DOI 10.

1007/s10517-019-04558-1
496 Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  167,  No.  4,  August,  2019

BIOTECHNOLOGIES

Study of Biointegration and Elastic-Strength Properties

of a New Xenopericardium-Based Biomaterial
for Reconstructive Cardiovascular Surgery
I. S. Fadeeva1, M. N. Sorkomov2, A. I. Zvyagina1, D. V. Britikov2,
A. S. Sachkov2, Ya. V. Evstratova1, R. S Fadeev1, R. M. Muratov2,
and V. S. Akatov1
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 167, No. 4, pp. 483-487, April, 2019
Original article submitted October 22, 2018

We analyzed biocompatibility, elastic-strength properties, and biointegration potential of a

new biomaterial made of xenopericardium for reconstructive cardiovascular surgery. The
biomaterial manufactured by the proposed technology demonstrated high biocompatibility and
biointegration potential and its elastic-strength properties 2-4-fold surpassed that of native
pericardium. The obtained results attested to good prospects of using the proposed technology
for preparing biomaterials for reconstructive cardiovascular surgery.
Key Words: xenopericardium-based biomaterials; decellularization; biointegration; elastic-
strength characteristics; repopulation

For many decades, materials based on the donor peri-
cardium have been widely used in reconstructive in-
terventions on the heart and blood vessels. The ex-
perience of the use of biological pericardial cardiac
prostheses fixed by cross-linking agents indicates that
allo- and xenogeneic cell-free extracellular matrixes
can provide extended life of the biomaterial and do not
induce immune response of the recipient [5]. At the
same time, the main limitation of wide use of xeno-
pericardial materials is the risk of pathological calci-
fication and structural degeneration at delayed terms
after surgery, especially in young patients [4,7]. This
fact is explained, first, by the suppression of repopu-
lation of biomaterials fixed by cross-linking agents
and, consequently, the impossibility of biointegration
1
Institute of Theoretical and Experimental Biophysics, Russian Acad-
emy of Sciences, Pushchino, Moscow region; 2A. N. Bakulev National
Medical Research Center of Cardiovascular Surgery, Moscow, Russia.
Address for correspondence: aurin.fad@gmail.com. I. S. Fadeeva
and following remodeling of the implanted material
along with the development of aseptic calcification
of the structurally-damaged extracellular matrix of
the biomaterial [7]. Considering the fact that bioma-
terials used in reconstructive cardiovascular surgery
should not only be biocompatible and durable, but also
have mechanical properties close to those of replaced
tissues (valves, cardiac septa, etc.), the creation of a
durable material based on decellularized extracellular
matrix of the pericardium with reduced calcification
potential and a high capacity for biointegration is a
pressing task of modern reconstructive cardiovascular
surgery.
Earlier, we have developed and introduced into
clinical practice the technology of devitalization and
anti-calcification processing of biomaterials with digi-
tonin and EDTA. This approach demonstrated high
efficiency in preparing allografts [1,2,6], but not for
xenomaterials. A new technology proposed for multi-
stage pre-implantation decellularization and delipidi-

0007­-4888/19/16740496 © 2019 Springer Science+Business Media, LLC

I. S. Fadeeva, M. N. Sorkomov, et al.
zation of allo- and xenogeneic biomaterials using li-
pophilic detergent demonstrated high efficiency for
heart valve and vascular transplants [7]. At the same
time, the effectiveness of the proposed new method of
manufacturing biomaterials from xenopericardium for
their use in reconstructive cardiovascular surgery has
not been studied.
Here we studied biocompatibility, biointegration
potential, and elastic and strength properties of a bio-
material produced by the proposed technology from
xenopericardium tissue for reconstructive cardiovas-
cular surgery.
MATERIALS AND METHODS
The pericardium of healthy mature calves from ani-
mals with appropriate veterinary certificates was used
in the study. We used the anterior part of the peri-
cardium from the site of its attachment to the main
vessels to the pericardial-diaphragmatic ligament. The
material was delivered to the laboratory no later than
in 4 h after slaughtering at 4oC in RPMI-1640 me-
dium containing 400 μg/ml gentamycin and 50 μg/ml
fluconazole and treated in accordance with [2]. Mul-
tistage treatment of the donor material included 2-h
incubation with 2 mM EDTA followed by treatment
in 0.5% sodium deoxycholate monohydrate (SDM,
D5670, Sigma-Aldrich) (pH 8.0) at 37oC, and repeated
washing from SDM in 150 mM NaCl solution (pH 7.8)
containing 20% ethanol; complete cycle of decellular-
ization took 10 days.
The pericardial samples and the resultant pericar-
dial biomaterial (BM) before and after implantation to
laboratory animals were fixed routinely (NBF, 24 h,
20-22oC) and histological sections (9 μ) were prepared
by cryotomy (Shandon E620, Thermo Fisher Scien-
tific). For histochemical analysis of BM before and
after implantation, we used Weigert and van Gieson
differential staining (for elastin components), Lillie’s
trichrome staining (for collagen components), Sudan
III and Mayer’s hematoxylin (for lipid components),
von Kossa staining (for calcium deposits), alcian blue
and PAS reaction (detection of glycosaminoglycans
and mast cells), and standard survey staining with he-
matoxylin and eosin for detection of remaining cell
nuclei and the degree and nature of materials repopu-
lation in the body.
For evaluation of the potential toxic effect of BM,
in vitro cytotoxicity was studied on human skin fibro-
blast culture (passages 1-4 after isolation). The cells
were seeded on serous surface of the BM and cultured
in αMEM medium (Sigma-Aldrich) supplemented with
10% fetal calf serum (Gibco) and 40 μg/ml gentamicin
sulphate (Sigma-Aldrich) at 37oC and 5% CO2 in an
incubator. Live and dead cells were counted in 24 and
497
96 h after inoculation after staining with fluorescent
dyes calcein AM and PI (both from Sigma-Aldrich).
The cells were detached from the substrate with ac-
cutase cocktail (Sigma-Aldrich) and stained in L-15
medium with 1% fetal calf serum containing 1 μg/ml
calcein AM and 2 μg/ml propidium iodide for 25 min
at 37oC. Analysis of live and dead cells was performed
using an Accuri C6 flow cytometer (BD Bioscience).
The degree of calcification of BM samples (μg
Ca/mg dry sample weight) before and after implanta-
tion was assessed by absorption spectroscopy using
standard calcium assay kit (Calcium AS FS Arsenazo
III, DiaSys) and an Infinite F200 microplate spectro-
fluorometer (Tecan) [1].
Mechanical tests of BM were performed on a
Zwick/Roell Z0.5 instrument. Pericardial and BM
samples (10×50 mm; n=20) were subjected to uniax-
ial tension until complete rupture (initial load 0.05 N
and velocity 50 mm/min) to determine the tensile and
tear strength (MPa) at relative elongation at rupture
(%) and elastic modulus (Young’s modulus, N/mm2)
in the longitudinal (relative to pericardial fibers) and
transverse direction.
The biocompatibility and degree of biointegra-
tion of the studied xenopericardium samples were
evaluated in a model of heterotopic (subcutaneous)
implantation in male Wistar rats weighing 180-200 g
under xylazine and Zoletil anesthesia. BM fragments
(1×1 cm; n=96 per test group) were implanted under
conditions of complete interstitial contact. In 4, 8 and
13 weeks, the implants were removed together with
surrounding tissues and analyzed in comparison with
untreated (native) xenopericardium that is known to
cause rejection in the model of heterotopic implanta-
tion. All manipulations were carried out in accordance
with the requirements of GOST R ISO 10993-2-2009.
Histological analysis of stained BM cryosections was
carried out using a Nikon Eclipse Ti-E microscop-
ic fluorescence station; survey histotopograms were
obtained in all cases using a NIS Elements software
(Nikon).
Statistical processing of the results was performed
using Mann—Whitney U test. The differences were
significant at p<0.05. All experiments were carried out
in at least three repeats.
RESULTS
Histological analysis of BM fragments before implan-
tation confirmed efficient elimination of donor cells,
the absence of lipid components, and preserved struc-
ture and architectonics of the extracellular matrix after
treatment of xenopericardium (Fig. 1) and showed that
BM was mostly presented by collagenous extracellular
matrix.
498 Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  167,  No.  4,  August,  2019 BIOTECHNOLOGIES
Fig. 1. Histological preparation of native pericardium (a) and BM (b). Light microscopy. Longitudinal sections of pericardium samples,
hematoxylin and eosin staining, matrix components colored gray and cell nuclei are black; inserts: cross sections at higher magnification
(Lillie’s trichrome staining), ECM collagen components and cell nuclei are stained from dark gray to black, cell nuclei are deep black, other
components of ECM are not stained.
The studied BM produced no cytotoxic effect on
cells. The cells adhered to the material, spread out, and
grew on BM. The number of cells in the control (cul-
ture plastic) in 96 h after seeding increased from 5000
to 35,000 per 1 cm2. Cell growth on BM surpassed the
control by 95±5%. The percentage of live cells on BM
and in in control cultures was the same: 96±2%. The
obtained results attest to high biocompatibility of the
manufactured biomaterial.
The comparison of the elastic-strength character-
istics of BM and native pericardium showed that BM
could withstand 2-fold higher loads than native pericar-
dium. In transverse direction, native pericardium has
maximum tensile strength (average, σB) of 3.77±0.95
MPa at average elongation of 35.84±6.15%; for BM
the corresponding parameters were 12.2±1.84 MPa at
76.5±10.49%. In the longitudinal direction, these char-
acteristics were 11.16±2.08 MPa at 24.35±5.73%for the
native pericardium and 34.29±3.78 MPa at 51.99±9.37%
for BM. These results show that the proposed method
of manufacturing BM from xenopericardium provides
2-4-fold higher elastic-strength properties in compari-
son with the native material.
Histological analysis of the samples revealed no
signs of immune response to the implant in the recipi-
ent at all terms after implantation (Fig. 2). Intensive
repopulation of the BM matrix by recipient cells was
observed in 4 weeks after implantation (Fig. 2, a).
All samples retained structural integrity and were not
resorbed throughout the experiment. Selective stain-
ing for calcium phosphates revealed no mineralized
calcium in the samples at all terms after implantation
(Fig. 2, b). The absence of the inflammatory reaction
and signs of aseptic calcification attested to high bio-
compatibility of BM.
By the 13th week, we observed significant remod-
eling of the matrix with de novo formation of elastin
fibers (Fig. 2, c) and glycosoaminoglycans and active
migration of tissue basophils to the samples from the
contact tissues of the subcutaneous space (Fig. 2, d).
These findings indicate active biointegration of the
samples into surrounding tissues.
Quantitative assay of mineralized calcium showed
that its content in the native pericardium and BM be-
fore implantation was 0.09±0.03 μg/mg dry tissue.
Analysis of BM samples on the 13th week after im-
plantation showed that the content of mineralized cal-
cium was 0.14±0.10 μg/mg dry tissue, which attested
to suppression of sample calcification in the recipient
body.
On the basis of this complex study, we can con-
clude that the method of decellularization/delipidi-
zation of biological tissues proposed by us ensures
complete destruction and removal of donor cells and
lipid components and preserves the structure and ar-
chitectonics of the extracellular matrix. The experi-
mental biomaterials produced by the proposed tech-
nique are characterized by high biocompatibility both
in vitro and in vivo, are actively repopulated by re-
cipient cells as soon as in 4 weeks after implantation,
do not induce mineralization even at delayed terms
after implantation, and undergo active remodeling of
the extracellular matrix, which indicates significant
potential for biointegration of these materials in the
recipient body. These properties together with high
elastic-strength characteristics of the examined BM
opens good prospects of their use in reconstructive
cardiovascular surgery.
The study was carried out using the material and
technical resources of the Common Use Center “Cen-
I. S. Fadeeva, M. N. Sorkomov, et al. 499

Fig. 2. Histological preparation of BM cross-sections in 4 (a) and 13 (b-d) weeks after heterotopic implantation to rats. Light microscopy. a)
Hematoxylin and eosin staining: nuclei of recipient cells migrated to BM matrix (black) against the background of ECM (gray); b) von Kossa
staining: the absence of black calcium deposits; c) Weigert and van Gieson staining: black nuclei and newly formed elastin components
of the BM matrix (arrows) against the background of other ECM components (gray); d) alcian blue and PAS staining: large mast cells that
migrated to the BM matrix of from subcutaneous adipose tissue (black, arrows); formation of acidic GAG centripetal from the serous layer
(delineated by a dotted line), and neutral GAG from the fibrous layer (delineated by a long dot-dash line).

3. Fadeeva IS, Akatov VS, Muratov RM, Britikov DV, Sachkov

ter for In Vitro Testing”, the Institute of Theoretical
and Experimental Biophysics, and was supported by
the Program of the Presidium of Russian Academy of
Sciences “Basic Research for Development of Bio-
medical Technologies” and by the Innovation Promo-
tion Fund.
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