Capsaicin reduces PLGA-induced fibrosis by promoting M2

macrophages and suppressing overall inflammatory Response

T. Truong,1 K. S. Jones1,2
1
Department of Chemical Engineering, McMaster University, Hamilton, ON, Canada
2
School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada

Received 30 October 2017; revised 29 March 2018; accepted 5 April 2018

Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.36436

Abstract: Capsaicin reduced poly(lactic-co-glycolic) acid
(PLGA)-induced fibrosis by promoting IL-10 secretion and
suppressing alpha-smooth muscle actin (a-SMA) expression.
The lifetime and efficacy of tissue engineering scaffolds are
determined by the foreign body response. In this study, we
investigated the in vitro and in vivo effects of capsaicin to
reduce biomaterial-induced fibrosis. RAW 264.7 cells cultured
on PLGA films with capsaicin responded with significant
(p < 0.05) upregulation in M2 markers arginase-1 and IL-10
and downregulation of M1 markers iNOS and IL-12, demon-
than PLGA-only implants. Capsaicin caused a 35% increase in
IL-10 which played a key role in suppressing fibrosis. Macro-
phage phenotype markers in peritoneal cells and adherent
cells were unaffected by capsaicin; however, capsaicin sup-
pressed the myofibroblast marker a-SMA in adherent cells by
day 14. Overall, our results revealed that capsaicin reduced
biomaterial-induced fibrosis and demonstrates that capsaicin
has the potential to extend the lifetime of a tissue engineer-
ing scaffold when used in long-term drug release applica-
tions from hydrophobic biomaterials. V C 2018 Wiley Periodicals,

strating the potential of capsaicin to reduce PLGA-induced
inflammation. Subsequent animal studies were conducted
where PLGA and capsaicin-embedded PLGA discs were
implanted in C57BL/6 mice for 2 and 14 days. Explanted
capsaicin-embedded PLGA implants had 40% less collagen
Inc. J Biomed Mater Res Part A: 00A:000–000, 2018.
Key Words: capsaicin, fibrosis, macrophage phenotype, pol-
y(lactic-co-glycolic) acid, myofibroblast

How to cite this article: Truong T, Jones KS. 2018. Capsaicin reduces PLGA-induced fibrosis by promoting M2 macrophages
and suppressing overall inflammatory Response. J Biomed Mater Res Part A 2018:00A:000–000.

INTRODUCTION (reviewed by Anderson6). However, macrophages are the key

We used capsaicin released from poly(lactic-co-glycolic) acid
(PLGA) in an attempt to mitigate fibrotic responses elicited by
the biomaterial. Capsaicin is a pungent capsinoid that has
been used as a therapeutic to treat chronic inflammatory
diseases, such as rheumatoid arthritis.1 The foreign body
response to biomaterials is often detrimental to the efficacy of
a device. For example, fibrous encapsulation of continuous
glucose monitoring sensors decreases the sensitivity of the
signal and reduces the longevity of the device.2 A scarring
response to a scaffold precludes regeneration in tissue engi-
neering. There is extensive research in designing coatings to
minimize inflammation, but fibrosis is nearly inevitable.3–5
We propose a targeted approach to reduce the influence from
pro-inflammatory and pro-fibrotic cell types such as macro-
phages and myofibroblasts, by promoting alternative pheno-
types or preventing differentiation, respectively.
Many cell types are involved in the host response to biomate-
rials. Neutrophils appear early, followed by macrophages, then
fibroblasts and myofibroblasts. Cells of the immune system such
as lymphocytes and mast cells also interact in the response
mediators of the inflammatory response. Macrophages migrate
to the implant and phagocytose or degrade the implant. Failure
to digest the implant results in granuloma formation and the
release of toxic radicals, inflammatory cytokines and inflamma-
tory lipid mediators in an attempt to degrade the material.7
However, inflammatory agents exacerbate the response and
promote fibrosis. Macrophages can express two phenotypes, the
M1, or “classically activated,” phenotype and the M2, or
“alternatively activated,” phenotype.
M1 macrophages are identified by their high expression of
inducible nitric oxide synthase (iNOS) and high production
of interleukin-12 (IL-12) and IL-23.7 The phenotype can be
polarized with lipopolysaccharide (LPS) and interferon
gamma (IFNg) and its activity can be downregulated, or
controlled, by transforming growth factor beta-1 (TGF-b1)
and IL-10 stimulation.7
M2 phenotype macrophages are involved in mediating
wound healing and angiogenesis. These cells express arginase-1
mRNA, produce IL-10 and IL-23, and can be differentiated from
monocytes by IL-4 and IL-10 stimulation.7 M2 macrophages can

Additional Supporting Information may be found in the online version of this article.
Correspondence to: K. S. Jones; e-mail: kjones@mcmaster.ca
Contract grant sponsor: Natural Sciences and Engineering Research Council of Canada

C 2018 WILEY PERIODICALS, INC.

V 1
control extracellular material deposition by producing arginase-
1, for collagen production, and TGF-b1 to regulate the M1 phe-
notype.7 It has been shown that macrophage phenotype plays a
critical role in fibrotic response to biomaterials where early tran-
sition to an M2 phenotype can lead to resolved wound healing,8
though unregulated M2 macrophage functionality can lead to
peritoneal dialysis failure.9
Myofibroblasts are crucial for the final stages of wound
healing and remodeling. Their ability to produce collagen and
contract provides the necessary characteristics to close
wounds. Prolonged stimulation from TGF-b1 will induce the
myofibroblast phenotype and cause severe fibrosis. These cells
can be derived from many cell types including locally residing
mesenchymal cells, epithelium and endothelium cells and bone
marrow derived fibrocytes.10 Myofibroblasts are characterized
by their high expression of alpha smooth muscle actin (a-SMA)
and low expression of cadherin-1 (E-cadherin).11,12
Recently there has been development in using flavo-
noids, such as capsaicin, to suppress inflammation. Capsai-
cin has been shown to have anti-inflammatory properties
and to promote macrophage phenotype transition from M1
to M2.13 This pungent active ingredient in chili peppers
reduces the severity of autoinflammatory disease such as
arthritis, osteoarthritis, and rheumatoid arthritis.1,14,15 To
our knowledge, there has never been an investigation on
the effects of this plant extract on biomaterial-induced
inflammation and fibrosis.
Brown et al. speculated that an early M2 phenotype pro-
motes good wound healing.8 As such, we were interested in
using capsaicin to promote early M2 phenotype transition
and reduce biomaterial induced inflammation and fibrosis.
We hypothesized that capsaicin would downregulate the
inflammatory and fibrotic response by promoting the M2
phenotype and by preventing myofibroblast differentiation,
respectively. To test this hypothesis, we used PLGA discs to
induce inflammation and fibrosis because PLGA has been
demonstrated to cause an in vitro M1 response16 and an
in vivo fibrotic response.17 We tested in vitro macrophage
phenotype response by culturing RAW 264.7 cells on PLGA
films and by implanting capsaicin-embedded PLGA discs
into C57BL/6 mice. Our results showed that capsaicin
caused an upregulation in M2 markers both in vitro and
in vivo. More importantly, histological analysis revealed that
capsaicin-embedded implants had significantly less collagen
deposition around the disc than PLGA control implants
(p < 0.05).
MATERIALS AND METHODS
Casting PLGA-lined petri dishes
PLGA-lined Petri dishes were made using solvent casting.
Poly(lactic-co-glycolic) acid (PLGA, 75:25) (Sigma-Aldrich,
Milwaukee) was dissolved in ethyl acetate (Sigma-Aldrich,
Oakville). One milliliter of the 0.01 g/mL solution was
added to autoclaved 60 mm glass Petri dishes and placed in
a sterile vacuum chamber for 24 h with gentle rocking. The
dishes were removed aseptically from the vacuum chamber
and stored in a biosafety cabinet before use. The average
thickness of the films was 0.1729 mm, had no openings
2 TRUONG AND JONES
when viewed under bright field microscopy and a 24 h ste-
rility assay revealed no contamination. Capsaicin-embedded
PLGA films were cast by mixing 100 lg of capsaicin (Sigma
Aldrich, Milwaukee) in the PLGA/ethyl acetate solution prior
to casting. We do not expect that adding capsaicin altered
the surface roughness of the films. Wang et al. showed that
solvent cast films were smooth at the nanometer level.18
Cell culture
Murine macrophage-like cells (RAW 264.7) (ATCC TIB-71)
were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
(Thermo Fisher Scientific, Burlington) supplemented with
10% fetal bovine serum (FBS) (Thermo Fisher Scientific,
Burlington) and 100 U/mL penicillin–streptomycin (Thermo
Fisher Scientific, Burlington). Cells were maintained in a 378C
and 5% CO2 environment and subcultured at 80%–90%
confluency using phosphate buffered saline (PBS) (Mg--, Ca--,
pH 7.4) and a cell scraper.
Cellular response to PLGA films
RAW 264.7 cells were seeded at a population of 1 3 106
cells on PLGA-lined Petri dishes at three different conditions
for 24 h: pure PLGA films, PLGA films with 60 lM of capsai-
cin in the medium, and PLGA films blended with 0.18 lmol
of capsaicin. We confirmed that the working concentrations
were not toxic (Supporting Information, Fig. S1). Dose
responses were also done to choose an optimal concentra-
tion (Supporting Information, Fig. S2). Capsaicin was sus-
pended in DMSO before adding to the medium where the
volume of DMSO used was <1% of the medium. The
medium was collected for IL-10 and IL-12 quantification
and the cells were lysed to extract total RNA for relative
quantification using qPCR.
Casting capsaicin-embedded PLGA implants
PLGA implants were made by solvent casting 1 mL of 0.011 g/mL
of PLGA/ethyl acetate in autoclaved silicone molds (McMaster–
Carr) with 3/8” diameter wells. The total solution was added in
12 intervals with 5 min of vacuuming between each interval. Discs
were vacuumed for an additional hour before being removed
aseptically and stored in a sterile environment at 48C.
Capsaicin-embedded PLGA discs were made using the pre-
viously mentioned method but with 100 lg of capsaicin dis-
solved in the PLGA mixture. Solvent extraction from the discs
suggested that implants had a 71% (wt/wt) loading efficiency
which might show some of the capsaicin was bound to the
silicone molds, or (more likely) that the solvent extraction
process was not complete. The discs were stored in sterile
Petri dishes at 48C before use.
Animals and implantation procedure
Canadian Council on Animal (CCAC) guidelines for the care
and use of laboratory animals were followed throughout the
study. All animal experimental protocols were approved by
the Animal Research Ethics Board at McMaster University.
Male C57BL/6, 6–8 week old mice were purchased from
Charles River (Montreal, QC) and housed in the Central Animal
PLGA-INDUCED FIBROSIS

Facility at McMaster University for one week to acclimatize to
the facility.
Mice were anesthetized with isoflurane and given a subcu-
taneous injection of buprenorphine (0.03 mg/mL) before
undergoing surgery. PLGA discs were implanted into the peri-
toneal cavity and placed in the right lower abdominal quad-
rant and away from the omentum. The muscle wall was closed
using degradable 4-0 Vicryl suture (Ethicon) before the mice
were taken off isoflurane and placed in a recovery cage. Mice
were housed individually for 2 or 14 days before they were
sacrificed. No additional analgesics or anti-inflammatory
drugs were provided to the mice for the duration of the study.
Animal sample collection and isolation
A 2 mL peritoneal lavage was done using 0.9% saline prior to
sacrificing the mice. Lavages were collected in DNase/RNase
free microcentrifuge tubes and immediately placed on ice
before they were centrifuged at 1000g for 5 min at 48C. The
supernatant was collected for IL-10, IL-12, and IL-23 ELISA
assay and the pellet was lysed using 1 mL of TRIzol.
Fibrotic capsules formed around all implants by day 14.
Adherent cell RNA was extracted by placing explanted PLGA
discs (with the capsule still attached) directly in 1 mL of
TRIzol (Thermo Fisher Scientific, Burlington). The solution
was immediately vortexed at high speeds for 30 s and left
to incubate at room temperature for 1 h before total RNA
was extracted by following the manufacturer’s protocol.
Capsaicin release from PLGA implants
To quantify capsaicin release profile, individual capsaicin-
embedded PLGA discs were incubated at 378C with 20 mL of
PBS (Mg11, Ca11, pH 7.4) for two weeks. One milliliter sam-
ples were collected and replaced with fresh PBS at various
time intervals. Samples were filtered using 0.22 mm PVDF
hydrophobic syringe filters (EMD Millipore) and stored at 48C
before being measured at 237 nm using HPLC. Degassed and
0.2 mm filtered ddH2O and HPLC grade methanol (Sigma-
Aldrich, Oakville) were used as the mobile phases for HPLC.
Samples were fed through a Luna C18 column (Acclaim) using
a gradient of 70%–100% methanol over a period of 13 min.
Capsaicin concentration was quantified using a standard
curve generated from a serial dilution of capsaicin in HPLC
grade methanol (0.1–7 mg/mL) with R2 5 0.99.
RNA isolation and quantitative PCR (qPCR)
RNA was isolated from cells using TRIzol (Thermo Fisher
Scientific, Burlington) and by following the manufacturer’s
protocol. Nanodrop was used to ensure RNA 260/280 nm
ratio was above 1.8 (the average was 1.82 6 0.03). Total
RNA was diluted to 50 ng/mL and converted to cDNA using
the High-Capacity cDNA Reverse Transcription Kit with
RNase inhibitor (Thermo Fisher Scientific, Burlington), as
outlined in the manufacturer’s protocol. cDNA samples were
quantified using SYBR Green (Thermo Fisher Scientific, Bur-
lington), 500 ng of cDNA and 200 nM of forward and
reverse primers (Supporting Information, Table S1). Primer
efficiency was tested for glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH), iNOS and arginase-1 using total RNA
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2018 VOL 00A, ISSUE 00
ORIGINAL ARTICLE
from untreated RAW 264.7 cells and a range of primer con-
centrations to determine the optimum primer concentration.
Genes of interest were measured using the one-step pro-
cess on the Mx3000 qPCR system and semi-quantified using
the 2-DCt method described by Livak and Schmittgen.19 Glyc-
eraldehyde 3-phosphate dehydrogenase (GAPDH) was used
as a reference gene, as it is commonly used to quantify fold
changes in murine cells.20 The GAPDH levels in test samples
were within 6 5% between control and treatment samples
both in vivo and in vitro.
ELISA
IL-10, IL-12, and IL-23 cytokine concentrations in the super-
natant of peritoneal lavages were quantified using ELISA
kits (catalog numbers 88–7105-22, 88–7230-22 and 88–
7121-22, respectively from eBioscience) and by following
the manufacturer’s protocol.
Histology
Explanted PLGA discs were immediately fixed in 10% for-
malin for 48 h. Samples were sent to the McMaster Immu-
nology Research Center histology lab for processing where
the samples were dehydrated through ascending grades of
alcohol and xylene and impregnated with paraffin wax.
Paraffin-embedded sections were stained with picrosirius
red (PSR) (Sigma-Aldrich, Oakville) and hematoxylin and
eosin (H&E) (Sigma-Aldrich, Oakville).
Imaging
Bright field micrographs were imaged using a Zeiss Axiovert
200M inverted microscope and Axiovision software (ver.
4.8.2.0). Polarized micrographs were imaged using an Olym-
pus BX41 inverted microscope with an analyzer and polar-
izer set at 100% degree of polarization.
Collagen thickness quantification
Collagen thickness was quantified in a single blinded study. Col-
lagen thickness was measured using a lab developed ImageJ
macro21 where measurements were made perpendicularly
from the edge of the disc to the outer edge of collagen. Meas-
urements were taken at five pixel intervals along the perimeter
of the disc after which the measurements were averaged.
Statistics
The data were assumed to be normally distributed and pre-
sented as mean 6 standard error of the mean (SEM). R was
used for statistical analysis between treatments. Multiple com-
parisons between treatments and genes of interest were com-
pared using a two-way ANOVA with Welch-like corrections for
non-homogeneity and a post hoc Tukey’s HSD if significance
was detected. A value of p < 0.05 was considered statistically
significant and only select comparisons are shown in figures.
RESULTS AND DISCUSSION
Capsaicin upregulates arginase-1 in PLGA-induced
macrophages
First, we wanted to confirm that RAW 264.7 cells grown on
PLGA films induced an M1 phenotype, and then whether
3

FIGURE 1. The effect of PLGA and capsaicin on iNOS and arginase
gene expression in RAW 264.7 cells. Cells were either grown for 24 h
on TCPS (-/-), on PLGA films (1/-), on TCPS and treated with capsaicin
in the medium (-/suspension) or on PLGA films in which capsaicin
was embedded (1/embedded). Capsaicin downregulated iNOS mRNA
and upregulated arginase-1 mRNA. Embedded capsaicin was more
effective at promoting the M2 phenotype than when in suspension.
Values were normalized to GAPDH and expressed as mean 6 SEM for
five independent experiments.
capsaicin, either in suspension or blended with the PLGA
film, could alter the phenotype to M2. Cells seeded on PLGA
had significantly lower (p < 0.05) arginase expression than
from cells seeded on tissue culture polystyrene (TCPS), sug-
gesting that PLGA alone induced an M1 bias (Fig. 1). There
was no significant effect due to introducing capsaicin in the
medium (Fig. 1). Capsaicin was not toxic to the cells at the
concentrations studied (Supporting Information, Fig. S1).
Embedded capsaicin downregulated M1 marker iNOS by
almost 90% and upregulated M2 marker arginase-1 levels
by 70%. These results showed that capsaicin altered macro-
phage phenotype change from M1 and M2, and embedded
capsaicin was surprisingly more effective than capsaicin in
suspension. The effectiveness of embedded capsaicin is
likely due to its hydrophobicity and poor solubility in water.
Rather than being rapidly released into the medium, the
capsaicin was likely taken up by the cells from the surface
of the PLGA.
Increasing concentrations of capsaicin in the medium
suppressed iNOS and upregulated arginase-1 in IFNg and
LPS-stimulated macrophages (Supporting Information, Fig.
2). Kim et al. have also demonstrated the anti-inflammatory
effects of capsaicin, showing that LPS-stimulated macro-
phages had lower iNOS mRNA with increasing capsaicin
dosages.20 In our case, we also used IFNg, which enhances
activation of the NF-jB inflammation pathway22,23 to pro-
duce a polarized M1 macrophage phenotype that is more
difficult to alter than just LPS stimulation. Our results show
that capsaicin is highly effective at altering phenotype
regardless of the stimulating factors.
PLGA induces the M1 phenotype, with similar effects to
those reported after exposure to LPS and IFNg.24 We found
a positive relationship between PLGA exposure time and M1
phenotype polarization: iNOS was expressed two times
more than arginase-1 after 24 h and three times more than
arginase-1 after 72 h (Supporting Information, Fig. S3), and
4 TRUONG AND JONES
this was not associated with changes in viability (Support-
ing Information, Fig. S1). Previous studies have also shown
that capsaicin has no cytotoxic effects at dosages up to 200
lM.14,20,25
Capsaicin in suspension upregulates IL-10 production
Capsaicin added in suspension to cells on PLGA films showed
a significant (two-fold, p < 0.05) increase in the M2 marker,
IL-10, compared to PLGA alone (Fig. 2). This is in contrast to
gene expression data that showed no significant effect.
From genetic and cytokine analyses, we demonstrated
that capsaicin had anti-inflammatory effects on PLGA-
stimulated RAW 264.7 cells and upregulated M2 markers.
Macrophages that interacted with capsaicin on the film and
in the medium expressed high levels of the M2 markers
arginase-1 and IL-10 and low M1 markers iNOS and IL-12.
We used a unicellular model that might not be predictive of
in vivo results, but these findings show that capsaicin is a
promising anti-inflammatory compound, when blended with
a polymer carrier, and we were interested in investigating
its effects in an animal model.
Capsaicin reduces collagen from PLGA-induced fibrosis
PLGA discs or capsaicin-embedded PLGA discs were
implanted intraperitoneally in C57BL/6 mice for 2 and 14
days to study the fibrotic response.26 The discs were
explanted and stained with PSR and H&E for collagen and
cellularity analysis.
Using polarized light microscopy, PSR stained sections
showed no birefringent areas around any discs on day 2 (Fig.
3a). This was expected as foreign body giant cell formation
and fibrous encapsulation does not initiate until 5–7 days
post-implantation.5 By day 14, there were large birefringent
regions around both types of implants where PLGA discs had
a thicker layer of collagen than capsaicin-embedded PLGA
implants. Upon quantification, capsaicin-embedded PLGA
implants resulted in a significantly thinner collagen capsule
FIGURE 2. The effect of PLGA and capsaicin on IL-12 and IL-10 cytokine
production in RAW 264.7 cells. Cells were either grown for 24 h on TCPS
(-/-), on PLGA films (1/-) or on TCPS and treated with capsaicin in the
medium (-/suspension). The medium was collected and IL-12 and IL-10
concentrations were quantified. Capsaicin caused significantly more
IL-10 secretion than cells cultured on PLGA films (p < 0.05). Each value
represents mean 6 SEM for three independent experiments.
PLGA-INDUCED FIBROSIS
 ORIGINAL ARTICLE

FIGURE 3. (a) Representative polarized micrographs of PSR stained PLGA and capsaicin-embedded PLGA discs explanted from mice after 2 and
14 days of incubation. No collagen was present until day 14 where PLGA implants had more birefringent regions than capsaicin-embedded
implants. (b) Birefringent regions were quantified and revealed no detectable collagen on day 2 but significantly more collagen on PLGA
implants than capsaicin-embedded implants (p < 0.05). Each value represents mean 6 SEM for three independent experiments.

(p < 0.05) than PLGA only discs (Fig. 3b). Capsaicin reduced
collagen thickness by 40%, demonstrating that capsaicin has
in vivo anti-fibrotic effects on PLGA-induced fibrosis.
H&E micrographs showed cell recruitment and adhesion
2 days after implantation, with more cells on day 14 (Sup-
porting Information, Fig. S4). PLGA implants on day 14 had
a thicker layer of cells than capsaicin-embedded PLGA
implants which suggests that capsaicin reduced cell recruit-
ment and thus decreased collagen deposition.
Cell recruitment and stimulation might have been
reduced through inhibiting nociceptor innervation and NO
release. In a rat wound model, capsaicin-treated rats showed
less pain sensation and poorer wound healing.27 Capsaicin-
treated rats had 30%–40% reduced wound healing and
increased presence of a-smooth muscle actin (a-SMA).27
This was also seen in a rabbit wound model where wounds
with surgically excised nerves exhibited poor wound healing
and decreased macrophage presence than controls.28 It is
possible that capsaicin’s analgesic effects are inhibiting cel-
lular activity resulting in poor wound healing but it is uncer-
tain what cell type capsaicin is affecting. In the context of
implanted biomaterials, we might expect a reduced foreign
body response.
Capsaicin upregulates M2 cytokines but not M2 genetic
markers from peritoneal cells
To understand how cells were responding to the implants,
we looked at macrophage markers iNOS and arginase-1
mRNA of cells in the peritoneal cavity where there is a high
population of macrophages.20,29 This is, however, a mixed
population that includes lymphocytes and neutrophils.
There were no significant treatment differences in the
M1 marker, iNOS. PLGA in this context appeared to induce
early increases in arginase (M2) whether or not capsaicin
was delivered (Fig. 4).
Supernatant from lavages were collected and M1
cytokine markers IL-12and IL-23 and M2 cytokine
marker IL-10 were quantified using ELISA. IL-12 was
not detectable in any of the lavages but there were
measureable levels of IL-23 and IL-10 (Fig. 5). The sur-
gery itself induced an early M1 response (IL-23) which
transitioned to an M2 (IL-10) response by day 14.
Implanting PLGA moderated the M1/M2 bias compared
to a sham surgery, inducing similar levels of IL-23 and
IL-10. Capsaicin further increased the release of IL-10 at
both times, and showed no detectable arginase release
at day 2 (compared to PLGA alone), suggesting an early
an sustained M2 bias.
Capsaicin might be decreasing the fibrotic response by
upregulating early IL-10 levels to suppress TNF-a mediated
inflammation and fibrosis. Previous studies have demon-
strated that IL-10 reduces TNF-a activity in LPS and IFNg-
activated macrophages.30,31 In clinical studies, healthy
patients express higher levels of IL-10 in respiratory epithe-
lial lining fluid than patients with cystic fibrosis31 and there
is a strong positive correlation between TNF-a and IL-23.
Slavov et al. discovered that healthy patients have lower lev-
els of both TNF-a and IL-23 when compared with patients
who have silicosis.32 This coincides with our results where
we saw significantly less IL-23 than IL-10 on day 14 when
capsaicin was used (p < 0.05), which was linked with thin-
ner fibrous capsule formation.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2018 VOL 00A, ISSUE 00 5

FIGURE 4. (a) iNOS and (b) arginase-1 gene expressions from peritoneal cells. Cells were collected from mice with PLGA and capsaicin-
embedded PLGA discs incubated for 2 and 14 days. There was no significant change in iNOS mRNA levels on day 14, however cells exposed to
implants expressed less arginase-1 on day 14 (p < 0.05). Each value was normalized to GAPDH and expressed as mean 6 SEM for three inde-
pendent experiments.

Capsaicin does not affect cells adhered to the implant
We were interested to see what effect the capsaicin had on
cells in the tissue capsule that consistently formed around
the implant. We looked at gene expression of M1 (iNOS)
and M2 (arginase) markers, and also at markers linked with
myofibroblast differentiation (a-SMA and collagen-1) or lack
thereof (E-cadherin).
Capsaicin had no significant effects on iNOS, arginase-1,
a-SMA, E-cadherin and collagen-1 on day 2 and 14 (Fig. 6).
All genes of interest, except for arginase-1, showed a
decreased expression on day 14. Interestingly, capsaicin
caused a nearly significant increase (p 5 0.06) in arginase-1
(M2) expression from day 2 to day 14.
IL-10 secretion regulates macrophage and myofibroblast
activity to reduce fibrosis. As previously mentioned, we saw
high concentrations of IL-10 the peritoneal lavage. Boehler
et al. have shown that IL-10 is capable of suppressing LPS and
IFNg stimulated macrophages by suppressing TNF-a expres-
sion33 through activation of STAT3, which is thought to be the
anti-inflammatory transcriptional regulator.34,35 Hung et al.
also showed that IL-10 is able to reduce liver thioacetamide-
induced liver fibrogenesis where mice treated with IL-10
human plasmids had significant reduction in TNF-a, TGF-b1,
and collagen-1 while attenuated a-SMA activation.36
Capsaicin might have both a multicellular effect on mac-
rophages, fibroblasts and myofibroblasts to promote expres-
sion of anti-fibrotic mediators such as prostaglandin E-2
(PGE-2). As previously mentioned, capsaicin’s analgesic
effects inhibit pain mediators that are essential for proper
macrophage function and myofibroblast control. Widgerow
and Kalaria reviewed that sensory nerve innervation is
needed to stimulate macrophage activity and promote
proper wound healing.37 Many pain mediators (e.g., sero-
tonin, PGE2, bradykinin, etc.) have important effects on
wound healing.37 Nociceptor innervation stimulation causes
macrophages to produce nitric oxide, which can lead to an
abundance of inflammatory mediators to be released, such
as prostaglandin E-2 (PGE2), to suppress a-SMA.38 Our
results showed that both adhered and peritoneal cells
expressed iNOS, which indicate NO was being produced.

FIGURE 5. IL-23 and IL-10 concentrations in peritoneal lavages from mice implanted with PLGA and capsaicin-embedded PLGA discs. (a) Capsaicin
caused a significant increase in IL-10 on day 2 and on (b) day 14 (p < 0.05). Undetectable levels of IL-10 and IL-23 in sham mice and mice
with capsaicin-embedded PLGA disc, respectively, on day 2 and no IL-12 was detected in any samples. Each value represents mean 6 SEM for three
independent experiments.

6 TRUONG AND JONES PLGA-INDUCED FIBROSIS

 ORIGINAL ARTICLE

FIGURE 6. (a) iNOS, (b) arginase-1, (c) a-SMA, (d) E-cadherin and (e) collagen-1 gene expression from adherent cells on PLGA and capsaicin-embedded
PLGA discs. Capsaicin upregulated arginase-1 while downregulating iNOS. Values were normalized to GAPDH and expressed as mean 6 SEM for three
independent experiments.

However, additional experiments are required to eluci-
date how capsaicin affected each cell type and which genes
played an important role in mediating fibrosis.
Capsaicin release is predicted to continue long-term
To predict if capsaicin was present on day 14, in vitro
release kinetics of capsaicin-embedded PLGA implants
were determined by incubating the discs in PBS and
measuring capsaicin concentration in the medium using
HPLC.
Capsaicin followed first order release kinetics from the
implant and only 20% of the loaded drug was release by day
14 (Fig. 7a). On day 14, we extracted all of the remaining capsa-
icin from the implant and collected approximately 63 lg (Fig.
7b). This slow release has been previously demonstrated in a
similar system where a hydrophobic drug was released from

FIGURE 7. Cumulative release kinetics of capsaicin from capsaicin-embedded PLGA discs. Capsaicin was embedded in 0.01 g of PLGA and incu-
bated in PBS for 14 days. (a) Capsaicin followed first order release kinetics during the 14 days of incubation. (b) By day 14, there was 63 lg of
capsaicin remaining in the disc. Each value represents mean 6 SEM for three independent experiments.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2018 VOL 00A, ISSUE 00 7

poly(lactic acid) modified nanoparticles.39 The slow release
kinetics is attributed to the capsaicin’s extremely low solubility
and PLGA’s degradation being driven by solubility-controlled
bulk erosion process.40,41
This prolonged release makes it likely that capsaicin will
be present until the PLGA is fully degraded which can take
at minimum six months.41 It is possible that increased
mechanical stimulation or altered pH in vivo might hasten
release. If capsaicin-embedded PLGA discs were implanted
beyond 14 days, we expect capsaicin to not cause chronic
inflammation in mice because there was no upregulation in
white blood cells (WBC) in whole blood on day 14 (Sup-
porting Information, Fig. S5).42 This shows that capsaicin is
a promising compound for long term use beyond 14 days.
CONCLUSION
We demonstrated that capsaicin promoted the M2 pheno-
type from PLGA-induced macrophages in vitro and PLGA-
induced peritoneal cells in vivo. Capsaicin reduced collagen
thickness by 40% around PLGA discs implanted in mice
after 14 days of incubation in the peritoneal cavity. The
decreased fibrotic response is evidently driven by the
increased IL-10 production while peritoneal cells and adher-
ent cells showed no genetic responses to capsaicin among
the genes we tested. Capsaicin was released from PLGA at a
slow rate and approximately 80% of capsaicin remained in
PLGA by day 14. This demonstrates that capsaicin is a
promising drug for long term anti-inflammatory and anti-
fibrotic applications to extend the lifetime of the biomate-
rial. Further work is needed to determine the cell types in
the peritoneum and on the implants to determine the cells
and pathways that are responsible for mediating fibrosis.
ACKNOWLEDGMENTS
The authors would like to acknowledge that this work was
supported by the Natural Sciences and Engineering Research
Council. We thank Emma Buller, Ugonwa Echendu and
Anthony Saraco for their assistance with animal studies and
blinded collagen thickness and blood smear quantification
studies.
DISCLOSURE STATEMENT
The authors declare that there were no conflicts of interests
regarding the publication of this article.
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