J Mater Sci: Mater Med (2016)27:84
DOI 10.1007/s10856-016-5689-2
TISSUE ENGINEERING CONSTRUCTS AND CELL SUBSTRATES
1,4-Butanediol diglycidyl ether-cross-linked hyaluronan inhibits
fibrosis in rat primary tenocytes by down-regulating autophagy
modulation
Dur-Zong Hsu1 • I-Ming Jou1,2
Received: 17 July 2015 / Accepted: 17 February 2016
Ó Springer Science+Business Media New York 2016
Abstract Epidural fibrosis, an inevitable part of the
postoperative healing process, is one of the important
causes of failed back surgery syndrome after spinal sur-
gery. The aim of this study was to examine the inhibitory
effect of a novel material 1,4-butanediol diglycidyl ether-
cross-linked hyaluronan (cHA) on fibrosis in primary
tenocytes. cHA inhibited migration, cell proliferation, and
suppressed the expression of fibronectin, but not trans-
forming growth factor-b, in primary tenocytes. cHA sig-
nificantly increased matrix metalloproteinase-3 but
decreased collagen-1 and microtubule-associated protein
light chain 3-II expression in a dose-dependent manner
compared with control groups. We therefore concluded that
suppressing autophagy activity may be involved in the anti-
fibrotic effect of cHA in primary tenocytes. Further, cHA
may have the potential for preventing epidural fibrosis and
subsequent failed back syndrome in patients with
laminectomy in the future.
1 Introduction
Epidural fibrosis describes extradural fibrous tissue for-
mation after a laminectomy procedure. Epidural fibrosis
often produces adhesions tethering the nerve root to adja-
cent tissues, restricts the mobility of the nerve roots, and
& I-Ming Jou
jming@mail.ncku.edu.tw
1 fibrillar collagen by MMPs is a key event in the resolution
Department of Orthopedics, National Cheng Kung University
Hospital, Tainan, Taiwan
2 responsible for mediating crucial functions in repair pro-
Department of Orthopedics, College of Medicine, National
Cheng Kung University, 138 Sheng-Li Road, Tainan 704,
Taiwan
Original Research
increases tension on the nerve during motion, which leads
to nerve injury, and causes recurrent local and radicular
pain [1]. Further, epidural fibrosis, as an inevitable part of
the postoperative healing process, is one of the important
causes of failed back syndrome after spinal surgery [2–4].
Hyaluronic acid (HA), an anti-adhesive and anti-fibrotic
agent [5–9], has being used to inhibit peripheral nerve
adhesion, scar formation, and post-laminectomy fibrosis
formation [10–13]. Recently, to improve the mechanical
properties and lengthen the residence time, the polymer
chains of HA are cross-linked with linker molecules, such as
divinyl sulfone, homobifunctional glycidyl ethers, glu-
taraldehyde, and formaldehyde [14, 15]. However, these
toxic, irritative, or corrosive linker molecules may be
released onto surrounding viable tissues during cross-linked
HA degradation. In contrast, 1,4-butanediol diglycidyl
ether-cross-linked hyaluronan (abbreviated as cHA) is a
non-resorbable cross-linked hyaluronan-derived polymer
without cytotoxicity and adverse changes in electrophysi-
ology and neurobehavior [16]. cHA has better mechanical
stability and viscoelastic properties than HA. Further, cHA
hydrogel is a more uniform hydrogel [17]. Despite, the anti-
fibrotic effect of cHA has never been investigated.
The components in extracellular matrix (ECM) includ-
ing matrix metalloproteinases (MMPs), collagen, trans-
forming growth factor (TGF)-b, and fibronectin, play an
important role in the regulating ECM turnover in fibrotic
tissues [18, 19]. Degradation of normal ECM components
in the early stages of fibrosis promotes deposition of newly
synthesized collagen [20]. The degradation of pathological
of fibrosis. Recently, MMPs are recognized as being
cess, such as cell migration, leukocyte activation, antimi-
crobial defense, chemokine processing [21, 22]. TGF-b
123
84 Page 2 of 7
plays an important role in wound healing and tissue fibrosis
[23]. TGF-b stimulates the production of various ECM
proteins and inhibits the degradation of these matrix pro-
teins. In many diseases, excessive TGF-b contributes to a
pathologic excess of tissue fibrosis [24]. Fibronectin, syn-
thesized by fibroblasts, endothelial cells, chondrocytes,
synovial cells and myocytes [25], is vital for establishing
and maintaining tissue architecture and for regulating cel-
lular processes including proliferation [26–29] and migra-
tion [30–32]. In addition, fibronectin has been used as a
fibroblast-specific marker [33].
Autophagy plays a crucial role in fibrosis process by regu-
lating the activities of ECM components, including MMPs and
collagens [34, 35]. Autophagy is a genetically programmed,
evolutionarily conserved catabolic process that occurs in
response to stress and is manifested as degradation of cellular
proteins and damaged organelles in an effort to promote cell
survival [36]. The autophagosome formation is regulated by 2
ubiquitin-like conjugation systems Atg5-12-16 and microtubule
light chain 3 (LC3), is a soluble protein distributed ubiquitously
in mammalian tissues and cultured cells [37]. During autophagy,
a cytosolic form of LC3 (LC3-I) is conjugated to phos-
phatidylethanolamine to form LC3-phosphatidylethanolamine
conjugate (LC3-II), which is recruited to autophagosomal
membranes [38]. In addition, LC3-II has been suggested to be
one of a specific indicator for evaluating the autophagy levels
[39]. The aim of the present study is to examine the inhibitory
effects of cHA on fibrosis in primary tenocytes.
2 Materials and methods
2.1 Materials
cHA was purchased from Maxigen Biotech Inc., New
Taipei City, Taiwan). There is no free HA in the cHA.
According to our previous studies, cHA has better
mechanical stability and viscoelastic properties than HA.
Further, cHA hydrogel is a more uniform hydrogel [17].
2.2 Animals
Male Sprague–Dawley rats weighing 300 to 400 g were
obtained from and housed in the Laboratory Animal Center
of National Cheng Kung University in Taiwan. Rats were
housed individually in a room with a 12-h light/dark cycle
and central air conditioning (25 °C, 70 % humidity). Rats
were allowed free access to tap water and were fed a rodent
diet from Richmond Standard, PMI Feeds, Inc. (St. Louis,
MO). The animal care and experimental protocols were in
accordance with nationally approved guidelines.
123
J Mater Sci: Mater Med (2016)27:84
2.3 Primary rat tenocyte culture
Achilles tendons from adult rats were used in this study.
The tendon samples were washed with sterile PBS and cut
into small pieces then placed in 10 cm dishes for explants
culture in Dulbecco’s modified Eagle medium (DMEM)
(Sigma) supplemented with 10 % fetal calf serum and
penicillin/streptomycin (100 U/mL). The tenocytes main-
tained in a humidified incubator containing 5 % CO2 at
37 °C. The medium was changed every 3 to 4 days until
confluent growth was achieved.
2.4 Experimental design
2.4.1 Experiment I
The inhibitory effect of cHA on fibrosis primary tenocytes.
Primary tenocytes were divided into three groups; various
concentrations of cHA ranging from 0, 0.06, and 0.25 %
were treated onto the cells, respectively. Cell proliferation,
migration, and fibronectin protein expressions were deter-
mined 48 h after.
2.4.2 Experiment II
The possible mechanism underlying the anti-fibrosis effect
of cHA in primary tenocytes. Primary tenocytes were
divided into three groups; various concentrations of cHA
ranging from 0, 0.03, 0.06, 0.12, and 0.25 % were treated
onto the cells, respectively. The protein expressions of
MMP3, collagen-1, and LC3-II were determined 48 h after.
2.5 Cell proliferation assay
A colorimetric WST-1 assay (TaKaRa, Shiga, Japan) was
used to assess cell viability after 3 days of incubation
according to the instructions of the manufacturer.
2.6 Fibroblast migration
The effect of cHA on synovial fibroblasts cell migration
was assessed by using wound healing assay. Cells were
grown in 6-well plates at a density of 3 9 106/mL, and a
small linear scratch was created in the confluent monolayer
by gently scraping with sterile cell scrapper. Cells were
extensively rinsed with medium to remove cellular debris
before treating with PBS or cHA. Forty-eight hours later,
images of the migrated cells were taken using digital
camera, connected to the inverted microscope (Nikon,
TMS-F, Japan).
J Mater Sci: Mater Med (2016)27:84
2.7 Immunoblot analysis
Fibronectin, TFG-b, MMP3, collagen-1, LC3-1, and LC3-
II expressions were determined by immunoblot analysis
with their monoclonal antibody (Santa Cruz). The blot was
reprobed with anti-b-actin monoclonal antibody (Sigma)
served as the loading control. After incubation with
horseradish peroxidase-conjugated secondary antibodies
(Santa Cruz), the protein bands were detected using
Immobilon Western Chemiluminescent HRP substrate
(Millipore) and visualized with a Biospectrum AC imaging
system (UVP).
2.8 Statistical analysis
Data are means ± standard deviation (SD). One-way
analysis of variance (ANOVA) followed by Student’s t test
were used for pairwise comparisons between treatments.
Significance was set at P \ 0.05.
3 Results
3.1 Effect of cHA on cell migration in primary
tenocytes
To examine the anti-fibrotic effect of cHA, the cell
migration and proliferation in primary tenocytes were
assessed. Compared to control group (Fig. 1a), migration
area was significantly decreased in 0.06 % (Fig. 1b) and
0.25 % (Fig. 1c) cHA-treated groups (Fig. 1d) in primary
tenocytes. Further, cHA at the dose of 0.25 %, but not
0.1 %, significantly decreased cell proliferation (Fig. 2)
without inducing cell death (data not shown) compared
with that in non-cHA control group.
3.2 Effect of cHA on fibronectin expression
in primary tenocytes
To examine the mechanism involved in cHA-exerted
effects of anti-migration and anti-proliferation, the fibro-
nectin expression was assessed in primary tenocytes. cHA
at the dose of 0.25 %, but not 0.1 %, significantly
decreased fibronectin protein expression compared with
that in non-cHA control group (Fig. 3a).
3.3 Effect of cHA on TGF-b expression in primary
tenocytes
To examine the role of TGF-b in cHA-exerted effects, the
TGF-b expression was assessed in primary tenocytes. cHA
at the dose of 0.25 % shows the trend to decrease TGF-b
Page 3 of 7 84
expression (P = 0.08). However, there is no significant
difference (Fig. 3b).
3.4 Effect of cHA on MMP3 and collagen-1
expressions in primary tenocytes
To examine the role of ECM components in cHA-exerted
anti-fibrotic effect, MMP3 and collagen-1 protein expres-
sions in were assessed in primary tenocytes. cHA signifi-
cantly increased MMP3 (Fig. 4a) and decreased collagen-1
(Fig. 4b) protein expressions in a dose-dependent manner
compared that with non-cHA control group.
3.5 Effects of cHA on LC3-I and LC3-II expression
in primary tenocytes
To examine the involvement of autophagy, LC3-I and
LC3-II expression in primary tenocytes was also deter-
mined. However, the changes of LC3-I were not as obvious
as the changes of LC3-II. It is reported that LC3-II, a
dominant autophagy marker, tends to be much more sen-
sitive to be detected by immunoblotting than LC3-I. To
evaluate autophagy, comparison of LC3-II expression is
more appropriate than comparison of LC3-I and LC3-II for
ratio [40]. Therefore, we calculated and compared the LC3-
II expressions in the present study. cHA at the concentra-
tions of 0.12 and 0.25 % significantly inhibited LC3-II
protein expression compared with non-cHA control group
(Fig. 5).
4 Discussion
In the present study, we have demonstrated the effect of
cHA on fibrosis in primary tenocytes. cHA inhibited
migration, cell proliferation, and suppressed the expression
of fibronectin in primary tenocytes. cHA significantly
increased MMP3, but decreased collagen-1 and LC3-II
expression. We suggested that suppressing autophagy
activity is involved in the anti-fibrotic effect of cHA in
primary tenocytes.
Inhibiting fibronectin-associated migration and cell
proliferation may be involved in cHA’s anti-fibrotic effect.
Fibroblast proliferation and migration into the wound are
the key factors in the pathogenesis and development of
fibrosis. Preventing fibroblasts migration from perispinal
muscles related to surgery prevents or reduces epidural
fibrosis formation [41]. Fibronectin, a specific fibroblast
marker, plays a crucial role in the process of fibroblast
proliferation and migration [33, 42, 43]. Fibronectin
functions both as a regulator of cellular processes and an
important scaffolding protein to maintain and direct tissue
123
84 Page 4 of 7 J Mater Sci: Mater Med (2016)27:84

Fig. 1 Effects of cHA on cell

migration in rat primary
tenocytes. Primary tenocytes
were divided into three groups,
various concentrations of cHA
ranging from 0 (a), 0.06 (b), and
0.25 % (c) were treated onto the
cells, respectively. Cell
migration area was determined
48 h after (a–d). Data were
expressed as mean ± SD.
*P \ 0.05 compared with
control group

TGF-b plays an important role in wound healing and

Fig. 2 Effects of cHA on cell proliferation in rat primary tenocytes.
Primary tenocytes were divided into three groups, various concen-
trations of cHA ranging from 0, 0.06, and 0.25 were treated onto the
cells, respectively. Cell proliferation was determined 48 h after. Data
were expressed as mean ± SD. *P \ 0.05 compared with control
group
organization [44]. In the present study, cHA significantly
inhibited fibronectin expression as well as tenocyte pro-
liferation and migration. It is likely that cHA may decrease
fibrosis by inhibiting fibronectin-associated cell prolifera-
tion and migration, at least partially.
tissue fibrosis [23]. TGF-b stimulates the production of
various ECM proteins and inhibits the degradation of these
matrix proteins. In many diseases, excessive TGF-b con-
tributes to a pathologic excess of tissue fibrosis [24]. In the
present study, cHA at the dose of 0.25 % show the trend to
decrease TGF-b expression (P = 0.08). However, there is
no significant difference. It is likely that the degradation of
ECM by inducing MMP expression may play a more
important role in the anti-fibrotic effect of cHA. We have
provided the results in our revision.
Autophagy modulation-associated ECM component
alteration may be involved in the mechanism underlying
the anti-fibrotic effect of cHA. Fibrosis is characterized by
excess deposition of a collagen-rich ECM, which can be
down-regulated by MMPs [20]. The main constituents of
fibrotic lesions are interstitial collagens and excessive
deposition of these durable fibers can result in disruption of
proper tissue structure and function [45]. Further, increas-
ing MMPs [20, 46] or inhibiting collagen [47, 48]
expression leads to a decrease of ECM formation and
subsequent fibrosis process. Although many studies have
demonstrated a correlation between dysregulated autop-
hagy and fibrotic diseases, both up- and down-regulation of
autophagy have been hypothesized to be involved in

123
J Mater Sci: Mater Med (2016)27:84 Page 5 of 7 84

Fig. 3 Effects of cHA on

fibronectin expression in rat
primary tenocytes. Primary
tenocytes were divided into
three groups, various
concentrations of cHA ranging
from 0, 0.06, and 0.25 % were
treated onto the cells,
respectively. Fibronectin (a) and
TGF-b (b) expressions was
determined 48 h after. Data
were expressed as mean ± SD.
*P \ 0.05 compared with
control group

Fig. 4 Effects of cHA on

MMP3 and collagen-1
expressions in rat primary
tenocytes. Primary tenocytes
were divided into three groups,
various concentrations of cHA
ranging from 0, 0.03, 0.06, 0.12,
and 0.25 % were treated onto
the cells, respectively. The
protein expressions of MMP3
(a) and collagen-1 (b) were
determined 48 h after. Data
were expressed as mean ± SD.
*P \ 0.05 compared with
control group

fibrosis progression [34, 35, 49]. However, some studies
indicated that autophagy is required for fibrogenesis while
loss of autophagic function in stellate cells and in mice
reduced fibrogenesis and matrix accumulation [50]. Fur-
ther, pharmacological inhibition of autophagy by various
autophagy inhibitors including bafilomycin-A1, 3-methy-
ladenine, and rapamycin also inhibits fibrotic response in
fibroblasts [51, 52]. In the present study, cHA significantly
inhibited autophagy, increased MMP3, and decreased col-
lagen-1 expressions. It is likely that cHA may inhibit
tenocyte fibrosis via modulating autophagy-associated
ECM component alteration. However, more investigation
will be needed.
In addition, cHA hydrogel is safe and effective for
reducing adhesions, biocompatible, and nontoxic [53–56].
Unlike traditional HA, cHA is highly viscous and con-
formable to the surfaces of any laminectomy defect. It
remains adherent to the tissue, even when the surface is
vertical rather than horizontal, before it degrades. cHA
hydrogel can act as an efficacious barrier to block the
invasion of epidural fibrosis to the spinal canal space.
Therefore, we suggested that cHA hydrogel, other than
solid interpositional materials, could be a useful agent for
preventing epidural fibrosis in the patients with laminec-
tomy. We concluded that cHA may inhibit fibrosis by
down-regulating autophagy modulation in primary

123
84 Page 6 of 7
Fig. 5 Effects of cHA on LC3-I and LC3-II expression in rat primary
tenocytes. Primary tenocytes were divided into three groups, various
concentrations of cHA ranging from 0, 0.03, 0.06, 0.12, and 0.25 %
were treated onto the cells, respectively. The protein expressions of
LC3-I and LC3-II were determined 48 h after. Data were expressed as
mean ± SD. *P \ 0.05 compared with control group
tenocytes. Further, cHA may have the clinical potential for
preventing against epidural fibrosis; however, more inves-
tigations will be needed.
Acknowledgments This study was supported by Grants NSC100-
2622-B-006-003-CC2 and NSC 101-2622-B-006-004-CC2 from the
National Science Council, Taiwan.
Compliance with Ethical Standards
Conflict of interest The authors declare no conflicts of interest.
References
1. Key JA, Ford LT. Experimental intervertebral-disc lesions.
J Bone Joint Surg Am. 1948;30A:621–30.
2. Djukic S, Lang P, Morris J, Hoaglund F, Genant HK. The post-
operative spine. Magnetic resonance imaging. Orthop Clin N Am.
1990;21:603–24.
3. Rabb CH. Failed back syndrome and epidural fibrosis. Spine J.
2010;10:454–5.
4. Ross JS. Magnetic resonance imaging of the postoperative spine.
Semin Musculoskelet Radiol. 2000;4:281–91.
5. Asch HL, Lewis PJ, Moreland DB, Egnatchik JG, Yu YJ, Cla-
beaux DE, Hyland AH. Prospective multiple outcomes study of
outpatient lumbar microdiscectomy: should 75 to 80% success
rates be the norm? J Neurosurg. 2002;96:34–44.
6. Atlas SJ, Deyo RA, Keller RB, Chapin AM, Patrick DL, Long
JM, Singer DE. The Maine Lumbar Spine Study, Part III: 1-year
outcomes of surgical and nonsurgical management of lumbar
spinal stenosis. Spine. 1996;21:1787–94.
123
J Mater Sci: Mater Med (2016)27:84
7. Atlas SJ, Keller RB, Chang Y, Deyo RA, Singer DE. Surgical and
nonsurgical management of sciatica secondary to a lumbar disc
herniation: five-year outcomes from the Maine Lumbar Spine
Study. Spine. 2001;26:1179–87.
8. Fritsch EW, Heisel J, Rupp S. The failed back surgery syndrome:
reasons, intraoperative findings, and long-term results: a report of
182 operative treatments. Spine. 1996;21:626–33.
9. Jonsson B, Stromqvist B. Repeat decompression of lumbar nerve
roots. A prospective two-year evaluation. J Bone Joint Surg Br.
1993;75:894–7.
10. Akeson WH, Massie JB, Huang B, Giurea A, Sah R, Garfin SR,
Kim CW. Topical high-molecular-weight hyaluronan and a
roofing barrier sheet equally inhibit postlaminectomy fibrosis.
Spine J. 2005;5:180–90.
11. Massie JB, Schimizzi AL, Huang B, Kim CW, Garfin SR, Akeson
WH. Topical high molecular weight hyaluronan reduces radicular
pain post laminectomy in a rat model. Spine. 2005;J5:494–502.
12. Schimizzi AL, Massie JB, Murphy M, Perry A, Kim CW, Garfin
SR, Akeson WH. High-molecular-weight hyaluronan inhibits
macrophage proliferation and cytokine release in the early wound
of a preclinical postlaminectomy rat model. Spine J. 2006;6:550–6.
13. Shih HN, Fang JF, Chen JH, Yang CL, Chen YH, Sung TH, Shih
LY. Reduction in experimental peridural adhesion with the use of
a crosslinked hyaluronate/collagen membrane. J Biomed Mater
Res B Appl Biomater. 2004;71:421–8.
14. Kim SS, Michelsen CB. Revision surgery for failed back surgery
syndrome. Spine. 1992;17:957–60.
15. Pheasant H. Sources of failure in laminectomies. Orthop Clin N
Am. 1975;6:319–29.
16. Lan SM, Jou IM, Wu PT, Wu CY, Chen SC. Investigation into
the safety of perineural application of 1,4-butanediol diglycidyl
ether-crosslinked hyaluronan in a rat model. J Biomed Mater Res
B Appl Biomater. 2015;103:718–26.
17. Wu CY, Huang YH, Lee JS, Tai TW, Wu PT, Jou IM. Efficacy of
topical cross-linked hyaluronic acid hydrogel in preventing post
laminectomy/laminotomy fibrosis in a rat model. J Orthop Res.
2016;34:299–306.
18. Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix
degradation and remodeling in development and disease. Cold
Spring Harb Perspect Biol. 2011;3:a005058.
19. Smith HW, Marshall CJ. Regulation of cell signalling by uPAR.
Nat Rev Mol Cell Biol. 2010;11:23–36.
20. Hemmann S, Graf J, Roderfeld M, Roeb E. Expression of MMPs
and TIMPs in liver fibrosis—a systematic review with special
emphasis on anti-fibrotic strategies. J Hepatol. 2007;46:955–75.
21. ManiconeAM McGuire JK. Matrix metalloproteinases as modu-
lators of inflammation. Semin Cell Dev Biol. 2008;19:34–41.
22. Parks WC, Wilson CL, López-Boado YS. Matrix metallopro-
teinases as modulators of inflammation and innate immunity. Nat
Rev Immunol. 2004;4:617–29.
23. Leask A, Abraham DJ. TGF-beta signaling and the fibrotic
response. FASEB J. 2004;18:816–27.
24. Branton MH, Kopp JB. TGF-beta and fibrosis. Microbes Infect.
1999;1:1349–65.
25. Mao Y, Schwarzbauer JE. Fibronectin fibrillogenesis, a cell-
mediated matrix assembly process. Matrix Biol. 2005;24:389–99.
26. Gui L, Wojciechowski K, Gildner CD, Nedelkovska H, Hocking
DC. Identification of the heparin-binding determinants within
fibronectin repeat III1: role in cell spreading and growth. J Biol
Chem. 2006;281:34816–25.
27. Jiang ST, Chiang HC, Cheng MH, Yang TP, Chuang WJ, Tang
MJ. Role of fibronectin deposition in cystogenesis of Madin–
Darby canine kidney cells. Kidney Int. 1999;56:92–103.
28. Sechler JL, Schwarzbauer JE. Control of cell cycle progression
by fibronectin matrix architecture. J Biol Chem. 1998;273:
25533–6.
J Mater Sci: Mater Med (2016)27:84
29. Sottile J, Mosher DF. N-terminal type I modules required for
fibronectin binding to fibroblasts and to fibronectin’s III1 module.
Biochem J. 1997;323:51–60.
30. Clark RA, An JQ, Greiling D, Khan A, Schwarzbauer JE.
Fibroblast migration on fibronectin requires three distinct func-
tional domains. J Invest Dermatol. 2003;121:695–705.
31. Couchman JR, Rees DA, Green MR, Smith CG. Fibronectin has a
dual role in locomotion and anchorage of primary chick fibrob-
lasts and can promote entry into the division cycle. J Cell Biol.
1982;93:402–10.
32. Hocking DC, Chang CH. Fibronectin matrix polymerization
regulates small airway epithelial cell migration. Am J Physiol
Lung Cell Mol Physiol. 2003;285:L169–79.
33. Lim R, O’Connell MM. Effect of glia maturation factor on glial
fibrillary acidic protein and fibronectin: a comparative study on
glioblasts and fibroblasts using immunofluorescence. Brain Res.
1982;281:29–39.
34. Kim SI, Na HJ, Ding Y, Wang Z, Lee SJ, Choi ME. Autophagy
promotes intracellular degradation of type I collagen induced by
transforming growth factor (TGF)-b1. J Biol Chem. 2012;287:
11677–88.
35. Mallat A, Lodder J, Teixeira-Clerc F, Moreau R, Codogno P,
Lotersztajn S. Autophagy: a multifaceted partner in liver fibrosis.
Biomed Res Int. 2014;2014:869390.
36. Dolganiuc A, Thomes PG, Ding WX, Lemasters JJ, Donohue TM
Jr. Autophagy in alcohol-induced liver diseases. Alcohol Clin
Exp Res. 2012;36:1301–8.
37. Fujita N, Itoh T, Omori H, Fukuda M, Noda T, Yoshimori T. The
Atg16L complex specifies the site of LC3 lipidation for mem-
brane biogenesis in autophagy. Mol Biol Cell. 2008;19:2092–
100.
38. Tanida I, Ueno T, Kominami E. LC3 and autophagy. Methods
Mol Biol. 2008;445:77–88.
39. Myeku N, Figueiredo-Pereira ME. Dynamics of the degradation
of ubiquitinated proteins by proteasomes and autophagy: asso-
ciation with sequestosome 1/p62. J Biol Chem. 2011;286:
22426–40.
40. Mizushima N, Yoshimori T. How to interpret LC3 immunoblot-
ting. Autophagy. 2007;3:542–5.
41. Bottegaro NB, Kos J, Pirkic B, Smolec O, Grabarevic Z,
Hohsteter M, Selanec J, Vrbanac Z. Reduction of epidural fibrosis
after laminectomy in rabbits by omental free graft. Vet Med.
2013;58:25–31.
42. Briggs SL. The role of fibronectin in fibroblast migration during
tissue repair. J Wound Care. 2005;14:284–7.
43. Tanaka M, Abe T, Hara Y. Roles of focal adhesions and fibro-
nectin-mediated cohesion in proliferation of confluent fibroblasts.
J Cell Physiol. 2009;219:194–201.
Page 7 of 7 84
44. To WS, Midwood KS. Plasma and cellular fibronectin: distinct
and independent functions during tissue repair. Fibrogenesis
Tissue Repair. 2011;4:21.
45. Giannandrea M, Parks WC. Diverse functions of matrix metal-
loproteinases during fibrosis. Dis Model Mech. 2014;7:193–203.
46. Schuppan D, Ruehl M, Somasundaram R, Hahn EG. Matrix as a
modulator of hepatic fibrogenesis. Semin Liver Dis. 2001;21:
351–72.
47. Challa AA, Vukmirovic M, Blackmon J, Stefanovic B. With-
aferin-A reduces type I collagen expression in vitro and inhibits
development of myocardial fibrosis in vivo. PLoS One.
2012;7:e42989.
48. Zhang C, Kong X, Liu C, Liang Z, Zhao H, Tong W, Ning G,
Shen W, Yao L, Feng S. ERK2 small interfering RNAs prevent
epidural fibrosis via the efficient inhibition of collagen expression
and inflammation in laminectomy rats. Biochem Biophys Res
Commun. 2014;444:395–400.
49. Araya J, Kojima J, Takasaka N, Ito S, Fujii S, Hara H, Yanagi-
sawa H, Kobayashi K, Tsurushige C, Kawaishi M, Kamiya N,
Hirano J, Odaka M, Morikawa T, Nishimura SL, Kawabata Y,
Hano H, Nakayama K, Kuwano K. Insufficient autophagy in
idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol
Physiol. 2013;304:L56–69.
50. Hernández-Gea V, Ghiassi-Nejad Z, Rozenfeld R, Gordon R, Fiel
MI, Yue Z, Czaja MJ, Friedman SL. Autophagy releases lipid that
promotes fibrogenesis by activated hepatic stellate cells in mice
and in human tissues. Gastroenterology. 2012;142:938–46.
51. Ghavami S, Cunnington RH, Gupta S, Yeganeh B, Filomeno KL,
Freed DH, Chen S, Klonisch T, Halayko AJ, Ambrose E, Singal
R, Dixon IM. Autophagy is a regulator of TGF-b1-induced
fibrogenesis in primary human atrial myofibroblasts. Cell Death
Dis. 2014;6:e1696.
52. Patel AS, Lin L, Geyer A, Haspel JA, An CH, Cao J, Rosas IO,
Morse D. Autophagy in idiopathic pulmonary fibrosis. PLoS One.
2012;7:e41394.
53. Falabella CA, Melendez MM, Weng L, Chen W. Novel macro-
molecular crosslinking hydrogel to reduce intra-abdominal
adhesions. J Surg Res. 2010;159:772–8.
54. Kocak I, Unlu C, Akcan Y, Yakin K. Reduction of adhesion
formation with cross-linked hyaluronic acid after peritoneal sur-
gery in rats. Fertil Steril. 1999;72:873–8.
55. Law JD, Lehman RA, Kirsch WM. Reoperation after lumbar
intervertebral disc surgery. J Neurosurg. 1978;48:259–63.
56. Tsai SW, Fang JF, Yang CL, Chen JH, Su LT, Jan SH. Prepa-
ration and evaluation of a hyaluronate-collagen film for pre-
venting post-surgical adhesion. J Int Med Res. 2005;33:68–76.
123