the ELISA were 3% and 5%, respectively.

RT–PCR

After 3 weeks’ treatment, the cell mass was collected from the cell-culture well and transferred into an Eppendorf tube containing 500 µl of 1 mg/ml proteinase K (Sigma-Aldrich) for digestion at 56°C for 1 h, followed by centrifugation at 260 g for 5 min. The pellets were dissolved in 1 ml of TRIzol® reagent (Takara Biotechnology [Dalian] Co. Ltd, Dalian, China) at room temperature for 5 min, followed by the addition of 0.2 ml chloroform with oscillation for 15 s, incubation at room temperature for 5 min and centrifugation at 4°C, 20,000 g for 20 min. The upper aqueous phase was carefully transferred into a new diethylpyrocarbonate (DEPC)-treated Eppendorf tube (Takara Biotechnology [Dalian] Co. Ltd), and mixed with an equal volume of isopropanol at room temperature for 15 min, followed by centrifugation at 4°C, 20,000 g for 20 min. The pellets were washed with 1 ml of 75% ethanol/DEPC-treated water, followed by centrifugation at 4°C, 20,000 g for 10 min. The pellets were air dried to obtain the RNA, followed by the addition of 20 µl of DEPC-treated water. RNA purity and concentration were determined using an ultraviolet (UV) spectrophotometer to measure the optical density (OD) 260/OD280 absorbance values of a 100-fold dilution in 1.0 M Tris-EDTA buffer (pH 9.0).

For the reverse transcription–polymerase chain reaction (RT–PCR), DNase I (Takara Biotechnology) was added to remove the genomic DNA. The cDNA was synthesized using an RT kit (Takara Biotechnology [Dalian] Co. Ltd) in accordance with the manufacturer’s instructions. The cDNA concentration was determined using a full-function spectrophotometer (TECAN M200 F200; Tecan Group Ltd, Zurich, Switzerland). The PCR reaction mixture included: 10 µl of 10 × buffer (Dingguo Biotechnology Development Centre, Beijing, China), 200 µmol/l of a mixture of deoxynucleotide triphosphates (Promega [Beijing] Biotech Co., Ltd, Beijing, China), 20 pmol of each primer (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, Shanghai, China), 1 µg of cDNA template, Taq DNA polymerase 2.5 U (TaqNa polymerase; Promega [Beijing] Biotech Co., Ltd), 1.5 mmol/l Mg²⁺ (Dingguo Biotechnology Development Centre), and triple-distilled water, to a final volume of 100 µl. The PCR cycling programme involved preliminary denaturation at 94°C for 10 s, followed by 30 cycles of denaturation at 94°C for 60 s, annealing at 57°C for 60 s and elongation at 72°C for 60 s, and a final elongation step at 72°C for 60 s. The primer sequences for PCR, which were designed to amplify three chondrogenic differentiation-related mRNAs (peroxisome proliferator-activated receptor-γ coactivator 1 -α [PGC1-α], sex determining region Y-box 9 [SOX9], and collagen type II [COL2A1]) and ACTB mRNA (β-actin; internal control), are shown in Table 1. The primers were designed using Beacon Designer™ 7 software (PREMIER Biosoft, Palo Alto, CA, USA), based on NCBI GenBank® sequences (available at: http://www.ncbi.nlm.nih.gov/genbank/). PCR primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China). PCR products were separated on 1.5% agarose gel (containing 0.5 µg/ml ethidium bromide), with an electrophoresis sample volume of 5 µl. The PCR products were visualized by scanning the agarose gel using a UVP gel imaging analysis system (UVP, Upland, CA, USA), followed by a semiquantitative analysis by calculating the target genes versus β-actin (internal control) grey-scale ratio, using the same UVP gel imaging analysis system.

Table 1.

Primer sequences used in polymerase chain reaction to investigate the effects of 7,8-dihydroxy coumarin and transforming growth factor-β₁ on the chondrogenic differentiation of rat adipose-derived mesenchymal stem cells.

mRNA, gene product	Primer sequence	Product  size,  base  pairs
ACTB, β-actin	Upstream 5′-GTGGACATCCGCAAAGAC-3′	302
	Downstream 5′-AAAGGGTGTAACGCAACTAA-3′
PGC1-α, peroxisome  proliferator-activated  receptor γ coactivator 1 α	Upstream 5′-ACTATTGAACGCACCTT-3′	575
	Downstream 5′-CTGGGATGACCGAAGT-3′
SOX9, sex determining  region Y-box 9	Upstream 5′-GAACGCACATCAAGACGGAG-3′	631
	Downstream 5′-TCTCGTTGATTTCGCTGCTC-3′
COL2A1, collagen type II	Upstream 5′-CTGGCTCCCAACACTGCCAACGTC-3′	414
	Downstream 5′-TCCTTTGGGTTTGCAACGGATTGT-3′

Statistical Analyses

All statistical analyses were performed using the SPSS® statistical software package, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Mean values of the GAG concentrations were compared using Student’s t-test and a P-value < 0.05 was considered to be statistically significant.

Results

Histological staining of the sections of ADMSC cell mass using alcian blue showed that the negative control sample (Figure 1A), compared with the positive control cells treated with TGF-β₁ alone (Figure. 1B), demonstrated a reduced level of acid mucopolysaccharides, similar to the levels in the three groups that were treated with 7,8-dihydroxy coumarin alone (Figures 1C–E). Cells treated with both 7,8-dihydroxy coumarin and TGF-β₁ (Figures 1F–H) were more intensely stained than the negative control sample (Figure 1A), especially the cells treated with 50 and 100 µg/ml 7,8-dihydroxy coumarin (Figures 1G and H).

Figure 1.

Representative photomicrographs showing alcian blue staining results for acid mucopolysaccharides (blue stain) after 3 weeks’ treatment of rat adipose-derived mesenchymal stem cells with two experimental drugs, tested for their ability to induce chondrogenic differentiation (scale bars, 20 µm). The negative control group (A) did not receive either experimental drug; the positive control group (B) received 10 ng/ml transforming growth factor (TGF)-β₁; three groups (C, D and E) received 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively; three groups (F, G and H) received 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively, with 10 ng/ml TGF-β₁.

Histological staining of the sections of ADMSC cell mass using alcian blue and periodic acid-Schiff to stain for both acid (blue stained) and neutral (magenta stained) mucopolysaccharides showed that there were low levels of acid mucopolysaccharides and high levels of neutral mucopolysaccharides in the negative control cells (Figure 2A). In contrast, positive control cells treated with TGF-β₁ alone demonstrated stronger staining for acid mucopolysaccharides than neutral mucopolysaccharides (Figure 2B). The levels of neutral mucopolysaccharides were higher in the three groups of cells treated with 7,8-dihydroxy coumarin alone (Figures 2C–E) compared with the negative control cells (Figure 2A), and all three groups of cells treated with 7,8-dihydroxy coumarin alone demonstrated very low levels of staining for acid mucopolysaccharides. The cells treated with both 7,8-dihydroxy coumarin and TGF-β₁ demonstrated intense staining for acid mucopolysaccharides, in addition to being positive for neutral mucopolysaccharides (Figures 2F–H).

Figure 2.

Representative photomicrographs showing the alcian blue/periodic acid-Schiff staining results for acid (blue stain) and neutral (magenta stain) mucopolysaccharides after 3 weeks’ treatment of rat adipose-derived mesenchymal stem cells with two experimental drugs, tested for their ability to induce chondrogenic differentiation (scale bars, 20 µm). The negative control group (A) did not receive either experimental drug; the positive control group (B) received 10 ng/ml transforming growth factor (TGF)-β₁; three groups (C, D and E) received 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively; three groups (F, G and H) received 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively, with 10 ng/ml TGF-β₁.

Immunohistochemical staining of sections of ADMSC cell mass for type II collagen showed no staining in the negative control cells (Figure 3A) compared with the positive control cells treated with TGF-β₁ alone (Figure 3B), which were stained positive for type II collagen protein. Cells treated with 7,8-dihydroxy coumarin alone were very weakly positive compared with the negative control (25 and 50 µg/ml groups; Figures 3C and D) or only had very low levels of staining for type II collagen (100 µg/ml group; Figure 3E). Cells treated with both 7,8-dihydroxy coumarin and TGF-β₁ demonstrated increased levels of type II collagen (Figures 3F–H), compared with both the negative control cells (Figure 3A) and the TGF-β₁-treated positive control cells (Figure 3B).

Figure 3.

Representative photomicrographs showing immunohistochemical staining of type II collagen after 3 weeks’ treatment of rat adipose-derived mesenchymal stem cells, with two experimental drugs tested for their ability to induce chondrogenic differentiation (scale bars, 15 µm). The negative control group (A) did not receive either experimental drug; the positive control group (B) received 10 ng/ml transforming growth factor (TGF)-β₁; three groups (C, D and E) received 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively; three groups (F, G and H) received 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively, with 10 ng/ml TGF-β₁.

The ELISA analysis demonstrated that treatment of the ADMSC cell masses for 3 weeks with TGF-β₁ alone (group B) or with 7,8-dihydroxy coumarin alone (groups C, D and E) significantly increased the levels of GAG measured in the cell culture supernatant, compared with the negative control group (group A) (P < 0.001 for all comparisons) (Table 2). The combination treatment of 7,8-dihydroxy coumarin and TGF-β₁ for 3 weeks (groups F, G and H) significantly increased levels of GAG in the cell culture supernatant, compared with both the negative and positive control groups (groups A and B, respectively) (P < 0.001 for all comparisons).

Table 2.

The amount of glycosaminoglycan (GAG) secreted into the cell culture supernatant by cultured rat adipose-derived mesenchymal stem cells treated for 3 weeks with 7,8-dihydroxy coumarin, with or without transforming growth factor-β₁.

Group	Group A negative control	Group B positive control 10 ng/ml TGF-β1	Group C 25 µg/ml 7,8-dihydroxy coumarin	Group D 50 µg/ml 7,8-dihydroxy coumarin	Group E 100 µg/ml 7,8-dihydroxy coumarin	Group F 25 µg/ml 7,8-dihydroxy coumarin + 10 ng/ml TGF-β1	Group G 50 µg/ml 7,8-dihydroxy coumarin + 10 ng/ml TGF-β1	Group H 100 µg/ml 7,8-dihydroxy coumarin + 10 ng/ml TGF-β1
GAG (µg/ml)	0.31 ± 0.05	6.72 ± 0.07  a	1.79 ± 0.11  a	2.83 ± 0.14  a	3.59 ± 0.09  a	10.10 ± 0.01 a,b	16.10 ± 0.05 a,b	18.20 ± 0.12 a,b

Data presented as mean ± SD.

a

P < 0.001 compared with negative control (no experimental drug); ^bP < 0.001 compared with positive control using Student’s t-test.

The effects of 3 weeks’ treatment of the ADMSC cell masses with 7,8-dihydroxy coumarin, with or without TGF-β₁, on the mRNA levels of three chondrogenic genes (PGC1-α, SOX9, and COL2A1) are shown in Figure 4. Treatment with 7,8-dihydroxy coumarin alone had little effect on the levels of PGC1-α, SOX9 and COL2A1 mRNA compared with the negative control group. Treatment with TGF-β₁ alone (positive control group) increased the levels of PGC1-α, SOX9 and COL2A1 mRNA compared with the negative control group, as did treatment with 7,8-dihydroxy coumarin in combination with TGF-β₁. The level of PGC1-α mRNA was 1.5-fold higher in the three groups treated with 7,8-dihydroxy coumarin in combination with TGF-β₁, compared with the group treated with TGF-β₁ alone. SOX9 and COL2A1 mRNA levels demonstrated a dose-dependent increase in the three groups treated with 7,8-dihydroxy coumarin (25, 50 and 100 µg/ml) in combination with TGF-β₁. The SOX9 mRNA levels were 4.4-fold, 4.7-fold and 5.0-fold higher than TGF-β₁ alone, respectively, and the COL2A1 mRNA levels were 4.0-fold, 4.4-fold and 4.6-fold higher than TGF-β₁ alone, respectively.

Figure 4.

Representative images showing results of reverse transcription–polymerase chain reaction (PCR) experiments undertaken to measure levels of mRNA after 3 weeks’ treatment of rat adipose-derived mesenchymal stem cells with two experimental drugs, tested for their ability to induce chondrogenic differentiation. PCR products were separated on a 1.5% agarose gel: (A) β-actin, internal control (ACTB; 302 base pairs [bp]); (B) peroxisome proliferator-activated receptor-γ coactivator 1 -α (PGC1-α; 575 bp); (C) sex determining region Y-box 9 (SOX9; 631 bp); (D) collagen type II (COL2A1; 414 bp). For each gel, lane 1: DNA markers; lane 2: negative control group; lane 3: positive control group treated with 10 ng/ml transforming growth factor (TGF)-β₁; lanes 4, 5 and 6: 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin, respectively; lanes 7, 8 and 9: 25, 50, or 100 µg/ml 7,8-dihydroxy coumarin alone, respectively, with 10 ng/ml TGF-β₁.

Discussion

It would be extremely beneficial if a naturally occurring agent could be identified that could induce ADMSCs to undergo chondrogenic differentiation, as it might be expected to help these cells to repair injured cartilage. The present study used routine histological staining (with alcian blue alone and in combination with periodic acid-Schiff) to identify acid and neutral mucopolysaccharides, immunohistochemical staining of type II collagen protein, an ELISA (to measure GAG secretion) and RT–PCR (to detect chondrogenic differentiation-related mRNA levels), in order to measure the extent of chondrogenic differentiation in rat ADMSCs treated with 7,8-dihydroxy coumarin (with or without TGF-β₁) for 3 weeks in cell culture. When used alone, 7,8-dihydroxy coumarin was only a weak inducer of chondrogenic differentiation of rat ADMSCs, but the findings suggested that it synergized with TGF-β₁ to more strongly promote the chondrogenic differentiation above that achieved by TGF-β₁ treatment alone.

The present study used the micromass suspension method to culture rat ADMSCs; this method has been shown to facilitate cell aggregation and chondrogenic differentiation. ²³ The chondrogenic differentiation markers used in the current study included two components: the production of extracellular matrix proteins (i.e. type II collagen, acid and neutral mucopolysaccharides and GAG) and the expression of chondrogenic differentiation-related genes. ¹⁰ The present study demonstrated markers indicating chondrogenic differentiation in the ADMSC cell mass and the secretion of GAG into the culture supernatant, suggesting that this culture system was able to support chondrogenic differentiation.

Coumarin is cytotoxic to human peripheral blood mononuclear cells and bone marrow progenitor stem cells; ²⁴ there is no toxicity for doses up to 100 µg/ml, but higher doses (up to 200 µg/ml) have obvious inhibitory effects. The present study used 7,8-dihydroxy coumarin at three concentrations (25, 50 and 100 µg/ml) that are within the range associated with better safety. Treatment of rat ADMSC cell masses with those three doses of 7,8-dihydroxy coumarin was only a weak inducer of chondrogenic differentiation. Levels of GAG that were secreted were low; cells synthesized predominantly neutral rather than acid mucopolysaccharides, and produced very low levels of type II collagen protein. In terms of inducing expression of three chondrogenic differentiation-related genes, PGC1-α, SOX9 and COL2A1 mRNA levels in ADMSC cell masses treated with the three doses of 7,8-dihydroxy coumarin were identical to the levels observed with negative control cells (treated with neither of the two experimental drugs). These findings suggested that the doses of 7,8-dihydroxy coumarin were insufficient to induce chondrogenic differentiation, or that 7,8-dihydroxy coumarin might not induce the expression of genes involved in chondrogenic differentiation, in rat ADMSCs.

When used alone, TGF-β₁ (10 ng/ml) was able to induce chondrogenic differentiation, as demonstrated by increased levels of GAG secreted into the supernatant, the production of predominantly acid (rather than neutral) mucopolysaccharides, and the production of increased levels of type II collagen protein. TGF-β₁ treatment also upregulated the levels of PGC1-α, SOX9 and COL2A1 mRNA, which can be considered indicative of chondrogenic differentiation.

When 7,8-dihydroxy coumarin and TGF-β₁ were used in combination, the effect on the induction of chondrogenic differentiation of ADMSCs increased markedly in a dose-dependent manner, based on the dose of 7,8-dihydroxy coumarin. This was particularly evident in terms of the secretion of GAG into the cell-culture supernatant and the results of the RT–PCR analyses undertaken for the levels of chondrogenic gene expression. PGC-1α, a member of a family of transcription coactivators, plays a key role in cellular energy metabolism. ²⁵ As part of its role as a transcriptional activator, PGC-1α can combine with the transcription factor SOX9 to regulate expression of the cartilage-specific gene COL2A1, ²⁶ thereby regulating cartilage formation. The present study showed that the combination of 7,8-dihydroxy coumarin and TGF-β₁ upregulated PGC-1α, with treatment with the three concentrations of 7,8-dihydroxy coumarin resulting in similar levels of PGC-1α mRNA, which were 1.5-fold higher than the level seen with TGF-β₁ alone. SOX9 is also one of the key transcription factors for cartilage formation. ²⁷ SOX9 can regulate the cartilage-specific genes that encode for collagen type II and BMP-2, promoting cartilage formation. ²⁷ The present study showed that 7,8-dihydroxy coumarin and TGF-β₁ upregulated SOX9 in a dose-dependent manner, based on the 7,8-dihydroxy coumarin dose (25, 50 and 100 µg/ml). COL2A1 is a cartilage-specific gene that encodes the type II collagen α-1 chain and is a cartilage-specific marker. ²⁶ COL2A1 mRNA was also upregulated in a dose-dependent manner, based on the 7,8-dihydroxy coumarin dose (25, 50 and 100 µg/ml) in the present study. Upregulation of mRNA levels of these three genes demonstrated that the ADMSCs had differentiated (or were in the process of differentiating) into cartilage, as a result of treatment with 7,8-dihydroxy coumarin and TGF-β₁.

Studies have shown that the coumarin used alone can promote the growth of osteoblasts and bone formation in mice.^21,22 The present study found that 7,8-dihydroxy coumarin (used alone) lacked the ability to induce strongly the chondrogenic differentiation of rat ADMSCs, which suggests that osteoblasts and ADMSCs might have important differences. Osteoblasts are destined to form bones, whereas ADMSCs are pluripotent stem cells. When exposed to coumarin, osteoblasts grow and ultimately form bone tissue. In contrast, ADMSCs differentiate into chondroblasts, which then form cartilage. These contrasting findings suggest that coumarin may be able to promote bone formation strongly by inducing the differentiation of osteoblasts, but that its actions are limited in terms of being able to promote the differentiation of pluripotent stem cells into chondroblasts.

Some studies have reported the opposite effects for coumarin. For example, a histological study found that the anticoagulants heparin and coumarin reduced the number of cells of the epiphyseal cartilage in mice. ²⁸ A possible explanation for this is that the two anticoagulants were used at doses that were cytotoxic, causing inhibition of cell proliferation, which would have adversely affected cartilage formation. Another study reported that heparin and coumarin reduced bone density. ²⁹ Coumarin is used as an anticoagulant thrombolytic agent, and the clinical dose is usually well above the safe range of 100 µg/ml. Thus, the clinical use of coumarin may inhibit bone formation by osteoblasts, causing a decrease in bone mineral density.

In conclusion, 7,8-dihydroxy coumarin used alone was a weak inducer of the chondrogenic differentiation of rat ADMSCs, but was able to synergize with TGF-β₁ to induce chondrogenic differentiation strongly, with the 50 and 100 µg/ml doses being particularly effective at inducing GAG secretion, type II collagen deposition and upregulation of three chondrogenic differentiation-related mRNAs.