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The Fasciacytes: A New Cell Devoted To Fascial Gliding Regulation

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Introduction: Hyaluronan occurs between deep fascia and muscle, facilitating gliding between these two structures, and also within the loose connective tissue of the fascia, guaranteeing the smooth sliding of adjacent fibrous fascial layers. It also promotes the functions of the deep fascia. In this study a new class of cells in fasciae is identified, which we have termed fasciacytes, devoted to producing the hyaluronan‐rich extracellular matrix. Materials and methods: Synthesis of the hyaluronan‐rich matrix by these new cells was demonstrated by Alcian Blue staining, anti‐HABP (hyaluronic acid binding protein) immunohistochemistry, and transmission electron microscopy. Strong expression of HAS2 (hyaluronan synthase 2) mRNA by these cells was detected and quantified using real time RT‐PCR. Results: This new cell type has some features similar to fibroblasts: they are positive for the fibroblast marker vimentin and negative for CD68, a marker for the monocyte‐macrophage lineage. However, they have morphological features distinct from classical fibroblasts and they express the marker for chondroid metaplasia, S‐100A4. Conclusions: The authors suggest that these cells represent a new cell type devoted to the production of hyaluronan. Since hyaluronan is essential for fascial gliding, regulation of these cells could affect the functions of fasciae so they could be implicated in myofascial pain. This article is protected by copyright. All rights reserved.
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ORIGINAL COMMUNICATION
The Fasciacytes: A New Cell Devoted to Fascial Gliding
Regulation
CARLA STECCO ,
1
* CATERINA FEDE,
1
VERONICA MACCHI,
1
ANDREA PORZIONATO,
1
LUCIA PETRELLI,
1
CARLO BIZ,
2
ROBERT STERN,
3
AND RAFFAELE DE CARO
1
1
Department of Neuroscience, University of Padova, via Gabelli 65, Padova, 35121, Italy
2
Department of Surgery, Oncology and Gastroenterology DiSCOG, Orthopedic Clinic, University of Padova,
via Giustiniani 2, Padova, 35121, Italy
3
Division of Basic Biomedical Sciences, Touro College of Osteopathic Medicine, 230 West-125th Street,
New York, New York, 10027
Hyaluronan occurs between deep fascia and muscle, facilitating gliding between
thesetwostructures,andalsowithinthelooseconnectivetissueofthefascia,
guaranteeing the smooth sliding of adjacent fibrous fascial layers. It also pro-
motes the functions of the deep fascia. In this study a new class of cells in fasciae
is identified, which we have termed fasciacytes, devoted to producing the
hyaluronan-rich extracellular matrix. Synthesis of the hyaluronan-rich matrix by
these new cells was demonstrated by Alcian Blue staining, anti-HABP (hyaluronic
acid binding protein) immunohistochemistry, and transmission electron micros-
copy. Expression of HAS2 (hyaluronan synthase 2) mRNA by these cells was
detected and quantified using real time RT-PCR. This new cell type has some fea-
tures similar to fibroblasts: they are positive for the fibroblast marker vimentin
and negative for CD68, a marker for the monocyte-macrophage lineage. However,
they have morphological features distinct from classical fibroblasts and they
express the marker for chondroid metaplasia, S-100A4. The authors suggest that
these cells represent a new cell type devoted to the production of hyaluronan.
Since hyaluronan is essential for fascial gliding, regulation of these cells could
affect the functions of fasciae so they could be implicated in myofascial pain. Clin.
Anat. 00:000–000, 2018. V
C2018 Wiley Periodicals, Inc.
Key words: fasciacytes; hyaluronan; extracellular matrix; fascia
INTRODUCTION
The deep fascia is a multilayered structure formed
by two to three layers of densely-packed collagen
fibers with a few elastic fibers, separated by layers of
loose connective tissue (Benetazzo et al., 2011), rich
in hyaluronan (HA). It is widely recognized that fasciae
have an active role in tissue maintenance and repair.
They are abundantly innervated and rich in proprio-
ceptors, vascular channels, and lymphatic channels
(Stecco et al., 2007; Bhattacharya et al., 2011). Fascia
has a multitude of functions including force transmis-
sion, movement, stability, proprioceptive communica-
tion throughout the body, and in promoting sliding and
reducing the friction associated with motion (Kumka
and Bonar, 2012). To ensure proper function it is
important that the loose connective tissue within
fascial sublayers has the correct status, including ori-
entation and organization.
Thickening and a densification of the loose connec-
tive tissue and its extracellular matrix (ECM) corre-
spond to the reduction or loss of fascial sliding ability
(Langevin et al., 2011; Chaitow, 2014). If the connec-
tive tissue is lost or its density is altered, the behavior
of the deep fascia and underlying muscle becomes
compromised. This could be the source of myofascial
*Correspondence to: Carla Stecco, Section of Anatomy, Depart-
ment of Neuroscience, University of Padova, via A. Gabelli 65,
35121 Padova, Italy. E-mail: carla.stecco@unipd.it
Received 21 February 2018; Accepted 13 March 2018
Published online in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/ca.23072
V
V
C2018 Wiley Periodicals, Inc.
Clinical Anatomy 00:00–00 (2018)
pain in many cases. One key element affecting fascia
density seems to be HA (Stecco et al., 2014).
HA is synthesized within the plasma membranes of
most cell types by membrane-bound synthases. The
chains are extruded into the extracellular space
through pore-like structures (Jiang et al., 2007). The
resulting HA serves a range of functions including
space filling, lubrication of joints, water homeostasis,
and provision of a matrix that facilitates cell migration
(Fraser et al., 1997; Cowman et al., 2015). In recent
years, it has also been demonstrated that HA provides
a substrate for the smooth gliding of different motor
units between and within muscle (McCombe et al.,
2001).
Previous work from this laboratory revealed
fibroblast-like cells on the inferior surface of the deep
fascia that stained prominently with Alcian Blue. We
suggested that these cells were related to other
fibroblast-like cells in the vertebrate body but were
specialized for HA synthesis and secretion (Stecco
et al., 2011), similar to the synoviocytes on the inner
surfaces of joint capsules (Chang et al., 2010) that
synthesize the HA-rich synovial fluid, and the hyalo-
cytes in the eye that elaborate the HA-rich humors of
the anterior and posterior chambers (Sakamoto and
Ishibashi, 2011). We suggested that they represent a
previously-unidentified class of cells that we called
“fasciacytes.” Fasciacytes have not yet been analyzed
morphologically and immunohistochemically; this is the
purpose of the current study. Here, we characterize this
new cell type, demonstrating that they differ in mor-
phological features and immunoreactivity from classical
fascial fibroblasts. Showing that they are specialized for
the biosynthesis of a HA-rich matrix helps to elucidate
the role of HA in conferring viscoelasticity of fascial tis-
sue under healthy conditions, and in a myriad of patho-
physiological conditions that include myofascial pain,
disuse atrophy, hypertrophy, and the fascia associated
with muscle scarring, contractures, and fibrosis.
MATERIALS AND METHODS
Sample Collection
This study was approved by our Institutional Ethical
Review Board (approval no. 3722/AO/16). The insti-
tute’s ethical regulations for research conducted on
human tissues were followed. Written informed con-
sent was obtained from each donor.
Samples of fascia lata (1 cm 31 cm) were col-
lected from 12 volunteer patients, five males and
seven females, average age 76 611 years. They were
all undergoing elective surgery at the Orthopedic
Clinic of Padova University (total hip prosthesis, ante-
rolateral approach). The samples were transported to
the laboratory in phosphate-buffered saline (PBS)
within a few hours of collection.
Each sample was divided into three sections: one
was frozen or maintained in PBS and used fresh for
real-time PCR; the second was formalin-fixed for his-
tology; and the third was fixed in 2.5% glutaralde-
hyde for transmission electron microscopy.
Morphological Analysis and Differential
Staining of Glycosaminoglycans
Each of the formalin-fixed human fascia specimens
(0.5 cm 30.5 cm) was dehydrated in graded ethanol,
embedded in paraffin and cut into 6 lm-thick sections.
The following histochemical stains were applied to
each dewaxed section: Hematoxylin and eosin (H&E),
and 0.05% Alcian Blue pH 5.8, which stains acidic pol-
ysaccharides such as glycosaminoglycans (GAGs). In
parallel, dewaxed sections were pretreated overnight
at 378C with 2 mg/mL hyaluronidase (from bovine tes-
tes, Sigma Aldrich) and then subjected to the protocols
described. As positive controls, human umbilical cords
from volunteer women were used (data not shown).
The critical electrolyte concentration (CEC) principle
was applied for the Alcian Blue staining procedure:
different concentrations of MgCl
2
(0.025 M to 2 M)
cause differences in staining because the cations of
the salt compete with those of the dye for the polya-
nionic sites in the tissue (Scott and Dorling, 1965).
Dewaxed sections were incubated for 1 hr in sodium
acetate buffer, pH 5.8, with 0.05 M or 2 M MgCl
2
,and
then stained with 0.05% Alcian Blue with the same
concentrations of MgCl
2
as in the buffer solution for a
total of 4 hr. The tissues were then washed in 0.01 N
HCl for 10 min and in distilled water. The nuclei in tissue
sections were counterstained with ready-to-use hema-
toxylin (Dako). Dehydrated samples were mounted
with a coverslip using Eukitt (Agar Scientific).
Images were obtained using a Leica DMR micro-
scope (Leica Microsystems, Wetzlar, Germany; objec-
tives 203and 403, Leica) and analyzed using Image
Jsoftware.
Immunohistochemistry to Detect HABP
(Hyaluronic Acid Binding Protein)
All the formalin-fixed specimens (0.5 cm 30.5 cm)
were dehydrated in graded ethanol, embedded in par-
affin and cut into 6 lm-thick sections as described
above. In parallel, dewaxed sections were pretreated
overnight at 378C with 2 mg/mL hyaluronidase. After-
wards, all samples were subjected to the protocols
described below.
Dewaxed sections were treated with avidin and bio-
tin solutions (20 min each at RT) to block endogenous
avidin biotin activity. Endogenous peroxidase was
blocked with 0.5% H
2
O
2
in PBS for 5 min at RT, and
then the specimens were washed with PBS, incubated
in 0.1% BSA for 1 hr at RT, treated with biotinylated 2
mg/mL HABP (Amsbio), diluted with the same pre-
incubation buffer, and incubated overnight at 48C. After
repeated PBS washing, the samples were incubated
with the secondary antibody HRP-conjugated Streptavi-
din (1:250) for 30 min (Jackson ImmunoResearch) and
washed in PBS. The reaction was then developed with
3,30-diaminobenzidine (Liquid DAB 1substrate Chromo-
gen System kit, Dako Corp, Carpinteria, CA) and the
reaction was terminated with distilled water.
For negative controls, similarly-treated sections were
used omitting the primary antibody. This confirmed the
specificity of the immunostaining reaction. As positive
2 Stecco et al.
controls we used human umbilical cords from female
volunteers. The nuclei in tissue sections were counter-
stained with ready-to-use hematoxylin (Dako Corp, Car-
pinteria, CA).
Images were recorded using a Leica DMR micro-
scope (Leica Microsystems, Wetzlar, Germany).
TEM (Transmission Electron Microscopy)
Analysis
Four specimens of human fascia lata (dimensions:
0.5 cm 30.5 cm) were fixed in 0.1 M phosphate-
buffered 2.5% glutaraldehyde (Serva Electrophoresis,
Heidelberg, Germany), post-fixed in 1% osmium
tetroxide (OsO
4
) (Agar Scientific Elektron Technology,
Stansted, UK) in 0.1 M phosphate buffer, dehydrated
in a graded ethanol series, and embedded in Epoxy
Embedding Medium Kit (45349, Sigma-Aldrich, St.
Gallen, Switzerland). Semithin (0.5 mm) and ultrathin
(60 nm) sections were cut with an RMC Power-Tome
ultramicrotome (Boeckeler Instruments, AZ). Semi-
thin sections were stained with 1% Toluidine blue;
ultrathin sections were collected on 300-mesh copper
grids and counterstained with 1% uranyl acetate and
then Sato’s lead. The specimens were examined with
a Hitachi H-300 Transmission Electron Microscope.
Characterization of Fasciacytes
The following immunohistochemical stains were
applied to each of the formalin-fixed human fascia lata
specimens: anti-vimentin (monoclonal mouse anti-
body, Invitrogen; dilution 1:250), anti-CD68 (monoclo-
nal mouse antibody, DakoCytomation; dilution 1:100),
and anti-S-100A4 (polyclonal rabbit antibody, Abcam;
dilution 1:100). The marker S-100A4 was selected to
demonstrate that fasciacytes are cells undergoing
chondroid metaplasia.
Indeed, Klein et al. (1999) demonstrated that in
the extensor retinaculum of the wrist and ankle there
are scattered HA-secreting cells, positive for S-100
protein. S-100A4 belongs to the family of calcium-
binding S-100 proteins that are not only specific for
neuronal and glial elements (Castagna et al., 2003)
but also occur in cells of different origins and functions
(Mohr et al., 1985). S-100 is a multifunctional protein
involved in matrix remodeling (Yammani et al., 2009)
and in regulating several cellular processes such as
motility, growth and differentiation, structural organiza-
tion of membranes, protection against oxidative dam-
age, protein phosphorylation, and secretion (Sedaghat
and Notopoulos, 2008). Recently, S-100A4 was used
as a marker to follow cancer progression (Fei et al.,
2017), as a mesenchymal marker (Seccia et al., 2016),
and as a marker of chondroid metaplasia (Klein et al.,
1999; Tas¸demir et al., 2014).
Briefly, dewaxed sections were incubated with
0.1% trypsin in 0.05%-calcium chloride, pH 7.8, for
15 min at 378C to unmask the antigens (for the S-
100A4 stain), or maintained at 968C for 15 min in
sodium citrate, pH 6.0, for heat-induced antigen
retrieval (for vimentin and CD68). Endogenous peroxi-
dases were blocked with 0.5% H
2
O
2
in distilled water
for 5 min at room temperature and then washed.
Specimens were then incubated in 1% BSA-0.2% Tri-
ton-X for 1 hr at RT, and then incubated overnight at
48C with the primary antibody diluted in the same
pre-incubation buffer. After repeated PBS washing,
samples were incubated with the secondary antibody
Advance HRP Detection System (Dako Corp., Carpin-
teria, CA), ready-to-use, for mouse and rabbit, and
washed in PBS. The reaction was then developed with
3,30-diaminobenzidine (Liquid DAB 1substrate Chro-
mogen System kit Dako Corp, Carpinteria, CA) and
stopped with distilled water. Sections incubated with-
out primary antibodies showed no immunoreactivity,
confirming the specificity of the immunostaining.
The images were acquired using a Leica DMR
microscope (Leica Microsystems, Wetzlar, Germany)
and S-100A4 positive cells were counted manually
(total number of cells counted >3,500).
Real-Time PCR
The biosynthesis of HA is dictated by three mem-
bers of the hyaluronan synthase family (HAS1, HAS2,
and HAS3), of which HAS2 is the most important and
most highly expressed (Camenisch et al., 2000). We
therefore detected and studied the expression of
HAS2 mRNA by real time RT-PCR.
Briefly, four tissue specimens (1 cm 30.5 cm)
were homogenized in RNA lysis buffer, and total RNA
was extracted using the SV Total RNA Isolation Sys-
tem (Promega Corporation, Madison, WI) and purified.
DNAse was used during RNA extraction to remove
genomic DNA contamination. The total RNA was then
reverse-transcribed to cDNA using the iScript cDNA
Synthesis Kit (BioRad Laboratories, Milan, Italy). RT-
PCR was performed in an I-Cycler iQ detection system
(BioRad), using the primers reported in Table 1, and a
30 ng starting amount of cDNA denaturation step at
958C for 3 min, 45 cycles of three amplification steps
(15s 958C / 15s 608C for RPS18 and RPLP0, 15s 648C
for HAS2 /15s 728C), and a melting curve (60–908C
with a heating rate of 0.58C/10 s).
TABLE 1. Sense and Antisense Sequences used as RT-PCR Primers
Sequence name Acc. number BP Primers
RPS18 F142
RPS18 R252
NM_022551 111 50-ATT AAG GGT GTG GGC CGA AG 230
50-GGT GAT CAC ACG TTC CAC CT 230
RPLP0 F824
RPLP0 R918
NM_053275 95 50- GCA GCA TCT ACA ACC CTG AA-30
50-CAG ACA GAC ACT GCC AAC AT-30
HAS2 F1233
HAS2 R1485
NM_005328 253 50-ATC CCA TGG TTG GAG GTG TT-30
50-TGC CTG TCA TCA CCA AAG CT 230
The Fasciacytes 3
Fig. 1. Hematoxylin & Eosin staining (A,B) and Alcian
blue staining of paraffin sections of human fascia lata. C,D
are stained with 0.05% Alcian Blue/MgCl
2
0.05 M in sodium
acetate buffer for GAGs; E,F are stained with 0.05% Alcian
Blue/2 M MgCl
2
specifically for HA and chondroitin sulfate;
and G,H are incubated with hyaluronidase (overnight at
378C) and then stained with 0.05% Alcian Blue/0.05 M
MgCl
2
. Arrows indicate prominent rounded nuclei with abun-
dant extracellular matrix. Scale bars: (A–H), 50 lm. [Color
figure can be viewed at wileyonlinelibrary.com]
4 Stecco et al.
During the exponential phase, the fluorescence signal
threshold was calculated and the number of PCR cycles
required to reach the threshold (cycle threshold, Ct) was
determined. Ct values decreased linearly with increasing
quantity of input target. All samples were amplified in
duplicate and expressions of RPS18 (Ribosomal Protein
S18) and RPLP0 (ribosomal protein lateral stalk subunit
P0) were used as a reference. The specificity of the
amplification was tested at the end of each run by melt-
ing curve analysis, using I-Cycler software 3.0.
RESULTS
We found some rounded cells in all the fascial sam-
ples, already evident using H&E staining: they are
Fig. 3. Semithin sections stained with 1% Toluidine blue. Left image: fasciacytes
in the external loose connective tissue; right image: fasciacytes in the loose connec-
tive tissue between the fibrous fascial layers. Scale bars: 100 mm. [Color figure can be
viewed at wileyonlinelibrary.com]
Fig. 2. HABP expression in human umbilical cord (A,B,C) and in human fascia lata
(D,E,F). C and F are incubated with hyaluronidase (overnight at 378C) before the immunos-
taining. Scale bars: (A–F), 50 lm. [Color figure can be viewed at wileyonlinelibrary.com]
The Fasciacytes 5
indicated by arrows in the Figure 1B. At greater enlarge-
ment they are clearly round, completely different from
the classic elongated spindle-shaped fascial fibroblasts.
Human fascia lata stains prominently with Alcian
Blue using 0.05 M MgCl
2
(Fig. 1C,D), and by using the
CEC principle we confirmed that the major GAG depos-
ited in the ECM is HA (Scott and Dorling, 1965). When
the MgCl
2
concentration was increased to 2 M (Fig.
1E,F) there was a marked reduction in staining, and
cells became clearer in the surrounding areas: the
decreased staining associated with increased electro-
lyte concentration lends support to the concept that
fascia is rich in HA and chondroitin sulfate (Schofield
et al., 1975). Such staining is lost when the tissue sec-
tions undergo preliminary digestion with hyaluronidase
(Fig. 1G,H). These cells do not have the typical elon-
gated organization of fascial fibroblasts, but are more
rounded, with cytoplasm restricted to the perinuclear
region. Around them there is abundant ECM positive
foralltheHA-specicstainsusedinthiswork.
The HA nature of the ECM around these cells was
confirmed using biotinylated HABP, which is highly spe-
cific for HA: Figure 2D,E shows the positive reaction in
the cytoplasm and around some fibroblast-like cells.
These areas became destained after digestion with hyal-
uronidase (Fig. 2F). Negative controls confirmed the
specificity of the immunostaining (data not shown),
whereas positive tissue controls evidenced positive reac-
tions, especially around muscular cells of the tunica
media of umbilical cord blood vessels (Fig. 2A,B), and
negative reaction following digestion of the HA (Fig. 2C).
These positive-staining fibroblast-like cells were
examined in semithin (Fig. 3) and ultrathin (Fig. 4) sec-
tions. Both images are distinguished from the loose
connective tissue that separates from the fibrous fascial
layer. These cells presented prominent nuclei, rounder
in shape, and cytoplasm restricted to the perinuclear
region with smaller and less elongated cellular pro-
cesses than in fibroblasts. The matrix around them also
appeared different from collagen fiber organization and
Fig. 4. TEM images of classical elongated fibroblasts (A) and prominent rounded
cells (B, C, D) with the HA-rich matrix around them. Both cells are located in the loose
connective tissue on the border with the fibrous layer. Scale bars: 3 mm.
6 Stecco et al.
the typical aspect of the glycosaminoglycans could be
recognized (Fig. 4). This matrix corresponded to that
stained with Alcian Blue. In all the samples of human
fascia lata, the cells were located mostly at the
boundaries of the fibrous fascial sublayers, forming
clusters of 3–4 cells. These clusters seem to demarcate
the boundaries between the loose and fibrous connec-
tive tissue sublayers (Fig. 3A).
Fig. 5. Vimentin expression in human fascia lata (A), negative immunoreaction for
CD68 (B), and S-100A4 positivity in fasciacytes (C,D). Scale bars: 50 mm. [Color figure
can be viewed at wileyonlinelibrary.com]
Fig. 6. Specific melting point plots of the PCR products using primers for RPS18
and RPLP0 (A), and for HAS2 (B) in human fascia lata. The baseline curves are the
results using water, indicating the negative reactions. Insets show the real time PCR
plots with cDNA, used to determine the Ct values. [Color figure can be viewed at
wileyonlinelibrary.com]
The Fasciacytes 7
Immunocharacterization of these cells demon-
strated that they are modified fibroblasts, positive for
the common intermediate filament fibroblast marker
vimentin (Fig. 5A), but also positive for S-100A4, doc-
umenting a subclass of chondroid metaplasia (Masani
et al., 1986; Klein et al., 1999) (Fig. 5C,D). The classi-
cal elongated fascial fibroblasts were not immunore-
active for S-100 (Fig. 5C,D). By manual counting,
29.4 64.2% of the total cells were immunopositive for
S-100A4 and were therefore involved in chondroid
metaplasia. Furthermore, immunohistochemical anal-
ysis with anti-CD68 proved that these fasciacytes
were not derived from the monocyte/macrophage lin-
eage (Bartok and Firestein, 2010). All the fascial cells
were negative, except for the occasional macrophage
and monocyte observed near vessels (Fig. 5B).
Finally, we studied the expression of HA synthase-2
by real time RT-PCR. We verified the specificity of the
reactions and the absence of false amplicons; all sam-
ples had the same specific melting temperature for
each amplified sequence, except for the water sam-
ples (the baseline curves) that indicate negative reac-
tions (Fig. 6). The Ct values from PCR plots are
reported in Table 2: the results confirmed the pres-
ence of HA synthase in fascial tissue, although with
lower expression than the housekeeping genes RPS18
and RPLP0 (Ragni et al., 2013), as indicated by the
higher Ct values: RPS18 had a mean Ct value of 20.7
(with more variability than the other housekeeping
gene, 61.1 vs.60.15); the mean Ct of RPLP0 was
21.9 60.15; the mean Ct for HAS2 was 30.4 61.3.
We used more than one verified reference gene to
monitor any significant changes in Ct values continu-
ously under our experimental conditions to avoid the
reporting of uncorrected data (Svingen et al., 2015).
DISCUSSION
In this work, we have characterized for the first
time fibroblast-like cells in fasciae specialized for the
biosynthesis of the HA-rich matrix. We have termed
this new class of cells “fasciacytes.” They are morpho-
logically distinct from classical fibroblasts: the firsts
have rounder nuclei, cytoplasm restricted to the peri-
nuclear region, and smaller and less elongated cellular
processes. Also, the location of these new cells is
peculiar: the fibroblasts lie among the collagen fibrous
bundles in the fibrous fascial sublayers, while the fas-
ciacytes form small clusters along the surface of each
fascial sublayer, defining the boundary between
the fibrous sublayer and the loose connective tissue
(Fig. 7). This peculiar location suggests that precise
regulation of the activity of these cells in HA produc-
tion could modulate gliding between adjacent fibrous
fascial sublayers, which seems to be strongly related
to myofascial pain and range of movement (Langevin
et al., 2011).
Our results clearly demonstrate that these
fibroblast-like cells produce different elements of the
ECM from classical fibroblasts. Fibroblasts produce
collagen and elastic fibers; fasciacytes produce hya-
luronan. Consequently, we can affirm that there are
two different cell types in fasciae: one devoted to pro-
ducing/regulating the fibrous component of the ECM,
the other to producing/regulating the loose compo-
nent. The fibrous component has a major role in
transmitting force at a distance, while hyaluronan per-
mits fascial gliding and autonomy among the various
fibrous sublayers. It is evident that fasciae are appear-
ing more and more complex, formed by various cells
with different roles and probably responding to different
inputs. The possibility of controlling/regulating the vari-
ous cells precisely will probably make fascial alterations
more responsive to more focused treatments and
drugs. Indeed, it is already known that changes in HA
concentration are associated with inflammatory and
degenerative arthropathies (Temple-Wong et al., 2016)
and that defects in fascial gliding can compromise the
entire functionality of tissues and can generate pain
(Bordoni and Zanier, 2014). Understanding what stimuli
affect fascial cell activity will be fundamental to a better
comprehension of the influence of the HA-rich matrix
on tissue hydration and the lubrication of sliding surfa-
ces (Stecco et al., 2011; Fakhari and Berkland, 2013),
and to understanding the changes in the HA-rich matrix
that occur in myofascial pain, as well as in other patho-
physiologies affecting fascial tissue.
We demonstrated the expression of HA synthase in
homogenates of fascial cells, and found that about
30% of fascial fibroblasts are fasciacytes. We think that
the percentage of fasciacytes could vary depending on
TABLE 2. Comparison of Ct Values for Real Time
PCR
C
t
RPS18 20.7 61.1
RPLP0 21.9 60.15
HAS2 30.4 61.3
The values are the means of at least three different
experiments in triplicate.
Fig. 7. Diagram of the layers of the deep fascia and
the main location of the fasciacytes. [Color figure can be
viewed at wileyonlinelibrary.com]
8 Stecco et al.
the stimuli that these cells undergo conducting them in
the metaplasic phase.
Other tissues also contain fibroblast-like cells spe-
cialized for synthesizing and secreting high levels of
HA. These include the synovial lining cells of joint cas-
pules, where there are two distinct cell types: bone
marrow–derived cells of the monocyte system (A
cells) and specialized fibroblasts (B cells) (Iwanaga
et al., 2000); and the hyalocytes in the eye (Saka-
moto and Ishibashi, 2011), which express tissue mac-
rophage marker (Holness and Simmons, 1993). The
cells we have described in fasciae are negative for
CD68, demonstrating that they are not of the mono-
cyte/macrophage lineage. They are therefore likely to
be related to chondrocytes, which are known to be S-
100 protein-positive and specialized for HA synthesis.
In summary, the cells described seem to have spe-
cific morphology and location and a well defined func-
tion (Table 3). For these reasons we propose to call
them “fasciacytes” as new class of fascia-associated
cells.
ACKOWLEDGMENTS
We thank the Orthopedic Clinic of Padua for patient
data and sample collection. The ultramicrotome was
purchased through the funding for scientific equipment
of the University of Padova (2013). Work by Caterina
Fede was partially supported by a Fascial Manipulation
Association grant.
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TABLE 3. Comparison Between the Main Features
of Classical Fibroblasts and Fasciacytes
Fascial fibroblasts Fasciacytes
Morphology Elongated Rounded shape
Location Inside the loose
connective tissue
fascial layers
Especially between
the fascial layers,
at the borders
Alcian blue
staining
Not all positive More pronounced,
inside the cells
and around them
HABP Not all positive Positive
CD68 Negative Negative
Vimentin Positive Positive
S100-A4 Negative Positive
TEM Extracellular matrix
around cells has
a collagen fibril
architectures
Extracellular matrix
around cells is
enriched in
proteoglycans
The Fasciacytes 9
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10 Stecco et al.
... In addition, slides were incubated in Alcian blue solutions (0.05%, pH 5.8) with different MgCl 2 concentrations (0.05 M; 0.3 M; 2 M) to stain the acidic polysaccharides and glycosaminoglycans (blue in color). As described in the literature [22,53], different concentrations of MgCl 2 cause differences in the staining: the cations of the salt compete with those of the dye for the polyanionic sites in the tissue, permitting a selective stain of specific glycosamynoglycans. ...
... The Purple-Jelley HA assay (Biocolor Ltd.) was used to measure the HA content in human and mouse skeletal muscles [53]. Briefly, 250 mg ± 50 mg human and mouse muscle tissues were cut into small fragments with a surgical scalpel and then transferred to 2.0 mL microcentrifuge tubes and digested at 55 • C in 400 µL TRIS-HCl (50 mM, pH 7.6) containing Proteinase K (Sigma) overnight. ...
... Finally, after the calculation of the µg HA extracted from the starting tissues samples, the average µg HA per gram of wet muscle tissue was obtained for each sample (mean of at least two measurements ± standard deviation). A reference was made to the results obtained previously from four adult human skin samples as control data to validate the extraction method [53]. ...
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The intramuscular connective tissue plays a critical role in maintaining the structural integrity of the muscle and in providing mechanical support. The current study investigates age-related changes that may contribute to passive stiffness and functional impairment of skeletal muscles. Variations in the extracellular matrix in human quadriceps femoris muscles in 10 young men, 12 elderly males and 16 elderly females, and in the hindlimb muscles of 6 week old, 8 month old and 2 year old C57BL/6J male mice, were evaluated. Picrosirius red, Alcian blue and Weigert Van Gieson stainings were performed to evaluate collagen, glycosamynoglycans and elastic fibers. Immunohistochemistry analyses were carried out to assess collagen I, collagen III and hyaluronan. The percentage area of collagen was significantly higher with aging (p < 0.01 in humans, p < 0.001 in mice), mainly due to an increase in collagen I, with no differences in collagen III (p > 0.05). The percentage area of elastic fibers in the perimysium was significantly lower (p < 0.01) in elderly men, together with a significant decrease in hyaluronan content both in humans and in mice. No significant differences were detected according to gender. The accumulation of collagen I and the lower levels of hyaluronan and elastic fibers with aging could cause a stiffening of the muscles and a reduction of their adaptability.
... Abundant myofibroblasts can give hypertrophic scarring and contractures if they work for too long [41]. Other important cellular actors are the fasciacytes, a small cluster of rounded cells along the surface of each deep fascial layer specialized in hyaluronan (HA) synthesis [42], and the telocytes, called a "network in network" by Dawidowics et al. [43], due to their thin extensions (telopodes) that form a 3D communication system in the interstitial ECM. Both cell types may have a role in diabetes that will need to be studied in future research. ...
... Some years ago, a new cell devoted to HA production was described in the fasciae, including in the plantar fascia, and for that reason it was called a fasciacyte. It is a rounded cell specialized in hyaluronan (HA) synthesis, placed along the surface of each deep fascial layer [42]. The production of hyaluronan in the fascial ECM can be affected by many physical, mechanical and metabolic factors, and these can affect the hydration level of the fasciae [42]. ...
... It is a rounded cell specialized in hyaluronan (HA) synthesis, placed along the surface of each deep fascial layer [42]. The production of hyaluronan in the fascial ECM can be affected by many physical, mechanical and metabolic factors, and these can affect the hydration level of the fasciae [42]. "The amount of HA varies according to the anatomical site and to the fascial type: in the aponeurotic fasciae it is about 43 μg/g, but it drastically decreases (about 6 μg/g) in epimysial fasciae, and it increases in the retinacula (90.4 μg/g)" [62]. ...
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... Such homeostatic cytoskeletal responses appear to be specific to fascial tissue and do not occur in more densely packed dermal fibroblasts [12]. Fascial fibroblasts are positive for vimentin, fibroblast-specific protein 1 (FSP1; S100A4), CD26, and Sca1 and are negative for monocytic marker CD68, demonstrating their fibroblastic nature [22,23]. Recent single-cell RNA-sequencing studies have revealed two additional fascial fibroblast markers: GPX3 (encoding Glutathione peroxidase 3) [24,25] and MSX1 (encoding Msh homeobox 1) [26] that mark the fibroblasts of mouse subcutaneous fascia. ...
... Subcutaneous fascia is rich in glycosaminoglycans, with a prevalence of hyaluronic acid, which plays a key role in providing hydration and viscosity to the fascial tissue, since it has the unique capacity of binding large quantities of water [7,33,34]. Extracellular hyaluronic acid is synthesized predominantly by fascial fibroblasts, as evidenced by Alcian blue staining, anti-hyaluronic acid binding protein (HABP) immunohistochemistry, transmission electron microscopy, and the expression of hyaluronan synthase 2 (HAS2) [20,22,35]. Fascial hyaluronic acid levels remain constant throughout life [35]. ...
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... There are also thin elastic fibres within the PF and the lubricating glycosaminoglycan (hyaluronan) is located amongst the collagen fibres which is probably produced by fibroblastic-like cells (fasciacytes) (Stecco et al., 2018). Nerve endings and the mechanoreceptors, Pacini and Ruffini corpuscles, are also found in the PF and respond to the mechanical deformation of the skin and PF (Fleming & Luo, 2013), and therefore, they would provide a sensory feedback system via the myofascial network to the brain for the locomotory system. ...
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Background One potential mechanism for early superficial cartilage wear in normal joints is alteration of the lubricant content and quality of synovial fluid. The purpose of this study was to determine if the concentration and quality of the lubricant, hyaluronan, in synovial fluid: (1) was similar in left and right knees; (2) exhibited similar age-associated trends, whether collected postmortem or antemortem; and (3) varied with age and grade of joint degeneration. Methods Human synovial fluid of donors (23–91 years) without osteoarthritis was analyzed for the concentrations of protein, hyaluronan, and hyaluronan in the molecular weight ranges of 2.5–7 MDa, 1–2.5 MDa, 0.5–1 MDa, and 0.03–0.5 MDa. Similarity of data between left and right knees was assessed by reduced major axis regression, paired t-test, and Bland-Altman analysis. The effect of antemortem versus postmortem collection on biochemical properties was assessed for age-matched samples by unpaired t-test. The relationships between age, joint grade, and each biochemical component were assessed by regression analysis. Results Joint grade and the concentrations of protein, hyaluronan, and hyaluronan in the molecular weight ranges of 2.5–7 MDa, 1–2.5 MDa, and 0.5–1 MDa in human synovial fluid showed good agreement between left and right knees and were similar between age-matched patient and cadaver knee joints. There was an age-associated decrease in overall joint grade (–15 %/decade) and concentrations of hyaluronan (–10.5 %/decade), and hyaluronan in the molecular weight ranges of 2.5–7 MDa (–9.4 %/decade), 1–2.5 MDa (–11.3 %/decade), 0.5–1 MDa (–12.5 %/decade), and 0.03–0.5 MDa (–13.0 %/decade). Hyaluronan concentration and quality was more strongly associated with age than with joint grade. Conclusions The age-related increase in cartilage wear in non-osteoarthritic joints may be related to the altered hyaluronan content and quality of synovial fluid.
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