504 Bulletin of Experimental Biology and Medicine, Vol. 156, No.

4, February, 2014 EXPERIMENTAL BIOLOGY

Scleractinium Coral Aquaculture Skeleton: a Possible 3D

Scaffold for Cell Cultures and Bone Tissue Engineering
N. S. Sergeeva*,**, T. A. Britaev***, I. K. Sviridova*, S. A. Akhmedova*,
V. A. Kirsanova*, A. A. Popov**, A. I. Antokhin**,
G. A. Frank*, and A. D. Kaprin*

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 156, No. 10, pp. 494-498, October, 2013
Original article submitted July 15, 2012

Cytocompatibility of 5 coral aquaculture skeleton species derived from two families (Acro-
poridae and Pocilloporidae) was studied over the course of in vitro culturing in continuous
human fibroblast culture by the MMT test. Biocompatibility and capacity of scaffold to
“transfer” cell cultures (specifically, multipotent mesenchymal stromal cells) to sites of im-
plantation were studied in vivo by subcutaneous implantation of skeletal fragments to rats. All
coral skeleton aquaculture specimens were cytocompatible (nontoxic and with surface matrix
characteristics satisfactory for cells), biocompatible, and could be tried as 3D matrices for
bone tissue engineering.

Key Words: coral aquacultures; cytocompatibility; biocompatibility; tissue engineering

construct; multipotent mesenchymal stromal cells

The development and introduction of modern bioma-
terials into medical practice for plastic repair of bone
tissue defects remains a pressing problem, despite the
progress attained in this sphere. The main disadvan-
tages of synthetic calcium phosphate materials, slow
bioresorption and poor strength [1,8], prompted the
search among objects of natural origin, for example,
natural coral skeleton (NCS) [7,9,10].
Due to unique microtopography of the surface,
pronounced matrix characteristics for cells, biocompat-
ibility, bioactivity, osteoconduction, and good agree-
ment between the biodegradation and neo-osteogenesis
rates, NCS largely meet the modern requirements to
materials intended for osteoplasty. This fact suggests
regarding this material as the implant for bone de-
fect reconstruction and as 3D matrices for cultured
cells (e.g., multipotent mesenchymal stromal cells) for
*P. A. Hertsen Moscow Research Cancer Institute, Ministry of Health
of the Russian Federation; **N. I. Pirogov Russian National Research
Medical University; ***A. N. Severtsov Institute of Ecology and Evo-
lution, Russian Academy of Sciences, Moscow, Russia. Address for
correspondence: prognoz.06@mail.ru. I. K. Sviridova
bone tissue engineering [3-5,12]. However, introduc-
tion of NCS in clinical medicine is impeded because
of limitations in their collection and differences in
admixtures determined by their habitation conditions.
Skeleton from cultured corals grown under stan-
dard conditions of coral farms could become an alter-
native to NCS.
We studied matrix characteristics and biocompat-
ibility of coral skeleton aquaculture (CSAC) speci-
mens in comparison with their natural equivalents for
prospective use in bone tissue engineering.
MATERIALS AND METHODS
The study was carried out on CSAC specimens derived
from families Acroporidae (A. nobilis, A. hyacinthus,
A. abrotanoides, and A. samoensis) and Pocilloporidae
(P. verrucosa) obtained from the Russian-Vietnamese
Tropical Research Technological Center and on speci-
mens of A. cervicornis NCS from natural populations.
Coral skeleton was cleansed from organic resi-
dues by a previously described method [2]. Before

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N. S. Sergeeva, T. A. Britaev, et al. 505

the experiment, all specimens were pulverized on a
planetary-type ball mill; the fraction of 200-600-μ
particles was used.
Experimental in vitro studies of cytocompatibility
of all coral skeleton specimens were carried out on hu-
man immortalized fibroblast cultures (V. A. Engelgardt
Institute of Molecular Biology, Russian Academy of
Sciences) by the MTT test [11] in 96-well culturing
plates (Corning).
Acute cytotoxicity was evaluated after 24-h cul-
turing and matrix characteristics of coral surface were
evaluated during the course of culturing over 10 days.
Samples with human fibroblast cultures in polystyrene
wells without studied materials served as the control.
All manipulations with corals and human fibroblasts
were carried out under sterile conditions, culturing was
carried out in humid atmosphere with 5% CO2 at 37oC.
The pool of viable cells (PVC) was evaluated at
different stages of the experiment (days 1, 4, 7, 10) as
the ratio of optical density of formazan solution (MTT
reaction product) on a certain day of the experiment
to optical density in the control. A coral specimen was
assumed to be cytocompatible in the absence of acute
cytotoxicity (PVC ≥70% after 24 h in culture) and
if its surface matrix characteristics were satisfactory
(PVC ≥100% during each period of observation).
Biocompatibility was studied on the model of sub-
cutaneous implantation of coral skeleton fragments to
rats (male Wistar rats, 180-220 g) [6]. Six groups of
animals (6 per group) received implantations of coral
skeleton particles: 1) A. nobilis; 2) A. hyacinthus; 3)
A. abrotanoides; 4) A. samoensis; 5) Pocillopora ver-
rucosa CSAC; and 6) A. cervicornis NCS. The rats
were sacrificed 4, 8, and 12 weeks after the operation
(2 rats per term), the specimens were removed, exam-
ined visually and photographed, fixed in 10% forma-
lin, decalcified (0.3 M EDTA, 37oC, ~30 days), after
which paraffin blocks were prepared and histological
sections were stained with hematoxylin and eosin.
Tissue engineering constructs were created with
the following variants of 3D scaffolds: A. abrotanoides
CSAC and a culture of multipotent mesenchymal stro-
mal cells derived from Wistar rat bone marrow (pas-
sage 3), characterized by morphology, proliferative ac-
tivity, colony formation, and induced differentiation in
the osteo- and adipogenic direction. Multipotent mes-
enchymal stromal cells in the tissue engineering con-
struct after subcutaneous implantation were visualized
by life-time CM-Dil stain (3 μg/ml). The suspension
of fluorochrome-stained cells (90-95% labeled cells)
was inoculated onto coral skeleton particles (400,000
cells per well in 2.0 ml complete growth medium) in
24-well plates (Cornig) and cultured for 3 days. Pre-
fabricated tissue engineering construct was subcutane-
ously implanted to rats. The animals were sacrificed
after 1, 2, 4, and 5 weeks (2 animals per term) and
histological preparations were made from the implants
by the above method.

TABLE 1. Optical Density of Formazan Solution (arb. units, MTT Test) and PVC (% of Control) over the Course of Human
Fibroblast Culturing

Day of experiment
Coral specimens
1 4 7 10

Polystyrene (control) 0.424±0.012 0.900±0.028 1.121±0.073 1.580±0.068

A. nobilis (No 1) 0.448±0.048 1.281±0.030* 1.625±0.128* 2.061±0.127*
(105.7) (142.3) (142.3) (130.40)
A. hyacinthus (No 2) 0.596±0.016* 1.157±0.087* 1.227±0.007 1.611±0.080
(140.6) (128.6) (109) (102)
A. abrotanoides (No 3) 0.583±0.012* 1.363±0.039* 1.788±0.069* 2.14±0.04*
(137.5) (151.4) (159.5) (135.4)
A. samoensis (No 4) 0.662±0.018* 0.938±0.021 1.245±0.094 1.420±0.021
(156.1) (104.2) (111.1) (84.2)
Pocillopora verrucosa (No 5) 0.529±0.025* 1.381±0.038* 1.622±0.077* 2.024±0.058*
124.8% (153.4) (144.7) (128.1)
A. cervicornis (No 6) 0.386±0.017 0.967±0.023 1.441±0.074* 1.942±0.073*
(91) (107.4) (128.5) (122.9)

Note. *p<0.05 in comparison with the control. In parentheses: % of control.

506 Bulletin of Experimental Biology and Medicine, Vol. 156, No. 4, February, 2014 EXPERIMENTAL BIOLOGY

All manipulations on animals were performed in

accordance with the International Bioethics Philoso-
phy presented in the Helsinki Declaration and with
regulations of humane handling of laboratory animals.
The results were statistically processed by BioStat
software. The significance of differences was evalua-
ted by Student’s test, the differences were considered
significant at p<0.05.

RESULTS
None of the studied CSAC specimens exhibited acute
cytotoxicity towards human fibroblast test culture. Op-
tical density of formazan solution and PVC after 24-h
culturing were the same in the experimental and con-
trol wells (samples Nos. 1 and 6) or were significantly
higher (samples Nos. 2-5; Table 1). Longer cultur-
ing resulted in an increase of fibroblast population in
CSAC specimens Nos. 1, 3, and 5 and NCS specimen
No. 6, in comparison with the control: optical density
of formazan solution in these experimental groups on
day 10 was 2.061, 2.140, 2.024, and 1.942 arb. units,
respectively, vs. 1.580 arb. units in the control, PVC
was by 22-35% higher than in the control (Table 1).
Only on two CSAC specimens (Nos. 2 and 4), the
fibroblast pool by the end of experiment virtually did
not differ from the control by the end of experiment:
optical densities of formazan solution in these groups
were 1.611 and 1.420 vs. 1.580 arb. units in the con-
trol, PVC 102.0 and 84.2%, respectively.
These findings attest to cytocompatibility of the
studied CSAC and NCS specimens: all the studied
coral specimens were not toxic towards human fibro-
blast test culture and exhibited matrix surface proper-
ties, more pronounced in CSAC No. 1 (A. nobilis), No.
3 (A. abrotanoides), No. 5 (P. verrucosa), and NCS
No. 6 (A. cervicornis).
The dynamics of biocompatibility of these mate-
rials was studied in vivo over 12 weeks after implan-
tation to rats. Examination of histological sections in
4 weeks showed no signs of inflammation and/or re-
jection of coral particles. The granule spaces (empty
spaces after decalcification) and the entire implant
were enveloped in properly organized vascularized
connective tissue. Spaces between the granules were
filled with loose connective tissue with few cells and
solitary capillaries (Fig. 1, a). The volume of con- Fig. 1. A. nobilis coral granules after subcutaneous implantation
nective tissue between the granules and the number to rats. Hematoxylin and eosin staining, ×100. a) 4 weeks; b) 8
weeks; c) 12 weeks.
of capillaries increased later after implantation of
CSAC and NCS specimens (8 and 12 weeks), foreign
body cells (macrophages) appeared at the interface and biologically with participation of the foreign
between the granules and the connective tissue. The body cells.
spaces occupied by the coral granules flattened and No macro- and microscopic signs of rejection or
shrank in the course of observation (Fig. 1, b, c). cellular inflammatory reaction were detected in histo-
Hence, the granules gradually resorbed – passively logical preparations over the course of experiment
N. S. Sergeeva, T. A. Britaev, et al. 507

Fig. 2. Tissue engineering construct based on A. abrotanoides CSAC and rat autologous multipotent mesenchymal cell culture (passage
3) at different terms after subcutaneous implantation to Wistar rats. a, c, e, g: phase contrast; b, d, f, h: fluorescent microscopy, ×400. a,
b) 1 week; c, d) 2 weeks; e, f) 4 weeks; g, h) 5 weeks.
508 Bulletin of Experimental Biology and Medicine, Vol. 156, No. 4, February, 2014 EXPERIMENTAL BIOLOGY
(weeks 4-12 after subcutaneous implantation), i.e.
CSAC and NCS specimens were biocompatible. No
crucial differences between the five CSAC and A. cer-
vicornis NCS specimens were detected.
The behavior of tissue engineering construct based
on A. abrotanoides CSAC with immobilized rat autol-
ogous multipotent mesenchymal stromal cells (passage
3) and labeled with fluorochrome CM-Dil and the re-
action to the construct implanted subcutaneously were
evaluated at the final stage of the study. Phase contrast
microscopy picture of histological preparations was
similar to that in common microscopy: empty spaces
at the site of granules filled by vascularized loose con-
nective tissue. Fluorescent microscopy during weeks
1-4 showed bright fluorescence in fibroblast-like con-
nective tissue cells and cells adjacent to the capillaries;
by the end of week 5, fluorescence was less bright
(Fig. 2). Hence, the rat multipotent mesenchymal stro-
mal cells in the tissue engineering construct based
on A. abrotanoides CSAC in an autologous system
remained viable and functionally active during at least
4 weeks.
The data indicated good prospects of further stu-
dies of CSAC intended for use as 3D matrices for bone
tissue engineering.
The study was supported by the Russian Founda-
tion for Basic Research (grant No. 13-03-12127ofi-m).
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