KAUNAS UNIVERSITY OF TECHNOLOGY
ODETA BANIUKAITIENĖ
CELLULOSE/HYDROXYAPATITE COMPOSITES FOR
BONE TISSUE ENGINEERING
Summary of Doctoral Dissertation
Physical Sciences, Chemistry (03P)
2014, Kaunas
1
The research was carried out at Kaunas University of Technology, Faculty of
Chemical Technology, Department of Polymer Chemistry and Technology in
the period of 2010–2014 under support of the Lithuanian State Science and
Studies Foundation, the Science Council of Lithuania Foundation.
Scientific supervisor:
Prof. Dr. Jolanta LIESIENĖ (Kaunas University of Technology, Physical
Sciences, Chemistry – 03P).
Board of the Chemical Science Field:
Prof. Dr. Habil. Algirdas ŠAČKUS (Kaunas University of Technology,
Physical Sciences, Chemistry – 03P) – chairman;
Prof. Dr. Habil. Marek BRYJAK (Wroclaw University of Technology,
Poland, Physical Sciences, Chemistry – 03P);
Prof. Dr. Saulius GRIGALEVIČIUS (Kaunas University of Technology,
Physical Sciences, Chemistry – 03P);
Assoc. Prof. Dr. Jolita OSTRAUSKAITĖ (Kaunas University of
Technology, Physical Sciences, Chemistry – 03P);
Dr. Arvydas ŪSAS (Lithuanian University of Health Sciences, Biomedical
Sciences, Medicine – 06B).
The official defence of the Dissertation will be held at the public meeting of
Board of Chemical Science Field at 1 p.m. on December 12, 2014 in the
Rectorate Hall at the Central Building of Kaunas University of Technology.
Adress: K. Donelaičio St. 73–402, LT-44029, Kaunas, Lithuania.
Tel. (370) 37 300042; fax (370) 37 324144; e-mail: [email protected]
The send out date of the Summary of the Dissertation is on 12th of November,
2014.
The Dissertation is available at the library of Kaunas University of
Technology (K. Donelaičio St. 20, LT-44239, Kaunas).
2
KAUNO TECHNOLOGIJOS UNIVERSITETAS
ODETA BANIUKAITIENĖ
CELIULIOZĖS/HIDROKSIAPATITO KOMPOZITAI KAULO
AUDINIO INŽINERIJAI
Daktaro disertacijos santrauka
Fiziniai mokslai, chemija (03P)
2014, Kaunas
3
Disertacija rengta 2010–2014 metais Kauno technologijos universitete,
Cheminės technologijos fakultete, Polimerų chemijos ir tecnologijos
katedroje, remiant Lietuvos valstybiniam mokslo ir studijų fondui ir Lietuvos
mokslo tarybai.
Mokslinė vadovė:
Prof. dr. Jolanta LIESIENĖ (Kauno technologijos universitetas, fiziniai
mokslai, chemija – 03P).
Chemijos mokslo krypties taryba:
Prof. habil. dr. Algirdas ŠAČKUS (Kauno technologijos
fiziniai mokslai, chemija, 03P) – tarybos pirmininkas;
Prof. habil. dr. Marek BRYJAK (Vroclavo technologijos
fiziniai mokslai, chemija – 03P);
Prof. dr. Saulius GRIGALEVIČIUS (Kauno technologijos
fiziniai mokslai, chemija, 03P);
Doc. dr. Jolita OSTRAUSKAITĖ (Kauno technologijos
fiziniai mokslai, chemija, 03P);
Dr. Arvydas ŪSAS (Lietuvos sveikatos mokslų universitetas,
mokslai, medicina – 06B).
universitetas,
universitetas,
universitetas,
universitetas,
biomedicinos
Disertacija bus ginama viešame Chemijos mokslo krypties tarybos posėdyje
2014 m. gruodžio 12 d. 13 val. Kauno technologijos universiteto centrinių
rūmų Rektorato salėje.
Adresas: K. Donelaičio g. 73–402, LT-44029, Kaunas, Lietuva.
Tel. (370) 37 300042; faksas (370) 37 324144; el.paštas: [email protected]
Disertacijos santrauka išsiųsta 2014 m. lapkričio 12 d.
Disertaciją galima peržiūrėti Kauno technologijos universiteto bibliotekoje
(K. Donelaičio g. 20, LT-44239, Kaunas).
4
1.
INTRODUCTION
Relevance of the work
The loss of teeth is associated not only with changes in the esthetical
appearance, but also with chewing dysfunction, which leads to a rapid bone
atrophy. The artificial implants can be an alternative for teeth. It is the teeth
size and shape imitating construction, which is placed in the jaw. The
implantation of such constructions which replace the teeth roots and together
with fixed crowns perform the function of the teeth is one of the fastest
growing areas of dentistry. However, the clinical experience shows that one
in five patients have bone loss; thus, it would be impossible to screw in an
implant. According to the statistics, about 20% of implantation procedures
require the augmentation of the jaw bone.
Autogenic bone is known as the best suitable for bone tissue
transplantation. Despite additional operation and possible infections,
autogenic bone due to its excellent osteoinductive, osteoconductive, and
osteogenic properties is still the standard and has no better suitable
alternatives.
For bone defects augmentation, allogenic bone (donor) or xenogenic
bone are also used. However, these are generally associated with disease
transmission. In addition, xenogenic bone transplantation is limited due to the
ethical predeterminations.
The synthetic materials such as calcium phosphates including βtricalcium phosphate (β-TCP), hydroxyapatite (HA) and their composites are
also used. However, the clinical practice shows that sometimes after
implantation particles agglomerate, and the blood vessels can not grow in.
Such construction should be removed.
Nowadays, the development of three-dimensional (3D) scaffolds for
bone regeneration is one of the current challenges in tissue engineering. A
variety of materials are proposed for the fabrication of 3D scaffolds. Usually,
synthetic polymers are used, especially polycaprolactone.
Significant attention is focused on natural polymers due to their
advantages over synthetic polymers, such as biocompatibility, non-toxicity,
hydrophilicity, and controllable biodegradability. It is supposed that in this
area cellulose is very promising. Although cellulose has long been used in
various industries (e.g., paper, textile, pharmaceutical, cosmetic), its use for
bone scaffolds due to the specific morphology has not yet been studied.
5
The aim of the study was to create cellulose/hydroxyapatite
composites and to investigate their possibility to be used for bone tissue
engineering.
In order to achieve the aim set out above, the following tasks
should be carried out:
 to select a suitable method for the preparation of well-designed
cellulose-based scaffolds;
 to mineralize the cellulose matrix by imitating biomineralization in
vitro in a simulated body fluid;
 to develop cellulose/hydroxyapatite composite scaffolds by
mechanically immobilizing hydroxyapatite particles of different size
during cellulose regeneration;
 to study the elemental composition, morphology and mechanical
properties of the cellulose matrix and composite scaffolds;
 to assess the mineralization process within scaffolds in vitro in a
simulated body fluid;
 to investigate the diffusion kinetics of glucose through the porous
scaffolds;
 to assess the cytotoxicity of the scaffolds in vitro;
 to assess the biocompatibility of the scaffolds in vivo.
Scientific novelty and practical value of the work
For the first time, cellulose and its composites with hydroxyapatite
were prepared for bone tissue engineering applications. The application of a
natural polymer which is non-cytotoxic, biocompatible and slowly
absorbable in the human organism is the main advantage of this technology.
For the first time it has been shown that the morphology of cellulose
xerogel obtained by lyophilization can be varied by changing the solvent
inside the gel prior lyophilization and its freezing temperature. The prepared
composite scaffolds exhibited the morphology suitable for bone tissue
regeneration. The size of pores is suitable for cell adhesion, proliferation, and
angiogenesis. The pores are interconnected, revealing nutrients diffusion.
During biomimetic mineralization the mineral layer occurs within scaffolds.
The results of cytotoxicity in vitro and biocompatibility in vivo have shown
that cellulose/hydroxyapatite composites have a great potential for bone
tissue renegeration.
6
Statements presented for defense:
1. The morphology of regenerated cellulose xerogel, obtained by
lyophilization, depends on the solvent inside the gel prior lyophilization
and its freezing temperature.
2. Macroporous cellulose/hydroxyapatite composites are suitable for bone
tisue regeneration.
Approbation of the research results
The results of the research were presented in 21 publications. Among
them, 2 articles were published in journals included in the list of Thomson
Reuters Web of Knowledge: “Cellulose Chemistry and Technology”
(accepted for publication), “BioMed Research International”. Two
publications were in other Thomson Reuters Web of Knowledge databases of
refereed journals (Proceedings). Also, the results were reported in 17
scientific conferences, among them 15 International ones.
Structure and contents of the dissertation
The dissertation consists of the introduction including the research
objectives, literature overview, experimental part, results and discussion,
conclusions, list of references (163), and a list of publications on the
dissertation topic. The material of the dissertation is presented on 97 pages
including 5 tables and 46 figures.
2.
EXPERIMENTAL
Materials. For the present study, cellulose diacetate (substitution
degree 2.4) was obtained from Roshal (Russia). Hydroxyapatite was
purchased from Sigma-Aldrich (USA). MG-63 (ATCC® CRL-1427™) cells
were obtained from the American Type Culture Collection (USA).
Methods. The cellulose-based gel was prepared by the regeneration of
cellulose from cellulose diacetate. The gel samples were washed with
ethanol–water solutions and lyophilized in a Christ ALPHA 2-4 LSC (Martin
Christ Gefriertrocknungsanlagen GmbH, Germany) freeze dryer for up to 24
h. Their morphology was studied using a high resolution field emission
scanning electron microscope Quanta 200 FEG (FEI Company,
Netherlands). The pore size was calculated using ImageJ version 1.47
software (National Institutes of Health, USA).
The microcomputer tomography analysis was performed at a b-cube
company (Switzerland) using a µCT40 system (Scanco Medical AG,
Netherlands) for the morphological characterisation of the scaffolds.
7
The in vitro mimetic biomineralization was carried out by immersing
the samples into a simulated body fluid (SBF). The electrolyte solution was
refreshed once a week. Changes in the weight of the scaffolds after
biomineralization were presented as percentages of mass increase.
X-ray diffraction (XRD) was used for the identification of the calcium
phosphate phase. Diffraction patterns were recorded on a DRON-6
(Bourevestnik Inc., Russia) using a Cu Kα radiation at 30 kV and 20 mA.
Infrared spectroscopy (IR) was used to analyse the chemical structure
of cellulose before and after modification; 4 mg of cellulose was mixed with
200 mg of KBr for the preparation of transparent pellets. All spectra were
recorded in the range from 4000 to 400 cm-1 on a Perkin Elmer FT-IR
spectrometer (Perkin Elmer, England).
The SEM equipped with the energy dispersive spectrometer Quanta
200 with a detector XFlash 4030 (Bruker AXS Microanalysis GmbH,
Germany) was used for the elemental analysis.
The biological characterization of the scaffolds was conducted with
human osteoblastic cells (ATCC® CRL-1427™) for a period of 7 days.
Colonized materials were evaluated throughout the culture time by DNA
content and by SEM in order to address the cell proliferation. DNA was
analyzed by the PicoGreen DNA quantification assay according to the
manufacturer’s instructions.
The mechanical properties of the cellulose matrix and composite
scaffolds were studied using the H25KT system (Tinus Olsen, England).
Scaffolds were loaded under compression using a crosshead speed of 1
mm/min with a 5000 N loud cell.
Glucose diffusion within scaffolds was evaluated using “side-by-side”
cell. The amount of diffused glucose was determined by the phenol-sulfuric
acid method. According to this method samples were analyzed with a
spectrofotometer Varian Cary 50 UV-VIS (Varian, Germany) reading the
absorbance at λ = 490 nm.
In vivo studies were approved by the Committee of the State Food and
Veterinary Service of Lithuania (No. 0208). The scaffolds were implanted
subcutaneously in the back of mice and harvested after 2 weeks, 1 and 3
months of implantation for histological examination.
3.
3.1
RESULTS AND DISCUSSION
Preparation of the cellulose-based matrix
By saponification of cellulose acetate in solution, a homogeneous
semi-rigid gel was prepared. Its pores, determined by the gel-inversion
8
chromatography are accessible to molecules of the molecular mass approx.
500000 Da. However, such pores are too small for the formation of bone
tissue as pores from 100 µm up to 1000 µm are required.
Lyophilization was chosen to create a highly porous matrix with an
optimal pore size required for a successful bone regeneration.
Before lyophilization, samples of the gel were washed with ethanol–
water solutions (ethanol concentration ranged from 0 to 40%).
It was noticed that the gel changed its volume depending on the
ethanol concentration. When the gel was in water it was swollen. However,
when ethanol was added into the system the affinity of the hydrophilic
polymer to the solvent decreased, what caused the structural collapse with an
increased density of the polymer network. Such samples were frozen at
different temperatures (-25 ºC or -80 ºC) and lyophilized.
The scanning electron microscopy (SEM) photographs (Fig. 1 and Fig.
2) showed that the structure of the gel depended on the solvent in its
discontinuous phase and on its freezing rate prior to the drying process. The
morphology of the obtained scaffolds varied from the macroporous to dense
structure.
9
Fig. 1. SEM photographs of the lyophilized cellulose matrix* from: (a) water; (b) 10%
ethanol; (c) 15% ethanol; (d) 20% ethanol; (e) 25% ethanol; (f) 40% ethanol
*
The matrix was pre-frozen at -25 ºC
As demonstrated by the SEM photograph (Fig. 1 a), the effect of
expanded frozen water destroyed the structure of the gel after it had been
filled with water, frozen at -25 ºC, and lyophilized. The obtained matrix was
expanded by about 5%. The majority of pores were larger than 1000 µm.
This kind of a matrix would be unsuitable for the bone scaffold as very large
pores decrease the surface area and limit the cell attachment.
10
The use of 15% ethanol solution shrunk the gel slightly; thus, the
matrix contained pores still rather large in the diameter (Fig. 1 b). The ideal
morphology for the bone tissue growth was obtained by filling the cellulose
gel before its freeze-drying with 20% ethanol solution. The pore size ranged
from micro to macro scale (Fig. 1 d). The SEM showed that pores distributed
through the matrix homogeneously. Lyophilization of gels with 25% or 40%
ethanol solutions gave opposite results (Fig. 1 e, f).
Fig. 2. SEM photographs of the lyophilized cellulose matrix* from: (a) water; (b) 10%
ethanol; (c) 15% ethanol; (d) 20% ethanol; (e) 25% ethanol; (f) 40% ethanol
*
The matrix was pre-frozen at -80 ºC
11
Figure 2 demonstrates the morphology of the scaffolds obtained by
lyophilization of the samples which had been pre-frozen at -80 ºC.
Comparing the SEM photographs (Fig. 1 and Fig. 2) it was assumed that the
faster freezing (at a lower temperature) results in smaller pores. In our case,
faster freezing is inexpedient as clearly large or very small pores are formed
(Fig. 2 a, b, c, d, e). The lyophilization of the gel from 40% of ethanol gave
an almost non-porous matrix (Fig. 2 f).
It can be concluded that the matrix of an optimal structure was
obtained by lyophilization of cellulose gel fulfilled with 20% ethanol and
frozen at -25 ºC.
3.2
Cellulose/hydroxyapatite composites
In order to enhance the bioactivity of the cellulose matrix with the
bone tissue, the composites of cellulose with hydroxyapatite were prepared
by two different methods: (i) mimicking bone biomineralization in a
simulated body fluid, and (ii) reinforcing the cellulose gel with the synthetic
hydroxyapatite (HA).
3.2.1 Cellulose-based composites obtained by biomimetic mineralization
For the development of a mineral layer by means of biomimetic
mineralization, an increased concentration of calcium ions is required as they
accelerate the nucleation rate of the crystals. Polymers with, e.g., hydroxyl,
carboxyl and silanol groups are used due to their capacity to induce apatite
nucleation. Thus, the cellulose matrix had been pre-treated using three
different methods: (i) treating freeze-dried cellulose samples with CaCl2 and
(NH4)2HPO4 solutions; (ii) storing the carboxymethylated cellulose matrix in
a saturated Ca(OH)2 solution; (iii) treating the lyophilized samples with
Si(OC2H5)4 : H2O : C2H5OH : HCl : CaCl2 solution.
The XRD, IR, SEM and EDS confirmed that the mineral phase forms
on the cellulose-based scaffolds after a few days of immersion in a simulated
body fluid. The highest mineralization rate was achieved on the
carboxymethylated cellulose matrix which was activated with the Ca(OH)2
solution. The mass percentage of the mineral phase was two times larger
(12%) after exposure to 1.5×SBF for 14 days as compared with the others.
The largest aggregates appeared on this kind of scaffold. This was clearly
visible in a SEM micrograph (Fig. 3 d).
12
Fig. 3. SEM micrographs of the surface of: (a) the cellulose matrix treated with CaCl2
and (NH4)2HPO4 solutions and then (b) soaked in 1.5×SBF; (c) the matrix activated
with Ca(OH)2 and then (d) soaked in 1.5×SBF; (e) the matrix treated with the
Si(OC2H5)4 : H2O : C2H5OH : HCl : CaCl2 solution and then (f) soaked in 1.5×SBF
The biological characterization of the cellulose matrix (control
scaffold) and the three developed mineralized cellulose samples was
conducted with human osteoblastic cells.
Assessment of DNA content showed that the cellulose matrix supports
the proliferation of the osteoblastic cells, with increasing values throughout
the 7 day culture period. Comparatively, significantly increased values were
observed for the mineralized cellulose matrices, suggesting a higher number
of attached cells over these substrates.
Cultures grown on carboxymethylated cellulose presented, throughout
the culture time, the highest values for the total DNA content. However,
differences among the three mineralized samples were not statistically
significant (Fig. 4).
13
Fig. 4. Osteoblastic cells proliferation over the cellulose and the mineralized cellulose
scaffolds
The observed results prove that the developed cellulose porous matrix
is not cytotoxic and can be used in contact with biological systems. In
addition, the presence of a calcium phosphate layer in the originally prepared
cellulose matrix clearly improves cell adhesion and proliferation.
3.2.2 Cellulose reinforced with hydroxyapatite
Cellulose/HA scaffolds were produced by the regeneration of cellulose
from its acetylated derivative and mechanically immobilizing
nanohydroxyapatite (nHA) and microhydroxyapatite (µHA) particles. Highly
porous cellulose/nHA and cellulose/µHA scaffolds were produced by freezedrying hydrogel filled with 20% of ethanol and frozen at -25 ºC.
It was found that HA improved the mechanical properties of the
cellulose matrix. The Young's modulus increased up to 9 MPa.
For the morphological characterisation of the prepared scaffolds,
micro-computed tomography was chosen. 2D images showed that HA
particles affected the morphology of the prepared scaffolds: differences in
pore size, framework thickness, and their distribution appeared (Fig. 5, Table
1).
14
The cellulose matrix as well as composite scaffolds comprised nonsymmetrical interconnected pores. Such arrangement of the pores is
particularly important for cellular activity and achieving the optimum rate of
the new tissue growth.
Fig. 5. 2D images: (a) cellulose matrix; (b) cellulose/nHA scaffold; (c) cellulose/µHA
scaffold
The structural parameters, such as the percent framework volume (Xv),
porosity (P), specific surface (SS), mean framework thickness (L), mean pore
diameter (D) within the scaffold were determined from 3D images (Fig. 6).
Fig.6. 3D images: (a) cellulose matrix; (b) cellulose/nHA scaffold; (c) cellulose/µHA
scaffold
The structural parameters of the regenerated cellulose matrix and its
composites with nHA and µHA are summarized in Table 1. For comparison,
the structural parameters of the human jaw bone are included.
15
Table 1. Structural parameters of the scaffolds and natural bone
Structural parameters
Samples
Xv, %
P, %
SS, mm-1
L, mm
D, mm
Cellulose scaffold
25
75
15
0,21
0,75
Cellulose/nHA scaffold
28
72
19
0,12
0,49
Cellulose/µHA scaffold
34
66
13
0,21
0,54
Anterior maxilla
1228
7288
1226
0,140,31
0,540,96
Anterior mandible
1447
5386
914
0,280,40
0,630,99
Posterior maxilla
730
7093
1228
0,120,32
0,501,03
Posterior mandible
749
5193
830
0,130,41
0,441,77
The microcomputer tomography data showed that the porosity of
cellulose scaffold was larger, leading to a reduced percentage of the
framework volume, as compared with its composites with nHA and µHA.
The largest pores also appeared within the cellulose scaffold. The mean
diameter of the pores was of 750 µm (Table 1).
The framework thickness with immobilized HA particles of different
composite scaffolds was different (Table 1). The frameworks of
cellulose/µHA scaffold were almost twice thicker, as compared with
frameworks of the cellulose/nHA composite.
The specific scaffold surface was found to be larger of cellulose/nHA
due to thinner frameworks and a higher number of them per millimeter, as
supposed. Thus, the scaffold had smaller pores. The mean pore diameter was
of 490 µm (Table 1).
Generally, the morphology of the prepared scaffolds for bone tissue
engineering meets the requirements of materials which are implanted in the
bone defect and have the possibility to provide space for the cells to
synthesize collagen and mineralize it.
The structural parameters (Xv, P, SS, L, D) of composite scaffolds were
compared with those of the human jaw bone. It was found that the structural
parameters of the cellulose scaffold with nHA particles were in the range of
the anterior maxilla, posterior maxilla and posterior mandible values. The
specific surface of the cellulose/nHA scaffold was significantly increased, as
compared with the anterior mandible. The porosity of the cellulose/µHA
scaffold was not in the range of the anterior and the posterior maxilla. Thus,
16
this kind of composite scaffold would be suitable only for the regeneration of
bone tissue in the mandible area.
The morphology of the scaffold should be appropriate for the nutrients
and waste exchange; otherwise, necrotic regions within the material could
appear. In order to ensure an excellent morphology of the scaffolds, a glucose
diffusion through the scaffold layer was evaluated using a “side-by-side” cell.
60
Diffused glucose (%)
50
1
2
40
3
30
20
10
0
0
4
8
12
16
20
24
28
32
Time (h)
Fig. 7. Glucose diffusion through: 1 – cellulose scaffold, 2 – cellulose/μHA scaffold,
3 – cellulose/nHA scaffold
It should be noted that the diffusion of glucose within cellulose-based
scaffolds was influenced by the morphology of the composites (Fig. 7). The
faster rate of diffusion was observed within cellulose scaffold. After 0.5 h,
the diffused glucose reached up to 15%. After 24 h, the concentration of
glucose was equal in both chambers.
As the porosity of the cellulose/nHA scaffold was higher than of
cellulose/µHA, the faster rate of diffusion was expected. However, the results
were opposite, due to larger pores of cellulose/µHA than expected. It is
important to note that both scaffolds were permeable to the glucose solution.
After 24 h, the concentration of glucose was approximately equal in both
chambers, revealing that the structure of cellulose-based scaffolds is suitable
for nutrients transport.
Studies in biomimetic mineralization showed that the size of HA
particles as well as the morphology of the scaffold effected the mineralization
17
process. It was found that the mass percentage of the mineral phase was
largest on the cellulose/nHA scaffold (Table 2).
Table 2. Mass increase on scaffolds after mineralization
Time,
weeks
Mass increase, %
Cellulose/nHA
scaffold
Cellulose/µHA scaffold
1
4 ± 0.5
1± 0.3
3
5 ± 0.2
5± 0.6
6
13 ± 0.7
11± 0.6
9
18 ± 0.3
13 ± 0.7
12
22 ± 0.7
15 ± 0.5
The cellulose/nHA composite accelerated the nucleation of crystals
more than cellulose/µHA due to the presence of more crystalization centers
than supposed. Moreover, the specific surface of cellulose/nHA was larger
than of cellulose/µHA, thus, there was more space for mineral formation.
In order investigate the cytotoxicity of the cellulose/nHA and
cellulose/µHA scaffolds, human osteoblastic cells were cultured on novel
scaffolds. The presence of nHA or µHA in the scaffolds improved cell
adhesion, as shown by the higher DNA content at day 1, found in these
scaffolds. Also, a higher cell proliferation was evident throughout the culture
time as compared with the control cellulose matrix (comp. Fig. 4 and Fig. 8).
18
Fig. 8. Osteoblastic cell culture proliferation over the cellulose/nHA and
cellulose/µHA scaffolds
The DNA content after 3 and 7 days was higher in the cellulose/nHA
scaffold. More cells were found in this kind of scaffold due to the better
morphology of the scaffold for cell adhesion and proliferation. Further, there
is an evidence that the presence of nHA promotes cell adhesion and growth,
due to the biomimetic nature of the resulting surface as in the bone there is
also apatite of nanosize.
In vitro studies with human osteoblastic cells have confirmed that
scaffolds are not cytotoxic and can be used in contact with biological
systems. However, a cellulose/nHA scaffold can be a better candidate for
bone tissue engineering than cellulose/µHA. Moreover, the scaffold with
nHA displayed the morpholocical parameters closer to those of the native
bone. Thus, it was decided to use this kind of scaffol for in vivo studies.
Cellulose/nHA scaffolds were implanted subcutaneously in the back of
mice. After 2 weeks, 1 and 3 months of implantation the scaffolds were
harvested for histological examination.
It should be noted that all mice remained alive after the implantation
periods as well as after the surgery. No wound complications were observed.
After 2 weeks of implantation, the histological analysis showed that
there was the inflammatory response of the surrounding tissue. The scaffold
was surrounded with a fibrous and collagenous tissue capsule. However, the
histological analysis of specimens harvested after 1 and 3 months revealed
19
the biocompatibility of the scaffold (Fig. 9). The connective tissue
proliferated within the scaffold, angiogenesis was also expressed.
Fig. 9. Histological analysis of the scaffold
after 3 months of implantation (40 ×)
From the in vivo results it can be concluded that the cellulose/nHA
scaffold has a possibility to be used as an implantable material in bone
defects.
CONCLUSIONS
1. It has been shown that the cellulose gel is suitable for the formation of
frameworks for bone tissue engineering. The morphology of the scaffold
could be varied changing conditions before lyophilization, e.g., changing
the solvent inside the gel prior to lyophilization and its freezing rate. The
morphology suitable for bone regeneration was successfully created by
the lyophilization of the regenerated cellulose gel filled with 20% ethanol
and frozen at -25 ºC.
2. It was found that mineral layer formation on the cellulose matrix (treated
with a solution of CaCl2 and (NH4)2HPO4; carboxymethylated and stored
in a saturated Ca(OH)2 solution; treated with Si(OC2H5)4 : H2O : C2H5OH
: HCl : CaCl2 solution) could be achieved in a simulated body fluid. The
highest mineralization rate was achieved on the matrix of
carboxymethylated cellulose activated with Ca(OH)2 solution. Such
mineralized scaffold showed best osteoconductive properties.
3. A new method was developed for the preparation of cellulose composites
with the micro- and nanosize particles of hydroxyapatite. The composites
of cellulose with embedded hydroxyapatite particles were prepared by
20
4.
5.
6.
7.
8.
mechanically immobilizing hydroxyapatite particles of different size
during the regeneration of cellulose. The cellulose scaffold with
nanohydroxyapatite displayed the morpholocical parameters closer to the
native bone than did cellulose with microhydroxyapatite.
It was found that a faster mineralization of cellulose occurs with
embedded nanohydroxyapatite. After 12 weeks the mass increased by
22%.
The mechanical properties of cellulose-based frameworks increase with
immobilizing hydroxyapatite particles. The prepared composites
exhibited Young᾽s modulus of 6–9 MPa.
Despite structural differences, both scaffolds with nano- and
microhydroxyapatite are permeable to the glucose solution. The rate of
diffusion is faster within the cellulose scaffold with embedded
microhydroxyapatite due to larger pores.
In vitro studies with human osteoblastic cells have confirmed that
scaffolds are not cytotoxic. The higher cell growth rate was evident
throughout the culture time on cellulose with embedded
nanohydroxyapatite particles.
In vivo results with mice have revealed the biological properties of the
cellulose scaffold with immobilized nanohydroxyapatite. The histological
analysis confirms that the proliferation of connective tissue and
angiogenesis processes occurs within scaffold.
LIST OF SCIENTIFIC PUBLICATIONS ON THE TOPIC OF THE
DISSERTATION
Articles in the journals included in the list of Thomson Reuters Web of
Knowledge
1. Petrauskaite, Odeta; Juodzbalys, Gintaras; Viskelis, Pranas; Liesiene,
Jolanta. Control of a porous structure of cellulose-based tissue
engineering scaffolds by means of lyophilization // Cellulose Chemistry
and Technology. 2014. [Thomson Reuters Web of Knowledge] [Impact
factor: 0.833] (accepted).
2. Petrauskaite, Odeta; Gomes, Pedro de Sousa; Fernandes, Maria Helena;
Juodzbalys, Gintaras; Stumbras, Arturas; Maminskas, Julius; Liesiene,
Jolanta; Cicciu, Marco. Biomimetic mineralization on a macroporous
cellulose-based matrix for bone regeneration // BioMed Research
International. ISSN 2314-6133. 2013, vol. 2013, p. 1 –9. [Thomson
Reuters Web of Knowledge]. [Impact factor: 2.880].
21
Articles in the other reviewed Thomson Reuters Web of Knowledge
Database [Proceedings and others]
1. Petrauskaitė, Odeta; Liesienė, Jolanta; Vitkauskas, Vytas; Jaraminaitė,
Monika; Juodžbalys, Gintaras; Daugėla, Povilas; Stumbras, Artūtas;
Maminskas, Julius. Cellulose reinforced with hydroxyapatite for bone
tissue engineering // Polymer Chemistry and Technology: Proceedings of
Scientific Conference Chemistry and Chemical Technology. 25 April,
2012, Kaunas University of Technology. Kaunas: Technologija. ISSN
2029-2457. 2012, p. 93–96.
2. Petrauskaitė, Odeta; Vitkauskas, Vytas; Liesienė, Jolanta; Stumbras,
Artūras; Maminskas, Julius; Juodžbalys, Gintaras. Tissue engineering:
cellulose scaffolds for bone replacement // Polymer Chemistry and
Technology: Proceedings of Scientific Conference Chemistry and
Chemical Technology. 27 May 2011, Kaunas University of Technology.
Kaunas: Technologija. ISSN 2029-2457. 2011, p. 37–40.
Papers of scientific conferences
1. Petrauskaite, Odeta; Juodzbalys, Gintaras; Stumbras, Artūras;
Maminskas, Julius; Daugela, Povilas; Gomes, Pedro de Sousa; Liesiene,
Jolanta. In vitro and in vivo biological analysis of cellulose-based
scaffolds for bone tissue engineering // 8th International Conference on
Polymer and Fiber Biotechnology (IPFB), May 24–28, 2014, Braga,
Portugal: book of abstracts, 2014, p. 95.
2. Jaraminaite, Monika; Petrauskaite, Odeta; Jaruseviciute, Ieva; Liesiene,
Jolanta. Glucose diffusion within cellulose/hydroxyapatite scaffolds //
Chemistry and Chemical Technology: Proceedings of International
Conference of Lithuanian Chemical Society, Chemistry and Chemical
Technology. 25 May 2014, Kaunas University of Technology. ISSN
2351-5643. 2014, p. 317–320.
3. Petrauskaite, Odeta; Liesiene, Jolanta; Juodzbalys, Gintaras; Maminskas,
Julius; Stumbras, Artūras; Gomes, Pedro de Sousa; Fernandes, Maria
Helena; Costa, Maria Elisabete Jorge Vieira. Cellulose with embedded
hydroxyapatite nanoparticles for bone tissue in-growth // MiMe-Materials
in Medicine: International Conference 1st edition, October 8–11, 2013,
Faenza, Italy: final programme and abstract book. National Research
Council of Italy, Institute of science and technology for Ceramics. 2013,
p. 216.
4. Petrauskaite, Odeta; Liesiene, Jolanta; Jaruseviciute, Ieva; Juodzbalys,
Gintaras; Maminskas, Julius; Stumbras, Artūras; Daugela, Povilas.
22
Cellulose/hydroxyapatite frameworks for bone tissue regeneration //
Baltic Polymer Symposium 2013, September 18–21, 2013, Kaunas,
Lithuania: programme and abstracts. Vilnius University. ISBN978-609459-227-0. 2013, p. 153.
5. Maminskas, Julius; Stumbras, Artūras; Juodzbalys, Gintaras; Liesiene,
Jolanta; Petrauskaite, Odeta; Gomes, Pedro de Sousa; Costa, Maria
Elisabete Jorge Vieira. Tissue engineering perspective in bone tissue
regeneration: cellulose-hydroxyapatite composite scaffold // The Fifth
International BOA Congress, September 13–14, 2013, Kaunas, Lithuania:
programme and abstract book. Baltic Osseointegration Academy. ISBN
978-609-95550-0-3. 2013, p. 24–25.
6. Petrauskaitė, Odeta; Liesienė, Jolanta; Juodžbalys, Gintaras; Gomes,
Pedro de Sousa; Costa, Maria Elisabete Jorge Vieira. Nanohydroxyapatite/cellulose scaffolds // 3rd International Conference on
Multifunctional, Hybrid and Nanomaterials, March 3–7, 2013, Sorrento,
Italy: abstracts. Oxford: Elsevier, 2013. p. 1.
7. Jaraminaitė, Monika; Vitkauskas, Vytas; Petrauskaitė, Odeta. Cellulose
matrix for bone tissue regeneration in defect size // Chemija ir cheminė
technologija, gegužės 4, 2012, Klaipėda, Lietuva: pranešimų medžiaga.
Klaipėdos universitetas / Klaipėdos universiteto leidykla. ISBN978995518-651-9. 2012, p. 91–93.
8. Petrauskaite, Odeta; Liesiene, Jolanta; Catarina, Santos; Gomes, Pedro de
Sousa; Garcia, Monika; Fernandes, Maria Helena; Almeida, Maria
Margarida; Costa, Maria Elisabete Jorge Vieira; Juodzbalys Gintaras;
Daugela, Povilas. Nano-hydroxyapatite/cellulose composite scaffold for
bone tissue engineering // Journal of Tissue Engineering and Regenerative
Medicine. Special Issue: 3rd TERMIS World Congress 2012. ISSN:
1932-6254. 2012, vol. 6, p. 34.
9. Daugela, Povilas; Juodzbalys, Gintaras; Liesiene, Jolanta; Petrauskaite,
Odeta; Gomes, Pedro; Costa, Elisabete. A novel cellulose-hydroxyapatite
scaffold for bone tissue regeneration // Clinical Oral Implants Research.
Special Issue: EAO 18 th Annual Scientific Meeting. ISSN 09057161.
2012, vol. 23, p. 230.
10. Stumbras, Artūras; Maminskas, Julius; Juodžbalys, Gintaras; Liesienė,
Jolanta; Petrauskaitė, Odeta; Darinskas, Adas. Aloplastinio celiuliozėshidroksiapatito kaulo karkaso biologinių savybių analizė: tyrimai in vitro
ir in vivo // LSMU SMD Jaunųjų tyrėjų ir mokslininkų konferencija 2012,
gegužės 24–25, 2012, Kaunas, Lietuva: LSMU SMD JMTK tezių knyga,
II dalis. Lietuvos sveikatos mokslų universitetas. 2012, p. 295.
23
11. Petrauskaite, Odeta; Liesiene, Jolanta. 3D matrix based on regenerated
cellulose for bone tissue regeneration // Baltic Polymer Symposium 2012,
September 19–22, 2012, Liepaja, Latvia: programme and proceedings.
Tallinn University of Technology. 2012, p. 73.
12. Maminskas, Julius; Stumbras, Artūras; Juodzbalys, Gintaras; Liesiene,
Jolanta; Petrauskaite, Odeta; Gomes, Pedro; Costa, Maria Elisabete Jorge
Vieira. Analysis of aloplastic cellulose-hydroxyapatite bone scaffold
biological features // The Fourth International BOA Congress, September
7–8, 2012, Kaunas, Lithuania: programme and abstract book. Baltic
Osseointegration Academy. ISBN 978-9955-905-11-0. 2012, p. 33.
13. Petrauskaitė, Odeta; Jaraminaitė, Monika; Liesienė, Jolanta. Porous
cellulose matrix for bone tissue engineering // EUPOC 2012,
Europolymer Conference, June 3–7, 2012, Gargnano, Italy: booklet of
abstracts. Gargnano: European Polymer Federation, 2012. p. 101.
14. Juodzbalys, Gintaras; Daugela, Povilas; Liesiene, Jolanta; Petrauskaite,
Odeta; Gomes, Pedro; Costa, Elisabete. Bone tissue regeneration in
cellulose/hydroxyapatite scaffold // The 6th International ACBID
Congress, May 30 - June 3, 2012, Antalya, Turkey: abstract book, p. 91.
15. Petrauskaite, Odeta; Vitkauskas, Vytas; Liesiene, Jolanta; Stumbras,
Artūras;
Maminskas,
Julius;
Juodzbalys,
Gintaras.
Cellulose/hydroxyapatite composites for bone tissue engineering // 10th
International Conference of Lithuanian Chemists "Chemistry 2011",
October 14–15, 2011, Vilnius, Lithuania: programme and abstracts.
Lithuanian Academy of Sciences. ISBN 9789955634652. 2011, p. 86.
16. Maminskas, Julius; Stumbras, Artūras; Juodzbalys, Gintaras; Liesiene,
Jolanta; Petrauskaite, Odeta. Bone tissue engineering: polimeric cellulosehydroxyapatite scaffold as bone grafting material. Synthesis and analysis
// The Third International BOA Congress, September 29 - October 1,
2011, Kaunas, Lithuania: programme and abstract book. Baltic
Osseointegration Academy. ISBN 978-9955-905-08-0. 2011, p. 26–27.
17. Petrauskaitė, Odeta; Liesienė, Jolanta. Cellulose/hydroxyapatite
composites as scaffolds for replacement of demaged bone // Baltic
Polymer Symposium 2011, September 21–24, 2011, Pärnu, Estonia:
program and abstracts. Tallinn: Tallinn University of Technology, p. 91.
24
CURRICULUM VITAE
Name, surname:
Date of birth and place:
Edutation:
June 2008
Experience:
September 2010 – Present
September 2011 – Present
May 2007 – May 2011
Fellowships:
February 2006 – May 2006
Odeta Baniukaitiene
11th of November, 1982 in Šilalė
Kaunas University of Technology, Faculty of
Chemical Technology, Master of Science in
Applied Chemistry
PhD studies at Kaunas University of
Technology,
Department
of Polymer
Chemistry and Technology, Faculty of
Chemical Technology
Assistant
at
Kaunas
University of
Technology,
Department
of Polymer
Chemistry and Technology, Faculty of
Chemical Technology
Chemist–analyst, Company „Valentis“, Drug
Quality Control Department, Kaunas
Erasmus student at Wageningen University,
Laboratory of Physical Chemistry and
Colloid Science, Netherlands
ACKNOWLEDGEMENTS
I would like to express my deepest appreciation to Prof. Dr. Jolanta
Liesiene for being my supervisor, for her support throughout all this time. I
am very grateful to Prof. Dr. Gintaras Juodžbalys and his students for their
advices and help. I am grateful to Pedro de Sousa Gomes for the
collaboration. I would like to thank to all the colleagues with whom I have
had the pleasure to work. I am grateful to my family and friends for warm
support and understanding.
REZIUMĖ
Temos aktualumas
Dantų praradimas yra susijęs ne tik su estetinės išvaizdos pokyčiais,
bet ir su kramtymo funkcijos sutrikimu, kas sukelia greitą kaulinio audinio
atrofiją (tirpimą). Prarastų dantų alternatyva – implantai. Tai dantų dydį,
formą ir funkciją imituojančios konstrukcijos, kurios įsriegiamos
25
žandikaulyje buvusių dantų vietoje. Dantų implantavimas – tai bene
greičiausiai besivystanti odontologijos sritis. Senstanti populiacija, didelis
dantų implantavimo procedūrų sėkmės procentas (92–95 % dešimties metų
laikotarpyje), nuolatiniai moksliniai tyrimai, ieškant būdų kaip supaprastinti
dantų implantaciją bei sparti technologijų pažanga lemia augantį dantų
implantavimo procedūrų populiarumą visame pasaulyje. Tačiau praktika
rodo, jog maždaug vieno iš penkių pacientų žandikaulio kaulo nepakanka
implantui įsriegti, todėl apie 20 % dantų implantavimo procedūrų reikalauja
kaulo priauginimo operacijos.
Dažnai tam pasirenkamas autogeninis (nuosavas) kaulas. Nors
transplanto naudojimas susijęs su papildomomis operacijomis ir galimomis
komplikacijomis, bet dėl savo puikių osteoindukcinių, osteokondukcinių ir
osteogenetinių savybių jis vis dar laikomas standartu, kuriam iki šiol nėra
tinkamų alternatyvų.
Kaulo defekto vietai užpildyti naudojamas ir alogeninis (donoro)
kaulas, tačiau baiminamasi dėl ligų pernešimo, o ksenogeninės (gyvulinės)
kilmės kaulo ribotas naudojimas neretai susijęs su etiniais įsitikinimais.
Esama ir sintetinių augmentacinių medžiagų. Tarpe jų, populiariausi – βtrikalcio fosfatas (β-TCP), hidroksiapatitas (HA) ir jų kompozitai. Po
operacijos augmentacinė medžiaga turėtų peraugti tikru kaulu, tačiau neretai
užsitęsia uždegimai, įvyksta dalelių aglomeracija, neįauga kraujagyslės ir šis
darinys turi būti pašalintas. Siekiant išvengti šių problemų, neorganinės
medžiagos yra įkomponuojamos trimačiuose polimeriniuose karkasuose.
Trimačių karkasų, taikomų kaulo regeneracijai defektų srityse,
sukūrimas yra vienas iš dabartinių iššūkių audinių inžinerijoje. Karkasų
gamybai naudojamos įvairios medžiagos. Dažniausiai pasirenkami sintetiniai
polimerai, tarpe jų, plačiausiai naudojamas poli(ε-kaprolaktonas). Kadangi
sintetiniai polimerai pasižymi blogesniu biosuderinamumu nei gamtiniai,
pastaruoju metu didelis dėmesys skiriamas gamtinių polimerų panaudojimui
kaulo audinio inžinerijoje. Manoma, kad šioje srityje didelę perspektyvą turi
celiuliozė. Celiuliozė ir jos dariniai jau seniai naudojami įvairiose pramonės
šakose (popieriaus, tekstilės, farmacijos, kosmetikos ir kt.), tačiau jos
naudojimas kaulo audinio inžinerijoje dėl savitos morfologijos, tyrinėtas
mažai.
Disertacijos darbo tikslas – sukurti celiuliozės/hidroksiapatito
kompozitus ir ištirti jų panaudojimo kaulo audinio inžinerijai galimybes.
Tikslui pasiekti keliami uždaviniai:

parinkti metodą, kuris užtikrintų tinkamos morfologijos celiuliozės
karkaso gavimą;
26







mineralizuoti
celiuliozės
karkasą,
taikant
imitacinę
biomineralizaciją in vitro modeliniame kūno skystyje;
suformuoti celiuliozės kompozitus su skirtingo dydžio
hidroksiapatito dalelėmis celiuliozės gelio gavimo metu;
ištirti celiuliozės karkaso bei kompozitų elementinę sudėtį,
morfologiją bei mechanines savybes;
ištirti biomineralizacijos procesą karkasuose in vitro modeliniame
kūno skystyje;
ištirti gliukozės difuzijos kinetiką per akytus kompozitus in vitro;
įvertinti karkasų citotoksiškumą in vitro;
įvertinti kompozitų biosuderinamumą in vivo.
Mokslinio darbo naujumas ir praktinė vertė
Pirmą kartą kaulo audinio inžinerijoje pasiūlyta panaudoti regeneruotos
celiuliozės/hidroksiapatito kompozitus. Privalumai – naudojamas ne
sintetinis, o gamtinis polimeras, kuris yra necitotoksiškas, biologiškai
suderinamas, organizme rezorbuojasi lėtai.
Pirmą kartą parodyta, kad celiuliozės kserogelio, gaunamo
liofilizacijos būdu, morfologija priklauso tiek nuo tirpiklio, esančio
pertraukiamojoje gelio fazėje, tiek nuo šaldymo prieš liofilizaciją
temperatūros. Keičiant etilo alkoholio koncentraciją regeneruotos celiuliozės
gelyje, pagaminti trimačiai kompozitai, kurių morfologija atitinka kaulo
audinio regeneracijai keliamus reikalavimus. Akutės savo dydžiu tinkamos
ląstelių adhezijai, proliferacijai bei angeogenezei. Akutės tarpusavyje
susisiekiančios, todėl užtikrina maistinių medžiagų difuziją. Imitacinės
biomineralizacijos metu karkasuose vyksta mineralizacija. Citotoksiškumo
tyrimais in vitro ir biosuderinamumo tyrimais in vivo parodyta, kad
celiuliozės/hidroksiapatito kompozitai yra tinkami kaulo audinio inžinerijai.
Ginamieji disertacijos teiginiai
1. Regeneruotos celiuliozės kserogelio, gaunamo liofilizacijos būdu,
morfologija priklauso tiek nuo tirpiklio, esančio pertraukiamojoje gelio
fazėje, tiek nuo šaldymo prieš liofilizaciją temperatūros.
2. Makroporėtos struktūros celiuliozės/hidroksiapatito kompozitai tinka
kaulinio audinio regeneracijai.
IŠVADOS
1. Parodyta, kad regeneruotos celiuliozės gelis tinka karkasų, skirtų kaulo
audinio inžinerijai, formavimui. Nustatyta, kad celiuliozės gelio
27
2.
3.
4.
5.
6.
7.
8.
morfologija gali būti reguliuojama, keičiant mėginių apdorojimo sąlygas
prieš jų liofilizavimą t.y. keičiant tirpiklį gelyje ir jo užšalimo greitį.
Tinkamos morfologijos karkasas gautas gelį prieš liofilizaciją
inkliuduojant 20 % etilo alkoholiu ir šaldant -25 °C temperatūroje.
Parodyta, kad celiuliozės karkasą galima mineralizuoti imitacinės
biomineralizacijos metodu modeliniame kūno skystyje, prieš tai celiuliozę
apdorojant trimis būdais: (a) veikiant CaCl2 ir (NH4)2HPO4 tirpalais; (b)
karboksimetilinant ir po to veikiant sočiu Ca(OH) 2 tirpalu; (c) veikiant
Si(OC2H5)4 :H2O : C2H5OH : HCl : CaCl2 mišiniu. Nustatyta, kad
mineralizacija sparčiausiai vyksta ant karboksimetilintos celiuliozės,
veiktos Ca(OH)2 tirpalu. Šis karkasas pasižymi didžiausiu
biosuderinamumu ir geriausiomis osteokondukcinėmis savybėmis.
Pasiūlytas naujas metodas celiuliozės su nano- bei mikrodydžių
hidroksiapatito dalelėmis kompozitams gauti, mechaniškai įterpiant
hidroksiapatito daleles celiuliozės gelio formavimo metu. Nustatyta, kad
celiuliozės kompozitų su nanohidroksiapatito dalelėmis struktūrinių
parametrų vertės artimesnės kauliniam audiniui, negu kompozitai su
mikrohidroksiapatito dalelėmis.
Imituojant biomineralizaciją modeliniame kūno skystyje, nustatyta, kad
spartesnė mineralizacija vyksta celiuliozės kompozituose su nanodydžio
hidroksiapatito dalelėmis. Po 12 savaičių masės prieaugis siekia 22 %.
Nustatyta, kad hidroksiapatito imobilizavimas į celiuliozę pagerina
karkaso mechanines savybes. Pagamintų kompozitų Jungo modulis siekia
6–9 MPa.
Nustatyta, kad nepaisant struktūrinių skirtumų, abu kompozitai su nano- ir
mikrohidroksiapatitu yra pralaidūs gliukozės tirpalui. Spartesnė gliukozės
difuzija vyksta per celiuliozės su mikrohidroksiapatitu kompozitą dėl
didesnių akučių jame.
Tyrimais in vitro su žmogaus osteoblastų ląstelėmis patvirtintas
kompozitų cito-tolerantiškumas. Didesnė ląstelių proliferacija vyksta
celiuliozės kompozituose su nanohidroksiapatito dalelėmis.
Tyrimais in vivo su pelėmis patvirtintas celiuliozės kompozitų su
nanohidroksiapatito dalelėmis biosuderinamumas. Atlikus histologinę šių
mėginių analizę nustatyta, kad vyksta jungiamojo audinio proliferacija ir
angiogenezė.
28
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29