Article

Characterization of osteoarthritic human knees indicates potential sex differences

Abstract

Background: The prevalence of osteoarthritis is higher in women than in men in every age group, and overall prevalence increases with advancing age. Sex-specific differences in the properties of osteoarthritic joint tissues may permit the development of sex-specific therapies. Sex hormones regulate cartilage and bone development and homeostasis in a sex-dependent manner. Recent in vitro studies show that the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] also has sex-specific effects on musculoskeletal cells, suggesting that vitamin D3 metabolites may play a role in osteoarthritis-related sex-specific differences. The purpose of this study was to determine if sex-specific differences exist in synovial fluid and knee tissues isolated from male and female patients with severe knee osteoarthritis. We determined the presence of vitamin D3 metabolites, inflammatory cytokines, growth factors, and matrix metalloproteinases (MMPs) in synovial fluid and assessed responses of articular chondrocytes and subchondral osteoblasts to 17β-estradiol, dihydrotestosterone, and 1α,25(OH)2D3. Methods: Samples from knee joints of 10 Caucasian male and 10 Caucasian female patients with advanced osteoarthritis aged 65 to 75 years were obtained from total knee arthroplasty. Vitamin D metabolites, cytokines, MMPs, and growth factors in the synovial fluid were measured. Primary cultures of chondrocytes were isolated from fibrillated articular cartilage adjacent to osteoarthritis lesions and minimally affected cartilage distal to the lesion. Osteoblasts were isolated from the subchondral bone. Expression of receptors for 17β-estradiol and 1α,25(OH)2D3 was assessed by real-time PCR. Chondrocytes and osteoblasts were treated with 10(-8) M 17β-estradiol, dihydrotestosterone, or 1α,25(OH)2D3 and effects on gene expression and protein synthesis determined. Results: Histology of the articular cartilage confirmed advanced osteoarthritis. Sex differences were found in synovial fluid levels of vitamin D metabolites, cytokines, and metalloproteinases as well as in the cellular expression of receptors for 17β-estradiol and 1α,25(OH)2D3. Male cells were more responsive to 1α,25(OH)2D3 and dihydrotestosterone, whereas 17β-estradiol-affected female cells. Conclusions: These results demonstrate that there are underlying sex differences in knee tissues affected by osteoarthritis. Our findings do not address osteoarthritis etiology but have implications for different prevention methods and treatments for men and women. Further research is needed to better understand these sex-based differences.
R E S E A R C H Open Access
Characterization of osteoarthritic human
knees indicates potential sex differences
Qingfen Pan
1
, Mary I. OConnor
2
, Richard D. Coutts
3
, Sharon L. Hyzy
4
, Rene Olivares-Navarrete
4
, Zvi Schwartz
4,5
and Barbara D. Boyan
4,6,7*
Abstract
Background: The prevalence of osteoarthritis is higher in women than in men in every age group, and overall
prevalence increases with advancing age. Sex-specific differences in the properties of osteoarthritic joint tissues may
permit the development of sex-specific therapies. Sex hormones regulate cartilage and bone development and
homeostasis in a sex-dependent manner. Recent in vitro studies show that the vitamin D
3
metabolite
1α,25-dihydroxyvitamin D3 [1α,25(OH)
2
D
3
] also has sex-specific effects on musculoskeletal cells, suggesting that
vitamin D
3
metabolites may play a role in osteoarthritis-related sex-specific differences. The purpose of this study
was to determine if sex-specific differences exist in synovial fluid and knee tissues isolated from male and female
patients with severe knee osteoarthritis. We determined the presence of vitamin D
3
metabolites, inflammatory
cytokines, growth factors, and matrix metalloproteinases (MMPs) in synovial fluid and assessed responses of articular
chondrocytes and subchondral osteoblasts to 17β-estradiol, dihydrotestosterone, and 1α,25(OH)
2
D
3
.
Methods: Samples from knee joints of 10 Caucasian male and 10 Caucasian female patients with advanced
osteoarthritis aged 65 to 75 years were obtained from total knee arthroplasty. Vitamin D metabolites, cytokines,
MMPs, and growth factors in the synovial fluid were measured. Primary cultures of chondrocytes were isolated from
fibrillated articular cartilage adjacent to osteoarthritis lesions and minimally affected cartilage distal to the lesion.
Osteoblasts were isolated from the subchondral bone. Expression of receptors for 17β-estradiol and 1α,25(OH)
2
D
3
was assessed by real-time PCR. Chondrocytes and osteoblasts were treated with 10
8
M17β-estradiol,
dihydrotestosterone, or 1α,25(OH)
2
D
3
and effects on gene expression and protein synthesis determined.
Results: Histology of the articular cartilage confirmed advanced osteoarthritis. Sex differences were found in
synovial fluid levels of vitamin D metabolites, cytokines, and metalloproteinases as well as in the cellular expression
of receptors for 17β-estradiol and 1α,25(OH)
2
D
3
. Male cells were more responsive to 1α,25(OH)
2
D
3
and
dihydrotestosterone, whereas 17β-estradiol-affected female cells.
Conclusions: These results demonstrate that there are underlying sex differences in knee tissues affected by
osteoarthritis. Our findings do not address osteoarthritis etiology but have implications for different prevention
methods and treatments for men and women. Further research is needed to better understand these sex-based
differences.
Keywords: Osteoarthritis, Chondrocytes, Osteoblasts, Estrogen, Testosterone, Dihydrotestosterone, 1α,25(OH)
2
D
3
,
24R,25(OH)
2
D
3
, Vitamin D
* Correspondence: bboyan@vcu.edu
4
Department of Biomedical Engineering, Virginia Commonwealth University,
Richmond, VA, USA
6
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute
of Technology, Atlanta, GA, USA
Full list of author information is available at the end of the article
© 2016 Pan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Pan et al. Biology of Sex Differences (2016) 7:27
DOI 10.1186/s13293-016-0080-z
Background
Osteoarthritis (OA) is a leading cause of pain and dis-
ability in adults. Nearly one in two people may develop
symptomatic knee OA by age 85 [1]. OA affects the
whole joint, including the articular cartilage, subchon-
dral bone, muscle, tendons, meniscus, and synovium,
and impacts daily life and function. Symptoms may in-
clude joint pain, tenderness, stiffness, and locking [2]. A
variety of causes, including hereditary, developmental,
metabolic, and mechanical deficits, may initiate pro-
cesses leading to cartilage loss. Risk factors for deve-
loping OA include patient sex, race/ethnicity, joint
instability, obesity, joint trauma, and age. Epidemiologic
studies show sex-specific differences in the prevalence
and severity of OA [3, 4]. On average, the rate of arth-
ritis is 58 % higher in women than in men [5]. Women
report more pain due to OA than men report and are
more likely to have reductions in function and quality of
life [6]. Biomechanical differences between men and
women may contribute to differences in OA prevalence,
although data supporting this are not clear [7]; thus,
other sex differences may be involved.
Sex hormones are important regulators of cartilage
biology. Ovariectomized Cynomolgus monkeys exhibit
histopathologic features typical of OA, suggesting that
estrogen has a protective effect [8]. Studies using a
mouse model of OA support this; ovariectomized female
mice had more OA than intact mice, but orchidecto-
mized male mice had less severe OA than intact males
[9]. A number of studies have shown sexual dimorphism
in the response of chondrocytes and osteoblasts to 17β-
estradiol (E2) and dihydrotestosterone (DHT) at the cel-
lular level [1012]; however, the effects of sex hormones
on the incidence and progression of OA of the knee, as
well as on the regenerative potential of affected cartilage,
are poorly understood.
Studies indicate that bone and cartilage cells exhibit
sex-specific responses to the vitamin D metabolites
1α,25-dihydroxyvitamin D3 [1,25(OH)
2
D
3
] and 24R,25-
dihydroxyvitamin D3 [24,25(OH)
2
D
3
][1315]. In
addition to its role in maintaining calcium homeosta-
sis and bone metabolism [1618], recent evidence sug-
gests that 1,25(OH)
2
D
3
plays important roles in cartilage
remodeling and participates in the inflammatory response
[19, 20]. Epidemiologic studies have correlated low serum
levels of 25-hydroxyvitamin D
3
[25(OH)D
3
], the precursor
of 1,25(OH)
2
D
3
and 24,25(OH)
2
D
3
, with OA progression
[21]. Moreover, vitamin D supplementation had a pro-
tective effect against OA in rats by reducing messenger
RNAs (mRNAs) for matrix metalloproteinase (MMP)-3,
interleukin-1β(IL1β), and tumor necrosis factor alpha
(TNFα), factors associated with cartilage degradation and
inflammation [22]. Interestingly, 1α,25(OH)
2
D
3
induced
production of E2 in female rat chondrocytes but not in
male chondrocytes, further demonstrating that there is
sexual dimorphism at the cellular level [23].
As a first step in understanding how sex differences
might have an impact on OA, we investigated whether
differences were present in patients with OA sufficiently
severe to warrant total joint replacement. We reasoned
that the disease was comparable in patients of both
sexes, and while the etiology of the disease in any one
patient was unknown, all patients would receive the
same treatment. Thus, identified differences would be
more likely to be intrinsic to the sex of the patient, ra-
ther than random non-specific observations. We exam-
ined serum and synovial fluid obtained at the time of
surgery as well as the morphology of the articular cartil-
age. Also, we isolated chondrocytes and osteoblasts and
examined their responses to 1α,25(OH)
2
D
3
, E2, and
DHT.
Methods
Patient population
Surgeries and preclinical assessments were performed at
Mayo Clinic, Jacksonville, FL, USA. Bench studies were
conducted at Georgia Institute of Technology, Atlanta,
GA, USA, and Virginia Commonwealth University,
Richmond, VA, USA. Analyses for the studies were per-
formed at all locations listed under affiliations. Each au-
thor certifies that his or her institution approved the
human protocol for this investigation (Mayo Clinic - 11-
001468; Georgia Tech - H11239; VCU - HM15270), and
that all investigations were conducted in conformity with
ethical principles of research, and that informed consent
for participation in the study was obtained.
Twenty white, non-Hispanic patients (10 men/10
women) between the ages of 65 and 75 years, who were
undergoing total knee arthroplasty (TKA) due to severe
OA of the knee, were included in our study. Patients
with inflammatory arthritis, osteonecrosis, prior upper
tibial osteotomy, premenopausal women, patients aged
less than 65 years or aged greater than 75 years; who
were insulin-dependent or diabetic, with a BMI greater
than 30 kg/m
2
, or history of knee infection were ex-
cluded from our study (Table 1). The study size of 20
was powered based on serum 25(OH)D
3
levels, given an
alpha of 0.05, a standard deviation of 17, an effect size of
22.5, and power of 0.8.
Information collected at the Mayo Clinic before surgery
included patient demographics (age, sex, body mass index
[BMI]) and medication and supplement use (vitamin D
supplements, bisphosphonates, and estrogen). In addition,
we used several rating scales to provide baseline infor-
mation on the two patient cohorts in the event that sex
differences were observed. These included the Short
Form-12 (SF-12) Health Survey, the Western Ontario &
McMaster Universities Arthritis Index (WOMAC), and
Pan et al. Biology of Sex Differences (2016) 7:27 Page 2 of 15
the Physical Activity Scale for the Elderly (PASE) func-
tional scale tests. Serum 25(OH)D
3
levels and standard ra-
diographs were obtained for each patient. Within 2 weeks
of surgery, patients completed the Osteoarthritis Research
Society International-Outcome Measures in Rheumatol-
ogy (OARSI-OMERACT) pain scale [24], the knee pain
map [25], and pressurepain thresholds at the knee using
JTECH Tracker wireless software (V.5; JTECH Medical,
Midvale, UT, USA) [26, 27].
Although not statistically significant, there was some
evidence to suggest that previous or current vitamin D
use was more common in women patients than in men
patients (90 vs. 50 %; p= 0.14). As expected, only the fe-
male cohort reported previous or current estrogen use
(80 vs. 0 %; p< 0.001). There was no evidence of a differ-
ence in age, BMI, bisphosphonate use, prior knee sur-
gery, prior intraarticular steroid injections, or prior
intraarticular hyaluronic acid injections between men
and women patients (p0.21).
Per preoperative radiographic and knee pain assess-
ments, the pain-pressure threshold 1 cm above the
medial joint line was lower in women than men (me-
dian, 3.0 vs. 5.1 kg; p= 0.007) (Table 2). There was no
evidence of a difference in patient serum levels of
25(OH)D
3
, radiographic assessment, knee pain, or areas
of knee pain (p0.37). There was no evidence of a dif-
ference between male and female patients in the pain
and functional assessments before surgery, including the
WOMAC, OARSI-OMERACT, and SF-12 (p0.36)
(Table 3).
All patients exhibited similar morphologic and histo-
logical features. All patients had radiographic evidence
of joint narrowing and bone-bone contact (Fig. 1a).
Sample collection
Fresh human tissue was obtained from consenting patients
undergoing elective TKA at Mayo Clinic, Jacksonville,
FL, USA. Synovial fluid was aspirated from the knee
joint prior to skin incision, snap frozen, and stored
at 80 °C. Bone, with overlaying cartilage specimens
from the distal and posterior medial and lateral fem-
oral condyles, and the proximal tibial bone cut were
Table 1 Patient demographics and clinical history
Variable Female Male pvalue
a
Median age (IQR) - years 70 (68, 71) 69 (66, 72) 0.42
Median body mass index (IQR) - kg/m
2
26.2 (25.6, 28.1) 27.0 (24.3, 28.6) 0.34
Vitamin D use - no. (%)
b
0.14
Currently 6 (60 %) 3 (30 %)
Previously 3 (30 %) 2 (20 %)
Never 1 (10 %) 5 (50 %)
Median 25-hydroxy D3 (IQR) - ng/ml 42.1 (33.7, 47.2) 38.4 (31.0, 52.6) 0.79
Bisphosphonates use - no. (%)
b
0.47
Previously 2 (20 %) 0 (0 %)
Never 8 (80 %) 10 (100 %)
Estrogen use - no. (%)
b
<0.001
Currently 4 (40 %) 0 (0 %)
Previously 4 (40 %) 0 (0 %)
Never 2 (20 %) 10 (100 %)
Previously injured a knee so badly it was difficult to walk for at least a week - no. (%) 0 (0 %) 3 (30 %) 0.21
Prior knee surgery - no. (%) 6 (60 %) 6 (60 %) 1.00
Prior intra-articular injections of steroids - no. (%) 7 (70 %) 5 (50 %) 0.65
Median no. of steroid injections (IQR) 1 (0, 1)
c
1 (0, 3) 0.58
Prior intra-articular injection of hyaluronic acid - no. (%) 3 (30 %) 5 (50 %) 0.65
Median no. of hyaluronic injections (IQR) 0 (0, 1) 2 (0, 3) 0.30
Knee to be replaced - no. (%) 1.00
Right 6 (60 %) 6 (60 %)
Left 4 (40 %) 4 (40 %)
IQR interquartile range
a
pvalues result from a Wilcoxon rank sum test for numerical variables and Fishers exact test for categorical variables
b
pvalues are based on categorization as currently or previously used vs. never used
c
Information was not available for 1 patient
Pan et al. Biology of Sex Differences (2016) 7:27 Page 3 of 15
also obtained. Bone/cartilage specimens were shipped, on
ice, to the Institute for Bioengineering and Bioscience at
the Georgia Institute of Technology (Atlanta, GA, USA)
or the School of Engineering, Institute for Engineering
and Medicine at Virginia Commonwealth University
(VCU; Richmond, VA, USA). Upon receipt, knee tissues
were dissected, separating articular cartilage, subchondral
bone, synovial membrane, and meniscus. Tissues were
coded using de-identified patient numbers provided by
Mayo Clinic. Articulating surfaces of the tibia and femur
were photographed. Chondrocytes and osteoblasts were
isolated at the time of receipt; meniscus, synovium, bone,
and cartilage were processed for histology. Synovial
fluid was thawed and aliquoted, then stored at 80 °C
Table 2 Preoperative knee pain and radiographic assessment
Variable Female Male pvalue
a
Radiographic assessment
Kellgren-Lawrence Grading Scale - no. (%) 0.37
Grade 3 1 (10 %) 0 (0 %)
Grade 4 9 (90 %) 10 (100 %)
Median pain-pressure threshold (IQR) - kg
Medial joint line 4.5 (3.7, 5.1) 5.3 (4.1, 6.9) 0.16
1 cm above the medial joint line 3.0 (2.7, 3.8) 5.1 (4.5, 6.2) 0.007
1 cm below the medial joint line 4.0 (3.5, 4.1) 5.6 (4.0, 6.8) 0.10
Area(s) of knee pain - no. (%)
b
Localized
Superior lateral 0 (0 %) 1 (10 %) 1.00
Patella 0 (0 %) 1 (10 %) 1.00
Medial joint line 3 (30 %) 2 (20 %) 1.00
Inferior lateral 1 (10 %) 0 (0 %) 1.00
Inferior medial 1 (10 %) 0 (0 %) 1.00
Regional
Medial 1 (10 %) 1 (10 %) 1.00
Patella 1 (10 %) 2 (20 %) 1.00
Diffuse pain 4 (40 %) 3 (30 %) 1.00
a
pvalues result from a Wilcoxon rank sum test for numerical variables and Fishers exact test for categorical variables
b
More than one location per patient was possible. Pain was not reported at any of the following locations: superior medial, lateral joint line, back of knee, lateral
(regional), and back of knee (regional)
Table 3 Pain and functional assessment prior to surgery
Variable Female Male pvalue
a
WOMAC
Median total raw score (IQR) 39 (32, 47) 45 (27, 55) 0.76
Median pain score (IQR) 8.5 (7, 10) 9.0 (5, 12) 0.73
Median stiffness score (IQR) 4.0 (2, 5) 4.0 (3, 6) 0.64
Median difficulty performing daily activities score (IQR) 28 (21, 33) 32 (18, 38) 0.70
OARSI-OMERACT pain scale
Median total pain score (IQR) 49 (30, 59) 42 (39, 61) 0.88
Median constant pain subscore (IQR) 45 (20, 55) 38 (25, 45) 0.36
Median intermittent pain subscore (IQR) 48 (42, 63) 50 (42, 67) 0.70
SF-12
Median physical scale (IQR) 33 (29, 42)
b
38 (30, 39) 0.55
Median mental scale (IQR) 61 (54, 63)
b
57 (54, 66) 0.90
a
pvalues result from a Wilcoxon rank sum test
b
Information was not available for 1 patient
Pan et al. Biology of Sex Differences (2016) 7:27 Page 4 of 15
until used. All assays performed at VCU were blinded to
donor sex.
Histology
Following fixation in 10 % formalin, cartilage was decal-
cified (Decal Chemical Corporation, Tallman, NY, USA)
for 16 h on a rotating platform before undergoing dehy-
dration in a series of 95 and 100 % ethanol and xylene
washes. Samples were embedded in paraffin; 7-μmsec-
tions were stained with safranin-O to assess glycosami-
noglycan content.
Synovial fluid
Synovial fluid levels of 1,25(OH)
2
D
3
were measured ac-
cording to manufacturers instructions using a human
1,25(OH)
2
D
3
ELISA kit (DHVD
3
, Novatein Biosciences,
Cambridge, MA, USA). Levels of 25(OH)D
3
were mea-
sured according to manufacturersinstructionsusingahu-
man 25-hydroxyvitamin D
3
ELISA kit (25HVD
3
,Novatein
Biosciences). In addition, we used an ELISA kit from
Immunodiagnostic Systems (Gaithersburg, MD, USA) that
measured 25(OH)D
3
,25(OH)D
2
, and 24,25(OH)
2
D
3
.We
then approximated the amount of 24,25(OH)
2
D
3
plus
25(OH)D
2
by subtracting the values for 25(OH)D
3
that
had been obtained using the Novatein Biosciences assay.
Because other vitamin D metabolites might have also been
present, including 1,24,25(OH)
3
D
3
, mass spectrometry
(Thermo Vantage Triple Quadrupole Mass Spectrometer;
Biotrial, Quebec, Canada) was used to measure
24,25(OH)
2
D
3
specifically. Data were calculated per
milliliter of synovial fluid.
Inflammatory cytokines and MMPs present in the syn-
ovial fluid were measured using a Luminex screening
assay (R&D Systems, Minneapolis, MN, USA). This
assay uses superparamagnetic beads coated with analyte-
specific antibodies. Transforming growth factor beta-1
(TGFβ1), TGFβ2, and TGFβ3 levels were measured
using a magnetic bead-based multiplex assay (Bio-Rad,
Hercules, CA, USA). Sulfated glycosaminoglycan levels
were measured using the dimethyl methylene blue assay
(Sigma-Aldrich, St. Louis, MO, USA) [28]. In all cases,
data were determined per milliliter of synovial fluid.
Chondrocyte cultures
Articular cartilage was harvested by sharp dissection
from areas of minimal fibrillation and areas of maximum
erosion (Fig. 1b). Cartilage pieces were minced and
incubated in Dulbeccos modified Eagles medium
(DMEM; Thermo Fisher, Waltham, MA, USA) contain-
ing 0.25 % trypsin (Life Technologies, Carlsbad, CA,
USA) for 30 min at 37 ° C. After discarding the trypsin
digest, chondrocytes were isolated from cleaned cartilage
fragments by incubating them for 16 h in 0.03 % type-II
collagenase (Worthington Biosciences, Lakewood, NJ,
USA) in DMEM containing 100 U/mL penicillin and
100 μg/mL streptomycin. Cells were separated from
remaining cartilage pieces by straining the mixture
through a 40-μm filter. Cells were cultured in DMEM
supplemented with 10 % fetal bovine serum (Life
Technologies) and 100 U/mL penicillin and 100 μg/mL
streptomycin. A preliminary study demonstrated that
first-passage cells retained their chondrocyte phenotype,
whereas cells at later passages did not. Therefore, pas-
sage 1 cells were used for all experiments and cells were
seeded at 15,000/cm
2
for all experiments. We have suc-
cessfully identified differences at the cellular level with
as few as three donors per group [29], but we opted to
culture the cells from all 20 patients. Unfortunately,
there was not sufficient tissue available in some cases; in
other instances, cell growth was inadequate. The final
sample size for the cell culture studies was 6 males and
6 females.
In order to determine if chondrocytes were competent
to respond to hormone treatment, mRNA levels for re-
ceptors for 1,25(OH)
2
D
3
(vitamin D receptor [VDR] and
protein disulfide isomerase A3 [PDIA3]) or E2 (ERα66
[ESR1] and ERα36) were measured in chondrocytes from
both fibrillated and minimally affected OA tissues via
Fig. 1 Gross changes in osteoarthritic joints. aRadiograph showing joint narrowing and bone-to-bone contact. bArticular cartilage showing areas
of minimal fibrillation (arrow) and areas of maximum erosion (star). cSafranin-O-stained fibrillated cartilage showing loss of proteoglycan
Pan et al. Biology of Sex Differences (2016) 7:27 Page 5 of 15
real-time qPCR [30]. The cells were further character-
ized with respect to basal expression of inflammatory cy-
tokines (IL1A, IL1B, IL6, IL8, and IL10) [31]. Related
work has shown that differentiation of chondrocytes in
response to 1,25(OH)
2
D
3
involves signaling via wingless-
Int pathway molecules (WNT3A, WNT5A, CTNNB,
DKK1, and DKK2) [32]; therefore, we also measured
RNA expression for these proteins. Primers for each of
the mRNAs are shown in Additional file 1: Table S1.
RNA was harvested using a TRIzol (Invitrogen, Carlsbad,
CA, USA) extraction method following manufacturers
protocol. RNA was quantified using a NanoDrop spectro-
photometer (Thermo Scientific, Waltham, MA, USA) and
specific mRNAs (250 ng) were amplified using reverse
transcription (High-Capacity cDNA Reverse Transcription
Kit; Life Technologies). Starting quantities of mRNA were
determined using SybrGreen chemistry (Power SYBR;
Green PCR Master Mix, Life Technologies) in a StepOne
Plus imaging system (Life Technologies). All gene expres-
sion was normalized to glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH).
In order to examine if there were sex differences in re-
sponse to hormonal treatment, confluent first passage
chondrocytes were treated with 10
8
M1α,25(OH)
2
D
3
(Enzo Life Sciences, Plymouth, PA, USA) or 10
8
ME2
(Sigma-Aldrich, St. Louis, MO, USA). In addition, cells
were treated with 10
7
M 24R,25(OH)
2
D
3
(Enzo Life Sci-
ences) or 10
8
M5α-DHT (Sigma-Aldrich). mRNAs for
aggrecan (ACAN), type-II collagen (COL2A1), and car-
tilage oligomeric matrix protein (COMP) were measured
at 12 h using real-time qPCR. Primers used are listed in
Additional file 1: Table S1.
To measure alkaline phosphatase specific activity, con-
fluent first-passage cells were treated for 24 h with either
vehicle (0.01 % ethanol), or 10
8
M1α,25(OH)
2
D
3
or E2.
Cells were harvested by trypsin digestion and were lysed
in Triton X-100 (Sigma-Aldrich). Enzyme activity was
measured by assaying the release of p-nitrophenol from
p-nitrophenylphosphate at a pH of 10.2 in the cell ly-
sates and was normalized to total protein content of the
cell lysates (BCA Protein Assay; Thermo Fisher Scien-
tific, Waltham, MA, USA) [33].
Osteoblast cultures
Subchondral bone samples obtained from regions under
minimally fibrillated cartilage and regions of maximal
cartilage erosion were minced into small chips, incu-
bated with 0.25 % trypsin for 30 min at 37 °C, and then
washed with DMEM with 1 % penicillin-streptomycin
three times. After washing, bone chips were cultured for
2 weeks in Petri dishes containing DMEM with 1 %
penicillin-streptomycin and 10 % fetal bovine serum to
enable osteoprogenitor cells to migrate out onto the dish
surface. Media were changed once a week until cells
reached confluence. Cells were then subcultured by
seeding at 10,000 cells/cm
2
for all experiments.
The presence of receptors for 1α,25(OH)
2
D
3
and E2
was assessed as described for the chondrocyte cultures
above. Response to the hormones was examined using
confluent first-passage osteoblasts treated with either
vehicle (0.01 % ethanol) or 10
8
M1α,25(OH)
2
D
3
,E2or
DHT for 24 h. The conditioned media were collected,
and levels of secreted osteocalcin and osteoprotegerin
were determined. Osteocalcin was measured using a
commercially available radioimmunoassay following
manufacturers instructions (Biomedical Technologies,
Stoughton, MA, USA) [34]. Osteoprotegerin was mea-
sured by ELISA (R&D Systems, Minneapolis, MN,
USA) [35]. After decanting the conditioned media, cells
were harvested by trypsin digestion and alkaline
phosphatase-specific activity was determined in cell ly-
sates. DNA was measured using a fluorometric assay
from Promega (Madison, WI, USA). Levels of secreted
factors in the conditioned media were normalized to
total DNA.
Statistical analysis
Clinical data were summarized for each sex by the sam-
ple median and interquartile (IQR) range for numerical
variables whereas categorical variables were summarized
by frequency and percentage. Comparisons between
men and women were evaluated using a Wilcoxon rank
sum test for numerical variables, Cochran-Armitage test
for trend for ordered variables, and Fisher exact test for
categoric variables. Statistical significance was consid-
ered at p< 0.05 without adjustment for multiple testing,
owing to the exploratory and hypothesis-generating na-
ture of the study. The considerable number of tests per-
formed increased the chance of a type-I error, and
interpretation of study results should consider this.
Additionally, the small sample size increased the chance
of a type-II error (i.e., false-negative association). Statis-
tical analyses were performed using SAS statistical soft-
ware (V. 9.3; SAS Institute Inc.; Cary, NC, USA).
Data from the cell studies are presented as the mean ±
standard error (SEM) of six independent cultures for
each patient per variable. Six patients of each sex were
examined for the cell-hormone treatment study. Statis-
tical significance was determined by ANOVA with post
hoc Bonferroni modification of Studentsttest. For
graphs labeled as treatment over control, the value of
each sample from the treated group was divided by the
mean of the control group. Each data point represents
the mean ± SEM for six normalized values. The control
is represented by a dashed line with a value equal to 1.
Significance was determined by using the Mann-
Whitney test. Significance was reached when pvalue
was less than 0.05.
Pan et al. Biology of Sex Differences (2016) 7:27 Page 6 of 15
Results
Histology
Histology confirmed the radiographic diagnosis of severe
OA. The articular cartilage was fibrillated and exhibited
marked loss of sulfated glycosaminoglycans (sGAGs)
(Fig. 1c).
Synovial fluid
Synovial fluid composition differed between men and
women patients (Table 4). Men had increased levels of
MMPs 1, 7, 9, and 13. Hepatocyte growth factor (HGF),
stem cell factor (SCF), and stem cell growth factor beta
(SCGFβ) were all higher in male synovial fluid than in
the synovial fluid of women patients. The amounts of
sGAG, TGFβ1, and TGFβ2 were higher in men com-
pared with women. Women had higher levels of in-
flammatory cytokines, IL2α, IL3, IL12p40, IL16, IL18,
and TNFβ, and chondrocyte apoptosis-inducing factor
(TRAIL) compared with men. Higher levels of macro-
phage stimulators (leukemia inhibitory factor [LIF],
macrophage colony-stimulating factor [M-CSF], macrophage
migration inhibitory factor [MIF]), and pro-inflammatory
Table 4 Levels of inflammatory cytokines measured in synovial fluid
Mean value (pg/ml) ± SEM
Male Female pvalue
HGF 1075.00 ± 254.20 514.30 ± 46.10 0.0352
Males > females
IFN-α2 279.30 ± 50.09 159.80 ± 12.10 0.0207
IL1α389.00 ± 23.91 337.40 ± 9.80 0.0367
MMP-1 2219.00 ± 539.60 1270.00 ± 84.74 0.049
MMP-2 1981.00 ± 50.44 1811.00 ± 42.84 0.0248
MMP-7 1189.00 ± 15.21 1147.00 ± 7.63 0.0173
MMP-9 1565.00 ± 473.20 585.60 ± 135.50 0.0328
MMP-12 39.20 ± 0.87 36.90 ± 0.46 0.0227
MMP-13 441.90 ± 18.22 396.00 ± 7.241 0.0187
SCF 478.00 ± 116.2 239.80 ± 21.45 0.0487
SCGF-β5503.00 ± 1713.20 1928.00 ± 433.60 0.0487
TGFβ1 1723.00 ± 282.00 1070.00 ± 109.00 0.0464
TGFβ2 35.79 ± 4.72 23.69 ± 2.74 0.0415
sGAG 14.04 ± 2.02 6.25 ± 0.57 0.004
GRO-α459.40 ± 95.18 739.80 ± 61.36 0.0217
Females > males
IL2α843.90 ± 166.50 1321.00 ± 120.20 0.0306
IL3 806.90 ± 204.80 1289.00 ± 55.29 0.0395
IL12p40 380.60 ± 81.14 578.20 ± 26.41 0.0342
IL16 708.10 ± 148.50 1353.00 ± 247.00 0.0449
LIF 309.60 ± 60.31 480.80 ± 41.60 0.0294
MCP-3 147.70 ± 29.88 220.10 ± 17.42 0.0466
M-CSF 748.80 ± 161.60 1156.00 ± 107.00 0.0468
MIF 752.60 ± 172.70 1665.00 ± 357.50 0.0323
MIG 1793.00 ± 321.80 4252.00 ± 1018.00 0.0371
TNF-β148.00 ± 42.95 252.20 ± 8.99 0.0304
TRAIL 714.90 ± 156.20 1100.00 ± 79.97 0.0332
β-NGF 5.67 ± 2.76 3.42 ± 0.83 0.4791
Not significant
CTACK 613.00 ± 112.90 925.30 ± 172.90 0.1621
IL18 156.80 ± 8.56 160.90 ± 7.90 0.727
IL1β214.70 ± 99.25 128.80 ± 13.42 0.4113
MMP-3 2898.00 ± 81.25 2961.00 ± 100.4 0.6544
MMP-8 1183.00 ± 310.20 985.50 ± 103.40 0.4934
SDF-1α506.50 ± 79.08 461.70 ± 51.30 0.6259
Pan et al. Biology of Sex Differences (2016) 7:27 Page 7 of 15
mediators (growth-regulated oncogene α[GRO-α], mono-
cyte chemotactic protein-3 [MCP-3], and monokine in-
duced by gamma interferon [MIG]) were found in women
compared with men.
No sex-specific differences in the concentration of
1,25(OH)
2
D
3
in synovial fluid were detected (Fig. 2a). Com-
pared to men, women had less 25(OH)D
3
;25(OH)D
3
+
25(OH)D
2
+24,25(OH)
2
D
3
; and calculated 24,25(OH)
2
D
3
+ 25(OH)D
2
in the synovial fluid (Fig. 2bd); however, mass
spectroscopy detected no differences in the levels of
24,25(OH)
2
D
3
between women and men (Fig. 2e).
Chondrocyte cultures
Chondrocytes expressed mRNAs for both receptors for
1,25(OH)
2
D
3
(nVDR and PDIA3) and for ERα66 (ESR1)
and ERα36 (Fig. 3ad, respectively). Female chondro-
cytes from minimally fibrillated cartilage had less VDR
mRNA than male cells but comparable levels of mRNAs
for the other three receptors. Female cells isolated from
eroded cartilage had less nVDR mRNA, less PDIA3
mRNA, and more ESR1 mRNA than male cells.
mRNAs for inflammatory cytokines were elevated in
male cells from eroded cartilage compared to cells from
minimally fibrillated cartilage (Fig. 3eh). In contrast, fe-
male cells exhibited no differences in IL1A or IL1B
mRNAs and expression of IL6 and IL8 was reduced in
the female erosion cells compared with the minimally fi-
brillated cells. Female cells from minimally fibrillated
cartilage had higher expression of IL1A, IL6, and IL8
than male cells from similar cartilage. No difference in
expression of the anti-inflammatory cytokine IL10 was
evident between male and female cells or as a function
of the type of cartilage from which the cells were iso-
lated (Fig. 3i).
Sex-specific differences in expression of WNT signal-
ing molecules were observed, particularly in chondro-
cytes isolated from erosion cartilage. WNT3A, CTNNB,
DKK2, and DKK2 were all expressed to a greater extent
in male erosion cells than in cells from minimally eroded
Fig. 2 Vitamin D metabolites in synovial fluid. adLevels of vitamin D metabolites in the synovial fluid of 10 males and 10 females undergoing
total joint arthroplasty were measured using ELISA and (e) mass spectrometry.
*
p< 0.05 compared with male
Pan et al. Biology of Sex Differences (2016) 7:27 Page 8 of 15
tissues (Fig. 3j, ln). This was also the case for CTNNB and
DKK1 in cells from female erosion tissue compared with
minimally fibrillated female cells (Fig. 3l, m). However,
WNT5A (Fig. 3k) and DKK2 (Fig. 3n) were reduced in fe-
male erosion tissue compared with minimally fibrillated
tissue, and the levels of these two mRNAs were significantly
lower than seen in male cells from similar tissues.
Chondrocytes responded to stimulation by vitamin D
metabolites in a sex-specific manner. Male cells exhibited a
more robust increase in alkaline phosphatase activity in
Fig. 3 Phenotypic characteristics of female and male primary chondrocytes isolated from osteoarthritic knees. Primary chondrocytes were isolated
from minimally fibrillated or eroded cartilage surfaces. mRNAs for 1α,25(OH)
2
D
3
(VDR (a) and PDIA3 (b)); estrogen receptors (ERα66 (c) and ERα36
(d)); interleukins (IL1A (e), IL1B (f), IL6 (g), IL8 (h), and IL10 (i)); and WNT signaling molecules (WNT3A (j), WNT5A (k), β-catenin [CTNNB] (l), DKK1
(m), and DKK2 (n)) were measured using real-time qPCR. Data are the mean levels from 6 males and 6 females.
*
p< 0.05 compared with minimal
fibrillation;
p< 0.05 compared with male
ab cd
ef gh
Fig. 4 Phenotypic characteristics of female and male chondrocytes isolated from osteoarthritic knees. adPassage 1 chondrocytes were treated with 10
8
M1α,25(OH)
2
D
3
or 10
8
ME2.ehAlkaline phosphatase specific activity (a,e) was measured in whole cell lysates. mRNAs for chondrocyte genes
aggrecan (b,f), type-II collagen (c,g), and cartilage oligomatric matrix protein (d,h) were measured using real-time qPCR. Data show treatment versus
vehiclecontrolratiosof6maleand6femaledonors.Thedashed line represents the vehicle control (dashed line =1).
*
p< 0.05 vs. control;
p< 0.05 vs. male
Pan et al. Biology of Sex Differences (2016) 7:27 Page 9 of 15
response to 1α,25(OH)
2
D
3
than female cells (Fig. 4a), but
expression of ACAN was reduced (Fig.4b).Nosex-specific
differences in expression of COL2A1 or COMP were ob-
served (Fig. 4c, d). 24R,25(OH)
2
D
3
inhibited alkaline phos-
phatase activity in female chondrocyte cultures compared
with control cells and compared with male cells (Additional
file 2: Figure S1A). 24R,25(OH)
2
D
3
had a stimulatory effect
on male cells compared with control cultures and to female
cells treated with the vitamin D metabolite (Additional
file 2: Figure S1B). 24R,25(OH)
2
D
3
affected male and
female cells comparably with respect to COL2A1 (increase)
or COMP (decrease) (Additional file 2: Figure S1C, D).
E2 reduced alkaline phosphatase activity in male chon-
drocytes and stimulated it in female cells (Fig. 4e). E2 had
no effect on expression of ACAN, COL2A1, or COMP in
male cells, but it increased expression of all three mRNAs
in female cells (Fig. 4fh). DHT reduced alkaline phosphat-
ase activity in male cells but had no effect on the enzyme in
female cells (Additional file 2: Figure S1E). DHT had no ef-
fect on expression of ACAN, COL2A1, or COMP in cells
of either sex (Additional file 2: Figure S1FH).
Osteoblast cultures
Male osteoblasts from erosion tissue expressed high levels
of nVDR compared with minimally fibrillated male cells
or with female cells from either site (Fig. 5a). No differ-
ences in expression of PDIA3 were observed (Fig. 5b). Fe-
male erosion cells expressed more ESR1 (ERα) and female
minimally fibrillated osteoblasts expressed more ERα36
than male cells (Fig. 5c, d); however, no differences in ex-
pression of either E2 receptor were seen as a function of
the tissue source for either males or females.
In male osteoblasts, 1α,25(OH)
2
D
3
, increased alkaline
phosphatase activity (Fig. 6a) and osteocalcin production
(Fig. 6b) but decreased levels of osteoprotegerin (Fig. 6c).
1α,25(OH)
2
D
3
had no effect on alkaline phosphatase ac-
tivity in female cells, but its stimulatory effect on osteo-
calcin and inhibitory effect on osteoprotegerin were
comparable to that seen in male cells. E2 inhibited alka-
line phosphatase activity in male cells (Fig. 6d). It had
no effect on enzyme activity in female osteoblast, but it
stimulated osteocalcin production in the female cells
(Fig. 6e), and it inhibited osteoprotegerin production
(Fig. 6f). In contrast, DHT increased alkaline phos-
phatase activity in male osteoblasts (Additional file 3:
Figure S2A), increased osteocalcin production (Additional
file 3: Figure S2B), and reduced osteoprotegerin produc-
tion (Additional file 3: Figure S2C). Female cells exhib-
ited increased osteoprotegerin production in response
to DHT.
Fig. 5 Phenotypic characteristics of female and male primary osteoblasts isolated from osteoarthritic knees. Female and male primary osteoblasts
were isolated from subchondral bone. mRNAs for 1α,25(OH)
2
D
3
(VDR (a) and PDIA3 (b)) and estrogen receptors ERα66 (ERα(c)) and ERα36 (d)
were measured using real-time qPCR. Data are the mean ± SEM of 6 males and 6 females.
*
p< 0.05 vs. minimal fibrillation;
p< 0.05 vs. male
Pan et al. Biology of Sex Differences (2016) 7:27 Page 10 of 15
Discussion
Rather than investigating an underlying sex-dependent
mechanism in the etiology of OA, the goal of this study
was to screen a broad range of variables in tissues and
fluids obtained during TKA to investigate whether there
are sex differences in knee tissues affected by OA. The
severity of OA in men and women study patients was
comparable. Thus, detected differences could be as-
cribed to underlying sex-dependent traits rather than
differences in disease status. Our results demonstrated
that sex differences are present in OA-affected tissues.
Not only were there differences in the composition of
synovial fluid between the sexes, but chondrocytes and
osteoblasts isolated from OA-affected cartilage and bone
differed in a sex-dependent manner with respect to ex-
pression of hormone receptors and responded differen-
tially to 1α,25(OH)
2
D
3
, 24R,25(OH)
2
D
3
, E2, and DHT.
Moreover, articular chondrocytes from men and women
patients exhibited sex differences in their expression of
genes for inflammatory cytokines, enzymes involved in
matrix degradation, and WNT signaling molecules in-
volved in regulation of proliferation and differentiation.
Our patient population exhibited few clinical differ-
ences between men and women. There was no evidence
of a difference in serum 25(OH)D
3
levels, radiographic
assessment, knee pain, or areas of knee pain prior to sur-
gery between women and men. This could be due to the
advanced stage of disease in our study population. Stud-
ies of younger patients have shown sex differences in
knees with OA, with articular cartilage surface areas be-
ing 17.5 % to 23.5 % lower in women than in men [36],
which supports the idea that sex differences contribute
to disease progression, but once the advanced disease is
established, sex differences are less important.
We did observe some clinical differences in the study pa-
tient population that were sex dependent. The pain-
pressure threshold 1 cm above the medial joint line was
lower in women than in men; however, the clinical
significance of this is unclear. We did not test the pain-
pressure threshold of anatomic regions outside the medial
knee, and further study is warranted. We also found that
womenweremorelikelytohaveusedorwereusingvita-
minDandestrogensupplements compared with men.
Studies have linked estrogen supplementation with reduced
risk of OA, which could explain how a drop in estrogen in
postmenopausal women could contribute to the severity of
OA found in women [8, 37, 38] compared with males. To-
gether, these data suggest that future studies should include
younger postmenopausal women and age-matched men to
better assess sex differences in their disease severity.
The synovial fluid composition was affected by the
genetic sex of the patient in a number of ways. Men had
higher levels of MMPs, the primary enzymes responsible
for the degradation of cartilage, and HGF, which is syn-
thesized by osteoblasts from the subchondral bone plate
and produced at a higher rate by OA osteoblasts [39].
However, men had higher levels of growth factors,
including TGFβs, SCF (stem cell factor), and SCGFβ
Fig. 6 Phenotypic characteristics of female and male primary osteoblasts isolated from osteoarthritic knees. First-passage osteoblasts were treated
with 10
8
M1α,25(OH)
2
D
3
(ac)or10
8
ME2(df). Alkaline phosphatase-specific activity was measured in whole cell lysates (a,d).
Protein levels of osteocalcin (b,e) and osteoprotegerin (c,f) were measured in conditioned media. Data show treatment compared with
vehicle control ratios of the responses of 6 male and 6 female patients. The dashed line represents the vehicle control (dashed line =1).
*
p<0.05 vs. control;
p<0.05 vs. male
Pan et al. Biology of Sex Differences (2016) 7:27 Page 11 of 15
(stem cell growth factor beta), and sGAGs compared
with females, which have been shown to protect articular
cartilage from degradation [4042]. These results suggest
that men may be more resistant to OA; although men had
higher levels of matrix degradation enzymes, they also had
more protective and growth-inducing factors that could
aid in the repair and remodeling processes. Women had
higher levels of inflammatory cytokines, especially IL18,
which induces the production of PGE2 [43]. This is cor-
roborated by studies showing that women experience
more pain [6]. Women had less protective factors, which
could explain their increased disease severity as they lose
the protective effect of estrogen after menopause.
Vitamin D metabolites play important roles in skeletal
development [44] and in maintaining cartilage and bone
homeostasis [45, 46]. The ELISA kit used to measure
1,25(OH)
2
D
3
, a metabolite of 25(OH)D
3
associated with
Ca++ homeostasis as well as terminal differentiation of
chondrocytes and osteoblasts [4749], did not detect a
difference in the synovial fluid levels between men and
women. Because a suitable kit does not exist for measur-
ing 24,25(OH)
2
D
3
, a metabolite associated with chondro-
cyte proliferation and resistance to apoptotic stimuli [50],
we compared results using two kits, one that measured
25(OH)D
3
alone and one that measured 25(OH)D
3
,
25(OH)D
2
, and 24,25(OH)
2
D
3
. Both kits identified reduced
vitamin D metabolites in female synovial fluid, suggesting a
possible reduction in 24,25(OH)
2
D
3
. However, when
24,25(OH)
2
D
3
wasmeasureddirectlybyacommerciallab
using mass spectroscopy, no sex difference was found.
None of these methods is definitive, but taken together, they
show a clear and reproducible finding that synovial vitamin
D metabolites are regulated in a sex-dependent manner.
Studies using growth plate chondrocytes and healthy
human articular chondrocytes have shown that the num-
bers of receptors vary in a sex-specific manner [11, 29,
51]. Our results examining gene expression also demon-
strated sex-dependent differences. Chondrocytes from
male erosion tissue and minimally fibrillated tissue
expressed higher levels of mRNAs for the nuclear VDR
whereas receptors for ERα66 (ESR1) were lower in cells
from male erosion tissue than from female erosion tis-
sue. Osteoblasts from male erosion tissue subchondral
bone exhibited higher nuclear VDR expression, and fe-
male osteoblasts had higher ESR1 than male cells. We
also found that male chondrocytes and osteoblasts were
more responsive to 1α,25(OH)
2
D
3
treatment than female
cells. Previously, studies have shown that male osteo-
blasts have a more robust response to 1α,25(OH)
2
D
3
[52], which supports our findings. Female cells were
more responsive to estrogen treatment compared with
male cells, which is supported by their greater expres-
sion of estrogen receptor mRNAs. The study results are
supported by our previous observations using articular
chondrocytes from young healthy men without OA,
which showed that male cells had no response to E2
whereas female chondrocytes exhibited a marked re-
sponse to E2 [29]. Studies have shown that chondrogenic
progenitor cells respond differently to estrogen and tes-
tosterone in repairing OA cartilage, suggesting sexual di-
morphism exists in cell-repair mechanisms in OA [53].
Collectively, our data suggest that the difference in the
levels of VDR and PDIA3 and local and circulating
25(OH)D
3
between men and women, could contribute
to the more prevalent and severe cases of OA found in
women.
Chondrocytes isolated from both erosion cartilage and
minimally fibrillated cartilage exhibited sex-specific
phenotypic traits. In addition to differences in receptor
expression, there were a number of differences with re-
spect to expression of mRNAs for pro-inflammatory cy-
tokines. The level of these cytokines was comparable for
eroded cartilage cells regardless of sex; however, in min-
imally fibrillated cartilage cells, women had higher levels
of pro-inflammatory cytokines compared with men. This
suggests that female chondrocytes may produce higher
levels of inflammatory cytokines at an earlier stage of
OA. Interestingly, the anti-inflammatory cytokine IL10
was expressed at comparable levels in cells from men
and women irrespective of tissue source.
Expression of mRNAs for WNT family genes varied in a
sex-specific manner as well, but the interpretation of these
data will require further experimentation. Higher levels of
WNT antagonists are suggested to inhibit chondrocyte
hypertrophy. DKK1 inhibits MMP-13 and ADAMTS-4
expression in chondrocytes in response to WNT3a treat-
ment [54]. Activation of WNT/β-catenin signaling in pri-
mary human chondrocytes inhibits basal IL1β-stimulated
increases in MMP-1, -3, and -13 levels, possibly through
inhibition of NFκB signaling [55, 56]. Men had higher
levels of WNT signaling molecules, which is consistent
with our findings that although men had higher levels of
MMPs, they may be more resistant to OA as they also had
more factors that are protective.
The underlying etiology of OA is not well understood. It
has been hypothesized that mechanical instabilities lead to
changes in the biomechanical properties of the articular
cartilage and, ultimately, in the biochemical properties of
the cells and extracellular matrix [7]. Recent data indicate
that there are sex-specific differences at the molecular and
cellular level that may exacerbate the effects of altered
mechanical load [5760]. Our data do not address disease
etiology. While they do support the hypothesis that sex
differences exist, all patients had advanced OA. Thus, it is
not possible to attribute specific differences to a clinical
outcome. It is possible, however, that sex differences may
have contributed to differences in disease progression that
led to the need for TKA.
Pan et al. Biology of Sex Differences (2016) 7:27 Page 12 of 15
This study had a number of limitations. We used pas-
sage 1 cells in our study to investigate the effect of hor-
mone treatments on the cells. Human chondrocytes lose
phenotypic traits when subpassaged, so it is preferable
to use primary cells. Unfortunately, the number of pri-
mary cells was limited, necessitating the need for culture
expansion. In a series of preliminary studies, we found
that the first-passage cells expressed similar levels of
chondrocyte and osteoblast markers to primary cells
(data not shown). One of the limitations of our studies is
that we could not get tissues from healthy human knees
as a control; however, we addressed the limitation by
using cartilage and subchondral bone from areas that
showed minimal articular fibrillation.
Conclusions
In this study, we identified sex-specific differences in
synovial fluid, chondrocytes, and osteoblasts in advanced
human knee OA tissues. Our understanding of such sex-
specific differences is still very early, and additional re-
search is needed. Understanding such sex differences in
knee OA may allow for the introduction of sex-specific
therapies and new treatment avenues for patients experi-
encing this often disabling condition.
Additional files
Additional file 1: Table S1. Primers for real-time PCR. (DOCX 16 kb)
Additional file 2: Figure S1. Phenotypic characteristics of female and
male chondrocytes isolated from osteoarthritic knees. First-passage
chondrocytes were treated with 10
7
M 24R,25(OH)
2
D
3
(A-D) or 10
-8
M
DHT (EH). Alkaline phosphatase-specific activity (A, D) was measured in
whole cell lysates. mRNAs for chondrocyte genes aggrecan (B, F), type-II
collagen (C, G), and cartilage oligomeric matrix protein (D, H) were
measured using real-time qPCR. Data show treatment compared with
vehicle control ratios of the responses of 6 male and 6 female patients.
The dashed line represents the vehicle control (dashed line = 1).
*
p< 0.05
vs. control;
p< 0.05 vs. male. (TIF 1363 kb)
Additional file 3: Figure S2. Phenotypic characteristics of female and
male primary osteoblasts isolated from osteoarthritic knees. First-passage
osteoblasts were treated with 10
8
M DHT. Alkaline phosphatase-specific
activity (A) was measured in whole cell lysates. Protein levels of osteocalcin
(B) and osteoprotegerin (C) were measured in conditioned media. Data
show treatment compared with vehicle control ratios of the responses of 6
male and 6 female patients. The dashed line represents the vehicle control
(dashed line = 1).
*
p<0.05 vs.control;
p< 0.05 vs. male. (TIF 1003 kb)
Abbreviations
sGAG: sulfated glycosaminoglycan; E2: 17β-estradiol;
DHT: dihydrotestosterone; 1α,25(OH)
2
D
3
: 1-alpha,25-dihydroxyvitamin D3;
24R,25(OH)
2
D
3
: 24R,25-dihydroxyvitamin D3; VDR: canonical vitamin D
receptor; PDIA3: protein disulfide isomerase family A member 3;
ERα66: canonical estrogen receptor a; ERα36: shorter variant estrogen
receptor; OA: osteoarthritis; HGF: hepatocyte growth factor; SCF: stem cell
factor; SCGFβ: stem cell growth factor beta; TGFβ: transforming growth
factor beta; TNFαand TNFβ: tumor necrosis factor alpha and beta;
MIF: macrophage migration inhibitory factor; WNT: wingless-related
integrated site a family of signaling molecules; DKK1: Dickkopf-1; IL1α
etc.: interleukin-1 alpha; ACAN: mRNA for aggrecan; COL2A1: mRNA for type-II
collagen alpha 1 chain; COMP: mRNA for cartilage oligomeric matrix protein;
MMP: matrix metalloproteinase; TRAIL: TNF-related apoptosis-inducing ligand;
LIF: leukemia inhibitory factor; M-CSF: macrophage colony-stimulating factor;
GRO-α: growth-regulated oncogene α; MCP-3: monocyte chemotactic protein-
3; MIG: monokine induced by gamma interferon; CTNNB: mRNA for beta-
catenin; TKA: total knee arthroplasty; SF-12: Short Form-12 Health Survey;
WOMAC: Western Ontario & McMaster Universities Arthritis Index; PASE: Physical
Activity Scale for the Elderly; OARSI-OMERACT: Osteoarthritis Research Society
International-Outcome Measures in Rheumatology pain scale.
Acknowledgements
We thank Colleen Thomas at Mayo Clinic with help on statistical analyses of
the clinical data. We also thank Dr. David Hart (University of Calgary) and Dr.
Karen Berkley (Florida State University) for their contributions to the design
of the studies. This work was supported by a grant from the Society for
Womens Health Research ISIS Network for Musculoskeletal Health and
funding from Childrens Healthcare of Atlanta, the Price Gilbert, Jr.
Foundation, the US Department of Defense, and the National Institutes of
Health.
Authorscontributions
This work represents partial completion of the PhD thesis for QP. QP and RON
performed the cell culture assays; SLH catalogued all samples and assisted in
the data analysis; QP wrote the original draft of the manuscript; MIO performed
all clinical assessments and surgeries; RDC performed a review of the clinical
data; BDB and ZS analyzed the results; BDB, ZS, and MIO edited the manuscript.
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Mechanical Engineering, Georgia Institute of Technology,
Atlanta, GA, USA.
2
Center for Musculoskeletal Care, Yale University School of
Medicine, New Haven, CT, USA.
3
Department of Orthopaedics, University of
California at San Diego, San Diego, CA, USA.
4
Department of Biomedical
Engineering, Virginia Commonwealth University, Richmond, VA, USA.
5
Department of Periodontics, University of Texas Health Science Center at
San Antonio, San Antonio, TX, USA.
6
Wallace H. Coulter Department of
Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
7
School of Engineering, Virginia Commonwealth University, 601 West Main
Street, Suite 331, Richmond, VA 23284, USA.
Received: 17 February 2016 Accepted: 12 May 2016
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Pan et al. Biology of Sex Differences (2016) 7:27 Page 15 of 15
... However, the biomechanical component alone does not explain the sex differences in OA pain observed in clinical practice. Women reporting worse pain scenario (Perruccio et al., 2017), increased VAS score (Glass et al., 2014;Solheim et al., 2017), and lower pain threshold (Pan et al., 2016) have been linked to an enhanced inflammatory response. Yet, despite the increase in researchers investigating these sex differences, the biological mechanisms behind these differences in OA pain appear to be understudied (Tschon et al., 2021). ...
... In OA, pain initially has a strong biomechanical component. Abnormal loading from the damaged joint activates Knee OA -SF (10 men and 10 women) ↑ IL2α, IL3, IL12p40, IL16, and TNFβ in women; ↑ macrophage stimulators LIF, M-CSF, MIF in women; ↑ pro-inflammatory mediators GRO-α, MCP-3, MIG in women Pan et al. (2016) Knee OA -SF (21 men and 23 women) ↑ IL-8, CCL-4, and MCP-2 in men; ↑ Acute pain (VAS score) after knee arthroscopy in women compared with men Solheim et al. (2017) Knee OA -Serum, SF, CSF (23 men and 17 women) IL8 (synovial fluid) positive correlation with VAS pain in women, but not men ↑ CCL2 levels significantly higher in CSF than serum, with positive correlations for CCL2 across CSF, serum and SF, in women but not in men Kosek et al. (2018) Knee OA -SF (24 men and 29 women) ↑ macrophages and ↓ monocytes in women Kriegova et al. (2018) Knee OA -Serum (81 men and 115 women) ↑ knee pain scores in women; ↑ TNF-α in men than women IL8 and IL-1β positive correlation with knee pain (WOMAC knee pain score) in men and negative correlation for women IL6 negative correlation with knee pain in men and positive correlation in women Perruccio et al. ...
... The synovial fluid composition of end-stage OA patients shows that women have higher levels of inflammatory cytokines and other proinflammatory mediators than men (Pan et al., 2016;Kosek et al., 2018). Men show higher levels of catabolic enzymes (Solheim et al., 2017) and anabolic growth factors (Pan et al., 2016). ...
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Osteoarthritis (OA) is a progressive whole-joint disease; no disease-modifying drugs are currently available to stop or slow its process. Symptoms alleviation is the only treatment option. OA is the major cause of chronic pain in adults, with pain being the main symptom driving patients to seek medical help. OA pathophysiology is closely associated with the innate immune system, which is also closely linked to pain mediators leading to joint pain. Pain research has shown sex differences in the biology of pain, including sexually dimorphic responses from key cell types in the innate immune system. Not only is OA more prevalent in women than in men, but women patients also show worse OA outcomes, partially due to experiencing more pain symptoms despite having similar levels of structural damage. The cause of sex differences in OA and OA pain is poorly understood. This review provides an overview of the involvement of innate immunity in OA pain in joints and in the dorsal root ganglion. We summarize the emerging evidence of sex differences regarding innate immunity in OA pain. Our main goal with this review was to provide a scientific foundation for future research leading to alternative pain relief therapies targeting innate immunity that consider sex differences. This will ultimately lead to a more effective treatment of pain in both women and men.
... A validation of our results from other countries would be required to determine whether those proteins could indeed be further studied as biomarkers. Second, gender discrimination could also be performed as it is well known that there are sex-specific differences in OA [101][102][103]. In this study, we could not perform such a discrimination as we had a relatively modest sample size, which was limited by the methodology used. ...
Article
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Background Osteoarthritis (OA) is a slowly developing and debilitating disease, and there are no validated specific biomarkers for its early detection. To improve therapeutic approaches, identification of specific molecules/biomarkers enabling early determination of this disease is needed. This study aimed at identifying, with the use of proteomics/mass spectrometry, novel OA-specific serum biomarkers. As obesity is a major risk factor for OA, we discriminated obesity-regulated proteins to target only OA-specific proteins as biomarkers. Methods Serum from the Osteoarthritis Initiative cohort was used and divided into 3 groups: controls ( n =8), OA-obese ( n =10) and OA-non-obese ( n =10). Proteins were identified and quantified from the liquid chromatography–tandem mass spectrometry analyses using MaxQuant software. Statistical analysis used the Limma test followed by the Benjamini-Hochberg method. To compare the proteomic profiles, the multivariate unsupervised principal component analysis (PCA) followed by the pairwise comparison was used. To select the most predictive/discriminative features, the supervised linear classification model sparse partial least squares regression discriminant analysis (sPLS-DA) was employed. Validation of three differential proteins was performed with protein-specific assays using plasma from a cohort derived from the Newfoundland Osteoarthritis. Results In total, 509 proteins were identified, and 279 proteins were quantified. PCA-pairwise differential comparisons between the 3 groups revealed that 8 proteins were differentially regulated between the OA-obese and/or OA-non-obese with controls. Further experiments using the sPLS-DA revealed two components discriminating OA from controls (component 1, 9 proteins), and OA-obese from OA-non-obese (component 2, 23 proteins). Proteins from component 2 were considered related to obesity. In component 1, compared to controls, 7 proteins were significantly upregulated by both OA groups and 2 by the OA-obese. Among upregulated proteins from both OA groups, some of them alone would not be a suitable choice as specific OA biomarkers due to their rather non-specific role or their strong link to other pathological conditions. Altogether, data revealed that the protein CRTAC1 appears to be a strong OA biomarker candidate. Other potential new biomarker candidates are the proteins FBN1, VDBP, and possibly SERPINF1. Validation experiments revealed statistical differences between controls and OA for FBN1 ( p =0.044) and VDPB ( p =0.022), and a trend for SERPINF1 ( p =0.064). Conclusion Our study suggests that 4 proteins, CRTAC1, FBN1, VDBP, and possibly SERPINF1, warrant further investigation as potential new biomarker candidates for the whole OA population.
... Due to the fact that females have a higher prevalence of OA than males [1,2], 'sex differences' [2][3][4][5][6][7] and 'gender-specific' [8][9][10][11] started to attract more and more attention in the field of OA [12]. Women also experience more significant functional disability than men. ...
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As the leading cause of disability, osteoarthritis (OA) affects people of all ages, sexes, and races. With the increasing understanding of OA, the sex differences have attracted specific attention as the burden of OA is greater in women. There is no doubt that gender-specific OA management has great potential for precision treatment. On the other hand, from the marketing aspect, a medication targeting the OA-responsive biomarker(s) shared by both genders is more favorable for drug development. Thus, in the current study, a published transcriptome dataset of knee articular cartilage was used to compare OA and healthy samples for identifying the genes with the same significantly different expression trend in both males and females. With 128 genes upregulated and 143 genes downregulated in both OA males and females, 9 KEGG pathways have been enriched based on the current knowledge, including ‘renal cell carcinoma,’ ‘ECM-receptor interaction,’ ‘HIF-1 signaling pathway,’ ‘MicroRNAs in cancer,’ ‘focal adhesion,’ ‘Relaxin signaling pathway,’ ‘breast cancer,’ ‘PI3K-Akt signaling pathway,’ and ‘human papillomavirus infection.’ Here, we explore the potential impacts of these clusters in OA. We also analyze the identified ‘cell plasma membrane related genes’ in-depth to identify the potential chondrocyte cell surface target(s) of OA management.
... Cartilage volume is correlated with LBM and sex hormones [1]. Since men have higher LBM and slower decrease of serum testosterone concentrations, the osteoarthritis risk is not elevated as quickly as in women as they age [24]. Although elderly men have a lower risk of osteoarthritis than elderly women, they are still susceptible to osteoarthritis due to decreased serum testosterone concentrations [25]. ...
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Water extracts of both Morus alba L. root bark (MBW) and Cornus officinalis Siebold and Zucc fruit (CFW) have traditionally been used to promote men’s health in the elderly in Asia. We determined that the 12-week consumption of MBW and CFW could alleviate testosterone-deficiency syndrome and osteoarthritis (OA) symptoms in testosterone-deficient rats, and the action mechanisms were explored. Rats with bilateral orchiectomy (ORX) were fed a 45% fat diet containing either 0.5% MBW (ORX-MBW), 0.5% CFW(ORX-CFW), or 0.5% dextrin (ORX-CON). Sham-operated rats also received 0.5% dextrin (Non-ORX-CON). After 8 weeks of treatment, all rats had an injection of monoiodoacetate (MIA) into the left knee, and they continued the same diet for the additional 4 weeks. ORX-CFW and ORX-MBW partially prevented the reduction of serum testosterone concentrations and decreased insulin resistance, compared to the ORX-CON. ORX-CFW and ORX-MBW protected against the reduction of bone mineral density (BMD) and lean body mass (LBM) compared to the ORX-CON. The limping and edema scores were lower in the order of the ORX-CON, ORX-CRF = ORX-MBW, and Non-ORX-CON (p < 0.05). The scores for pain behaviors, measured by weight-distribution on the OA leg and maximum running velocity on a treadmill, significantly decreased in the same order as limping scores. ORX-MBW protected against the increased expression of matrix metalloproteinase (MMP)-3 and MMP-13 and reduced the production of inflammatory markers such as TNF-α and IL-1β, by MIA in the articular cartilage, compared to the ORX-CON (p < 0.05). The cartilage damage near the tidemark of the knee and proteoglycan loss was significantly less in ORX-MBW than ORX-CON. In conclusion, MBW, possibly CFW, could be effective alternative therapeutic agents for preventing osteoarthritis in testosterone-deficient elderly men.
... Firstly, 24R,25(OH) 2 D 3 has an established chondroprotective effect and may prevent bone resorption by limiting calcium efflux in cartilage and bone cells 48 . Post-menopausal women have a higher incidence of osteoarthritis than men of similar ages 49,50 , and the extremely low levels of circulating estradiol in post-menopausal women have been implicated in this discrepancy 50,51 . Treatment with 24R,25(OH) 2 D 3 has been proposed as a potential therapeutic for osteoarthritis patients, as 24R,25(OH) 2 D 3 prevents oxidative damage and apoptosis in chondrocytes 19 . ...
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Vitamin D3 and its metabolites have antitumorigenic properties in vitro and in vivo; however, clinical trials and retrospective studies on the effectiveness of vitamin D3 oral supplementation against cancer have been inconclusive. One reason for this may be that clinical trials ignore the complex vitamin D metabolome and the many active vitamin D3 metabolites present in the body. Recent work by our lab showed that 24R,25(OH)2D3, a vitamin D3 metabolite that is active in chondrocyte proliferation and differentiation, has antitumorigenic properties in estrogen receptor alpha-66 (ERα66)–positive (ER⁺) breast cancer, but not in ERα66-negative (ER⁻) breast cancer. Here we show that 24R,25(OH)2D3 is protumorigenic in an in vivo mouse model (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice) of ER⁻ breast cancer, causing greater tumor growth than in mice treated with vehicle alone. In vitro results indicate that the effect of 24R,25(OH)2D3 is via a membrane-associated mechanism involving ERs and phospholipase D. 24R,25(OH)2D3 increased proliferation and reduced apoptosis in ERα66-negative HCC38 breast cancer cells, and stimulated expression of metastatic markers. Overexpressing ESRI, which encodes ERα66, ERα46, and ERα36, reduced the proapoptotic response of ERα66⁻ cells to 24R,25(OH)2D3, possibly by upregulating ERα66. Silencing ESR1 in ERα66⁺ cells increased apoptosis. This suggests 24R,25(OH)2D3 is differentially tumorigenic in cancers with different ERα isoform profiles. Antiapoptotic actions of 24R,25(OH)2D3 require ERα36 and proapoptotic actions require ERα66. Implications: These results suggest that 24R,25(OH)2D3, which is a major circulating metabolite of vitamin D, is functionally active in breast cancer and that the regulatory properties of 24R,25(OH)2D3 are dependent upon the relative expression of ERα66 and ERα36.
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Background Cervical disc diseases have been treated by means of cervical disc arthroplasty (CDA). Nevertheless, some patients will experience a mobility failure in their cervical prostheses over time because of heterotopic ossification (HO). Aim of this study is to investigate the role of gender in long-term outcomes after CDA. Materials and Methods A retrospective, single-center study including patients who underwent single-level CDA with a Bryan prosthesis was performed, including a narrative review about gender differences in both structural and biomechanical features of the cervical spine. Results Study patients (14M, 30F) had an average follow-up period of 9,8 ± 3,2 years. Significant differences emerged between genders for specific items in Neck Disability Index (NDI) preoperative evaluation, with females reporting worse pain scores (p=0.05). After stratification by age we report a higher preoperative overall NDI score for female patients under 36 years of age (p=0.03). In an intergender, BMI-specific comparison we also found a significant difference in NDI preoperative score with normal-weight male patients faring worse than overweight ones (p= 0.05). At a radiological level, we found a tendency towards a higher HO incidence in male patients (62% males, 17% females, p=0.06). Women’s cervical spine has peculiar features including bone structure, muscular action, soft-tissue response, genetic and epigenetic response to osteoarthritis. Conclusion The incidence of mobility failure in our series of single-level CDA was lower in females. Several gender specific factors both in static and dynamic features may play a significant role in spinal pathology and CDA long-term radiological outcome.
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The development of microphysiological models is currently at the forefront of preclinical research. Although these 3D tissue models are being developed to mimic physiological organ function and diseases, which are often sexually dimorphic, sex is usually neglected as a biological variable. For decades, national research agencies have required government-funded clinical trials to include both male and female participants as a means of eliminating male bias. However, this is not the case in preclinical trials, which have been shown to favor male rodents in animal studies and male cell types in in vitro studies. In this Opinion, we highlight the importance of considering sex as a biological variable and outline five approaches for incorporating sex-specific features into current microphysiological models.
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Osteoarthritis (OA) is a common degenerative joint disease treated mostly symptomatically before approaching its definitive treatment, joint arthroplasty. The rapidly growing prevalence of OA highlights the urgent need for a more efficient treatment strategy and boosts research into the mechanisms of OA incidence and progression. As a multifactorial disease, many aspects have been investigated as contributors to OA onset and progression. Differences in gender appear to play a role in the natural history of the disease, since female sex is known to increase the susceptibility to its development. The aim of the present review is to investigate the cues associated with gender by analyzing various hormonal, anatomical, molecular, and biomechanical parameters, as well as their differences between sexes. Our findings reveal the possible implications of gender in OA onset and progression and provide evidence for gaps in the current state of art, thus suggesting future research directions.
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Background: Osteoarthritis (OA) primarily affects mechanical load-bearing joints. The knee joint is the most impacted by OA. Knee OA (KOA) occurs in almost all demographic groups, but the prevalence and severity are disproportionately higher in females. The molecular mechanism underlying the pathogenesis and progression of KOA is unknown. The molecular basis of biological sex matters of KOA is not fully understood. Mechanical stimulation plays a vital role in modulating OA-related responses of load-bearing tissues. Mechanical unloading by simulated microgravity (SMG) induced OA-like gene expression in engineered cartilage, while mechanical loading by cyclic hydrostatic pressure (CHP), on the other hand, exerted a pro-chondrogenic effect. This study aimed to evaluate the effects of mechanical loading and unloading via CHP and SMG, respectively, on the OA-related profile changes of engineered meniscus tissues and explore biological sex-related differences. Methods: Tissue-engineered menisci were made from female and male meniscus fibrochondrocytes (MFCs) under static conditions of normal gravity in chondrogenic media and subjected to SMG and CHP culture. Constructs were assayed via histology, immunofluorescence, GAG/DNA assays, RNA sequencing, and testing of mechanical properties. Results: The mRNA expression of ACAN and COL2A1 , was upregulated by CHP but downregulated by SMG. COL10A1 , a marker for chondrocyte hypertrophy, was downregulated by CHP compared to SMG. Furthermore, CHP increased GAG/DNA levels and wet weight in both female and male donors, but only significantly in females. From the transcriptomics, CHP and SMG significantly modulated genes related to the ossification, regulation of ossification, extracellular matrix, and angiogenesis Gene Ontology (GO) terms. A clear difference in fold-change magnitude and direction was seen between the two treatments for many of the genes. Furthermore, differences in fold-change magnitudes were seen between male and female donors within each treatment. SMG and CHP also significantly modulated genes in OA-related KEGG pathways, such as mineral absorption, Wnt signalling pathway, and HIF-1 signalling pathway. Conclusion: Engineered menisci responded to CHP and SMG in a sex-dependent manner. SMG may induce an OA-like profile, while CHP promotes chondrogenesis. The combination of SMG and CHP could serve as a model to study the early molecular events of KOA and potential drug-targetable pathways.
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Precision medicine aims to better individualize healthcare. It requires that biomaterials be designed for the physiological characteristics of a specific patient. To make this a reality, biomaterials research and development must address differences of biological sex. More specifically, biomaterials should be designed with properties optimized and appropriate for male and female patients. In analyzing research articles from seven prominent biomaterials journals, sex as a biological variable is missing from an overwhelming majority of in vitro biomaterial studies. From the survey, the reporting of the sex of primary cell cultures happened only 10.3% of the time. Contributing to this trend is that commercial vendors bias cell lines toward one sex or another by not disclosing information of cell line sex at the time of purchase; researchers do not communicate this pertinent information in published studies; and many journal policies have little to no requirements for reporting cell line characteristics. Omitting this valuable information leads to a gap in the understanding of sex‐specific cell‐biomaterial interactions and it creates a bias in research findings towards one sex or another. To curb this concerning trend and make precision biomaterials a reality will require the biomaterials field to “talk about sex” by reporting cell sex more broadly.
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