Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA) and 20%–39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.
Basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) crystals are the two most common forms of articular crystals. Earlier studies find that these calcium crystals are associated with end-stage osteoarthritis (OA) in approximately 60% of cases [
Increasing experimental evidence indicates that these calcium crystals may play a role in the disease process of OA. Injection of calcium crystals into the knee joints of dogs and mice induced severe inflammatory response [
Because the most striking pathological changes are found in articular cartilage, OA is originally considered as a cartilage disease. OA fibroblast-like synoviocytes (FLSs) have traditionally been considered to be normal and were used as controls in studies investigating the cellular alterations occurring in rheumatoid arthritis (RA) FLSs. However, it has been gradually realized that OA is a whole joint disease. A low degree of synovium inflammation is common in OA [
The presence of calcium crystals in OA articular cartilage is well-recognized and the chondrocyte-mediated calcification process has been studied extensively. However, calcium crystals were not only detected in the cartilage and synovial fluid but also detected in the synovial membranes of OA patients [
An early study examined arthritis patients who had no radiographic evidence of chondrocalcinosis; this work found that BCP crystals were present in 66% synovial fluid specimens derived from OA patients and in 20% synovial fluid specimens derived from RA patients [
These findings taken together suggest that OA synovial cells can play a role in the pathological calcification process associated with OA and may have a higher calcifying potential than RA synovial cells. In this study, we examined and compared the expression of genes implicated in the biomineral formation biological process in OA FLSs and RA FLSs, FLSs-mediated calcium mineral formation and deposition in the absence and presence of ATP in regular medium, and chondrogenesis differentiating medium and osteogenesis differentiation medium to test the hypothesis that OA FLSs are not bystanders but potentially active players in the pathological calcification process occurring in OA.
Dulbecco’s minimum essential medium (DMEM), STEMPro chondrogenesis differentiation medium, STEMPro osteogenesis differentiation medium, fetal bovine serum (FBS), and stock antibiotic/antimycotic mixture were obtained from Invitrogen (Carlsbad, CA, USA).
hTERT-OA 13A FLSs and hTERT-RA 516 FLSs, telomerase-immortalized human OA FLSs, and RA FLSs have been described previously [
hTERT-OA 13A FLSs and hTERT-RA 516 FLSs were plated at 85–90% confluence in 100 mm plates. The next day, DMEM containing 10% FBS was added. Forty-eight hour later, total RNA was extracted using Trizol reagent (Qiagen, Valencia, CA) and subjected to microarray analysis. Briefly, double stranded DNA was synthesized using SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA). The DNA product was purified using GeneChip sample cleanup module (Affymetrix, Santa Clara, CA). cRNA was synthesized and biotin labeled using BioArray high yield RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY). The cRNA product was purified using GeneChip sample cleanup module and subsequently chemically fragmented. The fragmented, biotinylated cRNA was hybridized to HG-U133_Plus_2 gene chip using Affymetrix Fluidics Station 400. Fluorescent signal was quantified during two scans using Agilent Gene Array Scanner G2500A (Agilent Technologies, Palo Alto, CA) and GeneChip operating Software (Affymetrix). Genesifter software (VizX Labs, Seattle, WA) was used for the analysis of fold changes.
RNA samples (1
Briefly, cDNA was synthesized using TaqMan Reverse Transcription reagents (Applied Biosystems, Inc., University Park, IL). Quantification of relative transcript levels for selected genes was performed using ABI7000 Real Time PCR system (Applied Biosystems, Inc.). TaqMan Gene Expression assay (Applied Biosystems, Inc.) was used, with FAM-MGB probes for fluorescent detection. cDNA samples were amplified with an initial Taq DNA polymerase activation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for one minute. Fold changes were calculated and the expression levels of genes were normalized to the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) according to the method described [
Cell-mediated calcium mineral formation and deposition were first investigated using a well-characterized ATP-induced CPPD crystal formation assay. It has been demonstrated that 45Calcium uptake in the monolayer cultures of chondrocytes is proportional to CPPD crystal formation [
Because the ATP-induced calcium mineral formation assay was mainly used to detect ATP-induced formation of CPPD crystals, we decided to further examine the FLSs-mediated calcium mineral formation and deposition in the absence of ATP using alizarin red, a method which can detect all types of calcium crystals. Briefly, hTERT-OA 13A FLSs, hTERT-RA 516 FLSs, primary OA FLSs, and primary RA FLSs were plated in forty-eight well plates at 85–90% confluence. The next day, culture medium was replaced with STEMPro chondrogenesis differentiation medium or STEMPro osteogenesis differentiation medium (triplicate). The cells were cultured for 9 days and fed with fresh STEMPro chondrogenesis differentiation medium or STEMPro osteogenesis differentiation medium every three days. At the end of the experimental period, medium was removed. Calcium mineral formation and deposition were examined using alizarin red. Alizarin red stains were extracted from each well with 200
Briefly, FLSs were harvested from 100 mm plates and suspended in DMEM containing 10% FBS. For preparation of a micromass, a droplet of the cell suspension containing
At the end of the experimental period, each well containing a micromass was rinsed twice with 500
The nonaqueous alcoholic eosin staining method was used to detect birefringent crystals [
The results of calcium mineral formation assays were expressed as the mean ± SD. The difference between two groups was analyzed using Student’s
We recently reported that numerous genes implicated in the inflammatory response biological process are differentially expressed in hTERT-OA 13A FLSs and hTERT-RA 516 FLSs [
Of the 31 differentially expressed genes (fold changes >1.8), eight genes displayed elevated expressions and 23 genes displayed decreased expressions in the hTERT-OA 13A FLSs compared with the hTERT-RA 516 FLSs (Table
Genes differentially expressed in hTERT-OA 13A FLSs compared with hTERT-RA 516 FLSs.
Biological process | Gene name | Differential expression (fold)* | Description |
---|---|---|---|
Biomineral formation | IGSF10 | 10.97 | Immunoglobulin superfamily, member 10 |
CHRDL1 | 8.40 | Chordin-like 1 | |
TWIST2 | 5.21 | Twist homolog 2 (Drosophila) | |
RUNX2 | 2.26 | Runt-related transcription factor 2 | |
ENPP1 | 1.95 | Ectonucleotide pyrophosphatase/phosphodiesterase 1 | |
BGLAP | 1.87 | Bone gamma-carboxyglutamate (gla) protein (osteocalcin) | |
BMPR1B | 1.87 | Bone morphogenetic protein receptor, type IB | |
COL13A1 | 1.85 | Collagen, type XIII, alpha 1 | |
FGF9 | −29.84 | Fibroblast growth factor 9 (glia-activating factor) | |
PTGER4 | −10.03 | Prostaglandin E receptor 4 (subtype EP4) | |
EGFR | −5.57 | Epidermal growth factor receptor | |
ANKH | −5.26 | Ankylosis, progressive homolog (mouse) | |
CTGF | −4.99 | Connective tissue growth factor | |
PTN | −4.67 | Pleiotrophin | |
ADRB2 | −4.45 | Adrenergic, beta-2-, receptor, surface | |
TGFB2 | −4.12 | Transforming growth factor, beta 2 | |
TGFB3 | −3.07 | Transforming growth factor, beta 3 | |
TGFB1 | −2.16 | Transforming growth factor, beta 1 | |
PTHLH | −3.61 | Parathyroid hormone-like hormone | |
BMP6 | −3.49 | Bone morphogenetic protein 6 | |
BMP4 | −2.86 | Bone morphogenetic protein 4 | |
MEF2C | −3.18 | Myocyte enhancer factor 2C | |
CDK6 | −3.13 | Cyclin-dependent kinase 6 | |
FOXC1 | −3.02 | Forkhead box C1 | |
TNFRSF11A | −2.35 | Tumor necrosis factor receptor superfamily, member 11a | |
AXIN2 | −2.31 | Axin 2 (conductin, axil) | |
GNAS | −2.13 | GNAS complex locus | |
SORT1 | −2.00 | Sortilin 1 | |
TUFT1 | −1.91 | Tuftelin 1 | |
SRGN | −1.90 | Serglycin | |
GLI2 | −1.82 | GLI-Kruppel family member GLI2 |
Negative number indicates decreased expression (fold) in hTERT-OA 13A FLSs compared with hTERT-RA 516 FLSs.
The genes which displayed decreased expression in hTERT-OA 13A FLSs included fibroblast growth factor 9 (FGF9: −29.84-fold), prostaglandin E receptor 4 (PTGER4: −10.03-fold), ANKH (−5.26-fold), pleiotrophin (PTN: −4.7-fold), parathyroid hormone-like hormone (PTHLH: −3.6-fold), and serglycin (SRGN: −1.9-fold). The genes displaying decreased expression also included epidermal growth factor receptor (EGFR: −5.57-fold), connective tissue growth factor (CTGF: −4.99-fold), adrenergic beta-2 receptor (ADRB2: −4.45-fold), transforming growth factor beta 1 (TGF-
To confirm the differential expression of ENPP1 and ANKH, we performed semiquantitative RT-PCR. As shown in Figure
Semiquantitative RT-PCR. RNA samples were extracted from hTERT-OA 13A FLSs and hTERT-RA 516 FLSs. The messenger levels of ENPP1, ANKH, and TNAP were determined by RT-PCR.
We also performed real time RT-PCR experiments to confirm the differential expression of selected genes. The results are listed in Table
Differential expression detected by microarray and real time RT-PCR*.
Gene name | Microarray | Real time RT-PCR | Description |
---|---|---|---|
RunX2 | 2.26 | 2.5 | Runt-related transcription factor 2 |
ENPP1 | 1.95 | 2.4 | Ectonucleotide pyrophosphatase/phosphodiesterase 1 |
BGLAP | 1.87 | 2.1 | Bone gamma-carboxyglutamate (gla) protein (osteocalcin) |
PTN | −4.67 | −5.2 | Pleiotrophin |
CTCG | −4.99 | −2.8 | Connective tissue growth factor |
SRGN | −1.90 | −1.7 | Serglycin |
Negative number indicates decreased expression (fold) in hTERT-OA 13A FLSs compared with hTERT-RA 516 FLSs.
First, we examined FLSs-mediated calcium mineral formation and deposition using an ATP-induced CPPD crystal formation and deposition assay. We found that ATP induced an ~40-fold increase in calcium mineral deposition in the monolayer culture of hTERT-OA 13A FLSs (Figure
ATP-induced calcium mineral formation and deposition in monolayer culture. Left bar group: untreated hTERT-OA 13A FLSs, ATP-treated hTERT-OA 13A FLSs, and
Next, we examined FLSs-mediated calcium mineral formation and deposition using primary OA FLSs derived from five OA patients (mean age 49 years) and primary RA FLSs derived from four RA patients (mean age 38 years). Consistently, we found that ATP-induced calcium mineral deposition (CPM=25,005) in the monolayer cultures of primary OA FLSs was collectively ~2-fold greater than that (CPM=14,606) in the monolayer cultures of primary RA FLSs (Figure
Primary OA FLSs-mediated and primary RA FLSs-mediated calcium mineral deposition.
OA FLSs | RA FLSs | ||||
---|---|---|---|---|---|
Age/gender of patients | Age/gender of patients | Age/gender of patients | Age/gender of patients | Control (CPM) | ATP (CPM) |
42 M | 27 F | 27 F | 27 F | 375 ± 91 | 28,580 ± 2,825 |
48 F | 39 F | 39 F | 39 F | 345 ± 123 | 19,790 ± 1005 |
50 F | 42 F | 42 F | 42 F | 290 ± 48 | 24,155 ± 1,987 |
52 F | 43 M | 43 M | 43 M | 365 ± 56 | 24,310 ± 1,105 |
54 F | 490 ± 89 | 28,325 ± 1,850 |
F: female; M: male; CMP: count per minute normalized against total protein levels; control—cultured in the absence of ATP; ATP—cultured in the presence of ATP.
ATP-induced calcium mineral formation and deposition in monolayer culture of primary FLSs. Left panel: untreated OA FLSs, ATP-treated OA FLSs, and
To further investigate OA FLSs- and RA FLS-mediated calcium mineral formation and deposition, we cultured OA FLSs and RA FLSs in STEMPro chondrogenesis differentiation medium for 14 days in the absence of ATP and examined calcium mineral formation and deposition using alizarin red. Representative alizarin red staining and readings are shown in Figure
Alizarin red staining and reading. (a) Alizarin red staining of hTERT-RA 516 FLSs, hTERT-OA 13A FLSs, primary OA FLSs, and primary RA FLSs cultured in STEMPro chondrogenesis differentiation medium. (b) Absorbance of alizarin red extract from the monolayer cultures of hTERT-RA 516 FLSs, hTERT-OA 13A FLSs, primary OA FLSs, and primary RA FLSs cultured in the basal medium and STEMPro chondrogenesis differentiation medium.
The alizarin red was extracted and quantified by reading at 405 nm. As shown in Figure
We also cultured hTERT-OA 13A FLSs and hTERT-RA 516 FLSs in STEMPro osteogenesis differentiation medium in the absence of ATP and examined calcium mineral formation and deposition using alizarin red. Representative alizarin red staining and results are shown in Figure
Alizarin red staining and analysis.(a) Alizarin red staining of hTERT-OA 13A FLSs and hTERT-RA 516 FLSs cultured in basal medium and STEMPro osteogenesis differentiation medium. (b)Absorbance of alizarin red extract from the monolayer cultures of hTERT-OA 13A FLSs and hTERT-RA 516 FLSs cultured in basal medium and STEMPro osteogenesis differentiation medium.
We cultured the micromasses of hTERT-OA 13A FLSs and hTERT-RA 516 FLSs in STEMPro chondrogenesis differentiation medium for 14 days and assessed calcium mineral formation and deposition using alizarin red staining. Representative images of alizarin red staining are shown in Figure
Alizarin red staining. Micromass of hTERT-RA 516 FLSs cultured in the absence of ATP. There were no calcium deposits detected (first photo from the left). Micromass of hTERT-OA 13A FLSs cultured in the absence of ATP. Small amounts of calcium deposits were detected within the micromass (second photo from the left). Micromass of hTERT-OA 13A FLSs cultured in the absence of ATP. Large amounts of calcium deposits were detected in the fragments of the micromass, but not within the micromass (third photo from the left). Micromass of hTERT-OA 13A FLSs cultured in the presence of ATP. Large amounts of calcium deposits were detected within the micromass (far right photo).
We examined the micromasses of hTERT-OA 13A FLSs using nonaqueous alcoholic eosin staining method. Representative images of the stained sections are shown in Figure
Nonaqueous alcoholic eosin staining. Birefringent crystals in the micromasses of hTERT-OA 13A FLSs were observed using polarizing light microscope (left photos). Enlarged images of the respective birefringent crystals are shown in the corresponding right hand images.
When performing ATP-induced calcium deposition assay, increased amounts of PC were added in select wells. Twenty-four hours later, calcium deposition was examined. The results are shown in Figure
Inhibition of calcium mineral formation and deposition. (a) PC inhibited ATP-induced calcium mineral formation and deposition in the monolayer culture of OA FLSs in a dose dependent manner (
The presence of BCP crystals in the synovial fluid of OA patients is well recognized; however, the sources of these crystals remain unclear. In this study, we found that many genes classified in the biomineral formation process were differently expressed in the hTERT-OA 13A FLSs and hTERT-RA 516 FLSs (Table
Many of the genes displaying lower expression levels in the hTERT-OA 13A FLSs have been previously implicated in the negative regulation of biomineral formation. For example, SRGN inhibited the growth of hydroxyapatite crystals [
Indeed, we found that FLSs were capable of mediating calcium mineral formation and deposition and that OA FLSs had a higher calcifying potential than RA FLSs. In addition, we found that the majority of calcium minerals formed in FLSs micromass culture were nonbirefringent. These findings are consistent with the clinical observations that calcium crystals are present in the synovial fluid and synovial membranes of OA patients and the majority of crystals presented in the OA synovial fluid are BCP crystals [
Previous studies demonstrated that CHRDL1 enhanced the proliferation of MSCs [
We found that more calcium minerals were formed in the monolayer cultures of OA FLSs and RA FLSs cultured in the STEMPro chondrogenesis differentiation medium than in the monolayer cultures of OA and RA FLSs cultured in STEMPro osteogenesis differentiation medium. This finding indicates that the differentiation of OA FLSs into hypertrophic chondrocytes-like cells, but not osteoblast-like cells, may play an important role in the OA FLSs-mediated calcium mineral formation and deposition.
The finding that ATP stimulated more calcium mineral formation and deposition in OA FLSs than in RA FLSs, together with the previous finding that OA synovial fluid contains higher concentrations of nucleoside triphosphate pyrophosphatase and ATP than RA synovial fluid [
The finding that more calcium minerals are formed and deposited in chondrogenesis condition, together with the previous finding that the protein level of BGLAP/osteocalcin is significantly higher in the OA synovial fluid than that in RA synovial fluid [
Finally, we demonstrate that PC significantly inhibits the OA FLSs-mediated calcium mineral formation and deposition. Interestingly, the morphology of OA FLSs (round and chondrocyte-like) within the micromasses in the absence of PC is different from the morphology of OA FLSs (long and fibroblast-like) in the presence of PC (Figure
Our study suggests that OA FLSs are not passive bystanders, but active players in the pathological calcification process occurring in OA. ATP and certain other unidentified differentiation-inducing factors presented within OA synovial fluid may play an important role in the OA FLSs-mediated calcium mineral formation and deposition. The FLSs-mediated calcification process is a valid target for the development of disease-modifying drug for OA therapy.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study is supported in part by a Charlotte-Mecklenburg Education and Research Foundation Grant and a Mecklenburg County Medical Society Smith Arthritis Fund Grant (to Yubo Sun). This study was performed at Carolinas Medical Center, Charlotte, NC, USA.