Mechanical Properties, Structure, Bioadhesion and Biocompatibility of Pectin

Mechanical properties, structure, bioadhesion and biocompatibility of pectin
hydrogels
Pavel A. Markov1, Nikita S. Krachkovsky1, Eugene A. Durnev2 , Ekaterina A. Martinson2,
Sergey G. Litvinets2 and 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
Corresponding author: Pavel A. Markov, p.a.markov@mail.ru
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/jbm.a.36116
This article is protected by copyright. All rights reserved.

Journal of Biomedical Materials Research: Part A Page 2 of 32
ABSTRACT
The surface structure, biocompatibility, textural and adhesive properties of calcium
hydrogels derived from 1, 2 and 4% solutions of apple pectin were examined in this study. An
increase in the pectin concentration in hydrogels was shown to improve their stability toward
elastic and plastic deformation. The elasticity of pectin hydrogels, measured as Young’s
modulus, ranged from 6 to 100 kPa. The mechanical properties of the pectin hydrogels were
shown to correspond to those of soft tissues. The characterization of surface roughness in terms
of the roughness profile (Ra) and the root-mean-square deviation of the roughness profile (Rq)
indicated an increased roughness profile for hydrogels depending on their pectin concentration.
The adhesion of AU2% and AU4% hydrogels to the serosa abdominal wall, liver and colon was
higher than that of the AU1% hydrogel. The adhesion of macrophages and the non-specific
adsorption of blood plasma proteins were found to increase as the pectin concentration in the
hydrogels increased. The rate of degradation of all hydrogels was higher in phosphate buffered
saline (PBS) than that in DMEM and a fibroblast cell monolayer. The pectin hydrogel was also
found to have a low cytotoxicity.
Keywords: biomaterials, pectin, hydrogels, texture, surface morphology, elemental
analysis, tissue and macrophage adhesion, protein adsorption, cytotoxicity.
INTRODUCTION
Currently, there is a need to develop biomaterials with novel properties for biomedical
applications, such as drug delivery, tissue engineering, and the creation of implantable devices.
Synthetic and natural polymers are used for the development of these materials. Many studies on
the functional properties of biomaterials were conducted using synthetic polymers to control the
polymer structure and to modify its functional properties. Furthermore, many reports have
demonstrated that the majority of synthetic polymers have a number of shortcomings, such as
high cytotoxicity and low biocompatibility.1-3 The main advantages of natural polymers are
biocompatibility, biodegradability, and low cost.
John Wiley & Sons, Inc. 2

Page 3 of 32 Journal of Biomedical Materials Research: Part A
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. A wide variety of hydrocolloid pectin-based wound dressings has been patented and
is commercially available. In addition, pectin materials provide improved systems of loading and
releasing drugs (i.e., antibiotics, pain relievers and/or tissue repair factors at the site of action).4
Potential applications of pectin hydrogels have been under intense study in the field of
biomaterials for wound healing, tissue engineering, dentistry, and skin-care products. Pectin
materials were found to have great potential for bone tissue engineering applications because
they promote the nucleation of a mineral phase if they are immersed in adequate physiological
solutions, forming biomimetic constructs that better mimic the natural architecture of the bone.5,
6
Pectin hydrogels may also be used as a barrier material for the prevention of post-operative
adhesions in surgery.7
The presence of a positive or negative charge on the pectin macromolecule determines
the characteristics of the intermolecular interaction of pectin with animal tissues and cells.8-12
However, when pectin hydrogels are formed, functional groups are partially or fully engaged,
thus impeding their ability to participate in further intermolecular interactions. Biofunctional
properties of synthetic hydrogels are assumed to be determined by their surface structural 13, 14, 15
and mechanical properties.16, 17

However, little is known about the influence of the surface
structural and mechanical properties of pectin hydrogels on biocompatibility and biofunctional
properties.
The aim of this study was to evaluate the effect of the structure of pectin hydrogels on
tissues, cells, plasma protein adhesion and biocompatibility in vitro.
MATERIALS AND METHODS
Materials
Low methyl-esterified commercial apple pectin AU701 (Herbstreith & Fox, Germany;
43% degree of methylation), alpha minimum essential medium (MEM, Biolot, Russia),

Dulbecco’s modifed eagle medium (DMEM, Biolot, Russia), HBSS (Hank’s balanced salt
solution), fetal bovine serum (HyClone), MTT (dimethylthiazol diphenyltetrazolium), 4′,6-
diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma), rhodamine (Sigma), bovine serum
albumin (BSA) and sodium dodecyl sulfate (SDS) were used in the study.
Hydrogel preparation and compression measurements
A volume of 1 ml of a calcium chloride (CaCl2) solution (0.1, 0.5, 1 and 2 M) was added
to the wells of a 12-well plate and frozen at -40°C. Then, 5 ml of a 1, 2 and 4% aqueous pectin
solution (+50°C) was poured into the wells. After the plates were incubated for 24 h, the calcium
solution was removed, and the resultant hydrogel was washed three times with distilled water.
Hydrogel samples (5-mm height, 20-mm diameter) were fixed on the platform of the
texture 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
samples were equilibrated to room temperature prior to analysis. The determination of the
mechanical properties of the hydrogels was performed at five points for each sample and was
performed for all five samples in each group. The method settings, including the pre-test, test
and post-test speeds, were 2.0, 1.0 and 2.0 mm/s, respectively. The distance (depth of insertion)
was set as 10%, 20%, 30% and 80% of the initial height of the samples. Several mechanical
parameters were extrapolated from the force-time curves: hardness, Young’s modulus, the work
of cohesion (cohesiveness), and the compressive strength of the hydrogels. Young’s modulus
was calculated using the following equation:
E= (F/A)/(∆H/H),
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 м2).

Surface morphology and elemental analysis
Dried gel samples were platinum coated under vacuum in an ion sputter for 30 s to render
them electrically conductive. Then, the surface morphology was observed using scanning
electron microscopy (SEM) (JEOL, JSM6510LV, USA) at 15 kV. The elemental analysis of the
cross-sections was performed using energy-dispersive X-ray spectroscopy (EDX).
Surface imaging was also performed using atomic force microscopy (AFM) (NTegra,
NT-MDT, Russia). An analysis of the surface roughness of the hydrogels was performed using
Nova PX image analysis software (Image Analysis-3.2, NT-MDT, Russia). The arithmetic mean
deviation of the roughness profile (Ra) and the root-mean-square deviation of the roughness
profile (Rq) were determined using horizontal profiles with steps of 2 µm (5 profiles for each
scan). The number of samples for each gel was 5. All of the experiments were performed in air
under ambient conditions.
Stability to degradation in vitro
Hydrogel samples (h=1 mm, d=20 mm) were placed in the wells of a 12-well plate and
incubated with 1 ml PBS or DMEM and a monolayer of fibroblast cells (NIH/3T3, concentration
8×104 - 105 cells/ml) in DMEM. The hydrogels were incubated at 37°C with 5% CO2. Hydrogels
were incubated in the indicated media for 14 days with daily replacement of the incubation
solution with fresh solution. The compressive strength of the hydrogels at 2, 4, 7 and 14 days
was evaluated using the above-described method.
Hydrogel adhesion to biological tissue
To evaluate the adhesive properties of the hydrogels, their adhesive force to the serosa of
the abdominal wall, colon and liver of white laboratory mice was measured. Adhesion strength
measurements were performed using a texture analyzer (TA-XT plus, Stable Micro Systems,
UK). A hydrogel sample measuring 5×5 mm was fixed using double-sided tape on a cylindrical
probe p/0.5R with a diameter of 12.7 mm. The adhesion was evaluated by the force of probe

separation from the tissue after 60 s of pressing with a load of 0.01 N. The force of adhesion was
calculated using Exponent Software (Version V6.1.5.0).
Plasma protein adsorption assay
The structure of the study and the human procedures were approved by the Ethical
Committee of the Komi Science Center of the Russian Academy of Sciences. Blood samples
were obtained from healthy volunteers, and plasma was prepared by centrifuging the blood
samples at 1500 rpm for 10 min and diluting 1×10 with PBS. Diluted plasma (200 µl) was added
to the hydrogel sample (h=1 mm, d=5 mm), and the mixture was incubated for 2 h at 37°C in a
shaker incubator. Then, the hydrogel samples were removed, and the remaining protein fraction
was determined by Lowry's method using a BSA standard.18 The percentage of proteins adsorbed
onto the hydrogels was quantified with respect to the total protein content.
Adhesion of macrophages to the hydrogel surface
J-774 murine macrophages (Collection of the Institute of Cytology, Russian Academy of
Sciences, St. Petersburg, Russia) were cultured in the DMEM that was supplemented with 10%
(v/v) fetal bovine serum, 50 µg/ml gentamicin and 50 µg/ml amphotericin (Sigma-Aldrich). The
cells were incubated at 37°C with 5% CO2.
The hydrogel samples (h=1 mm, d=20 mm) sterilized with UV radiation (1 h) were
placed in a 12-well plate containing 1 ml DMEM supplemented with 10% fetal bovine serum, 50
µg/ml gentamicin and 50 µg/ml amphotericin and were incubated for 1 h at 37°C with 5% CO2.
After the incubation was complete, the medium was replaced with 1 ml of a macrophages
suspension of line J774 (1×106 cell/ml) and incubated at 37°C with 5% CO2 for 24 h. Then, non-
adherent cells were removed by washing the wells with 3 ml of PBS three times. The hydrogel
samples were removed; the cells were fixed on the hydrogels with 2.5% glutaraldehyde for
15 min, washed with PBS three times and were stained with rhodamine and DAPI. Then, the
number of cells was visually counted on each hydrogel or plastic using a fluorescent microscope

(Altami, Russia) equipped with a digital camera (Canon). The number of adherent cells was
counted and was expressed as cells/100 mm2.
Cytotoxicity to fibroblasts
NIH3T3 murine fibroblasts (Collection of the Institute of Cytology, Russian Academy of
Sciences, St. Petersburg, Russia) were cultured in the MEM that was supplemented with 10%
(v/v) fetal bovine serum, 50 µg/ml gentamicin and 50 µg/ml amphotericin. Cells were plated in
96-well culture plates (100 µl, 5×104 cells/ml) and incubated for 12 h at 37°C with 5% CO2 to
allow attachment. After the incubation was complete, the medium was replaced with 100 µl of a
new medium, and the hydrogel samples (h=1 mm, d=5 mm) were added to the wells. The alpha
MEM solution (100 µl) was added to the control wells. An evaluation of the metabolic activity of
the cells was performed at 24, 48 and 96 h.
Cytotoxicity was assessed using the MTT assay; 10 µl of a solution of 5 mg/ml MTT in
PBS was added to each well and incubated at 37°C in air containing 5% CO2 for 4 h in the dark.
MTT was aspirated, 100 µl of solubilization solution (10% SDS/0.01 M HCl) was added, and the
plates were incubated overnight.19 The absorbance was read at 570 nm using a Power Wave-200
(BioTek Instruments, USA) spectrophotometer.
Hemolysis of red blood cells
Human blood samples from healthy donors were collected into vacutainer tubes
containing an anticoagulant. The samples were held for 2 h at room temperature, followed by
centrifugation at 350g for 15 min at 4°C. Subsequently, the red blood cells were removed,
washed 4 times in a 0.9% NaCl solution and diluted 10 fold. The hydrogels were placed in a 96-
well plate and 200 µl of red blood cells was added to each well and incubated for 1 h at 37°C
without stirring. The plate was then centrifuged (100g, 5 min), and 100 µl of the supernatant was
collected from the wells. The optical density (OD) was measured at a 540 nm wavelength using a
microplate reader (Power wave 200, BioTek instruments, USA). The positive control consisted
of blood with distilled water (20 µl), while blood with a 0.9% NaCl solution (20 µl) served as the

negative control. The level of hemolysis was calculated in accordance with the following
equation 20:
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 < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
Textural properties of the pectin hydrogel
The system hardness (i.e., the maximum positive force required to attain a given
deformation) and Young’s modulus were obtained from the initial slope of the stress–strain
curve at 10% strain. The compressive strength and cohesiveness (i.e., the proportion of the
positive area under the force-time curve from zero to maximum deformation imposed by the
hydrogel) were detected at the hydrogel deformation up to 80%. All these parameters are
reported in Table 1. The hydrogel obtained from a 1% pectin solution had a soft texture and was
incompatible with the strong mechanical loads. The increase in the pectin concentration in the
hydrogel was shown to improve its stability toward elastic and plastic deformation. The elasticity
of pectin hydrogel was measured as Young’s modulus and was found to range from 6 to 100 kPa
(Table 1), which is comparable to the mechanical properties of some soft tissues.
Pectin hydrogels have been used for the reconstruction of bone tissue, as delivery systems
for osteoblasts, osteoclasts, and stem cells and for increasing the biocompatibility of wound
surfaces.21, 8, 5, 22 In this mode of application, the textural properties of pectin hydrogels should
not match the mechanical properties of the bone tissue. The mechanical properties of pectin
hydrogels are comparable to the mechanical properties of some soft tissues, which decreases the
risk of implant rejection because of the mechanical incompatibility.

The elasticity of soft tissues has been recently reported to range from 0.1 to 1 kPa for
brain, 6 to 17 kPa for muscle, and to be approximately 100 kPa for cartilage and osteoids.1, 23, 24
The data obtained suggest that pectin hydrogels are appropriate for implantation into soft tissues
without mechanical incompatibility.
The maximum strength of pectin hydrogels was obtained using a 1 M CaCl2 solution. An
increase in the concentration of CaCl2 greater than 1 M failed to increase the mechanical strength
of the hydrogels (Table 1). Therefore, the pectin hydrogels that were prepared with the 1 M
CaCl2 solution were used in the remaining experiments.
[Table 1 near here]
Surface morphology and elemental analysis
The morphology of the dry gels determined by SEM at an increased magnification (x
5,000) revealed different surface appearances (Figure 1). The surface of the AU1% gel exhibited
smooth structures (Figure 1a). The gel prepared from 2% and 4% pectin solutions exhibited
fibrous and globular structures, respectively (Figure 1b, с). The surface topography of dry gels
determined by SEM was consistent with the results obtained using AFM. 3D AFM imaging of
the hydrogel surfaces revealed a smooth surface for the AU1% hydrogel (Figure 1d) and
globular domains of various sizes on the surface of AU2% and AU4% hydrogels (Figure 1e, f).
The characterization of surface roughness in terms of Ra and Rq indicated an increase in the
roughness profile for the hydrogels, depending on the pectin concentration. The surface of the
AU4% hydrogels exhibited parameters with higher values than those of hydrogels prepared from
1 and 2% pectin solutions (Figure 1d, e, f).
It was found that the surface morphology depends on the sugar composition and
macromolecular structure,25 as well as the polymer composition of the pectin gel.26 The surface
morphology of gels formed from pectin isolated from callus culture cells was dependent on the
conditions of the culture medium.27 The primary topographic elements of the surface of pectin

gels are globular domains of various sizes. For comparison, the features observed from the gels
of synthetic polymers have a smooth surface.14, 28
The elemental analysis obtained by EDX revealed that the external layers of the dry gels
were composed of oxygen, carbon, chlorine and calcium. The calcium content was found to be
16±1, 14±2 and 13±2 wt % for gels prepared from 1, 2 and 4% pectin solutions, respectively.
Our results showed that the calcium content in the hydrogels was independent of the
pectin concentration in the solution. A previous report 25 demonstrated that the calcium content
in gels prepared from TVF (the pectin of Tanacetum vulgare L.) and CU701 (citrus pectin
CU701, Herbstreith & Fox) was 14.0±1.0 and 12±0.4 wt.%, respectively, indicating that the
calcium concentration in the gel is not dependent on the type of pectin.
[Figure 1 near here]
Hydrogel adhesion to biological tissues
The bioadhesive properties of the pectin hydrogel have not been elucidated. There are
only a few studies describing the bioadhesive quality of pectin or pectin-containing gels.12, 29
However, the design of these studies does not allow for a conclusion regarding the effect of the
structural and mechanical properties on the bioadhesive properties of pectin hydrogels.
The measurement results for hydrogel adhesion to the serosa abdominal wall, liver and
colon of white laboratory mice are shown in Figure 2. The adhesion of the AU2% and AU4%
hydrogels was higher than that of the AU1% hydrogel. The adhesion of the pectin hydrogel to
biological tissues increased as the pectin concentration in the hydrogel increased.
The five theories that are most commonly presented in conjunction with bioadhesion are
the absorption, diffusion, electronic, fracture and wetting theories.30 Data from the literature have
shown that the bioadhesive properties of pectin conform to these theories. The force and pattern
interaction between pectin and colon mucin was shown to be dependent from the amount of
COOH and NH2 groups in the galacturonan backbone.10 The mucoadhesive performance of low
methyl-esterified pectin was significantly lower than that of high methyl-esterified pectin, and

the presence of amide groups in the structure enhanced the mucoadhesion of low methyl-
esterified pectin.9 The maximum adhesive force of rat and mouse peritoneal membranes
increased with increasing esterification of pectins. 11, 12
Our results showed that the values for the force of adhesion of pectin hydrogels to the
serosa abdominal wall, liver and colon are comparable. The adhesion force of hydrogels to
biological tissues increased with an increasing pectin concentration in the hydrogel. In our work,
we used hydrogels formed from low methyl-esterified pectin in which the amount of methyl-
esterified carboxylic groups was 43% of the total carboxylic groups in the galacturonan
backbone.
The Ca2+-pectin gel networks are generated via junction zones whose mechanism of
formation is mainly based on the “egg-box” model in which stretches of the non-methyl-
esterified groups of the galacturonan backbone are ionically cross-linked through Ca2+ bridges.
The methyl-esterified carboxylic groups are not involved in gel formation. We suppose that the
increased adhesion of AU2% and AU4% hydrogels to the tissue caused a change in the ratio of
methyl-esterified carboxylic groups to carboxyl groups, which reduces the negative potential of
the gel.
The low adhesion of the AU1% hydrogel may be explained by the small pectin
concentration in the hydrogel and the minor amount of methyl-esterified carboxylic groups.
Furthermore, the low adhesion of the AU1% hydrogel may be explained by a decrease in the
area of its contact with the serous membrane due to the partial destruction of the hydrogel under
compression. It is unclear why the adhesion force of the AU2% and AU4% hydrogels is
comparable.
Thus, we believe that the adhesion of hydrogels to biological tissues is due to the pectin
concentration in the hydrogels.
Adhesion of macrophages to the hydrogel surface

In the controls, macrophages efficiently colonized on the plastic surfaces (Figure 3a, b).
The number of cells that adhered to the plastic surface was 972±153 cells/100 mm2 after 24 h of
incubation. Approximately 50-60% of adherent cells had a spindle-shaped morphology; the
diameter of the cell body was 24±6 µm (n = 20). Adherent cells were not easily removed by
rinsing, and they exhibited motile phenotypes, often displaying lengthy filopodia. The length of
filopodia of these cells was 47±8 µm (n = 20). In addition, cells with filopodia lengths of 100 µm
were also found (Figure 3b). These results indicate that adherent macrophages are of a
functionally active state.
The results shown in Figure 3 indicate that the surface of the pectin hydrogels fails to
support macrophage adhesion and proliferation properties. The number of adherent macrophages
was 9±2, 43±7 and 91±15 cells/100 mm2 on hydrogels obtained from 1, 2 and 4% pectin
solutions, respectively (Figure 3с, e, g). All adherent cells had spherical-shaped morphologies,
with body diameters of 20±4 µm (Figure 3d, f, h). Note that no more than 10% of all
macrophages available from the cell suspension adhered to the hydrogel surface. For
comparison, the poly(ethylene glycol) hydrogel was found to adhere to more than 300
cells/mm2.31
It was previously shown that soft gels have diffuse and dynamic adhesion to epithelial
cells and fibroblasts. In contrast, stiff gels showed cells with stable focal adhesions, which are
typical of those observed in cells attached to glass.17 The stiffness of the gel can influence
16, 32
cellular processes such as adhesion, motility and differentiation, possibly because soft gels
might be perceived as being fluid by the cells, thereby not allowing the cells to show an
anchorage-dependent response. Our results agree with the data in the literature showing that the
adhesion of macrophages to the surface of hydrogels increases as the stiffness increases.
Furthermore, it is believed that the surface roughness and topography affect cell behavior.
A very rough surface can hamper the development of focal adhesion plaques and cell

spreading.13, 14 A comparison of the surface topography and the number of adhered cells showed
that increasing the magnitude of topographic elements of surface pectin hydrogels does not
prevent the attachment of macrophages.
It was previously shown that calcium ions mediate the adhesion of macrophages by
providing ligand binding lectin receptors.33, 34

In our study, hydrogels were formed
instantaneously by ionotropic gelation in which intermolecular crosslinks were formed between
divalent calcium ions and negatively charged carboxyl groups of pectin molecules. We
hypothesized that by increasing the amount of pectin in the hydrogel, the amount of calcium
would increase. However, the results of our energy-dispersive analysis found no difference in the
amount of calcium in the 1, 2 and 4% hydrogels.
The effect of pectin hydrogels on J-774 macrophages in vitro due to
monocyte/macrophage activation was studied and was assumed to represent a key stage of the
foreign body response (FBR).35 It was previously shown that after of subcutaneous implantation
of chitosan36, j-carrageenan and agarose37 hydrogels showed the presence of an inflammatory
infiltrate characterized by the presence of polymorphonuclear neutrophils (PMNs) and
macrophages. A similar reaction is caused by synthetic hydrogels. 38 We found that macrophages
had low adherence to the surface of pectin hydrogels. The adherent macrophages had a spherical
shape, which is not typical of cells in a functionally active state. Perhaps implantation of pectin
hydrogels induced minimal FBR through the inhibition of macrophage activity. The test results
may or may not be reproducible in vivo.
Plasma protein adsorption
The adsorption on the biomaterial surface of proteins, such as albumin, fibrinogen,
fibronectin, vitronectin, globulin, and others, has been known to activate host inflammatory cells,
thus inducing a subsequent FBR.35 Hydrogels obtained from the 1% pectin solution adsorbed
27±2% of blood plasma proteins. Hydrogels obtained from 2 and 4% pectin solutions were
shown to adsorb approximately 40% of blood plasma proteins (Figure 4).

For comparison, hydrogels prepared by crosslinking sodium alginate with methylene-bis-
acrylamide have been previously reported to adsorb 27% of blood plasma proteins.18 The
molecular mechanisms of high absorption plasma proteins of pectin hydrogels remain unclear.
Note that the adhesion of pectin hydrogels to tissues and the adhesion of proteins to
hydrogels have similarities, possibly due to a reduction in the negative potential of the hydrogel,
thus increasing the adsorption of proteins such as albumin.
Stability to degradation in vitro
The in vitro degradation profiles of pectin hydrogels are shown in Figure 5. The rate of
degradation of all hydrogels was higher in PBS than that in DMEM and a fibroblast monolayer.
The strength of hydrogels obtained from 1, 2 and 4% pectin solutions was reduced by 53, 30 and
20%, respectively, after a 48-h incubation in PBS (Figure 5a). The total destruction of hydrogels,
with a decrease of strength by 99%, was detected in the 1% pectin hydrogel after 4 days, whereas
2 and 4% pectin hydrogels disintegrated after 7 and 9 days of incubation in PBS, respectively.
Pectin hydrogels obtained from 1 and 2% pectin solutions were shown to disintegrate at a
very low rate in DMEM. The strength of 1 and 2% pectin hydrogels decreased by 40% after 2
and 7 days of incubation in DMEM, respectively. The mechanical properties of hydrogels
obtained from the 4% pectin solution did not change after 14 days of incubation in DMEM
(Figure 5b). The degradation behavior of pectin hydrogels in the fibroblast monolayer was
similar to that in DMEM (Figure 5c). To study hydrogel biodegradation, the hydrogels were
incubated with a monolayer of fibroblasts. In this study, we evaluated the effect of cellular
metabolites (ROS and hydrolytic enzymes) extracted by cells on the mechanical properties of the
hydrogels.
The data obtained demonstrated that hydrogel degradation was higher in saline than that
in DMEM. In PBS, the possible reason for hydrogel disintegration is 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.39 The low rate of disintegration
of the pectin hydrogels in the DMEM and fibroblast monolayer is due to protein adhesion on the
hydrogel substrate. Both the DMEM and the fibroblast culture medium contain fetal bovine
serum. The serum proteins may adhere to the surface of the hydrogel and can impede the elution
of calcium ions.
Cytotoxicity to fibroblasts
Cytotoxicity can be rated based on cell viability relative to controls, where an activity
level relative to controls of less than 30% is severe cytotoxicity, between 30 and 60% is
moderate cytotoxicity, between 60 and 90% is slight cytotoxicity, and greater than 90% is no
cytotoxicity. 40, 41
Figure 6 shows the results of fibroblast MTT assays after 24, 48 and 96 h of incubation
with pectin hydrogels. A reduction of cell viability compared to the control was detected for
hydrogels obtained from 1 and 2% pectin solutions after 48 h of incubation; these were
considered to have slight cytotoxicity. The cell viability recovered after 96 h of incubation with 1
and 2% pectin hydrogels, as shown in Figure 6. The effect of the 4% pectin hydrogel on
fibroblast viability was not time-dependent. The data obtained demonstrate that hydrogels from
pectin appear to possess low cytotoxicity, similar to hydrogels from other natural
polysaccharides such as alginate, carrageenan and chitosan.37, 42, 43

The slight cytotoxicity of
pectin hydrogels may explain, at least partially, the reduction in the functional activity of
macrophages.
Hemolysis of red blood cells
During contact with red blood cells, certain materials can cause cell degradation and the
release of hemoglobin. Hemolysis levels were evaluated by measuring the change in the optical
density of the solution as a result of the release of hemoglobin from red blood cells after

membrane destruction. The results of this test show whether a material is able to cause
hemolysis. The level of red blood cell hemolysis while interacting with pectin hydrogels is
shown in Table 2. The optical densities of the positive and negative controls were 3.8 and 0.04,
corresponding to 100 and 0% levels of hemolysis, respectively. The level of hemolysis during
the interaction with hydrogels ranged from 4 to 5.5%. These values are within the set range of
the standard ISO 10993-4: 2002.42 These results show that the developed pectin hydrogels cause
virtually no damage to the erythrocyte membrane during contact. Therefore, these hydrogels are
hemocompatible.
[Table 2 near here]
CONCLUSIONS
In our research, the biofunctional properties of pectin hydrogels with differing textural
and structural characteristics were investigated. An increase in the pectin concentration in
hydrogels was shown to enhance their cohesion, Young’s modulus, compressive strength and
stiffness. An increased concentration of pectin causes an increase in the roughness profile
surface of the pectin hydrogel.
The biofunctional properties of the pectin hydrogel, namely adhesion to serosa,
macrophage adherence, adhesion of plasma proteins and time of biodegradation, were
determined by the pectin concentration in the hydrogel. The correlation coefficient (Pearson's, r)
between the pectin concentration and these variables was calculated as 0.87-0.98. The surface of
the pectin hydrogel prevents adherence of the macrophages and inhibits their transformation to a
functionally active state. This finding indicates a potentially low immunogenicity of pectin
hydrogel-based implants. The textural properties of the pectin hydrogels were demonstrated to
correspond to those of soft tissues. The pectin hydrogel was also found to be hemocompatible in
vitro and to have a low cytotoxicity to fibroblast cells.
In biomedical applications, pectin hydrogels are primarily used as drug delivery systems,
such as those for pharmaceutical compounds 45, 46 and probiotics.47 In tissue engineering, pectin

21
hydrogels have been applied for the isolation of wound surfaces , as a barrier to prevent
postoperative adhesion, 48, 7 as an extracellular matrix for reconstructing bone tissue 5, 22 and as a
coating for hard tissue implants.8 To prepare hydrogels, low methyl-esterified pectins have been
used. To modify the biofunctional properties of hydrogels, pectins with a similar composition to
natural and synthetic polymers have been used. 39, 46
Our results showed that pectin hydrogels can be used as an extracellular matrix for the
reconstruction of soft tissue. The surface of the pectin hydrogel can inhibit the adherence of
macrophages, indicating a potentially low immunogenicity for scaffolds derived from pectin
hydrogels. Furthermore, a simple method can be used to modify the mechanical properties and
biocompatibility of pectin hydrogels. All these qualities represent an advantage for using pectin
hydrogels as a scaffold for tissue engineering.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research,
authorship, and/or publication of this article.
Funding
The study was supported the Program of UD RAS, project 15-3-4-38.

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Captions
FIGURE 1. Scanning electron micrographs (a, b, c) and 3D height AFM images (d, e, f) of the
surface AU1% (a, d), AU2% (b,e) and AU4% (c,f) pectic gels. Ra and Rq are represented as the
arithmetic mean ± SD (n = 5).
FIGURE 2. The adhesive properties of hydrogels. Data are presented as the arithmetic mean ±
SD (n = 6). * – p < 0.05 compared with AU1% hydrogel.
FIGURE 3. Microscopic images of J-774 macrophage adherent on the plastic (a, b) and pectin
hydrogel (c-h) surface after 24 h of incubation; a, c, e, g: phase contrast photomicrographs. Cell
number adhered on the plastic was equal to 972±153 cell/100 mm2 (×200); b, d, f, h:
corresponding fluorescent micrographs (×400), arrows indicate cell filopodia. C, d - AU1%; e, f
- AU2% and g, h - AU4%.
FIGURE 4. The adsorption of blood plasma proteins by hydrogels. Data shown are the
arithmetic mean ± SD; n=9. * – p < 0.05 compared with AU1% hydrogel.
FIGURE 5. In vitro degradation profiles of pectins hydrogels during incubation in PBS (a),
DMEM (b) and cell monolayer (c). Data shown are the arithmetic mean ± SD, n=9. * - p < 0.05
compared to zero point.
FIGURE 6. Viability of the NIH3T3 fibroblast cells in the presence of the pectin hydrogels.
Data shown are the arithmetic mean ± SD; n=9. a - p < 0.05 compared to 24 h, b - p < 0.05
compared to 48 h.

TABLE 1. Mechanical properties of pectin hydrogels
Young Compressive
Samples Cohesion, mJ Hardness, mN
modulus, kPa strength, kPa
AU 1%
0.1 М 0.26±0.0 6±1 15±3 34±2
0.5 М 1 8±1 33±4 45±3
1М 0.81±0.1 10±1 32±4 58±5
2М 8 9±1 34±4 50±7
0.79±0.0
6
0.82±0.1
3
AU 2%
0.1 М 1.32±0.1 13±2 39±3 75±10
0.5 М 9 28±2 96±3 165±12
1М 3.35±0.1 23±2 104±6 127±17
2М 9 30±3 106±7 171±16
3.64±0.2
3
3.56±0.3
3
AU 4%
0.1 М 1.69±0.4 20±3 51±8 118±23
0.5 М 1 70±15 204±20 424±70
1М 6.78±0.7 101±1 219±15 598±66
2М 7 1 236±10 570±67
8.34±0.6 96±11
2
9.31±0.6
9
Data shown are the arithmetic mean ± SD, n = 5.
John Wiley & Sons, Inc.

FIGURE 1. Scanning electron micrographs (a, b, c) and 3D height AFM images (d, e, f) of the surface AU1%
(a, d), AU2% (b,e) and AU4% (c,f) pectic gels. Ra and Rq are represented as the arithmetic mean ± SD (n
= 5).
119x117mm (300 x 300 DPI)

The adhesive properties of hydrogels. Data are presented as the arithmetic mean ± SD (n = 6). * – p <
0.05 compared with AU1% hydrogel.
68x43mm (600 x 600 DPI)

FIGURE 3. Microscopic images of J-774 macrophage adherent on the plastic (a, b) and pectin hydrogel (c-h)
surface after 24 h of incubation; a, c, e, g: phase contrast photomicrographs. Cell number adhered on the
plastic was equal to 972±153 cell/100 mm2 (×200); b, d, f, h: corresponding fluorescent micrographs
(×400), arrows indicate cell filopodia. C, d - AU1%; e, f - AU2% and g, h - AU4%.
119x180mm (300 x 300 DPI)

FIGURE 4. The adsorption of blood plasma proteins by hydrogels. Data shown are the arithmetic mean ±
SD; n=9. * – p < 0.05 compared with AU1% hydrogel.
67x46mm (600 x 600 DPI)

FIGURE 5. In vitro degradation profiles of pectins hydrogels during incubation in PBS (a), DMEM (b) and cell
monolayer (c). Data shown are the arithmetic mean ± SD, n=9. * - p < 0.05 compared to zero point.
130x249mm (600 x 600 DPI)

FIGURE 6. Viability of the NIH3T3 fibroblast cells in the presence of the pectin hydrogels. Data shown are
the arithmetic mean ± SD; n=9. a - p < 0.05 compared to 24 h, b - p < 0.05 compared to 48 h.
78x56mm (600 x 600 DPI)

TABLE 2. Hemolysis of red blood cells
Samples Optical densities, (545 nm) Hemolysis , %
DW (Positive) 3.7±0.85 100±22
0.9 % NaCl 0.04±0.01 1±0.18
(Negative)
AU1% 0.15±0.024 4.0±0.5
AU2% 0.19±0.031* 4.8±0.7*
AU4% 0.21±0.012* 5.5±0.3*
Data shown are the arithmetic mean ± SD, n=9. * - p < 0.05 compared to AU1%. DW –
distilled water.


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Mechanical properties, structure, bioadhesion and biocompatibility of pectin

Pavel A. Markov1, Nikita S. Krachkovsky1, Eugene A. Durnev2 , Ekaterina A. Martinson2,

Sergey G. Litvinets2 and Sergey V. Popov1

Sciences, Syktyvkar, Russia.

Corresponding author: Pavel A. Markov, p.a.markov@mail.ru

This article is protected by copyright. All rights reserved.

The surface structure, biocompatibility, textural and adhesive properties of calcium

found to have a low cytotoxicity.

Keywords: biomaterials, pectin, hydrogels, texture, surface morphology, elemental

analysis, tissue and macrophage adhesion, protein adsorption, cytotoxicity.

biocompatibility, biodegradability, and low cost.

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The presence of a positive or negative charge on the pectin macromolecule determines

thus impeding their ability to participate in further intermolecular interactions. Biofunctional

and mechanical properties.16, 17

structural and mechanical properties of pectin hydrogels on biocompatibility and biofunctional

tissues, cells, plasma protein adhesion and biocompatibility in vitro.

MATERIALS AND METHODS

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solution), fetal bovine serum (HyClone), MTT (dimethylthiazol diphenyltetrazolium), 4′,6-

diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma), rhodamine (Sigma), bovine serum

Hydrogel preparation and compression measurements

was calculated using the following equation:

Compressive strength was calculated as follows:

of the probe P/5 (0.000019 м2).

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Surface morphology and elemental analysis

cross-sections was performed using energy-dispersive X-ray spectroscopy (EDX).

under ambient conditions.

Stability to degradation in vitro

was evaluated using the above-described method.

Hydrogel adhesion to biological tissue

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calculated using Exponent Software (Version V6.1.5.0).

Plasma protein adsorption assay

Adhesion of macrophages to the hydrogel surface

J-774 murine macrophages (Collection of the Institute of Cytology, Russian Academy of

cells were incubated at 37°C with 5% CO2.

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counted and was expressed as cells/100 mm2.

NIH3T3 murine fibroblasts (Collection of the Institute of Cytology, Russian Academy of

the cells was performed at 24, 48 and 96 h.

(BioTek Instruments, USA) spectrophotometer.

Hemolysis of red blood cells

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value of p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Textural properties of the pectin hydrogel

risk of implant rejection because of the mechanical incompatibility.

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without mechanical incompatibility.