Received: 3 October 2018 Revised: 6 May 2019 Accepted: 9 May 2019

DOI: 10.1002/jbm.a.36721

ORIGINAL ARTICLE

Mechanical properties of the pectin hydrogels

and inflammation response to their subcutaneous implantation

Pavel A. Markov1 | Darya S. Khramova1 | Konstantin V. Shumikhin1 |

Ida R. Nikitina1 | Vladislav S. Beloserov2 | Ekaterina A. Martinson2 |
Sergey G. Litvinets2 | Sergey V. Popov1

1
Institute of Physiology, Komi Science Centre,
The Urals Branch of the Russian Academy of
Sciences, Syktyvkar, Russia
2
Department of Biotechnology, Vyatka State
University, Kirov, Russia
Correspondence
Pavel A. Markov, Institute of Physiology,
Komi Science Centre, The Urals Branch of the
Russian Academy of Sciences, Syktyvkar,
Russia.
Email: p.a.markov@mail.ru
Abstract
We studied the influence of the mechanical properties of pectin hydrogels on acute
inflammation and tissue repair after subcutaneous implantation. We used hard and
soft pectin hydrogels. The results of histology and the analysis of serum-level cyto-
kines demonstrated that the intensity of acute inflammation increased with increas-
ing hardness of the pectin hydrogels. We also showed that the pectin hydrogels did
not inhibit tissue repair. The results of the morphometric and texture analysis of the
pectin hydrogels showed that the in vivo biodegradation kinetics of hard hydrogels
were greater than those of soft pectin hydrogels. We also observed that on the sur-
face of the hard and soft pectin hydrogels, a network of collagen fibers was formed.
The surface of the pectin hydrogel was shown to prevent the adhesion of infiltrating
inflammatory cells. The results of the in vitro experiments demonstrated that pectin
hydrogels inhibited the functional activity of macrophages and minimally activated
the complement system. Therefore, we showed that soft pectin hydrogels have low
proinflammatory potential and can be used in surgery as a barrier material as preven-
tion of adhesions in the abdominal cavity. The hard pectin hydrogel can be used in
tissue engineering. The hard pectin hydrogels can be used in the reconstruction of
skin because are overpopulated with collagen fibers and contribute to the formation
of new connective tissue, their elasticity is comparable to the skin and can be
adjusted. They are biodegradable, and no additional manipulation is required to
remove them.

KEYWORDS
complement, cytokines, foreign body reaction, histology, hydrogel, implantation, inflammation,
macrophages, pectin, reactive oxygen species, texture

1 | I N T RO D UC T I O N The FBR composed of macrophages and foreign body giant cells is

The development of novel biomaterials, biomedical devices, or tissue-
engineered constructs necessitates a thorough understanding of the
biological responses to implanted materials. The implantation of syn-
thetic and natural biomaterials elicits a foreign body reaction (FBR).
the end-stage response of the inflammatory and wound healing
responses following the implantation of a medical device or biomate-
rial (Anderson, Rodriguez, & Chang, 2008).
The results of many studies have shown that the texture, structure
and surface chemistry of biomaterials can impact macrophage behaviors,

2088 © 2019 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/jbma J Biomed Mater Res. 2019;107A:2088–2098.
MARKOV ET AL.
such as adhesion, apoptosis, fusion, polarization, and cytokine secretion
(Barbosa, Amaral, Aguas, & Barbosa, 2010; Blakney, Swartzlander, &
Bryant, 2012; Vasconcelos et al., 2013). Our previous studies have
shown that some pectins are capable of inhibiting the functional activity
of macrophages and neutrophils and have an anti-inflammatory effect
(Markov, Popov, Nikitina, Ovodova, & Ovodov, 2011; Popov, Ovodova,
Popova, Nikitina, & Ovodov, 2005; Popov, Ovodova, Popova, Nikitina, &
Ovodov, 2007). Pectin is an anionic polysaccharide that constitutes the
cell walls of most plants, and it has been extensively employed in the
pharmaceutical and food industries due to its gelling properties. We
have shown that the mechanical properties of pectin hydrogels can
influence macrophage adhesion, the adsorption of blood plasma pro-
teins, and the kinetics of biodegradation in vitro (Markov et al., 2017).
However, the influence of the mechanical properties of pectin hydro-
gels on acute inflammation and tissue repair after subcutaneous implan-
tation of pectin hydrogels is not known.
The aim of this study was to evaluate the influence of the
mechanical properties of pectin hydrogels on the functional activity of
macrophages and the complement system in vitro and the inflamma-
tory responses of rats after the subcutaneous implantation of the
hydrogels.
2 | MATERIALS AND METHODS
2.1 | Materials
Low methyl-esterified commercial apple pectin AU701 (Herbstreith &
Fox, Germany; 43% degree of methylation), Dulbecco's modified eagle
medium (DMEM, Biolot, Russia), HBSS (Hank's balanced salt solution),
fetal bovine serum (HyClone), 4,6-diamidine-2-phenylindole dihydro-
chloride (DAPI, Sigma), rhodamine (Sigma), bovine serum albumin (BSA),
sodium dodecyl sulfate (SDS) zymosan A, ethylenediaminetetraacetic
acid (EDTA), luminol (BioRad), human C3a ELISA kit (Hycult biotech)
and recombinant rat cytokine, antigen affinity purified polyclonal
antibody rabbit, anti-rat antigen affinity purified polyclonal antibody
biotinylated rabbit anti-rat for assay cytokine IL-1β, IL-10, TNF-α (all
manufactured PeproTech) were used in the study.
2.2 | Hydrogel preparation and compression
measurements
The pectic hydrogels were made from 1 and 4% water pectin solution
as previously described (Markov et al., 2017). Hydrogel samples
(2-mm height, 8-mm diameter) were fixed on the platform of the tex-
ture analyzer (TA-XT plus, Stable Micro Systems, UK). The mechanical
characterization of the hydrogels was performed using a cylindrical
aluminum probe P/5 (5-mm diameter). The method settings, including
the pretest, test, and posttest speeds, were 2.0, 1.0, and 2.0 mm/s,
respectively. The distance (depth of insertion) was set as 10 and 80%
of the initial height of the samples. Several mechanical parameters
were extrapolated from the force-time curves: hardness, Young's
modulus, and the compressive strength of the hydrogels. Young's
modulus was calculated using the following equation:
2089
E = ðF=AÞ=ðΔH=HÞ
where F/A is the applied force (N) per surface unit and dH/H is the
uniaxial deformation.
Compressive strength was calculated as follows:
Qt = F=A
where F is the force (N) measured during compression and A is the
cross-sectional area of the probe P/5 (0.000019 m2). The mechanical
characterization of the tissue was performed using a cylindrical probe
P/2 (5-mm diameter, 0.000031 m2), at five points for each sample and
was performed for all rats in each group.
2.3 | In vitro test
2.3.1 | Reactive oxygen species quantification
Production of reactive oxygen species (ROS) by J774 macrophages
(Collection of the Institute of Cytology, Russian Academy of Sciences,
St. Petersburg, Russia) was determined using a chemiluminescence
method. Cells were plated in 96-well culture plates (100 μL, 1 × 106 cel-
ls/mL) in the presence or absence of hydrogel samples and incubated in
RPMI 1640 supplemented with 10% fetal bovine serum for 2 hr at
37 C with 5% CO2. Then 50 μL of luminol was added into the each well.
The phosphate buffer solution (PBS, 10 μL) and the opsonized zymosan
(OZ) solution (10 μL, 100 μg/mL) were used as a negative and positive
control, respectively. Chemiluminescence was monitored every 2 min
for a 30-min period using a multidetector Zenyth 3,100 (Anthos, Aus-
tria). OZ was obtained by incubation of washed commercial zymosan A
with 1:10 diluted human serum at 37 C for 30 min. After washing, the
opsonized product was resuspended in HBSS up to the final concentra-
tion of 0.8 mg/mL (Beukelman et al., 2008).
2.3.2 | Complement activation
A serum pool was obtained by drawing blood from five healthy volun-
teers. Serum was prepared by allowing the blood to clot at room tem-
perature for 2 hr following centrifugation at 2500 g for 15 min at 4 C.
Hydrogel sample was mixed with 100 μL of serum in the plate well
and, then, incubated for 2 hr at 37 C in a shaker incubator. EDTA was
then added to a final concentration of 20 mM. Analysis of the fluid-
phase C3a complement activation products was performed using
ELISA commercial kit. OZ was obtained by incubation of washed com-
mercial zymosan A with 1:10 diluted human serum at 37 C for 30 min.
After washing, the opsonized product was resuspended in HBSS up to
the final concentration of 0.8 mg/mL (Beukelman et al., 2008).
2.4 | In vivo test
2.4.1 | Hydrogels subcutaneous implantation
After a quarantine period of 7 days, the Wistar albino rats
(250–300 g) were randomly divided into four groups of seven animals
each, according to the following protocol: group I (Int.), intact rats
2090
FIGURE 1 Subcutaneous implantation of hydrogels in rats
(without skin longitudinal incisions); group II (Contr.), negative control
group was set for the time periods of implantation with empty
pockets; group III (AP 1%), rats with AP 1% hydrogel implantation;
and group IV (AP 4%), rats with AP 1% hydrogel implantation.
Animals were housed individually in standard cages with an ambi-
 
ent temperature of 24 C to 26 C, and a12/12 hr light/dark cycle. The
animals had free access to drinking water and standard food for ani-
mals. The diet was supplemented with a vitamin-mineral premix rec-
ommended by the American Institute of Nutrition (AIN-93M diet).
Rats were anesthetized by the administration of tiletamine/
zolazepam (Zoletil 100, 10 mg/kg, i.m.). An aseptic technique was used
throughout the experimental period. The pectic hydrogel discs were
prepared as described above. Under surgical sterile conditions, two full
thickness skin longitudinal incisions (about 2 cm) containing the subcu-
tis and the panniculus carnosus (skin and smooth muscle) were per-
formed in the dorsum of each animal. The hydrogel discs were inserted
into these pockets (two discs of the same materials per animal), and the
panniculus carnosus and the skin were carefully sutured (Figure 1).
The structure of the study and the animal experimental proce-
dures were approved by the Ethical Committee of Komi Science
Center of Russian Academy of Sciences on Animal Care and Use.
2.4.2 | Measurement of white blood cell numbers
and serum cytokine
After each predetermined implantation time period each animal was
anesthetized with diethyl ether. The rats were bled with cardiac punc-
ture using a heparinized syringe and the blood was separated into two
aliquots. In the first aliquot of blood the following parameters were
determined: the differential white cell percentage (neutrophils, lympho-
cytes, eosinophils and monocytes), using a hemocytometer and a light
microscope. The second aliquot of blood was centrifuged to collect
stored at −40 C. The levels of cytokines TNF-α, Il-1β and IL-10 in the
serum samples were determined using a sandwich ELISA according to
the manufacturer's protocol. Monoclonal affinity purified anti-rat
TNF-α, anti-rat IL-1β and anti-rat IL-10 antibodies were used as capture
antibodies, and biotin-conjugated anti-rat TNF-α, anti-rat IL-1β anti-
bodies were used as detection antibodies. Binding was detected using
horseradish peroxidase-labeled streptavidin. The color reaction was
MARKOV ET AL.
developed by the addition of H2O2 and orthophenylenediamine. The
reaction was stopped after 15 min by the addition of 0.1 mL of
2.5 mol/L hydrochloric acid, and the absorbance (OD) was read at
492 nm (Power Wave 200, BioTek Instruments, Winooski, VT).
2.4.3 | Collection of tissue and hydrogels samples
At each time point, hydrogels were retrieved from the implantation
sites of each animal together with the skin, liver, kidneys, lung, and
spleen. The tissue samples were treated with formaldehyde (10%) and
embedded in paraffin. Thereafter, the samples were cut into 4 μm
thick slices using cryomicrotome Leica RM 2145 RTS (Leica Bio-
systems, Germany). After the tissue sections were deparaffinized, they
were dehydrated and stained with hematoxylin–eosin and picrofuxin
by Van-Geison's method using a vacuum infiltration processor
(Donatello Open System, DiaPath, Italy). The stained samples were
examined by light microscopy (Altami, Russia).
The hydrogel samples were fixed with 2.5% glutaraldehyde for
15 min, washed with PBS and were stained with rhodamine and DAPI.
Then, the number of cells was visually counted on each hydrogel using
a light-fluorescent microscope (Altami, Russia) equipped with a digital
camera (Canon). The number of adherent cells was counted and was
expressed as cells/100 mm2.
2.4.4 | Hydrogel microstructure analysis
Dried gel samples were platinum coated under vacuum in an ion sput-
ter for 30 s to render them electrically conductive. Then, the surface
morphology was observed using scanning electron microscopy (SEM)
(JEOL, JSM6510LV) at 15 kV.
2.5 | Statistical analysis
The results were presented as the arithmetic mean standard deviation
(SD). The significance of the differences between mean values was
evaluated using the Student's t test. The value of p < .05 was consid-
ered statistically significant.
3 | RESULTS
3.1 | Production of ROS by macrophages and
activation of the complement system in vitro
Macrophage activity and activation of the complement system
(amount of C3a in the serum) have been used as the key of FBRs. In
the first part of this study, we investigated the immunogenicity of
pectin hydrogels in vitro by estimating the ROS generated by J774
macrophages and the activation of the complement system in the
serum of volunteers.
We analyzed the ROS products using a chemiluminescent method
and estimated the ROS amount based on the area under the curve.
ROS generation by nonstimulated J774 macrophages was 280 ± 20
RLU. OZ was added to the J774 macrophages and enhanced ROS
MARKOV ET AL.
F I G U R E 2 The production of reactive oxygen species (ROS) by
nonstimulated (PBS) and OZ-stimulated J774 macrophages after
incubation with 1% and 4% AP hydrogels. Data are presented as the
arithmetic mean ± SD of the area under the curve (AUC); n = 5.
(a) p < .05 compared with PBS; (b) p < .05 compared with OZ
production by 40–50%, indicating the functional activity of macro-
phages. After incubating the 1% and 4% AP hydrogels with J774 mac-
rophages, ROS generation was not enhanced in response to
stimulation with OZ (Figure 2).
In the control group, the serum concentration of the С3а compo-
nent of the complement system was 760 ± 86 ng/mL. After OZ was
added to the serum, the amount of the C3a fragment increased ten-
fold. After incubating the 1% and 4% AP hydrogels in serum, the
amount of the С3а formed was less than that in the control group
after the addition of PBS (Figure 3).
Therefore, the results of these experiments showed that the immu-
nogenicity of the hydrogels is insignificant and that they have good
prospects for being used as implants in tissue engineering applications.
3.2 | The effect of subcutaneously implanted hard
and soft hydrogels on inflammation and tissue repair
The aim of this part of the study was to determine changes in the
inflammatory response after subcutaneously implanting pectin hydro-
gels. We used the 4% AP hydrogel since we believed it had a profound
effect on inflammatory repair processes.
Tissue damage caused by surgical manipulation leads to inflamma-
tion in the incision area. At 24 hr, in control animals on the border
between the muscular layer and the hypodermis, inflammatory cell
infiltration was observed and included neutrophils, macrophages,
eosinophils, and plasmatic cells (Figure 4a). On day 3, inflammatory
cell infiltration increased (Figure 4b). On days 8–11, the amount of
foreign body giant cells (FBGCs) increased (Figure 4c), and new con-
nective tissue continued to proliferate (Figure 4d). Three weeks after
the surgery, the leukocyte infiltrate became focal (Figure 4e), and the
damage located in the incision area was repaired with a new connec-
tive tissue. Figure 4f shows a fragment of the incision restored by
new connective tissue containing collagen fibers, as indicated by the
picrofuchin staining.
2091
F I G U R E 3 C3a production in nonstimulated (PBS) and OZ-
stimulated (OZ) human serum and C3a formation after co-incubation
of 1% and 4% AP hydrogels with OZ-stimulated serum. Data are
presented as the arithmetic mean ± SD; n = 5. (a) p < .05 compared
with PBS; (b) p < .05 compared with OZ; (c) p < .05 compared with AP
1%; Sb—serum blood
The inflammatory response changed after implanting the 4% AP
hydrogel. After 24 hr, inflammatory cell infiltration was comparable to
that of the control group in its intensity and composition. After 3 days,
inflammatory cell infiltration increased, mainly due to the involvement
of neutrophils in the tissue. The cell infiltration intensity was consider-
ably greater than that in the control animals (Figure 5a). On day 8, post-
operative swelling and hemorrhaging resolved in most animals. All
hydrogels were divided into fragments, and the collagen fibers were
stained with picrofuchin (Figure 5b). The hydrogel fragments were sur-
rounded by FBGCs (Figure 5c). Macrophages, plasmatic cells, and fibro-
blasts were the predominant constituents of the leukocyte infiltrate.
Between days 8 and 11 of the experiment, there was no clear differ-
ence observed in the inflammation pattern. On day 17, the hydrogel
surface was completely covered with collagen fibers, forming a capsule
and isolating the implant from the surrounding tissues (Figure 5d).
Among the collagen fibers, proliferative inflammatory cells, including
macrophages, lymphocytes, histiocytes, and fibroblasts, were observed.
Three weeks after implantation, the proliferative inflammation phase
continued, and the infiltrating cells included lymphocytes, fibroblasts,
and FBGCs. As a result of biodegradation, the gel fragments were rep-
laced with empty cavities (Figure 5e). The newly formed connective tis-
sue proliferated and gradually filled the cavities where the hydrogel
fragments had degraded (Figure 5f).
Hydrogel surface imaging performed by scanning electron micros-
copy confirmed the histological data. On day 21 of the experiment,
the hydrogel surface was covered with collagen fibers, forming a
branched network (Figure 6).
The results of histological tests of the liver, kidneys, spleen, and
lungs showed that hydrogel implantation did not cause any pathologi-
cal changes in the tissue structure of these organs. There were some
insignificant changes in the liver. Three days after implantation, hepa-
tocytes with small-droplet vacuolar dystrophy were observed in the
2092 MARKOV ET AL.

F I G U R E 4 Micrographs of tissue sections of the negative control group on 1 (a), 3 (b), 8 (c, d), and 21 days (e, f) after surgical manipulations.
Arrows indicate the inflammatory infiltrate, hd—hypoderm, ml—muscular layer, gt—granulation tissue, FBGCs—Foreign body giant cells,
Cf—Collagen fibers (van Gieson stainings). The new connective tissue at the incision site (f)

F I G U R E 5 Micrographs of tissue sections obtained on 3 (a), 8 (b, c), 17 (d), and 21 (e, f) days after subcutaneous implantation of the 4% AP
hydrogels. Arrows indicate the inflammatory infiltrate; hd—hypodermis, ml—muscular layer, gt—granulation tissue, FBGCs—Foreign body giant
cells, Cf—collagen fibers (van Gieson's staining)

liver of the rats, but by day 8, the signs of degeneration disappeared 1. inflammatory cell infiltration in tissues at day 3 after
(data not shown). implantation;
Based on the obtained results, three periods were distinguished 2. inflammatory cell proliferation with an inflammatory response of
with the most typical phases of the inflammatory response as follows: giant cells on day 11; and
MARKOV ET AL. 2093

F I G U R E 6 Scanning electron
micrographs of the surface of the
4% AP hydrogel after 21 days
after subcutaneous implantation.
Cf—collagen fibers; (a) before
implantation, (b) (c, d) after
implantation; (a, b) ×1,000;
(c, d) ×2,500

F I G U R E 7 Micrographs of
tissue sections obtained on 3 (a,
b), 8 (c), and 21 (d) days after
subcutaneous implantation of the
1% AP hydrogels. Arrows indicate
the inflammatory infiltrate; ml—
muscular layer, FBGCs—Foreign
body giant cells, Cf—collagen
fibers (van Gieson's staining)

3. tissue repair stage involving gel implant enclosure in the connec-
tive tissue on day 21.
Further studies on the effects of the mechanical properties of pec-
tin hydrogel implants on acute inflammation and tissue repair were
conducted during the abovementioned periods.
The effects of the implantation of the 1% AP hydrogel on the
inflammatory response differed from the processes following the
implantation of the 4% AP hydrogel. The leukocyte infiltration intensity
in animals implanted with the 1% AP hydrogel was less than that in the
animals implanted with the 4% AP hydrogel (Figure 7a). On the third
day after implantation, the hydrogel was already divided by collagen
fibers into individual fragments (Figure 7b). Furthermore, by days 8–11
of the experiment, most of the fragments were degraded, and new cavi-
ties were formed. A proliferative giant cell reaction was observed
around the remaining fragments of the hydrogel (Figure 7c). Three
2094
weeks after implantation, the infiltrating cells included giant cells, fibro-
blasts, and lymphocytes. Van Gieson's staining showed proliferation of
new connective tissue that replaced the degraded implant (Figure 7d).
Therefore, within 21 days, the 1% AP hydrogel was completely
degraded and replaced with fresh connective tissue. Nevertheless,
the inflammatory infiltrate remained, which showed that the repair
process was not yet completed.
3.3 | Cell adhesion on pectin hydrogel implants
Cell adhesion was assessed by counting the number of cells on the
surface of the samples of implanted hydrogels at different time points
after implantation. After 3 days of implantation, the number of adher-
ent cells on the surface of the 1% and 4% AP hydrogel implants was
2
82 ± 18 and 70 ± 14 cells/100 mm , respectively (Figure 8a,b). After
8 days of implantation, the number of adherent cells increased to
2
110 ± 20 cells/100 mm for the 1% AP hydrogel and 125 ± 20
2
cells/100 mm for the 4% AP hydrogel (Figure 8c,d). At 21 days after
implantation, the 1% AP hydrogel was degraded (Figure 8e), and the
number of adherent cells on the AP hydrogel increased to 184 ± 40
2
cells/100 mm (Figure 8f).
As shown in Figure 8a-f, most of the cells did not adhere to the
hydrogel surface, but adhered mainly to the collagen fibers; therefore,
MARKOV ET AL.
F I G U R E 8 Fluorescent
micrographs of infiltrated
inflammatory cells adhered to the
surface of the 1% and 4% AP
hydrogels. Ic—inflammatory cell,
Cf—collagen fibers (rhodamine
and DAPI staining)
cell adhesion appeared to be determined by the extent of the collagen
fiber network formed on the surface of the hydrogels.
3.4 | In vivo biodegradation of the pectin hydrogel
implants
In this study, hydrogels with different mechanical properties were
used. The hardness of the 1% AP hydrogel was 10 times less than that
of the 4% AP hydrogel, and the 1% AP hydrogel was less elastic, that
is, its ability to resist elastic deformation was significantly less than
that of the 4% AP hydrogel. The tensile strength, which is the ability
to resist plastic deformation, of the 4% AP hydrogel was approxi-
mately 7 times greater than that of the 1% AP hydrogel (Table 1). We
evaluated how the mechanical properties of pectin hydrogels affect
the kinetic biodegradation after subcutaneous implantation in animals.
We observed that 1% AP hydrogel implants degraded two times
faster than 4% AP hydrogel implants. For example, the mechanical
properties of the 1% AP hydrogels, such as hardness, compressive
strength, and Young's modulus, decreased up to 12–15 times at 24 hr
postimplantation. Furthermore, the mechanical properties of the 4%
AP hydrogels decreased up to 6–8 times (Table 1). Hydrogel biodegra-
dation is accompanied by the fragmentation of hydrogels. At 3 days
postimplantation, the 1% AP hydrogel collapsed into fragments with
MARKOV ET AL. 2095

T A B L E 1 Mechanical properties and

Hardness Compressive Young modulus Area
size of pectin hydrogels before and after
Samples/day (mN) strength (kРа) (kPa) (mm2)
subcutaneous implantation
AP 1% 0d 58 ± 5 32 ± 2 14 ± 3 51 ± 1
1d 4 ± 1* 2.6 ± 0.8* 0.8 ± 0.1* 12 ± 3*
3d n. d. n. d. n. d. 3.4 ± 0.7*
11 d n. d. n. d. n. d. 0.8 ± 0.4*
21 d n. d. n. d. n. d. Destr.
AP 4% 0d 615 ± 16 215 ± 20 106 ± 21 50 ± 1
1d 114 ± 27* 25 ± 2* 13 ± 1.5* 47 ± 2
3d 8 ± 4* 1.4 ± 0.11* 0.7 ± 0.3* 38 ± 1*
11 d n. d. n. d. n. d. 6.1 ± 1.9*
21 d n. d. n. d. n. d. 1.3 ± 0.2*

Note: Data are presented as the arithmetic mean ± SD; n = 7.

*p < .05 compared with previous point.

an approximate are of 4 mm2; furthermore, the size of the 4% AP 3.5 | Whole blood leukocyte response after
hydrogel did not change (Table 1). The fragmentation of the 4% AP the implantation of pectin hydrogels
4% began at 3 days postimplantation. On day 21, the 1% AP hydrogel
Surgical operations cause successive changes in the stages of the
was completely degraded. The 4% AP hydrogel was observed in the
increase and decrease in the number of leukocytes in the blood
form of fragments with a size of approximately 1 mm2 (Table 1).
(Figure 9a). The results of the leukogram of control rats showed an
Unfortunately, the mechanical properties of the hydrogel implants
increase in neutrophils and eosinophils, which were elevated by
could not be analyzed after 24 hr and 11 days after implantation for
230 and 190%, respectively, compared to the baseline levels at 3 days
the 1% and 4% AP hydrogels, respectively, due to insufficient
after the operation (Figure 9b). The implantation of 1% and 4% AP
material size.

F I G U R E 9 White blood cells after subcutaneous implantation; (a) leukocytes in the blood, (b) cell percentage in the blood of control rats,
(c) cell percentage in the blood of rats with 1% AP hydrogel implants, (d) cell percentage in the blood of rats with 4% AP hydrogel implants.
N—neutrophils, L—lymphocytes, E—eosinophils, M—monocytes. Data are presented as the arithmetic mean ± SD; n = 5. *p < .05 compared with
the previous point
2096 MARKOV ET AL.

hydrogels resulted in the enhancement of the whole blood leukocyte

number and the prolongation of leucopoenia (Figure 9a). The leukocyte
number in rats administered 1% AP hydrogel implants peaked only after
11 days of implantation due to an increase in the number of neutrophils
and eosinophils (Figure 9c). The leukocyte number in rats implanted
with the 4% AP hydrogel did not increase after 3 days of implantation
compared to the baseline level, which appeared to be associated with a
nonsignificant increase in the monocyte and eosinophil number and a
reduction in the lymphocyte number (Figure 9d). The peak leukocyte
number was observed at 21 days after implantation in rats administered
4% AP hydrogel implants (Figure 9a). The differences in the leukocyte
response observed for the 4% and 1% AP hydrogel implants appeared
to be determined by the kinetic biodegradation of the hydrogels.

3.6 | Serum cytokine levels after the implantation of

pectin hydrogels
The surgical procedures resulted in the enhancement of proinflammatory
cytokine levels in the blood serum of control rats. TNF-α and IL-1β levels
in the blood increased after 3 days and peaked at 11 days after the oper-
ation, respectively, compared to the baseline levels. At 21 days after the
operation, the TNF-α concentration returned to the baseline values,
whereas the IL-1β level remained higher. In contrast, the Il-10 concentra-
tion in the blood decreased after the operation (Figure 10a). The implan-
tation of pectin hydrogels caused an increase in the proinflammatory
cytokine concentrations in the blood, especially for the 4% AP hydrogel
implants. TNF-α and IL-1β concentrations were significantly higher in rats
implanted with the 4% AP hydrogel than in rats implanted with the 1%
AP hydrogel (Figure 10b,c). In contrast to the 4% AP hydrogel, implanta-
tion of the 1% AP hydrogel only caused an enhancement in the IL-1β
blood level, which peaked at 3 days after implantation compared to the
control rats (Figure 10b).
Implantation of the 1% AP hydrogel did not affect the concentra-
tion of Il-10 (Figure 10b). The Il-10 concentration increased after
11 days and remained at a higher level after 21 days in the rats that
received the 4% AP hydrogel implants (Figure 10c). F I G U R E 1 0 TNF-α, IL-1β, and IL-10 cytokines in serum at 3, 11,
Therefore, at the initial stages, the production of proinflammatory and 21 days after subcutaneous hydrogel implantation; (a) control
rats, (b) cytokines in the serum of rats with 1% AP hydrogel implants,
cytokines was stimulated, and with the increasing hardness of the
(c) cytokines in the serum of rats with 4% AP hydrogel implants. Data
pectic hydrogel, the intensity of inflammation increased. are presented as the arithmetic mean ± SD; n = 5. *p < .05 compared
with the previous point

4 | DISCUSSION The complement system is a host recognition system that is

The data obtained in this study demonstrated that inhibition of the
functional activity of macrophages seems to be an advantage of pectin
hydrogel compared to other hydrogel materials. For example, hydro-
gels prepared from polydioxanone (Smith, White, Smith, & Bowlin,
2009), poly(ethylene glycol)diacrylate (Waldeck, Wang, Joyce, & Kao,
2012), and chitosan (Santos, Marques, Silva, et al., 2007) have not
been shown to generate ROS by stimulated macrophages and mono-
cytes. The decrease in ROS in the medium after co-incubation of
macrophages with hydrogels may be due to the absorption of ROS by
hydrogels or a decrease in the functional activity of macrophages.
activated by any foreign surface, including biomaterials, and therefore,
it is a useful tool to investigate biocompatibility at the molecular level
(Nilsson, Ekdahl, Mollnes, & Lambris, 2007). The results showed that
pectin hydrogels minimally activated the complement system. The
inhibition of C3a production by pectin hydrogels appeared to be com-
parable with that provided by an alginate hydrogel (Rokstad et al.,
2011). The decrease in the amount of the C3a fragment may be due
to the adhesion of components of the complement system on the
surface of the hydrogel. We have previously shown that when incu-
bating hydrogels with human serum, hydrogels adsorb 30–50% of
whey proteins on their surface (Markov et al., 2017).
MARKOV ET AL.
There are three main phases of tissue repair, including acute
inflammation, proliferation, and remodeling with complete closure of a
wound (Krzyszczyk, Schloss, Palmer, & Berthiaume, 2018; Sindrilaru &
Scharffetter-Kochanek, 2012). In general, we observed this pattern of
tissue repair in the control animals, that is, in animals that were sub-
jected to all surgical procedures except for hydrogel implantation.
Inflammation provoked by hydrogel implants is determined by the
type of biomaterial (Chen, Lianga, & Thouas, 2013), the chemical com-
position of the polymer (Barbosa et al., 2010; Vasconcelos et al.,
2013) or the component composition of the hydrogel (Nguyen,
Abueva, Ho, Lee, & Lee, 2018). We observed that pectic hydrogel
implantation stimulated an acute phase of inflammation. The acute
inflammatory response increased with increasing hardness, Young's
modulus, and compressive strength of the pectin hydrogels. It is possi-
ble that the acute inflammatory response was caused by a decreased
mechanical compatibility of the 4% AP hydrogel implants with the
host tissue and an increased inflammatory response. Additionally, it is
possible that the changes were due to a high concentration of pectin
in the gel, as a result of the prolonged release of the 4% AP hydrogel
implant degradation products into the surrounding tissues, which
could be either the pectin polysaccharides themselves or their com-
plexes with serum proteins, lipids, antibodies, and cytokines.
By comparing the results of the biodegradation kinetics of pectin
hydrogels in vitro (Markov et al., 2017) and in vivo, we concluded that
after subcutaneous implantation of the hydrogels, the intensity of their
biodegradation was much higher than that in PBS or DMEM. Pectin
hydrogel degradation was due to the displacement of calcium ions that
crosslink the hydrogel matrix with sodium, potassium, and phosphate
ions, along with the preferential dissolution of hydrophilic polymers. All
of these salts are present in blood plasma and in interstitial fluid. An
additional destructive factor that enhances the biodegradation of pectin
hydrogels could be ROS generated by neutrophils and macrophages.
We also observed that the tissue repair step began, similar to the
control animals. The tissue repair step was accompanied by a decrease
in the amount of TNF-α and IL-1β and an increase in IL-10 cytokines,
regardless of the mechanical properties of the implanted hydrogels.
The results of some studies have shown that the pectin-containing
hydrogel was potential for skin tissue engineering. Such composite
materials provide adhesion and proliferation of fibroblasts and
keratinocyte as well as de novo deposition of extracellular matrix com-
ponents on its surface (Lin, Chen, Chang, & Ni, 2013; Martins et al.,
2018; Naumenko, Guryanov, Yendluri, Lvov, & Fakhrullin, 2016; Pereira,
Barrias, Bartolo, & Grania, 2018). However, the action of the pectin-
containing biomaterials on skin regeneration was made in vitro
conditions.
In this study, it was shown that following subcutaneous implan-
tation, the pectin hydrogels were covered with collagen fibers for-
ming an insulating connective tissue capsule. It has been previously
shown that the subcutaneous implantation of hydrogels derived
from natural polysaccharides, such as chitosan (Linh, Abueva, & Lee,
2017; Moura, Brochado, Gil, & Figueiredo, 2017), dextrin (Silva et al.,
2016), carrageenan and agarose (Popa et al., 2014), do not induce
the formation of a fibrous capsule. Granulation tissue is formed by
2097
acidic mucopolysaccharides (hyaluronic and glucuronic acids, galac-
tosamine, glucosamine, etc.), which together with collagen fibers
form an intercellular matrix in which leukocytes, fibroblasts, histio-
cytes and mast cells are present (Franz, Rammelt, Scharnweber, &
Simon, 2011). Pectins, in contrast to chitosan, carrageenan, dextrin
and agarose, are polyanionic polysaccharides. It is possible that, as in
the case of glycosaminoglycans, the polyanionic charge of the
macromolecule of pectins facilitates the binding of collagen and the
formation of a connective tissue capsule around the hydrogel.
We have previously demonstrated that the surface of the pectic
hydrogel has qualities preventing the adhesion of J774 macrophages
(Markov et al., 2017). The results of this study also confirmed this
observation, and more than 90% of the adherent cells of the inflam-
matory infiltrate were not adhered to the surface of the hydrogels, but
adhered on the collagen fibers covering the surface of the implants.
5 | C O N CL U S I O N S
In this study, the effect of hard and soft pectin hydrogels after subcu-
taneous implantation on the inflammatory response and tissue repair
was investigated. The intensity of acute inflammation and the dura-
tion of biodegradation increased with increasing hardness, Young's
modulus, and compressive strength of the pectin hydrogels. The sur-
face of the pectin hydrogel prevented the adhesion of infiltrated
inflammatory cells. It was shown that after the subcutaneous implan-
tation of the pectin hydrogel, a network of collagen fibers formed, and
new connective tissue developed and grew on its surface.
The hard pectin hydrogel can be used in tissue engineering
because it has low cytotoxicity, their elasticity is comparable to the
skin and can be adjusted. The pectin hydrogel are biodegradable, and
no additional manipulation is required to remove them. These pectin
hydrogel are overpopulated with collagen fibers and contribute to the
formation of new connective tissue. The newly formed connective tis-
sue network can be used as a material for the extracellular matrix to
culture autotransplants under in vitro.
We have previously demonstrated that soft pectin hydrogels
inhibit postoperative adhesions in the abdominal cavity (Popov et al.,
2016). The results from this current study showed that soft pectin
hydrogels had low proinflammatory potential. In combination with a
nonprolonged period of biodegradation, this property of pectin hydro-
gels can be used in surgery as a barrier material.
OR CID
Pavel A. Markov https://orcid.org/0000-0002-4803-4803
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How to cite this article: Markov PA, Khramova DS,
Shumikhin KV, et al. Mechanical properties of the pectin
hydrogels and inflammation response to their subcutaneous
implantation. J Biomed Mater Res. 2019;107A:2088–2098.
https://doi.org/10.1002/jbm.a.36721