Scaffolds for bone tissue regeneration must be precisely designed as they support cell attachment, proliferation, differentiation and blood vessel in-growth. Moreover, the materials which are implanted in human body must be non-cytotoxic, absolutely harmless. In this work cellulose scaffolds with nanohydroxyapatite and microhydroxyapatite were prepared. The results obtained in this work revealed that the morphology of the scaffolds depended on the size of hydroxyapatite particles. The porosity of the scaffolds varied from 66 to 72%. The pores were interconnected with the average diameter 0.49 and 0.54 mm for scaffolds with nano- and microhydroxyapatite, respectively. Biocompatibility and potential toxicity of the experimental cellulose/hydroxyapatite scaffolds were tested. It was determined that the scaffolds containing nanohydroxyapatite particles showed slight cytotoxic effect

Babenko, N.

Baniukaitiene, O.

Harkavenko, V.

Kharchenko, V.

Liesiene, J.

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 4 No 2, 02NABM03(3pp) (2015) 
 
 
2304-1862/2015/4(2)02NABM03(3) 02NABM03-1  2015 Sumy State University 
Comparison of Morphology and Cytotoxicity of Cellulose Composites with Nano- and  
Microhydroxyapatite for Bone Tissue Engineering 
 
J. Liesiene1, O.Baniukaitiene1, N. Babenko2, V. Harkavenko2, V. Kharchenko2 
 
1 Kaunas University of Technology, Radvilenu pl. 19, 50254 Kaunas, Lithuania 
2 Kharkiv V.N. Karazin National University, m. Svobody 4, 61022 Kharkiv, Ukraine 
 
(Received 29 May 2015; published online 22 August 2015) 
 
Scaffolds for bone tissue regeneration must be precisely designed as they support cell attachment, pro-
liferation, differentiation and blood vessel in-growth. Moreover, the materials which are implanted in hu-
man body must be non-cytotoxic, absolutely harmless. 
In this work cellulose scaffolds with nanohydroxyapatite and microhydroxyapatite were prepared. The 
results obtained in this work revealed that the morphology of the scaffolds depended on the size of hydrox-
yapatite particles. The porosity of the scaffolds varied from 66 to 72%. The pores were interconnected with 
the average diameter 0.49 and 0.54 mm for scaffolds with nano- and microhydroxyapatite, respectively. Bi-
ocompatibility and potential toxicity of the experimental cellulose/hydroxyapatite scaffolds were tested. It 
was determined that the scaffolds containing nanohydroxyapatite particles showed slight cytotoxic effect. 
 
Keywords: Scaffolds, Cellulose, Hydroxyapatite, Cytotoxicity, Bone tissue engineering. 
 
 PACS number: 87.85.Jj 
 
 
1. INTRODUCTION 
 
Three-dimensional (3D) scaffolds fabricated from 
polymers are rapidly growing materials for bone regen-
eration procedures [1–3]. Saturated aliphatic polyes-
ters are usually applied for this purpose [4]. Promising 
results have been obtained using naturally derived 
polymers, especially chitosan, alginate, starch, agarose 
or bacterial cellulose [5–9]. In order to enhance me-
chanical properties and bioactivity, several materials, 
such as hydroxyapatite, β-tricalcium phosphate, bioac-
tive glass are embedded in polymer matrices [10–15]. 
Beside the choice of suitable material, the morphol-
ogy is also very important. The structure of the scaf-
folds must be accessible for cell attachment, prolifera-
tion, differentiation and for blood vessel in-growth. The 
permeable pore network is required for nutrient deliv-
ery and waste exchange [16].  
Hydroxyapatite being a component of natural bone 
is very often used for the preparation of bone scaffolds. 
Commercial hydroxyapatite is of different particle size, 
morphology and crystallinity.  
The aim of this work was to prepare 3D cellulose 
based scaffolds with nano- and microhydroxyapatite 
particles and to compare their morphology as well as 
cytotoxicity. 
 
2. MATERIALS AND METHODS 
 
Cellulose/hydroxyapatite composites were produced 
mechanically immobilizing nanohydroxyapatite (nHA, 
100 nm) and microhydroxyapatite (µHA, 20 µm) parti-
cles during the formation of cellulose scaffold. Compo-
sites were formulated with HA particles of ~ 50 wt. %, 
close to the percentage of inorganic part in natural 
bone. The micro-computed tomography (micro-CT) 
analysis was performed using a µCT40 micro-CT sys-
tem (Scanco Medical AG, Switzerland) in order to eval-
uate the morphology of the prepared scaffolds. 
Cytotoxicity of the investigated scaffolds was stud-
ied using ex-vivo hepatocytes isolated from 3-month old 
Wistar rats’ liver. Immediately after isolation, cell ali-
quots were stained with trypan blue and counted in 
hemacytometer. Viability of the isolated hepatocytes 
before incubation in all experiments was not less than 
95%. After isolation, cells were incubated in Petri dish-
es at 37°C through 90 minutes with milled composite 
samples 50 mg/ml of cell suspension for solid compo-
sites and 10 mg/ml for HA powders. After incubation 
aliquots for cell count and viability testing were dis-
pensed. Cell viability was valued by staining with try-
pan blue dye. ≥600 cells for every sample in the set of 
experiments were tested. In all experiments control 
cells viability was over than 92%. 
Membrane integrity was tested on isolated rat 
hepatocytes and extensor digitorum longus (EDL) 
muscle tissue by evaluating lactate dehydrogenase 
(LDH) and aldolase activity in incubation medium. 
LDH and aldolase outcome from the cells indicates 
membrane damage, and both these analyzes are wide-
ly used in clinics. For evaluating membrane damage 
in hepatocytes, cells after incubation with composite 
samples were centrifuged, and supernatants were 
collected for LDH assay. To investigate potential myo-
cyte membranes damage, isolated EDLs from 3-month 
old Wistar rats were incubated at 37°C for 90 minutes 
in O2 enriched Krebs saline buffer with composite 
samples. Incubation medium was used for aldolase 
assay. 
Metabolic effects of cellulose/ hydroxyapatite com-
posites were studied in isolated hepatocytes and EDLs 
of 3-month old rats by evaluating hormonal sensitivity 
and glucose uptake rate after 90 minutes incubation 
with composite samples. [3Н]-D-glucose uptake under 
the insulin action was investigated in the EDLs, prein-
cubated with composite samples. [14C]-glucose was 
added into the hepatocyte incubation medium after 
preincubation with samples to evaluate the basal levels 
of [14С]-glycogen synthesis, and insulin-stimulated 
[14С]-glycogen synthesis. 
 
 J. LIESIENE, O.BANIUKAITIENE, N. BABENKO, ET AL. PROC. NAP 4, 02NABM03 (2015) 
 
 
02NABM03-2 
3. RESULTS AND DISCUTION  
 
For the morphological characterisation of the pre-
pared scaffolds, micro-computed tomography was cho-
sen. 2D images showed that HA particles affected the 
morphology of the prepared scaffolds: differences in pore 
size, framework thickness, and their distribution ap-
peared (Fig. 1, Table 1). 
The scaffolds comprised non-symmetrical intercon-
nected pores. Such arrangement of the pores is particu-
larly important for cellular activity and achieving the 
optimum rate of the new tissue growth. 
 
 
 
Fig. 1 – 2D images: cellulose/nHA scaffold (a), cellulose/µHA 
scaffold (b) 
 
The structural parameters, such as the percent 
framework volume (Xv), porosity (P), specific surface 
(SS), mean framework thickness (L), mean pore diame-
ter (D) within the scaffold were determined from 3D 
images (Fig. 2). 
 
 
 
Fig. 2 – 3D images: cellulose/nHA scaffold (a), cellulose/µHA 
scaffold (b) 
 
The structural parameters of the scaffolds are 
summarized in Table 1. The microcomputer tomogra-
phy data showed that the porosity of cellulose/nHA 
scaffold was larger, leading to a reduced percentage 
of the framework volume, as compared with the cellu-
lose/µHA scaffold. The framework thickness with the 
immobilized HA particles of the different composite 
scaffolds was different (Table 1). The frameworks of 
the cellulose/µHA scaffold were almost twice thicker, 
as compared with the frameworks of the cellu-
lose/nHA composite. It was also observed in the 2D 
images (Fig. 1). The specific scaffold surface was 
found to be larger for the cellulose/nHA due to the 
thinner frameworks and a higher number of them per 
millimeter, as supposed. Thus, the scaffold had 
smaller pores. The mean pore diameter was of 
0.49 mm. 
 
Table 1 – Structural parameters of the scaffolds 
 
 
Samples 
Structural parameters 
Xv 
(%) 
P 
( %) 
SS 
(mm1) 
L 
( mm) 
D 
(mm) 
Cellulose/nHA 28 72 19 0.12 0.49 
Cellulose/µHA 34 66 13 0.21 0.54 
 
Generally, the morphology of the prepared cellu-
lose/HA scaffolds for bone tissue engineering meets the 
requirements of materials which are implanted in the 
bone defect [17]. 
Besides the suitable morphology, the scaffolds 
should be absolutely harmless to body. Thus, the bio-
compatibility and cytotoxicity of the cellulose/HA scaf-
folds were tested. Trials were divided into three main 
stages, where cytotoxicity, membrane integrity and 
metabolic effects of the scaffolds were studied. 
All investigated composites, including scaffolds con-
taining µHA particles have demonstrated insignificant 
changes in cell viability after incubation, except cellu-
lose/nHA scaffolds, where cell viability demonstrated 
significant 5 to 8% viability drop in comparison to con-
trol cells. Furthermore, hepatocyte incubation with HA 
powder have shown that µHA particles made no signif-
icant changes in cell viability, at the time when nHA 
decreased cell viability by about 10% (Fig 3).  
 
 
 
Fig. 3 – effect of cellulose/HA composite scaffolds and HA 
powdered particles on hepatocytes viability. 1 – untreated 
cells, 2 –control cells, 3 – nHA particles, 4 - µHA particles,  5 – 
cellulose/ nHA , 6 - cellulose/ µHA. *-p<0,05 
 
Comparison of nHA and µHA particles incorporated 
into cellulose also demonstrated cytotoxicity of nHA, 
while µHA were harmlessness. 
45% rise in LDH activity in hepatocyte incubation 
medium was found after preincubation of the cells with 
cellulose/nHA scaffolds. Other composite samples have 
demonstrated no significant LDH release from the cells 
in regard to control (Fig. 4a). When incubated with 
EDLs, cellulose/nHA scaffolds also significantly in-
creased aldolase activity in the culture medium: about 
3-fold compared to control (Fig. 4b). At the same time, 
whole-cellulose scaffolds without HA addition did not 
change the LDH or aldolase release from the cells (Fig. 
4). Enzymatic activity, elevated in incubation medium 
of cells and muscle tissue after preincubation with cel-
lulose scaffolds, containing nHA particles indicate 
membrane damage, caused by composite, which may 
lead to metabolic disregulation and cell death.  
Significant [14С]glycogen synthesis failure under 
the action of insulin, but not in basal glycogen synthe-
 COMPARISON OF MORPHOLOGY AND CYTOTOXICITY … PROC. NAP 4, 02NABM03 (2015) 
 
 
02NABM03-3 
sis rate, was found in cells incubated with cellu-
lose/nHA scaffolds and whole-cellulose scaffolds with-
out HA additives. Cellulose/nHA scaffolds have de-
creased [14С]glycogen synthesis rate by about 20%, 
compared to control at the time when whole-cellulose 
scaffolds decreased [14С]glycogen synthesis rate by 
about 10%. Other cellulose-based composites have 
demonstrated no significant decrease in [14С]glycogen 
synthesis under the insulin action. At the same time, 
cellulose scaffolds containing nHA induced 2-fold 
[3H]glucose uptake failure under the action of insulin 
in isolated EDLs in comparison to control, while other 
composite samples caused no significant changes in 
[3H]glucose uptake rate. 
 
4. CONCLUSIONS 
 
The prepared nanohydroxyapatite/cellulose and mi-
crohydroxyapatite/cellulose scaffolds showed well de-
signed structures. The morphological parameters were 
in the range of required. All composite samples, except 
nanohydroxyapatite carrying cellulose scaffolds, during 
incubation period have demonstrated acceptable levels of 
cell integrity and viability, and were not cytotoxic or 
damaging to native primarily isolated liver and muscle 
cells. At the same time cellulose/nanohydroxyapatite 
scaffolds, according to the set of assays, have demon-
strated slight cytotoxic influence and downregulation of 
insulin sensitivity in hepatocytes and EDL muscle tissue 
models. Moreover, nanohydroxyapatite powder, com-
pared to control and to microhydroxyapatite particles, 
have demonstrated cytotoxic effects. 
 
 
 
Fig. 4 – effect of cellulose/HA composite scaffolds on LDH and 
aldolase release in the incubation medium of cells. a – LDH 
release from hepatocytes; b – aldolase release from EDLs. *-
p<0,05 
 
AKNOWLEDGEMENTS 
 
The studies were performed in a frame of Lithua-
nia-Ukraine joint research program «Investigation of 
biocompatibility of cellulose/hydroxyapatite composites for 
bone tissue engineering» Ukr: № М/ 127-2014, Lithuanian 
No. TAP LU 01/2014. 
 
 
REFERENCES 
 
1. C. Delabarde, C.J.G. Plummer, P.E. Bourban, J.A.E. Man-
son, J. Mater. Sci. - Mater. Med. 23, 1371 (2012). 
2. A. Aravamudhan, D.M. Ramos, J. Nip, M.D. Harmon, R. 
James, M. Deng, C.T. Laurencin, X. Yu, S.G. Kumbar, J. 
Biomed. Nanotechnol. 9, 719 (2013). 
3. N. Ninan, Y. Grohens, A. Elain, N. Kalarikkal, S. Thomas, 
Eur. Polym. J. 49, 2433 (2013). 
4. I.S. Muhammad, X. Xiaoxue, L. Li, J. Mater. Sci. 44, 5713 
(2009). 
5. J. Seol, J.Y. Lee, Y.J. Park, Y.M. Lee, Young-Ku, I.C. 
Rhyu, S.J. Lee, S.B. Han, C.P. Chung, Biotechnol. Lett. 26, 
1037 (2004). 
6. Z. Li, H.R. Ramay, K.D. Hauch, D. Xiao, M. Zhang, Bio-
materials 26, 3919 (2005). 
7. A.J. Salgado, O.P. Coutinho, R.L. Reis, Tissue. Eng. 10, 
465 (2004). 
8. J.A. Puertolas, J.L. Vadillo, S. Sanchez-Salcedo, A. Nieto, E. 
Gomez-Barrena, M. Vallet-Regi, Acta Biomater. 7, 841 (2011).  
9. M. Zaborowska, A. Bodin, H. Backdahl, J. Popp, A. Gold-
stein, P. Gatenholm, Acta Biomater. 6, 2540 (2010). 
10. A.R. Boccaccini, V. Maquet, Compos. Sci. Technol. 63, 
2417 (2003). 
11. C.Y. Zhang, H. Lu, Z. Zhuang, X.P. Wang, Q.F. Fang, J. 
Mater. Sci. - Mater. Med. 21, 3077 (2010). 
12. N. Barbani, G.D. Guerra, C. Cristallini, P. Urciuoli, R. 
Avvisati, A. Sala, E. Rosellini, J. Mater. Sci. - Mater. Med. 
23, 51 (2012). 
13. J. Liuyan, L. Yubao, X. Chengdong, J. Biomed. Sci. 16, 
110 (2009). 
14. S. Sanchez-Salcedo, A. Nieto, M. Vallet-Regi, Chem. Eng. 
J. 137, 62 (2005). 
15. B.M. Chesnutt, A.M. Viano, Y. Yuan, Y. Yang, T. Guda, 
M.R. Appleford, J.L. Ong, W.O. Haggard, J.D. Bumgardner, 
J. Biomed. Mater. Res. Part A. 88, 491 (2009). 
16. K. Kim, A. Yeatts, D. Dean, J.P. Fisher, Tissue Eng. B 
Rev. 16, 523 (2010). 
17. S. Bose, S. Vahabzadeh, A. Bandyopadhyay, Materials 
Today 16, 496 (2013). 
 


Comparison of Morphology and Cytotoxicity of Cellulose Composites with Nano- and Microhydroxyapatite for Bone Tissue Engineering

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Comparison of Morphology and Cytotoxicity of Cellulose Composites with Nano- and Microhydroxyapatite for Bone Tissue Engineering

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