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International Journal of Biological Macromolecules 118 (2018) 938–944
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: http://www.elsevier.com/locate/ijbiomac
In vivo study on scaffolds based on chitosan, collagen, and hyaluronic
acid with hydroxyapatite
B. Kaczmarek a,⁎, A. Sionkowska a, M. Gołyńska b, I. Polkowska b, T. Szponder b, D. Nehrbass c, A.M. Osyczka d
a
Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Poland
Department and Clinic of Animal Surgery, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Głęboka 30 Street, 20-612 Lublin, Poland
AO Research Institute Davos (ARI), Switzerland
d
Department of Biology and Cell Imaging, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University in Kraków, Poland
b
c
a r t i c l e
i n f o
Article history:
Received 10 April 2018
Received in revised form 27 May 2018
Accepted 28 June 2018
Available online 30 June 2018
Keywords:
In vitro
In vivo
Hydroxyapatite
Scaffolds
a b s t r a c t
Scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with nano-hydroxyapatite were obtained with the use of the freeze-drying method. Composites swelling behavior was assessed by the liquid uptake
test. The adhesion and proliferation of human osteosarcoma SaOS-2 cells on the scaffolds were examined in 4day culture. The biocompatibility of the chosen scaffolds was further studied by in vivo implantation into subcutaneous tissue of rabbits.
The results showed low stability of the scaffolds based on chitosan, collagen, and hyaluronic acid supplemented
with hydroxyapatite. The addition of hydroxyapatite delayed the degradation process of the obtained scaffolds.
The X-ray images of the tissues surrounding the scaffolds showed that both, the control scaffold without hydroxyapatite (HAp) and those with addition of 50% wt. HAp underwent degradation after 6 months. However,
the scaffolds supplemented with 80% wt. HAp premained in the implanted place. The results showed satisfactory
tissue response on the implanted scaffolds.
© 2018 Elsevier B.V. All rights reserved.
1. Introduction
Functional materials for biomedical application in regenerative
medicine should be biocompatible and bioactive in order to create the
appropriate stimulating environment for cell attachment and differentiation towards the desired cell phenotype. The implant should also be
well ancored into the tissue to avoid implant loosening and adverse tissue reactions upon implant wear [1].
Based on the above reasons, it is necessary to develop new functional
materials which can easily be implanted into the human body and stay
there to support the target tissue, improve their healing process, and
prevent its further deterioration as a consequence of acute and chronic
inflammation. Several natural compounds are currently examined for
their biocompatibility, degradability, and non-cytotoxicity to eventually
obtain materials which may find use in tissue engineering [2–4]. Collagen and chitosan are among those extensively studied natural polymers
that may find broad range of applications in biomaterial science [5, 6].
The main goal of tissue engineering is to design implant materials
that can support and stimulate damaged tissue regeneration [7]. Several
material improvements are being investigated to obtain new biocompatible substrates and scaffolds. Porous scaffolds are able to create the
⁎ Corresponding author.
E-mail address: [email protected] (B. Kaczmarek).
https://doi.org/10.1016/j.ijbiomac.2018.06.175
0141-8130/© 2018 Elsevier B.V. All rights reserved.
appropriate environment for cells migration, proliferation, differentiation, and eventually for blood vessel invasion [8–10]. For regenerative
medicine applications, any material must be characterized by the physicochemical methods followed by the in vitro and in vivo biological evaluation [11].
Scaffolds obtained from chitosan and collagen have been previously
studied [12] upon their implantation into ear skin and cartilage of rabbits. The immunohistochemical staining of the sections showed that
the biodegrading behavior of those scaffolds was a long-termprocess.
Moreover, it was found that chitosan/collagen scaffolds promoted angiogenesis [13]. Finally, chitosan and collagen mixtures were also studied as materials potentially applicable for skin repair [14]. These results
showed that scaffolds were biocompatible and cells proliferated well on
the scaffolds. Moreover, wound healing was promoted. Based on these
reports, chitosan/collagen scaffolds seem good candidates for tissue engineering applications.
The aim of the study was to examine the chitosan/collagen/
hyaluronic acid scaffolds supplemented with hydroxyapatite for potential tissue engineering applications. The polymeric mixture was chosen
based on the previously performed miscibility studies of chitosan and
collagen [15]. The physicochemical properties of such scaffolds have already been examined [16, 17]. The hydroxyapatite addition improves
mechanical and thermal properties. Moreover, calcium release was detected, which is necessary for bone calcification. In the present study,
B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944
the liquid uptake ability as well as both in vitro and in vivo biological parameters were carried out to assess the scaffolds as potential candidates
to apply in regenerative medicine.
939
made with 200 μm resolution. The scaffolds structure before and after
immersion in PBS was compared.
2.3. Attachment and proliferation of human osteosarcoma SaOS-2 cells
2. Materials and methods
2.1. Sample preparation
Collagen (Coll) was isolated from rat tail tendons [18]. Chitosan
(CTS), hyaluronic acid (HA) and nano-hydroxyapatite (particle size
b 200 nm (BET); HAp) were purchased from Sigma-Aldrich company.
Chitosan was of low molecular weight (DD = 77%, Mv = 5.4
× 105 g/mol [19]) and hyaluronic acid molecular weight was 1.8
× 106 g/mol [20]. Collagen and chitosan solutions were prepared in
0.1 M acetic acid separately. Hyaluronic acid was dissolved in 0.1 M hydrochloric acid at 1% concentration. Chitosan and collagen were mixed
in the 50/50 weight ratio. Hyaluronic acid was added in ratio 1, 2 and
5% to the chitosan and collagen. The amount of collagen, chitosan and
hyaluronic acid were determined by miscibility studies [15]. The
nano-hydroxyapatite was added in ratio 50 and 80% to the polymeric
mixture. After the mixture stirring the samples were placed in the sterile 24-well polystyrene plates and frozen. Samples were then lyophilized at −55 °C and 5 Pa for 48 h (ALPHA 1-2LD plus, CHRIST, Germany).
2.2. Liquid uptake test
Scaffolds were immersed in 15 ml phosphate buffer solution (PBS)
and incubated in 37 °C for 1 h, 1, and 3 days. After the time intervals,
samples were removed. The weight of PBS before the sample immersion
and after the scaffold removal was determined. The liquid uptake was
then calculated:
Liquid uptake ¼
mt −m0
100%
m0
where mo is the scaffold weight before immersion (with dry surface)
and mt after the specific immersion time. The scaffolds after the time intervals were washed with distilled water to remove PBS salt. Then, they
were frozen and lyophilized. The liquid uptake analysis was repeated in
triplicate.
The morphology of the samples was studied using Scanning Electron
Microscope (SEM) (LEO Electron Microscopy Ltd., England). The scaffolds were frozen in liquid nitrogen for 3 min and gently cut with a
razor scalpel for the interior structure observation. The samples were
covered with gold and scanning electron microscope images were
For cell culture, scaffolds (0.5 cm height, 0.12 mm diameter) were
soaked in 70% EtOH (water solution) followed by washing in sterile
PBS (pH = 7.4). The scaffolds were then transferred to 24-well plates
and left for 24 h to dry out. For the preliminary assessment of cell attachment and proliferation, we have chosen human osteosarcoma cell line
SaOS-2. Although these cells are of tumorous origin, they are broadly
used in literature for the initial assessment of biological compatibility
of implant materials. Also, these cells resemble the osteoblast phenotype and respond similarly to normal human osteoblasts [21]. Cells
were seeded at the density of 15 × 104 cells/scaffold and cultured for
total of 4 days in alpha-MEM supplemented with 10% fetal bovine
serum (FBS) and antibiotics. At day 1 culture, cell-seeded scaffolds
were transferred to new culture plates and covered with fresh culture
medium. The initial plates were examined with the CellTiter96Aqueous
One Solution Cell Proliferation Assay (MTS, Promega, Poland) for any remaining cells that had not adhered to the scaffolds. Briefly, MTS solution
was diluted 10× in phenol-free alpha-MEM and 200-μl aliquots were
added per well. The absorbance at 490 nm was measured after 30 min
incubation at 37 °C in the dark. At day 4 of culture, the viability of cells
was examined directly on the scaffolds using similar procedure as of
day 1, except that the scaffolds were covered with 400-μl aliquots of
10× diluted MTS. This assay was successfully used by us previously to
examine cell viability on polyurethane [22] and PLGA-bioactive glass
scaffolds [23]. Results were expressed as relative change in cell viability
on HAp-modified scaffolds compared to results obtained for unmodified
scaffolds. For statistical analysis, the value of p b 0.05 was considered
significant.
2.4. In vivo implant studies
The study was conducted on a group of 8 male New Zealand White
rabbits, at an age of X-Y weeks and a body weight (BW) ranging from
2.6 to 3.7 kg. The rabbits were purchased from the Center for Experimental Medicine at the Medical University of Silesia in Katowice. Animals were placed in the animal house for a 1-week adaptation to the
new environment. All the animal surgical procedures were carried and
approved by the II Local Ethical Committee for Experiments on Animals
in Lublin (Agreement no. 16/2010).
On the surgery day, each animal was premeditated by intramuscular
injection of medetomidine at a dose of 0.5 mg/kg BW (Domitor, Orion
Fig. 1. The liquid uptake of different scaffolds (chitosan/collagen mixture with 1% wt. hyaluronic acid addition – CTS/Coll/1HA, with 50% - CTS/Coll/1HA + 50%HAp respectively with 80%
hydroxyapatite addition – CTS/Coll/1HA + 80%HAp, etc.).
940
B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944
The implantation site was prepared by shaving the thoracic hair in
half of the ribs length on the left side of the body, at the subscapular region. The skin was disinfected with alcohol and iodine, and prepared for
a sterile operation. A vertical 1 cm long cut was made with a scalpel between the 7th and 8th rib. Then, the sterile material was placed into the
subcutaneous pocket. The pocket was sutured with single absorbable
Pharma, Finland) and butorphanol at a dose of 0.2 mg/kg BW
(Butomidor, Richter Pharma, Austria). After 10 min, an intravenous
catheter was placed into the marginal vein of the ear. General anesthesia was maintained by the intravenous administration of ketamine
(Bioketan, Biowet, Poland) at a dose of 30–50 mg/kg BW. The anesthesia
period for each animal lasted 30 min on average.
dry
1h
2 4h
Fig. 2. SEM images with 200 μm resolution of a) CTS/Coll/2HA b) CTS/Coll/2HA + 80%HAp c) CTS/Coll/5HA d) CTS/Coll/5HA +80%HAp, respectively, for dry scaffold, scaffolds after 1 h and
24 h of immersion in PBS.
B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944
sutures made of PGLA Lactic 4- 0 (YAVO, Poland) and the skin - with
sealed with Nylon 3-0 (YAVO, Poland). The identical surgery procedures
were performed on the right side of the subscapular region. The following scaffolds were implanted separately: chitosan/collagen supplemented with 2% wt. hyaluronic acid and 80% wt. hydroxyapatite (CTS/
Coll/2HA + 80%HAp), CTS/Coll supplemented with 5% wt. hyaluronic
acid and 50% wt. HAp (CTS/Coll/5HA + 50%HAp) or 80% wt. HAp
(CTS/Coll/5HA + 80%HAp), or unmodified scaffolds without HAp
(CTS/Coll/5HA). Four rabbits (R1–R4) were implanted with CTS/Coll/
2HA + 80% HAp on the left side and with CTS/Coll/5HA + 80%HAp on
the right side. The other four rabbits (R5–R8) were implanted with
CTS/Coll/5HA + 50%HAp on the left side and with CTS/Coll/5HA on
the right side.
After surgery, all the rabbits were individually housed in cages. In
order to minimize the risk of infection, they were given enrofloxacin
at a dose of 5 mg/kg BW IM side (Enrobioflox 5%, Vetoquinol, Poland).
The antibiotic was administered for 3 days after surgery. The skin sutures were removed 10 days after the surgery.
Six months after implantation, blood samples were taken for hematological and biochemical studies and the animals were euthanized with
medetomidine at 0.5 mg/kg BW in combination with ketamine
25 mg/kg BW and finally pentobarbital (Morbital, Biowet, Poland) at a
dose of 2–5 mg/kg BW. Skin samples with the subcutaneous tissue
from the place where the material was initially implanted were taken
for further analyses.
2.5. Hematological and biochemical parameters
Blood tests included the assessment of white blood cells (WBC), red
blood cells (RBC), and platelets (PLT) amounts using a Scil Vet ABC Plus
+ hematology analyzer (Horiba Medical). Serum creatinine (CR) and
urea nitrogen (BUN) levels as well as alanine aminotransferase (ALT)
and aspartyl aminotransferase (AST) activities were assessed. The
markings were made with the use of the biochemical analyzer, Mindray
BS-120. The values given by Winnicka [24] were corrected for the above
parameters.
2.6. Radiological assessment
Radiological images of the harvested skin samples were taken with
Planmeca Romexis, Finland and exposure parameters of 40 kV, 30 mA,
300 mAs.
2.7. Histological assessment
The tissue samples (1.5 × 1.5 cm) harvested from the implant site
were trimmed and then fixed in a 10% buffered formalin solution.
Then, they underwent standard histological processing. Sections of
5–6 μm thickness were stained with hematoxylin and eosin (HE) and
examined under with a brightfield light microscope.
3. Results and discussion
Fig. 3. SaOs-2 cell viability at day 4 of culture comparing the different scaffolds (chitosan
and collagen mixed in 50/50 weight ratio with 1% wt. hyaluronic acid addition – CTS/
Coll/1HA, with 2% - CTS/Coll/2HA, and with 5% hyaluronic acid – CTS/Coll/5HA) with 50
and 80% wt. HAp addition (*p b 0.05 vs. CTS/Coll/1HA; **p b 0.05 vs. CTS/Coll/2HA; # p b
0.05 vs. CTS/Coll/5HA; Student t-test).
The scaffolds with 1% wt. HA show an increase in liquid uptake when
increasing the HAp content, most pronounced at 3 d. Compared to those
with 1% wt. HA, the scaffolds with 2 and 5% wt. HA content showed a decrease in liquid uptake once hydroxyapatite was added (50 or 80% wt.
HAp). Note the effects of erosion, compared to the scaffolds without
HAp the process starts earlier and is already visible after 5 d in the scaffolds with 80% wt. HAp. As the liquid uptake is related with the scaffolds
porosity – a higher value of the liquid uptake may suggest that the scaffold presents higher porosity [25].
The degradation of different scaffold types was detected by SEM observation of the material structure before immersion in PBS, after 1 h
and 24 h (Fig. 2). The observed scaffold pores diameter was bigger
when compared to that of the scaffolds with dry surface. The images
show that the erosion process resulting from PBS salt presence was initiated during the immersion of all the scaffold types with and without
HAp addition.
3.2. Attachment and proliferation of human osteosarcoma SaOS-2 cells
Human SaOS-2 cells attached similarly to all studied scaffolds although day 1 analyses showed that the scaffolds with HAp supported
cell attachment better when compared to the unmodified ones. This
was based on the observation that less cells remained on the plates
1 day post-cell seeding after removing the modified scaffolds vs. unmodified ones (results not shown). Day 4 analyses (Fig. 3) showed
that the addition of HAp significantly improved cell attachment to the
scaffolds and cells viability. We could actually observe that increasing
Table 1
Results of blood cell count and biochemical blood tests in the study group 6 months after
surgery.
Rabbits
3.1. Liquid uptake tests
The liquid uptake ability of the scaffold depended on its composition.
The scaffolds absorbed high amounts of water, in the range of
2659–5205% after 1 h of immersion, 3168–10,463% after 3 h, and
2671–4987% after 24 h (Fig. 1). The highest liquid uptake after 1 day is
related to the liquid flow through the scaffold and the pores filling.
After 3 days, the material degradation process caused by PBS salt has already been initiated, which is observed as liquid uptake decrease. However, the scaffolds of CTS/Coll with 1% wt. HA addition and high amount
of HAp are more stable in PBS solution when compared to the scaffolds
without inorganic particles.
941
R1
R2
R3
R4
R5
R6
R7
R8
Reference
values
Evaluated parameters
WBC
(109/l)
RBC
PLT
(1012/l) (109/l)
ALT
(U/l)
AST
(U/l)
CR
BUN
(mg/dl) (mg/dl)
14.1
12.6
10.3
11.0
8.9
11.2
7.6
7.8
2.0–15.0
8.7
8.0
7.6
7.5
6.8
7.4
8.1
8.0
4.0–9.0
43
32
38
33
38
43
44
37
31–53
48
56
74
45
50
51
50
68
42–98
70.8
189.0
146.3
208.3
107.1
91.0
166.8
201.0
44–234
698
632
586
735
574
623
716
528
120–800
18.0
19.0
17.6
24.5
21.2
17.8
14.2
14.0
13–30
Note: White blood cell count (WBC), red blood cell count (RBC), platelet level (PLT), creatinine (CR) and blood urea nitrogen (BUN) levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity evaluated for every implanted rabbit.
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B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944
Fig. 4. The radiological evaluation of A) CTS/Coll/2HA + 80%HAp; B)CTS/Coll/5HA + 50%HAp, C) CTS/Coll/5HA + 80%HAp 6 months after surgery. Arrowhead: implanted scaffold.
Fig. 5. The histological sections of the tissue surrounding A) CTS/Coll/5HA B) CTS/Coll/5HA + 50%HAp and C) CTS/Coll/5HA + 80%HAp 6 months after implantation (Scale bar indicates 100
μm (left) and with 12.6 × 20 magnification (right). T: marks the subcutismainly consisting of fibrous tissue, N: nerve, A: arteriole, C: capillary, S: skeletal muscle, E: elastic fibers).
B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944
amount of inorganic compounds in the scaffolds corresponded with increased cell attachment and viability. Similar results were presented by
Jain et al. [25], who showed that addition of HAp to polyamide improves
the MSCs cells attachment and proliferation. We have also showed that
the combination of bioactive glass and HAp increases human MSC viability and promotes their osteogenic differentiation [26]. On the other
hand side, we could observe that supplementing the scaffolds with
hyaluronic acid does not influence cells attachment and viability.
Based on the above in vitro studies, we have chosen the following
scaffolds for further in vivo analyses: chitosan and collagen mixture
supplemented with 2% wt. hyaluronic acid and 80% wt. hydroxyapatite
(CTS/Coll/2HA + 80%HAp); CTS/Coll enriched with 5% wt. hyaluronic
acid (CTS/Coll/5HA) and 50% wt. HAp (CTS/Coll/5HA + 50%HAp) or
80% wt. HAp (CTS/Coll/5HA + 80%HAp). We assumed these scaffolds
had the highest biocompatibility and could serve as potential regenerative medicine substitute.
3.3. In vivo studies
3.3.1. Macroscopic observation
The post-operative wounds were healed with no complications. In
the surrounding tissues, there were no inflammatory changes, swelling,
or congestion. Upon the 6-month implantation schedule, the rabbits
showed no signs of general or local health problems.
3.3.2. Hematological and biochemical parameters
Hematological (WBC, RBC, PLT) and biochemical blood test results
(ALP, AST, creatinine, urea nitrogen) were within the reference values
(Table 1).
The laboratory tests results 6 months after surgery were within the
reference range for all the implanted rabbits. Animals showed no symptoms of disease or general inflammation associated with the implantation of the materials used.
3.3.3. Radiological assessment
The results of the radiological examination are shown in Fig. 4. Six
months after the materials enriched with HAp had been implanted,
they were visible on X-rays. The scaffold of CTS/Coll/5HA was not captured by X-rays because it had degraded.
3.3.4. Histological assessment
The implantation period allowed us to evaluate the granulation and
tissue healing phase. The first one is usually identified by the presence of
angioblasts, fibroblasts, and collagen synthesis [27]. The images (Fig. 5)
allow to observe the physiological structures of the rabbit skin as lamina
subcutanea with elastic and collagen fibers and nerves. The scaffold
made of chitosan and collagen with 5% wt. hyaluronic acid addition
biodegraded because of its low stability. In contrast, scaffolds with addition of HAp were still visible at the implantation place after 6 months. In
the scaffold, the fibroblasts presence was noticed. They grew into the
scaffolds with the HAp addition (Fig. 5b and c). The scaffold morphology
was similar to the surrounding tissue [27]. For the scaffolds with HAp,
the formation of blood vessels (capillaries) was observed, which is essential for tissue regeneration. Moreover, the vascularization was observed as physiologic for subcutis consisting of mid-sized vessels often
in pairs as well in trias with nerve.
The results showed that scaffolds based solely on natural polymers
such as chitosan, collagen, and hyaluronic acid undergo degradation
and the HAp presence changes the long-term stability. Summarized,
the analyses of cell and tissue responses to the studied materials
showed no inflammation at the implantation sites. The implants did
not elicit any adverse or unexpected reactions at the adjacent soft
tissues.
Many reports have indicated the accelerating effects of chitosan and
its derivatives on wound healing [28]. Because of its antimicrobial properties, chitosan has been widely investigated as an antimicrobial agent
943
used for preventing infections, which is advantageous in the case of a
substance used as an implant component [29].
Host reactions following biomaterials implantation include injury,
blood-material interactions, acute or chronic inflammation, and granulation tissue development as a reaction of the host to foreign body. Depending on the extent of an injury, the acute inflammatory response to
biomaterials at the implant site usually occurs quickly, within a few
days. Then, chronic inflammation characterized by the presence of
mononuclear cells (i.e. monocytes and lymphocytes) at the implant
site develops. The presence of chronic inflammatory responses for a period longer than 3 weeks usually indicates an infection. After the chronic
inflammation stage, new healthy tissue can be identified, the granulation tissue characterized by macrophages presence, fibroblasts infiltration, and neovascularization [30].
4. Conclusions
In this study, we verified the biocompatibility of chitosan, collagen,
and hyaluronic acid-based scaffolds enriched with nanohydroxyapatite. Cell culture studies showed improved cell attachment
and cell growth on the scaffolds when enriched with HAp, whereas
the in vivo examination of the tissues surrounding the scaffolds
6 months after implantation showed the overall satisfactory wound
healing, lack of inflammation of the surrounding tissue caused by the
implants. We can assume that the HAp addition to the CTS/Coll/HA scaffolds slows down the implant biodegradation process and creates a scaffold that provides longer stability to the surrounding tissues. The
implantation for periods longer than 6 months should be studied further for the scaffolds with hydroxyapatite addition.
Acknowledgement
Financial support from the National Science Centre (NCN, Poland)
Grant no. UMO-2013/11/B/ST8/04444 is gratefully acknowledged.
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