ArticlePDF Available

Abstract

Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90, CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA (3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10 μg/mL) for 3 weeks, and cellular morphological changes were evaluated. IL-1β and IL-6 were determined by ELISA, mineralization was assessed by alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from SM and after immunomagnetic separation were positive for CD90 (53 ± 8%) and CD105 (52 ± 18%) antigens, with 53 ± 5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals did not cause morphological damage or affect cell viability, nor were indicators of inflammation detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5 μg/mL MSU crystals; however, the differences were not significant with respect to the control. The highest dose of MSU crystals (10 μg/mL) induced significant Runx2 expression with respect to the control (1.4 times greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate osteogenic differentiation of SM-MSCs through an increase in Runx2.
Citation: Martínez-Flores, K.;
Plata-Rodríguez, R.; Olivos-Meza, A.;
López-Macay, A.; Fernández-Torres,
J.; Landa-Solís, C.; Zamudio-Cuevas,
Y. Osteogenic Potential of
Monosodium Urate Crystals in
Synovial Mesenchymal Stem Cells.
Medicina 2022,58, 1724. https://
doi.org/10.3390/medicina58121724
Academic Editors: Hang Korng Ea
and Daniela Opris-Belinski
Received: 25 September 2022
Accepted: 18 November 2022
Published: 24 November 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
medicina
Article
Osteogenic Potential of Monosodium Urate Crystals in
Synovial Mesenchymal Stem Cells
Karina Martínez-Flores 1, Ricardo Plata-Rodríguez 2, Anell Olivos-Meza 3, Ambar López-Macay 1,
Javier Fernández-Torres 1, Carlos Landa-Solís4and Yessica Zamudio-Cuevas 1,*
1Laboratorio de Líquido Sinovial, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra,
Mexico City 14389, Mexico
2
Facultad de Química, UNAM, Circuito Exterior S/N, Coyoacán, Cd. Universitaria, Mexico City 04510, Mexico
3
Servicio de Ortopedia del Deporte y Artroscopía, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra
Ibarra, Mexico City 14389, Mexico
4Unidad de Ingeniería de Tejidos, Terapia Celular y Medicina Regenerativa,
Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, Mexico City 14389, Mexico
*Correspondence: yesszamudio@gmail.com; Tel.: +52-55-5999-1000 (ext. 19501)
Abstract:
Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased
levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with
chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal
stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through
SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90,
CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic
separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA
(3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10
µ
g/mL) for 3 weeks, and cellular morphological
changes were evaluated. IL-1
β
and IL-6 were determined by ELISA, mineralization was assessed by
alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from
SM and after immunomagnetic separation were positive for CD90 (53
±
8%) and CD105 (52
±
18%)
antigens, with 53
±
5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals
did not cause morphological damage or affect cell viability, nor were indicators of inflammation
detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5
µ
g/mL MSU crystals;
however, the differences were not significant with respect to the control. The highest dose of MSU
crystals (10
µ
g/mL) induced significant Runx2 expression with respect to the control (1.4 times
greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate
osteogenic differentiation of SM-MSCs through an increase in Runx2.
Keywords:
monosodium urate crystals; gout; osteodifferentiation; synovial membrane; mesenchymal
stem cells
1. Introduction
Joint damage is a characteristic of erosive arthropathies, such as tophaceous gout, and
is associated with musculoskeletal disability. Joint deformity and disability progress as
more joints are affected and the tophi increase in size and number [
1
,
2
]. The association
between monosodium urate (MSU) crystal deposition and joint damage in patients with
chronic gout has been determined by the presence of tophi adjacent to sites of cartilage
loss and joint destruction. Tophaceous deposits can erode bones, cartilage, and tendons,
causing significant structural damage [3].
During an acute gout attack, proinflammatory cytokines, such as IL-1
β
, are involved in
the promotion of bone damage and in the activation and differentiation of osteoclasts, and
induce the production of enzymes that degrade the extracellular matrix (ECM), including
metalloproteinases, because of chronic inflammation [4].
Medicina 2022,58, 1724. https://doi.org/10.3390/medicina58121724 https://www.mdpi.com/journal/medicina
Medicina 2022,58, 1724 2 of 14
The stimulation of osteoclast precursors by MSU crystals does not directly promote
the formation of osteoclasts; this process can occur indirectly through the stromal cells
of tophi. MSU crystals inhibit the gene and protein expression of osteoprotegerin (OPG)
or osteoclastogenesis inhibitor factor in stromal cells and fibroblast-like synoviocytes
(FLSs) without altering the gene expression of the receptor activator ligand for nuclear
factor kappa-B (RANKL); thus, an imbalance in RANKL–OPG may lead to osteoclast
development from precursors [5].
On the other hand, MSU crystals in osteoblasts promote the expression of RANKL–
OPG, inducing osteoclastogenesis and bone resorption, and there is less osteoblast differen-
tiation, since human osteoblasts stimulated by MSU crystals and IL-1
β
decrease osteocalcin
formation and alkaline phosphatase activity [6].
MSU crystals contribute to bone erosion in gout through the formation and activation
of osteoclasts and decreased viability, function, and differentiation of osteoblasts, due to a
reduction in mineralization and the expression of genes related to osteoblast differentiation,
such as Runx2, Osterix (Sp7), Bone Sialoprotein (Ibsp), and Osteocalcin (Bglap). This
suggests that bone erosion in gout occurs at the tophus–bone interface through alterations
in the physiological turnover of bone, with excessive new bone formation, and activation
of osteoclasts, leading to reduced osteoblast differentiation [7].
Currently, there are no studies on mesenchymal stem cells (MSCs) from the synovial
membrane (SM) evaluating the impact of uric acid (UA) and MSU crystals on osteod-
ifferentiation. Some reports indicate that hyperuricemia induces the differentiation of
placenta-derived MSCs in neuronal cells
in vitro
[
8
], and other studies have reported the
effect of UA on the osteogenic and adipogenic differentiation of human MSCs isolated
from bone marrow (hBMSCs) through the 11
β
-hydroxysteroid dehydrogenase type 1
(11
β
-HSD1), alpha-1 subunit of nucleus binding factor, Cbfa1, Runx2, and Wnt signaling
pathways [9,10].
To date, most gout models have been used to study inflammatory processes in order
to clarify the pathogenic and molecular mechanisms of MSU crystals in cells that comprise
the SM, such as FLSs. The participation of MSCs in this process has been poorly studied,
especially in osteodifferentiation induced by UA exposure or MSU crystal deposition.
There is little information about the mechanisms of osteogenesis induced by UA and
MSU crystals in gout in terms of modulating the expression of proteins that favor the
bone erosion and new bone formation in affected joints. Understanding the molecular
mechanisms of MSCs in terms of amplifying their response to soluble UA (sUA) and crystals
will help in better defining their role in the regulation of osteogenesis in chronic gout.
Therefore, we hypothesized that sUA and/or MSU crystals play a role in the regulation of
osteodifferentiation during the chronic phase of the disease.
2. Materials and Methods
A comparative, analytical, cross-sectional, and non-probabilistic experimental study
was carried out [11].
Samples were obtained from 6 patients (n= 6) with anterior cruciate ligament tear
confirmed by diagnostic anterior drawer and Lachman tests [
12
] by physicians at the
Arthroscopy Service of the Instituto Nacional de Rehabilitación. At the time of recruit-
ment, all the subjects gave informed consent for inclusion before participating in the
study. Patients who underwent arthroscopic surgery donated their remaining SM tissue.
The inclusion criteria for patients were as follows: a body mass index (BMI) lower than
25 kg/m
2
, no existing gout condition or diagnosed disease, and a serum UA level lower
than 416.36
µ
mol/L (7 mg/dL). Additionally, individuals with type 2 diabetes, chronic
renal failure, or any other rheumatic disease were not included in the study. The study was
conducted in accordance with the Declaration of Helsinki, and the protocol was approved
by the Ethics Committee CONBIOETICA-09-CEI-031-20171207 (Project identification 23/21)
of the INR-LGII, on 12 July 2021.
Medicina 2022,58, 1724 3 of 14
2.1. Cell Isolation, Culture, and Immunophenotyping
The collected synovial tissue was washed with 1
×
PBS supplemented with 10%
penicillin/streptomycin (PS), followed by mechanical disintegration with a scalpel in
60
×
15 mm Petri dishes. Seeding by explant was performed, in which the SM fragments
were added to DMEM-F12 culture medium (Gibco, Life Technologies, Carlsbad, CA, USA)
supplemented with 20% fetal bovine serum (FBS) and 1% PS. The explants were incubated
at 37
C in an atmosphere of 95% humidity and 5% CO
2
. At 24 h, nonadherent cells were
removed by replacing the medium with fresh culture medium; this process was conducted
every third day until the cells were 80–90% confluent. Subcultures were performed, and
in the second passage, the cells were immunophenotyped using flow cytometry (BD
FACSCalibur) with anti-CD90 antibodies coupled to fluorescein isothiocyanate (FITC; BD
Pharmingen 561988, San Diego, CA, USA), CD105 coupled to phycoerythrin (PE; BD
Pharmingen 560839), CD73 coupled to peridinin chlorophyll (PerCP-C; BD Pharmingen
581280), CD117 allophycocyanin (APC; BD Pharmingen 341106), CD14–FITC (Thermo
Scientific 1-82074, Waltham, MA, USA), CD34–PE (BD Pharmingen 555822), CD45–FITC,
(BD Pharmingen 555482), CD166–PE (BD Pharmingen 559263), CD271–FITC (Miltenyi
Biotec 130098, Bergisch Gladbach, Germany), and CD31–FITC (Thermo Scientific 1-80360).
2.2. Immunomagnetic Separation and Immunophenotyping Assay
From 2
×
10
6
cells in the third passage, the CD90+ subpopulation was isolated by
MidiMACS immunomagnetic separation (Miltenyi Biotec, Bergisch Gladbach, Germany)
using anti-CD90 (Miltenyi Biotec) coupled to magnetic beads (MACS MicroBeads, Miltenyi
Biotec) and separation columns (MS Columns, Miltenyi Biotec). Cells were incubated with
anti-CD90 conjugated to 50 nm superparamagnetic particles for 15 min at 4
C protected
from light. Then, the cells were passed through magnetic separation columns and recov-
ered by negative selection (CD90- cells); the column was subsequently removed from the
magnetic field to recover CD90+ cells by elution. CD90+ cells were expanded and used for
osteodifferentiation assays.
2.3. Characterization of CD90+ Cells and Colony-Forming Unit (CFU) Assay
CD90+ cells were cultured until confluent and were immunophenotyped using flow
cytometry (BD FACSCalibur). The cells were phenotyped with CD90–FITC, CD105–PE,
CD73–PerCP-Cy, and CD117–APC; other cells were labeled with CD14–FITC and CD34–PE,
CD45–FITC and CD166–PE, and CD271–FITC and CD31–FITC. To identify whether the
SM-MSCs had the capacity to form colonies, 1000 cells of the CD90+ subpopulation were
cultured in 6-well plates for 14 days [
13
] in DMEM-F12 supplemented with 20% FBS and
1% PS; the cells were incubated at 37
C in an atmosphere with 95% humidity and 5% CO
2
.
For the identification of CFUs, the crystal violet dye (Sigma-Aldrich C3886-25G, St. Louis,
MO, USA) incorporation technique was used [
14
]. CFUs were identified and quantified
under a SteReo Discovery V.20 Zeiss stereoscopic microscope.
2.4. Cell Stimulation
CD90+ cells were exposed to UA (Sigma-Aldrich U2625, St. Louis, MO, USA) at
concentrations of 3, 6.8, and 9 mg/dL, simulating states of hypouricemia, normouricemia,
and hyperuricemia [
15
], respectively. The MSU crystals were synthetized, characterized,
and sterilized according to [
16
]. The absence of microbial contaminants was confirmed by
negative cultures for microorganisms and endotoxins. Cells were exposed to MSU crystals
in concentrations of 1, 5, and 10
µ
g/mL in DMEM-F12 medium supplemented with 20%
FBS and 1% PS. Cells stimulated with osteogenic culture medium (StemMACS OsteoDiff
Media, human, Miltenyi Biotec, Friedrich-Ebert-Strasse, Bergish Gladbach, Germany) were
used as positive control. All experimental groups were compared with a control group
(cells without stimuli). All culture media were changed every third day for 3 weeks.
Medicina 2022,58, 1724 4 of 14
2.5. Analysis of Cell Viability
After stimulating the cells with the established concentrations of UA, MSU crystals,
and osteogenic induction medium for the established time, the supernatants were removed
from the culture dishes and stored at
20
C for further studies. The cells were washed
with PBS and fixed with 2.5% glutaraldehyde (ICN Biomedicals, Inc., Chillicothe Rd.,
Aurora, OH, USA) for 10 min. Then, the cells were washed again and stained with crystal
violet. The dye was quantified through dissolution with acetic acid glacial (Fermont 03011
Productos Químicos, Monterrey, S. A de C.V, Mexico) [
13
]. The absorbance was measured
with a microplate reader at 595 nm (iMark
TM
, Bio-Rad, serial no. 10176, Hercules, CA,
USA). Absorbance readings were normalized with respect to the control, converting the
values into 100% viable cells [17].
2.6. Determination of Calcium Nodules
To determine the mineralization of the ECM of SM-MSCs exposed to the established
conditions, staining with alizarin red (Sigma-Aldrich A5533, St. Louis, MO, USA) was
performed [
18
]. The formation of mineralized ECM was identified by intense red regions
corresponding to Ca
2+
nodules in pleomorphic clusters [
19
] under an Evos microscope
(L1113-178C-173, Life Technologies Corp., Bothell, WA, USA). The cells were washed with
PBS and fixed with 4% paraformaldehyde. Then, the cells were washed, allowed to dry, and
stained with 2% alizarin red (Sigma-Aldrich A5533-25G, St. Louis, MO, USA) for 20 min
in a shaker (Compact Rocker, Bio-Rad Labnet International Inc., Woodbridge, NY, USA).
Subsequently, alizarin red staining was quantified by removing the dye with isopropanol
for 20 min under orbital agitation, and the absorbance of the dye was measured using
a microplate reader (iMark
TM
, Bio-Rad, serial no. 10176, Tokyo, Japan) at 415 nm; the
absorbance of the positive control was used as a reference, based on the red regions, which
were quantified and used as a reference for 100% calcium nodules.
2.7. Analysis of Proinflammatory Cytokines
IL-1
β
and IL-6 were quantified by ELISA using the supernatants. The commercial
Human IL-1
β
Standard ABTS ELISA Development Kit (Peprotech, 900-K95, Cedarbrook
Drive, Cranbury, NJ, USA) was used, and IL-6 was quantified using the Human IL-6
Standard ABTS ELISA Development Kit (900-K16, Cedarbrook Drive, Cranbury, NJ, USA).
Avidin peroxidase, 2,2
0
-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ABTS (Sigma-
Aldrich A9941, St. Louis, MO, USA) was used as a chromogenic substrate. The samples
were read at 405 nm using a microplate reader (iMark
TM
, Bio-Rad, serial no. 10176, Tokyo,
Japan). The results were compared to the standard curve for IL-1
β
and IL-6 and expressed
in pg/mL.
2.8. Extraction and Quantification of Total Protein
The cells were washed with PBS (4–8
C) and then mechanically lysed. The lysate
was centrifuged at 23,000
×
gfor 15 min at 4
C, and the cell pellet was resuspended in
lysis buffer M-PER Mammalian protein extraction reagent (Thermo Fisher Scientific, Pierce
Biotechnology, Rockford, IL, USA) containing 1 M DTT, 1 M PMSF, protease inhibitor
(Complete, Roche Diagnostics GmbH, Mannheim, Germany), and phosphatase inhibitor
(PhosSTOP, Roche Diagnostics GmbH, Mannheim, Germany). Cells were sonicated (Tmish-
ion, 008 China, Rue de la Caille, Nuaillé, France) for 15 min and subsequently centrifuged at
23,000
×
gfor 10 min at 4
C. The supernatant containing the total protein was aliquoted and
stored at
80
C. The protein concentration was determined using Quick Start Bradford
1
×
dye reagent (#5000205 Bio-Rad Laboratories, Inc., Hercules, CA, USA). A bovine serum
albumin (BSA) (P6154-Biowest, Nuaillé-France) standard curve from 25 to 2000
µ
g/mL
was used. The proteins were incubated for 2 h at 37
C. Absorbance was measured using a
microplate reader (iMarkTM, Bio-Rad, 10176, Tokyo, Japan) at 595 nm.
Medicina 2022,58, 1724 5 of 14
2.9. Analysis of Runx2 Protein Expression by Western Blot
Separation of 20
µ
g total protein was carried out by electrophoresis (120 V for 120 min)
on 10% polyacrylamide gels containing 0.1% SDS. Subsequently, the protein was transferred
to nitrocellulose membranes for 10 min at 25 V and 1 ampere in a Trans-Blot Turbo Trans-
fer System (690BR4448 Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes
were blocked for 1 h under agitation in TBS-Tween containing 5% BSA. Subsequently, the
membranes were incubated overnight under agitation at 4
C with anti-Runx2 (mouse,
monoclonal, Abcam 76956, Boston, MA, USA) at a 1:1000 dilution and
β
-actin-peroxidase
(mouse, monoclonal, A3854; Sigma-Aldrich, St. Louis, MO, USA) at a 1:10,000 dilution. A
secondary antibody coupled to anti-mouse IgG peroxidase (Abcam 97200) was used at a
1:10,000 dilution. Immunodetection was performed using Immobilon Western Chemilumi-
nescent HRP Substrate (Millipore Corp., Billerica, MA, USA). The blots were visualized
by exposure to CL-XPOSURE radiographic plates (Pierce Chemical, Rockford, IL, USA)
and scanned with VueScan software (HP DeskJet 2135, Korea). Densitometric analysis was
performed with ImageJ version 1.53e (National Institutes of Health, Bethesda, MD, USA).
β-actin was used as a load control.
2.10. Statistical Analysis
Each experiment was performed independently at least 3 times using cells from
different patients (n= 6). Statistical analysis of the results was performed with GraphPad
Prism 9.1.2. One-way analysis of variance (ANOVA) was followed by Dunnett’s or Tukey’s
post hoc test. A p< 0.05 was considered statistically significant.
3. Results
Among the patients, 66% were male and 34% were female. The average age of the
participants was 37
±
13 years. None of them had a history of gout or hyperuricemia, none
had clinical evidence of gout, and their synovial fluid did not indicate inflammation at the
time of arthroscopy surgery nor contained MSU crystals. SM-derived cells presented a
fibroblastic, fusiform phenotype with cytoplasmic extensions. Growth in groups was ob-
served; a heterogeneous population of cells with oligo- and polydendritic morphology was
also identified. Microscopic observation showed that fibroblasts became the predominant
cells in culture after 10–15 days (Supplementary Figure S1).
3.1. Immunophenotypic Characterization of Cells Isolated from SM
Among the isolated cells, 77
±
17% expressed CD90, 73
±
15% expressed CD105,
71
±
18% expressed CD73, and 40
±
15% expressed CD117. To a lesser extent, 2
±
1%
expressed CD14, 2
±
1% expressed CD34, 1
±
1% expressed CD45, 3
±
1% expressed
CD166, 4
±
8% expressed CD271, and 2
±
2% expressed CD31 (Supplementary Figure S2).
More than 70% of cells expressed the cellular markers CD90, CD105, and CD73; less than
50% expressed CD117; and less than 5% expressed CD14, CD34, CD45, CD166, CD271, and
CD31 (Figure 1).
Medicina 2022,58, 1724 6 of 14
Medicina 2022, 58, x FOR PEER REVIEW 6 of 14
Figure 1. Immunophenotyping of SM-derived cells. SM-derived cells showed abundant CD90,
CD105, and CD73 (mesenchymal) expression and lower CD117, CD34, CD45 (hematopoietic),
CD271 (neural), and CD31 (endothelial) expression. Values represent the mean ± standard deviation
of independent experiments (n = 6).
3.2. Immunomagnetic Separation of CD90+ Cells
Cells isolated from the SM and separated by an immunomagnetic column were
shown to be mostly CD90+ cells. A total of 1,860,000 ± 922,395 CD90+ cells were isolated
from each synovial culture from different patients. Flow cytometry showed that cells de-
rived from the SM after immunomagnetic separation were positive for CD90 (53 ± 8%)
and CD105 (52 ± 18%) antigens, among which 53 ± 5 CFUs were identified in multiple
clusters (Supplementary Figure S3).
3.3. Effect of UA and MSU Crystals on the Viability of SM-MSCs
Different stimuli did not affect the morphology of the cells. The number of cells in-
creased significantly, by 38 and 19%, in media supplemented with UA at concentrations
of 3 and 6.8 mg/dL, respectively, compared to the control. The cell population decreased
by 7% with respect to the control using 9 mg/dL UA. MSU crystals at a dose of 5 μg/mL
increased the cell population by 8%. Likewise, the cells exposed to 1 and 10 μg/mL MSU
crystals exhibited a tendency toward increased viability (4 and 7%, respectively); how-
ever, these changes were not significant with respect to the control. The cells exposed to
the osteogenic medium showed a significant increase in cell viability (94%) with respect
to the control (Figure 2).
Figure 1.
Immunophenotyping of SM-derived cells. SM-derived cells showed abundant CD90,
CD105, and CD73 (mesenchymal) expression and lower CD117, CD34, CD45 (hematopoietic), CD271
(neural), and CD31 (endothelial) expression. Values represent the mean
±
standard deviation of
independent experiments (n= 6).
3.2. Immunomagnetic Separation of CD90+ Cells
Cells isolated from the SM and separated by an immunomagnetic column were shown
to be mostly CD90+ cells. A total of 1,860,000
±
922,395 CD90+ cells were isolated from
each synovial culture from different patients. Flow cytometry showed that cells derived
from the SM after immunomagnetic separation were positive for CD90 (53
±
8%) and
CD105 (52
±
18%) antigens, among which 53
±
5 CFUs were identified in multiple clusters
(Supplementary Figure S3).
3.3. Effect of UA and MSU Crystals on the Viability of SM-MSCs
Different stimuli did not affect the morphology of the cells. The number of cells
increased significantly, by 38 and 19%, in media supplemented with UA at concentrations
of 3 and 6.8 mg/dL, respectively, compared to the control. The cell population decreased
by 7% with respect to the control using 9 mg/dL UA. MSU crystals at a dose of 5
µ
g/mL
increased the cell population by 8%. Likewise, the cells exposed to 1 and 10
µ
g/mL MSU
crystals exhibited a tendency toward increased viability (4 and 7%, respectively); however,
these changes were not significant with respect to the control. The cells exposed to the
osteogenic medium showed a significant increase in cell viability (94%) with respect to the
control (Figure 2).
Medicina 2022,58, 1724 7 of 14
Medicina 2022, 58, x FOR PEER REVIEW 7 of 14
Figure 2. Morphology of SM-MSCs at different concentrations of UA and MSU crystals. Bright-
field microscopy of SM-MSCs cultured with UA and MSU crystals. (A) Control, (B) 3 mg/dL UA,
(C) 6.8 mg/dL UA, (D) 9 mg/dL UA, (E) 1 μg/mL MSU crystals, (F) 5 μg/mL MSU, (G) 10 μg/mL
MSU, and (H) osteogenic medium (20×). Representative images of independent experiments (n =
6). (I) Cell viability increased in cultures treated with 3 and 6.8 mg/dL UA and in cells cultured in
osteogenic medium. The data represent the mean ± SD of independent experiments (* p < 0.05 vs.
control; ** p < 0.001 vs. control).
3.4. Effect of UA and MSU Crystals on the Formation of Calcium Nodules
Few mineralized nodules were observed in the control with respect to cells stimu-
lated with osteogenic medium; in those cells stimulated with 3 mg/dL UA, greater miner-
alization was observed than in the control and was similar to the positive control. In cells
stimulated with 6.8 and 9 mg/dL, limited mineralization was observed with respect to the
positive control based on the intensity of alizarin red dye. In the cells exposed to MSU
crystals (1, 5, and 10 μg/mL), lower-intensity mineralization was observed, as determined
by the scarce red coloration, a finding that was observed in the control. Experiments with
UA at concentrations of 3, 6.8, and 9 mg/dL showed less calcium nodule formation com-
pared to the positive control. Likewise, in the cells exposed to MSU crystals, less nodule
Figure 2.
Morphology of SM-MSCs at different concentrations of UA and MSU crystals. Bright-
field microscopy of SM-MSCs cultured with UA and MSU crystals. (
A
) Control, (
B
) 3 mg/dL UA,
(
C
) 6.8 mg/dL UA, (
D
) 9 mg/dL UA, (
E
) 1
µ
g/mL MSU crystals, (
F
) 5
µ
g/mL MSU, (
G
) 10
µ
g/mL
MSU, and (
H
) osteogenic medium (20
×
). Representative images of independent experiments
(n= 6). (
I
) Cell viability increased in cultures treated with 3 and 6.8 mg/dL UA and in cells cultured
in osteogenic medium. The data represent the mean
±
SD of independent experiments (* p < 0.05 vs.
control; ** p < 0.001 vs. control).
3.4. Effect of UA and MSU Crystals on the Formation of Calcium Nodules
Few mineralized nodules were observed in the control with respect to cells stimulated
with osteogenic medium; in those cells stimulated with 3 mg/dL UA, greater mineralization
was observed than in the control and was similar to the positive control. In cells stimulated
with 6.8 and 9 mg/dL, limited mineralization was observed with respect to the positive
control based on the intensity of alizarin red dye. In the cells exposed to MSU crystals
(1, 5, and 10
µ
g/mL), lower-intensity mineralization was observed, as determined by the
scarce red coloration, a finding that was observed in the control. Experiments with UA at
concentrations of 3, 6.8, and 9 mg/dL showed less calcium nodule formation compared to
Medicina 2022,58, 1724 8 of 14
the positive control. Likewise, in the cells exposed to MSU crystals, less nodule formation
was observed with respect to the positive control: 1
µ
g/mL MSU crystals generated
2% mineralization, 5
µ
g/mL MSU crystals generated 5%, and 10
µ
g/mL generated 3%
mineralization. In the control cells, there was a smaller percentage of calcium nodules
(Figure 3).
Medicina 2022, 58, x FOR PEER REVIEW 8 of 14
formation was observed with respect to the positive control: 1 μg/mL MSU crystals gen-
erated 2% mineralization, 5 μg/mL MSU crystals generated 5%, and 10 μg/mL generated
3% mineralization. In the control cells, there was a smaller percentage of calcium nodules
(Figure 3).
Figure 3. Effect of UA and MSU crystals on the formation of calcium nodules. (A) Osteogenic in-
duction control, (B) 3 mg/dL UA, (C) 6.8 mg/dL UA, (D) 9 mg/dL UA, (E) 1 μg/mL MSU crystals,
(F) 5 μg/mL MSU crystals, (G) 10 μg/mL MSU, and (H) control. Bright-field microscopy; cell culture
with red-stained calcium nodules (20×), representative of independent experiments (n = 6). The for-
mation of calcium nodules was observed to be high in the positive control (osteogenic medium); in
the cells cultured in media containing UA and MSU crystals, the effect was null, as was that in the
control. (I) Alizarin red staining was quantified by removing the dye with isopropanol. The data
represent the mean ± SD of independent experiments (**** p < 0.00001 vs. control +).
3.5. Effect of UA and MSU Crystals on Inflammation in SM-MSCs
IL-1β levels were not detected at any UA and MSU crystal concentrations used or in
cells exposed to osteogenic differentiation medium (data not shown). For IL-6, in cells
exposed to 3 mg/dL UA, the concentration was 1119 ± 656 pg/mL; in cells exposed to 6.8
mg/dL UA, the concentration was 1013 ± 327 pg/mL; and in cells stimulated with 9 mg/dL
UA, the concentration was 793 ± 430 pg/mLall of which were lower than compared to
control cells (1712 ± 503 pg/mL). In SM-MSCs stimulated with 1, 5, and 10 μg/mL MSU
crystals, the IL-6 levels were 930 ± 352, 899 ± 397, and 1026 ± 302 pg/mL, respectively; in
Figure 3.
Effect of UA and MSU crystals on the formation of calcium nodules. (
A
) Osteogenic
induction control, (
B
) 3 mg/dL UA, (
C
) 6.8 mg/dL UA, (
D
) 9 mg/dL UA, (
E
) 1
µ
g/mL MSU crystals,
(
F
) 5
µ
g/mL MSU crystals, (
G
) 10
µ
g/mL MSU, and (
H
) control. Bright-field microscopy; cell culture
with red-stained calcium nodules (20
×
), representative of independent experiments (n= 6). The
formation of calcium nodules was observed to be high in the positive control (osteogenic medium);
in the cells cultured in media containing UA and MSU crystals, the effect was null, as was that in the
control. (
I
) Alizarin red staining was quantified by removing the dye with isopropanol. The data
represent the mean ±SD of independent experiments (**** p< 0.00001 vs. control +).
3.5. Effect of UA and MSU Crystals on Inflammation in SM-MSCs
IL-1
β
levels were not detected at any UA and MSU crystal concentrations used or
in cells exposed to osteogenic differentiation medium (data not shown). For IL-6, in cells
exposed to 3 mg/dL UA, the concentration was 1119
±
656 pg/mL; in cells exposed to
6.8 mg/dL UA, the concentration was 1013
±
327 pg/mL; and in cells stimulated with
9 mg/dL UA, the concentration was 793
±
430 pg/mL—all of which were lower than
compared to control cells (1712
±
503 pg/mL). In SM-MSCs stimulated with 1, 5, and
Medicina 2022,58, 1724 9 of 14
10
µ
g/mL MSU crystals, the IL-6 levels were 930
±
352, 899
±
397, and 1026
±
302 pg/mL,
respectively; in cells exposed to osteogenic differentiation medium, the IL-6 concentration
was 63 ±68 pg/mL (Figure 4).
Medicina 2022, 58, x FOR PEER REVIEW 9 of 14
cells exposed to osteogenic differentiation medium, the IL-6 concentration was 63 ± 68
pg/mL (Figure 4).
Figure 4. Effect of UA and MSU crystals on IL-6 production by SM-MSCs. Quantification of IL-6 in
SM-MSCs treated with different stimuli, i.e., UA (3, 6.8, and 9 mg/dL), MSU crystals (1, 5, and 10
μg/mL), osteogenic medium (control +), and control. The data represent the mean ± SD of independ-
ent experiments (n = 6) (* p < 0.05 vs. control; ** p < 0.001 vs. control).
3.6. Analysis of Runx2 Protein Expression
The expression of Runx2 in cells treated with 3 and 6.8 mg/dL of sUA increased by
20% with respect to the control, and in cells treated with 9 mg/dL UA, expression in-
creased by 30%; however, the difference was not significant. In cells stimulated with 9
mg/dL, an upward trend in Runx2 expression (8.3%) was detected with respect to cells
stimulated with 3 and 6.8 mg/dL sUA. Treatment with 10 μg/mL MSU crystals resulted in
a significant increase of 40% with respect to the control, which was similar to that in cells
stimulated with the osteogenic medium. Treatment with 1 and 5 μg/mL MSU crystals re-
sulted in an upward trend of 20% (Figure 5).
Figure 4.
Effect of UA and MSU crystals on IL-6 production by SM-MSCs. Quantification of IL-6
in SM-MSCs treated with different stimuli, i.e., UA (3, 6.8, and 9 mg/dL), MSU crystals (1, 5, and
10
µ
g/mL), osteogenic medium (control +), and control. The data represent the mean
±
SD of
independent experiments (n= 6) (* p< 0.05 vs. control; ** p< 0.001 vs. control).
3.6. Analysis of Runx2 Protein Expression
The expression of Runx2 in cells treated with 3 and 6.8 mg/dL of sUA increased by
20% with respect to the control, and in cells treated with 9 mg/dL UA, expression increased
by 30%; however, the difference was not significant. In cells stimulated with 9 mg/dL, an
upward trend in Runx2 expression (8.3%) was detected with respect to cells stimulated
with 3 and 6.8 mg/dL sUA. Treatment with 10
µ
g/mL MSU crystals resulted in a significant
increase of 40% with respect to the control, which was similar to that in cells stimulated
with the osteogenic medium. Treatment with 1 and 5
µ
g/mL MSU crystals resulted in an
upward trend of 20% (Figure 5).
Medicina 2022,58, 1724 10 of 14
Medicina 2022, 58, x FOR PEER REVIEW 10 of 14
Figure 5. Effect of UA and MSU crystals on Runx2 expression. (A) Blot representative of the levels
of Runx2 protein expression in relation to actin (internal control). (B) Graph of the densitometric
analysis of Runx2 expression in the control group and treatment groups. The values in the columns
are the average ± SD of at least 3 independent experiments; Dunnett’s test, ** p < 0.01.
4. Discussion
In diarthrodial joints, the SM acts as a semipermeable membrane, controlling molec-
ular traffic into and out of the joint space and maintaining the composition of synovial
fluid. Besides FLSs and macrophage-like synoviocytes, the SM contains a subpopulation
of MSCs [20].
SM can provide a heterogeneous source of MSCs for experimental studies. According
to the definition provided by the International Society of Cell Therapy, the cells that we
isolated from SM are considered MSCs because of their ability to adhere to plastic, their
expansion when cultured in vitro, their ability to form colonies, and their fibroblast-like
morphology [13].
The immunophenotypes of the SM-derived cells were CD90+, CD105+, and CD73+,
and CD34-, CD14-, and CD4-. CD90 is highly expressed in cells derived from SM, indicat-
ing stem cells with multilineage capacity. The immunophenotype corresponds to those
reported by Huang et al. [21], Sakaguchi et al. [22], Segawa et al. [23], Prado et al. [24], and
Hatakeyama et al. [25], which indicates that the MSCs are characterized by positive ex-
pression of CD90, CD105, CD73, and CD44 (specific for stem cells) and negative expres-
sion of markers associated with the hematopoietic cell lineages CD45, CD34, CD11, CD14,
and CD117, among the most common ones.
Immunomagnetic separation was used to enrich CD90+ cells. This tool provides an
adequate method to obtain a satisfactory number of cells from a small part of tissue in a
short time, allowing us to obtain homogeneous cultures of MSCs that expressed CD90 and
CD105. These results are consistent with those of Jia et al. [26], who performed isolation
and culture of MSCs derived from synovial fluid, and successfully purified them by
Figure 5.
Effect of UA and MSU crystals on Runx2 expression. (
A
) Blot representative of the levels
of Runx2 protein expression in relation to actin (internal control). (
B
) Graph of the densitometric
analysis of Runx2 expression in the control group and treatment groups. The values in the columns
are the average ±SD of at least 3 independent experiments; Dunnett’s test, ** p < 0.01.
4. Discussion
In diarthrodial joints, the SM acts as a semipermeable membrane, controlling molecular
traffic into and out of the joint space and maintaining the composition of synovial fluid.
Besides FLSs and macrophage-like synoviocytes, the SM contains a subpopulation of
MSCs [20].
SM can provide a heterogeneous source of MSCs for experimental studies. According
to the definition provided by the International Society of Cell Therapy, the cells that we
isolated from SM are considered MSCs because of their ability to adhere to plastic, their
expansion when cultured
in vitro
, their ability to form colonies, and their fibroblast-like
morphology [13].
The immunophenotypes of the SM-derived cells were CD90+, CD105+, and CD73+,
and CD34-, CD14-, and CD4-. CD90 is highly expressed in cells derived from SM, indicat-
ing stem cells with multilineage capacity. The immunophenotype corresponds to those
reported by Huang et al. [
21
], Sakaguchi et al. [
22
], Segawa et al. [
23
], Prado et al. [
24
], and
Hatakeyama et al. [
25
], which indicates that the MSCs are characterized by positive expres-
sion of CD90, CD105, CD73, and CD44 (specific for stem cells) and negative expression of
markers associated with the hematopoietic cell lineages CD45, CD34, CD11, CD14, and
CD117, among the most common ones.
Immunomagnetic separation was used to enrich CD90+ cells. This tool provides
an adequate method to obtain a satisfactory number of cells from a small part of tissue
in a short time, allowing us to obtain homogeneous cultures of MSCs that expressed
CD90 and CD105. These results are consistent with those of Jia et al. [
26
], who performed
isolation and culture of MSCs derived from synovial fluid, and successfully purified them
by MACS using the MSC surface marker CD90. Indeed, MACS is a useful technique for the
purification of MSCs from synovial fluid or SM.
Medicina 2022,58, 1724 11 of 14
In this study, sUA and MSU crystals did not affect the viability of SM-MSCs at any
of the doses for the prolonged times of cell stimulation. The sUA doses were used to
simulate states of hypouricemia, normouricemia, and hyperuricemia, and the micro-doses
of the MSU crystals were for long-term stimulation. In a previous report, using FLSs to
evaluate the dose–response curve of MSU crystals from 60 to 100 ug/mL during 24 h, a
non-significant decrease was observed with the lower dose [
27
]. For the current study, we
proposed minimal doses of MSU crystals in order to assure SM-MSC survival and identify
the long-term effects in the model.
Regarding inflammation, IL-1
β
was undetectable; nevertheless, IL-1
β
plays a crucial
role in driving the transition from the acute phase of arthritis to the irreversible chronic
phase [
28
]. Only low levels of IL-6 were quantified with respect to the control; therefore,
our system did not induce inflammation at those doses. Evidence by Zheng et al. [
28
]
shows that exposure of FLSs to MSU crystals (1 and 10 ug/mL) transiently induced a
significant increase in IL-1
β
expression in a culture medium, with a peak at 6 h. Changes
in IL-6 and TNF-
α
expression were not observed. Likewise, Braga et al. [
29
] incubated
bone marrow-derived macrophages in the presence of sUA alone or with LPS (sUA+LPS)
for 6, 24, and 72 h. The sUA+LPS induced IL-1
β
mRNA expression when compared to
non-stimulated cells and cells stimulated with sUA alone. IL-1
β
expression was higher at
6 h and decreased with time.
Our findings confirm that low levels of inflammation could be attributed to the micro-
doses of MSU crystals in the model, and the sUA needs an adjuvant to cause inflammation.
On the other hand, some authors attribute the self-limiting phases of inflammation in gout
to regulators such as miR-146a, because MSU crystals can be present within clinically non-
inflamed joints and extra-articular tissues in people with previous acute outbreaks and in
tophaceous gout, suggesting that there are additional self-limiting regulatory mechanisms
of inflammation [30].
Regarding the effect of UA and MSU crystals on mineralization, the alizarin red
technique is quite sensitive and does not strongly reflect calcium deposits; however, Runx2
expression was significantly increased in cells treated with 10
µ
g/mL of MSU crystals; i.e.,
similar to that observed in cells in the osteogenic medium. Runx2 is a relevant biomarker,
because medications such as allopurinol reduce osteoblast apoptosis, increase their viability,
and reduce the risk of vascular calcification by decreasing Runx2 in animal models of
hyperuricemia [
31
,
32
]. In a recent study by Naot et al. [
33
], factors such as PGE2 and
TNF-
α
, secreted by macrophages stimulated with MSU crystals, reduced the viability of
osteoblasts in a dose-dependent manner in long-term cultures (13 days); however, the doses
ranged from 100 to 500
µ
g/mL, which decreased the expression of Runx2, suggesting that
bone erosion is a result of direct and indirect effects, such as the stimulation of exosomes
derived from neutrophils by MSU crystals, which has a negative effect on the viability of
osteoblasts [34].
Therefore, our findings indicate that MSU crystals induce signals that could favor the
osteodifferentiation of SM-MSCs in the long term. Our model reflects the late events that
occur due to the signaling between crystals and SM-MSCs. The use of SM-MSCs in this
study is novel; this is one of the first studies to use this cell type to assess the long-term
effects of both sUA and MSU crystals, in order to identify their effects on the induction of
bone. However, we identified some limitations that must be addressed in future research.
It will be necessary to evaluate other markers of osteogenic activity, such as alkaline
phosphatase and bone morphogenetic proteins, among others. If possible, increasing the
doses of MSU crystals would allow an approximation of the severe conditions that occur in
chronic gout.
5. Conclusions
MSU crystals in SM-MSCs modulated osteogenic differentiation through an increase
in Runx2 expression; however, more studies are needed to corroborate these findings,
Medicina 2022,58, 1724 12 of 14
in which other osteodifferentiation markers should be considered to contribute to the
knowledge regarding the role of crystals in the chronic and erosive stages of gout.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/medicina58121724/s1, Figure S1, Figure S2 and Figure S3.
Author Contributions:
Conceptualization, C.L.-S. and Y.Z.-C.; data curation, K.M.-F., R.P.-R., A.L.-M.
and J.F.-T.; formal analysis, K.M.-F., R.P.-R., A.L.-M., J.F.-T., C.L.-S. and Y.Z.-C.; funding acquisition,
A.O.-M., C.L.-S. and Y.Z.-C.; investigation, K.M.-F., R.P.-R., A.L.-M., J.F.-T. and Y.Z.-C.; methodology,
K.M.-F., R.P.-R., A.O.-M., A.L.-M., J.F.-T., C.L.-S. and Y.Z.-C.; project administration, C.L.-S.; resources,
A.O.-M.; software, K.M.-F. and J.F.-T.; validation, K.M.-F., A.L.-M., J.F.-T. and C.L.-S.; visualization,
Y.Z.-C.; writing—original draft, K.M.-F., R.P.-R., A.L.-M., J.F.-T., C.L.-S. and Y.Z.-C.; writing—review
and editing, A.O.-M., C.L.-S. and Y.Z.-C. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was supported by Federal Resources from INR-LGII.
Institutional Review Board Statement:
The study was approved by the Ethics and Research Com-
mittee of the INR-LGII (INR 23/21) on 12 July 2021, under the criteria established in the Declaration
of Helsinki for studies involving humans.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the
study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol
was approved by the Ethics Committee CONBIOETICA-09-CEI-031-20171207 (Project identification
23/21) of the INR-LGII, on 12 July 2021.
Acknowledgments:
The authors thank Rebeca Elizabeth Franco y Bourland of the Instituto Nacional
de Rehabilitación Luis Guillermo Ibarra Ibarra for the sterilization method for the MSU crystals
used in this study, and Luis EsaúLópez Jácome for the confirmation of the absence of microbial
contaminants by negative cultures for microorganisms and endotoxins.
Conflicts of Interest:
The authors declare no conflict of interest with respect to the research, author-
ship, and/or publication of this study.
References
1.
Anghelescu, A. Multiarticular deforming and erosive tophaceous gout with severe comorbidities. J. Clin. Rheumatol.
2019
,26,
e269–e271. [CrossRef] [PubMed]
2.
Oh, Y.J.; Moon, K.W. Presence of tophi is associated with a rapid decline in the renal function in patients with gout. Sci. Rep.
2021
,
11, 5684. [CrossRef] [PubMed]
3.
Shi, D.; Chen, J.Y.; Wu, H.X.; Zhou, Q.J.; Chen, H.Y.; Lu, Y.F.; Yu, R.S. Relationship between urate within tophus and bone
erosion according to the anatomic location of urate deposition in gout: A quantitative analysis using dual-energy CT volume
measurements. Medicine 2019,98, e18431. [CrossRef] [PubMed]
4.
McQueen, F.M.; Doyle, A.; Reeves, Q.; Gao, A.; Tsai, A.; Gamble, G.D.; Curteis, B.; Williams, M.; Dalbeth, N. Bone erosions in
patients with chronic gouty arthropathy are associated with tophi but not bone oedema or synovitis: New insights from a 3 T
MRI study. Rheumatol 2014,53, 95–103. [CrossRef] [PubMed]
5.
Dalbeth, N.; Smith, T.; Nicolson, B.; Clark, B.; Callon, K.; Naot, D.; Haskard, D.O.; McQueen, F.M.; Reid, I.R.; Cornish, J. Enhanced
osteoclastogenesis in patients with tophaceous gout: Urate crystals promote osteoclast development through interactions with
stromal cells. Arthritis. Rheum. 2008,58, 1854–1865. [CrossRef]
6.
Chhana, A.; Callon, K.E.; Pool, B.; Naot, D.; Watson, M.; Gamble, G.D.; McQueen, F.M.; Cornish, J.; Dalbeth, N. Monosodium
urate monohydrate crystals inhibit osteoblast viability and function: Implications for development of bone erosion in gout. Ann.
Rheum. Dis. 2011,70, 1684–1691. [CrossRef]
7.
Chhana, A.; Pool, B.; Callon, K.E.; Tay, M.L.; Musson, D.; Naot, D.; McCarthy, G.; McGlashan, S.; Cornish, J.; Dalbeth, N.
Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflam-
matory and proresorptive state. Arthritis Res. Ther. 2018,20, 208. [CrossRef]
8.
Yang, N.; Xu, L.; Lin, P.; Cui, J. Uric acid promotes neuronal differentiation of human placenta-derived mesenchymal stem cells in
a time- and concentration-dependent manner. Neural. Regen. Res. 2012,7, 756–760. [CrossRef]
9.
Xu, L.; Han, Y.; Li, P.; Ma, L.; Xin, Y.; Hao, X.X.; Huang, H.; Liu, B.; Yang, N. The Effects of Uric Acid on Bone Mesenchymal Stem
Cells Osteogenic Differentiation. J. Appl. Sci. Eng. Innov.
2017
,4, 39–45. Available online: http://www.jasei.org/PDF/4-2/4-39-4
5.pdf (accessed on 22 August 2022).
10.
Li, H.Z.; Chen, Z.; Hou, C.L.; Tang, Y.X.; Wang, F.; Fu, Q.G. Uric Acid Promotes Osteogenic Differentiation and Inhibits Adipogenic
Differentiation of Human Bone Mesenchymal Stem Cells. J. Biochem. Mol. Toxicol. 2015,29, 382–387. [CrossRef]
Medicina 2022,58, 1724 13 of 14
11.
Von Elm, E.; Altman, D.G.; Egger, M.; Pocock, S.J.; Gotzsche, P.; Vandenbroucke, J.P. The Strengthening the Reporting of
Observational Studies in Epidemiology (STROBE) statement: Guidelines for reporting observational studies. J. Clin. Epidemiol.
2008,61, 344–349. [CrossRef] [PubMed]
12.
Makhmalbaf, H.; Moradi, A.; Ganji, S.; Omidi-Kashani, F. Accuracy of lachman and anterior drawer tests for anterior cruciate
ligament injuries. Arch. Bone Jt. Surg. 2013,1, 94–97. [PubMed]
13.
Stemcell Technologies. Technical Manual: Human Colony-Forming Unit (CFU) Assays Using MethoCultTM. 2019. (Document
#28404/Version 4.6.0). Available online: https://cdn.stemcell.com/media/files/manual/MA28404-Human_Colony_Forming_
Unit_Assays_Using_MethoCult.pdf (accessed on 22 August 2022).
14.
Flick, D.A.; Gifford, G.E. Comparison of
in vitro
cell cytotoxic assays for tumor necrosis factor. J. Immunol. Methods.
1984
,68,
167–175. [CrossRef] [PubMed]
15.
Sánchez-Lozada, L.G.; Lanaspa, M.A.; Cristóbal-García, M.; García-Arroyo, F.; Soto, V.; Cruz-Robles, D.; Nakagawa, T.; Yu, M.A.;
Kang, D.H.; Johnson, R.J. Uric acid-induced endothelial dysfunction is associated with mitochondrial alterations and decreased
intracellular ATP concentrations. Nephron Exp. Nephrol. 2013,121, 71–78. [CrossRef] [PubMed]
16.
Zamudio-Cuevas, Y.E.; Martínez-Flores, K.; Fernández-Torres, J.; Loissell-Baltazar, Y.A.; Medina-Luna, D.; López Macay, A.;
Camacho-Galindo, J.; Hernández-Díaz, C.; Santamaría-Olmedo, M.G.; López-Villegas, E.O.; et al. Monosodium urate crystals
induce oxidative stress in human synoviocytes. Arthritis Res. Ther. 2016,18, 117. [CrossRef] [PubMed]
17.
Krebs, A.; Nyffeler, J.; Rahnenführer, J.; Leist, M. Corrigendum to Normalization of data for viability and relative cell function
curves. ALTEX Altern to Anim Exp. 2019,36, 505. [CrossRef]
18.
Arufe, M.C.; De La Fuente, A.; Fuentes, I.; de Toro, F.J.; Blanco, F.J. Chondrogenic potential of subpopulations of cells expressing
mesenchymal stem cell markers derived from human synovial membranes. J. Cell Biochem. 2010,111, 834–845. [CrossRef]
19.
Harvanová, D.; Tóthová, T.; Šarišský, M.; Amrichová, J.; Rosocha, J. Isolation and characterization of synovial mesenchymal stem
cells. Folia Biol. 2011,57, 119–124.
20.
Zamudio-Cuevas, Y.; Plata-Rodríguez, R.; Fernández-Torres, J.; Martínez-Flores, K.; Cárdenas-Soria, V.H.; Olivos-Meza, A.;
Hernández-Rangel, A.; Landa-Solís, C. Synovial membrane mesenchymal stem cells for cartilaginous tissues repair. Mol. Biol.
Rep. 2022,49, 2503–2517. [CrossRef]
21.
Huang, Y.Z.; Xie, H.Q.; Silini, A.; Parolini, O.; Zhang, Y.; Deng, L.; Huang, Y.C. Mesenchymal Stem/Progenitor Cells Derived from
Articular Cartilage, Synovial Membrane and Synovial Fluid for Cartilage Regeneration: Current Status and Future Perspectives.
Stem. Cell Rev. Rep. 2017,13, 575–586. [CrossRef]
22.
Sakaguchi, Y.; Sekiya, I.; Yagishita, K.; Muneta, T. Comparison of human stem cells derived from various mesenchymal tissues:
Superiority of synovium as a cell source. Arthritis Rheum. 2005,52, 2521–2529. [CrossRef]
23.
Segawa, Y.; Muneta, T.; Makino, H.; Nimura, A.; Mochizuki, T.; Ju, Y.J.; Ezura, Y.; Umezawa, A.; Sekiya, I. Mesenchymal stem
cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression
profiles. J. Orthop. Res. 2009,27, 435–441. [CrossRef] [PubMed]
24.
Prado, A.A.F.; Favaron, P.O.; da Silva, L.C.L.C.; Baccarin, R.Y.A.; Miglino, M.A.; Maria, D.A. Characterization of mesenchymal
stem cells derived from the equine synovial fluid and membrane. BMC Vet. Res. 2015,11, 1–13. [CrossRef] [PubMed]
25.
Hatakeyama, A.; Uchida, S.; Utsunomiya, H.; Tsukamoto, M.; Nakashima, H.; Nakamura, E.; Pascual-Garrido, C.; Sekiya, I.;
Sakai, A. Isolation and Characterization of Synovial Mesenchymal Stem Cell Derived from Hip Joints: A Comparative Analysis
with a Matched Control Knee Group. Stem. Cells Int. 2017,2017, 8–10. [CrossRef] [PubMed]
26.
Jia, Z.; Liang, Y.; Xu, X.; Li, X.; Liu, Q.; Ou, Y.; Duan, L.; Zhu, W.; Lu, W.; Xiong, J.; et al. Isolation and characterization of human
mesenchymal stem cells derived from synovial fluid by magnetic-activated cell sorting (MACS). Cell Biol. Int. 2018,42, 262–271.
[CrossRef]
27.
Zamudio-Cuevas, Y.; Fernández-Torres, J.; Martínez-Nava, G.A.; Martínez-Flores, K.; Ramírez-Olvera, A.; Medina-Luna, D.;
Hernández-Pérez, A.D.; Landa-Solís, C.; López-Reyes, A. Phagocytosis of monosodium urate crystals by human synoviocytes
induces inflammation. Exp. Biol. Med. 2019,244, 344–351. [CrossRef]
28.
Zheng, S.C.; Zhu, X.X.; Xue, Y.; Zhang, L.H.; Zou, H.J.; Qiu, J.H.; Liu, Q. Role of the NLRP3 inflammasome in the transient release
of IL-1βinduced by monosodium urate crystals in human fibroblast-like synoviocytes. J. Inflamm. 2015,12, 30. [CrossRef]
29.
Braga, T.T.; Forni, M.F.; Correa-Costa, M.; Ramos, R.N.; Barbuto, J.A.; Branco, P.; Castoldi, A.; Hiyane, M.I.; Davanso, M.; Latz, E.;
et al. Soluble Uric Acid Activates the NLRP3 Inflammasome. Sci. Rep. 2017,7, 39884. [CrossRef]
30.
Dalbeth, N.; Pool, B.; Shaw, O.M.; Harper, J.L.; Tan, P.; Franklin, C.; House, M.E.; Cornish, J.; Naot, D. Role of miR-146a in
regulation of the acute inflammatory response to monosodium urate crystals. Ann. Rheum. Dis. 2015,74, 786–790. [CrossRef]
31.
Zhao, J.; Wei, K.; Jiang, P.; Chang, C.; Xu, L.; Xu, L.; Shi, Y.; Guo, S.; Xue, Y.; He, D. Inflammatory Response to Regulated Cell
Death in Gout and Its Functional Implications. Front. Immunol. 2022,13, 888306. [CrossRef]
32.
Yan, B.; Liu, D.; Zhu, J.; Pang, X. The effects of hyperuricemia on the differentiation and proliferation of osteoblasts and vascular
smooth muscle cells are implicated in the elevated risk of osteopenia and vascular calcification in gout: An
in vivo
and
in vitro
analysis. J. Cell Biochem. 2019,120, 660–662. [CrossRef] [PubMed]
Medicina 2022,58, 1724 14 of 14
33.
Naot, D.; Pool, B.; Chhana, A.; Gao, R.; Munro, J.; Cornish, J.; Dalbeth, N. Factors secreted by monosodium urate crystal-stimulated
macrophages promote a proinflammatory state in osteoblasts: A potential indirect mechanism of bone erosion in gout. Arthritis
Res. Ther. 2022,24, 212. [CrossRef] [PubMed]
34.
Jia, E.; Zhu, H.; Geng, H.; Zhong, L.; Qiu, X.; Xie, J.; Xiao, Y.; Jiang, Y.; Xiao, M.; Zhang, Y.; et al. The Inhibition of Osteoblast
Viability by Monosodium Urate Crystal-Stimulated Neutrophil-Derived Exosomes. Front. Immunol.
2022
,13, 809586. [CrossRef]
[PubMed]
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
Background Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts. Methods Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay. Results In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1 , Runx2 , Sp7 , Bglap , Ibsp , and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE 2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE 2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs. Conclusions Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.
Article
Full-text available
Background and Objective Bone erosion is common in patients with gout. The role of neutrophil-derived exosomes in gouty bone erosion remains elusive. This study aimed to investigate the functions of the neutrophil-derived exosomes in the development of bone erosion in gout. Methods Neutrophil-derived exosomes were collected and assessed by transmission electron microscopy and nanoparticle tracking analysis. Cell counting kit-8 assay was applied to evaluate cell viability, and cell apoptosis was assessed by flow cytometry. In addition, quantitative Real-time PCR and Western blotting were used to determine the expression levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL). Neutrophil-derived exosomes were tagged with PKH67. The miRNA expression profiles of exosomes and human fetal osteoblasts (hFOB) were compared using high-throughput sequencing. Functional miRNAs transfected into hFOB after co-incubation with exosomes were selected and validated by preliminary qPCR. Results Neutrophil-derived exosomes were stimulated by monosodium urate (MSU). The exosomes could inhibit the viability of the hFOB, and the expression levels of ALP and OPG were down-regulated, while the expression level of RANKL was up-regulated. However, there was no significant difference in the viability of osteoclasts and the expression of nuclear factor of activated T cells 1. Exosomes were observed in the cytoplasm under a confocal microscopy, confirming that exosomes could be taken up by hFOB. In total, 2590 miRNAs were found, of which 47 miRNAs were differentially expressed. Among the delivered miRNAs, miR-1246 exhibited the highest level of differential expression. The viability of hFOB was reduced by miR-1246 mimics and increased by miR-1246 inhibitors. There was no significant difference in hFOB apoptosis rate between the miR-1246 mimic and miR-1246 inhibitor group. MiR-1246 overexpression decreased the expression levels of ALP and OPG, whereas increasing the expression level of RANKL. In contrast, miR-1246 inhibitor increased the expression levels of ALP and OPG, while decreasing the expression level of RANKL. Neutrophil-derived exosomes stimulated by MSU could increase the expression of miR-1246. Conclusion Neutrophil-derived exosomes stimulated by MSU could inhibit the viability of osteoblasts.
Article
Full-text available
Gout, a chronic inflammatory arthritis disease, is characterized by hyperuricemia and caused by interactions between genetic, epigenetic, and metabolic factors. Acute gout symptoms are triggered by the inflammatory response to monosodium urate crystals, which is mediated by the innate immune system and immune cells (e.g., macrophages and neutrophils), the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation, and pro-inflammatory cytokine (e.g., IL-1β) release. Recent studies have indicated that the multiple programmed cell death pathways involved in the inflammatory response include pyroptosis, NETosis, necroptosis, and apoptosis, which initiate inflammatory reactions. In this review, we explore the correlation and interactions among these factors and their roles in the pathogenesis of gout to provide future research directions and possibilities for identifying potential novel therapeutic targets and enhancing our understanding of gout pathogenesis.
Article
Full-text available
Background The present review is focused on general aspects of the synovial membrane as well as specialized aspects of its cellular constituents, particularly the composition and location of synovial membrane mesenchymal stem cells (S-MSCs). S-MSC multipotency properties are currently at the center of translational medicine for the repair of multiple joint tissues, such as articular cartilage and meniscus lesions. Methods and results We reviewed the results of in vitro and in vivo research on the current clinical applications of S-MSCs, surface markers, cell culture techniques, regenerative properties, and immunomodulatory mechanisms of S-MSCs as well as the practical limitations of the last twenty-five years (1996 to 2021). Conclusions Despite the poor interest in the development of new clinical trials for the application of S-MSCs in joint tissue repair, we found evidence to support the clinical use of S-MSCs for cartilage repair. S-MSCs can be considered a valuable therapy for the treatment of repairing joint lesions.
Article
Full-text available
We aimed to compare clinical characteristics of patients with and without tophi at the time of the diagnosis of gout and investigate the association of tophi and renal function in gout patients. The patients who were first diagnosed with gout at the Kangwon National University Hospital were retrospectively studied. Patients were divided into 2 groups according to the presence of tophi at the diagnosis. We compared clinical characteristics and the progression of renal dysfunction between the two groups. Of 276 patients, 66 (25.5%) initially presented with tophi. Tophi group was older, had a longer symptom duration, and a higher prevalence of multiple joint involvement than those without tophi. In multivariate logistic regression analysis, prolonged symptom duration and multiple joint involvement were significantly associated with increased risk of formation of tophi. The decline in the eGFR was more prominent in patients with tophi than in those without (− 4.8 ± 14.5 vs. − 0.7 ± 11.9 ml/min/1.73 m ² /year, respectively; P = 0.039). The presence of tophi was significantly associated with a rapid decline in the eGFR (β = − 0.136; P = 0.042). In conclusion, the presence of tophi was associated with a rapid declining renal function. Therefore, an early diagnosis and closely monitoring of renal function might be important in gout patients with tophi.
Article
Full-text available
The aim of this study was to measure the urate volume within tophus and bone erosion volume using dual-energy computed tomography in patients with tophaceous gout. Furthermore, our study aims to quantitatively analyze the relationship between monosodium urate (MSU) crystal deposition and bone erosion according to the anatomic location of urate deposition.Seventy-seven subjects with chronic gout were positively identified for the presence of urate deposition. Only 27 subjects identified for the presence of urate in contact with bone erosion were included in this study. The urate volumes and associated erosion volumes were measured. The relationships between urate within tophus and bone erosion were separately analyzed according to the anatomic location of urate deposition.Twenty-seven subjects were all male (100%) with a median (interquartile range, IQR) age of 52 (45-61) years. From all the subjects, 103 tophi depositions were identified in contact with bone erosion, including 58/103 tophi that contained an intraosseous component and 45/103 nonintraosseous tophi. Tophi containing intraosseous components were larger than nonintraosseous tophi (urate volume: median [IQR] 45.64 [4.79-250.89] mm vs 19.32 [6.97-46.71] mm, P = .035) and caused greater bone erosion (erosion volume: 249.03 [147.08-845.33] mm vs 69.07 [32.88-111.24] mm, P < .001). Almost all erosion volumes were larger than urate volumes in nonperiarticular tophi, in contrast to most erosion volumes, which were less than urate volumes in the tophi that contained a periarticular component (odds ratio, 95% confidence interval: 74.00, 14.70-372.60; P < .001). Urate volume and erosion volume demonstrated positive correlations in intraosseous tophi, intraosseous-intra-articular-periarticular tophi, and intraosseous-intra-articular tophi (rs = 0.761, rs = 0.695, rs = 0.629, respectively, P < .05).MSU crystal deposition shows a promoting effect on the development of bone erosions in varying degrees, associated with the location of MSU crystals deposited in the joints. The intraosseous tophi contribute the most to bone erosions, followed by intra-articular tophi, and periarticular tophi.
Article
Full-text available
Background: Bone erosion is a frequent complication of gout and is strongly associated with tophi, which are lesions comprising inflammatory cells surrounding collections of monosodium urate (MSU) crystals. Osteocytes are important cellular mediators of bone remodeling. The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocytes. Methods: For direct assays, MSU crystals were added to MLO-Y4 osteocyte cell line cultures or primary mouse osteocyte cultures. For indirect assays, the RAW264.7 macrophage cell line was cultured with or without MSU crystals, and conditioned medium from these cultures was added to MLO-Y4 cells. MLO-Y4 cell viability was assessed using alamarBlue® and LIVE/DEAD® assays, and MLO-Y4 cell gene expression and protein expression were assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Histological analysis was used to examine the relationship between MSU crystals, inflammatory cells, and osteocytes in human joints affected by tophaceous gout. Results: In direct assays, MSU crystals reduced MLO-Y4 cell and primary mouse osteocyte viability but did not alter MLO-Y4 cell gene expression. In contrast, conditioned medium from MSU crystal-stimulated RAW264.7 macrophages did not affect MLO-Y4 cell viability but significantly increased MLO-Y4 cell expression of osteocyte-related factors including E11, connexin 43, and RANKL, and inflammatory mediators such as interleukin (IL)-6, IL-11, tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). Inhibition of COX-2 in MLO-Y4 cells significantly reduced the indirect effects of MSU crystals. In histological analysis, CD68+ macrophages and MSU crystals were identified in close proximity to osteocytes within bone. COX-2 expression was also observed in tophaceous joint samples. Conclusions: MSU crystals directly inhibit osteocyte viability and, through interactions with macrophages, indirectly promote a shift in osteocyte function that favors bone resorption and inflammation. These interactions may contribute to disordered bone remodeling in gout.
Article
Background: In this study, we established both an animal model and a cellular model of hyperuricemia (HUC). Subsequently, we treated these models with allopurinol (ALLO) to study the effect of uric acid (UA) and ALLO on the differentiation and proliferation of osteoblasts and vascular smooth muscle cells (VSMC). Methods: Western Blot, immunohistochemistry assay, and real-time polymerase chain reaction were conducted to measure the changes in the expression of differentiation-related factors in osteoblasts and VSMCs in HUC and HUC+ALLO groups. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry were utilized to observe the changes in the proliferation of osteoblasts in HUC and HUC+ALLO groups. Von Kossa staining was performed along with calcium content measurement to investigate the effect of HUC/ALLLO on vascular calcification. Results: In this study, the levels of Wnt3a and differentiation-related factors, including Runx2, Sp7, Ibsp, Bglap, Dmp1, and Col1a1, were all evidently decreased in HUC rats, while the presence of ALLO increased the levels of above factors. In addition, the viability of osteoblasts was reduced while their apoptosis was elevated in the HUC group, and ALLO treatment reduced the apoptosis and increased the viability of osteoblasts to a certain extent. Moreover, HUC elevated the levels of Wnt3a, Runx2, Sp7, Bglap, Col1a1, SM22a, and Acta2 in VSMCs of HUC rats, leading to greatly increased calcium content and obvious vascular calcification. In contrary, ALLO treatment reduced the effect of HUC. Furthermore, the effect of UA and ALLO on osteoblasts and VSMCs was also validated in cellular models treated with monosodium urate (MSU) crystals or MSU+ALLO. Conclusions: HUC can suppress the differentiation and proliferation of osteoblasts while promoting the differentiation of VSMCs both in vivo and in vitro. The treatment by ALLO exhibited a therapeutic effect on HUC by promoting the differentiation and proliferation of osteoblasts while reducing vascular calcification.
Article
Impact statement: Gout is distinguished by an inflammatory process that is mediated by phagocytosis of monosodium urate (MSU) crystals in synoviocytes by regulation of unknown mechanisms. Here we suggest that the synovial cells play a crucial role in gouty arthritis by activating inflammation by MSU uptake and increasing the secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, TNF-α, MCP-1, and the growth factors NGF and HGF. We discuss some co-existing features in synoviocytes, including anomalous morphologies of the cells, and microvesicle formation, dysregulation in VEGF gene expression. We provide evidence that phagocytosis of MSU crystals triggers an inflammatory cellular state in synoviocytes in the pathogenesis of crystal-induced arthritis.