Polymer Testing 65 (2018) 163–168

Contents lists available at ScienceDirect

Polymer Testing
journal homepage: www.elsevier.com/locate/polytest

Scaffolds based on chitosan and collagen with glycosaminoglycans cross- T

linked by tannic acid
Beata Kaczmareka,∗, Alina Sionkowskaa, Anna Maria Osyczkab
a
Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University, Toruń, Poland
b
Department of Biology and Cell Imaging, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University in Kraków, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Scaffolds based on chitosan, collagen, and glycosaminoglycans-enriched ones, cross-linked by tannic acid can be
Glycosaminoglycans obtained with the use of the freeze-drying method. Composites were characterized by different analyses, e.g.
Tannic acid SEM images, porosity and density measurements, swelling, liquid uptake, mechanical tests in wet conditions,
Scaffolds and enzymatic degradation by collagenase and hyaluronidase. In addition, the viability of human osteosarcoma
SaOS proliferation
SaOS-2 cells was examined on the obtained scaffolds.
The results showed that the scaffolds based on chitosan, collagen, and glycosaminoglycans cross-linked by
tannic acid are not cytotoxic. Scaffolds are stable in aqueous environment and show high swelling behavior.
Each material porosity is above 90% which is appropriate for the tissue engineering applications. The me-
chanical parameters of the scaffolds decrease with increasing immersion time in PBS. SEM images showed the
homogeneous scaffold structure with interconnected pores. Due to their stability and biocompatibility, the
scaffolds presented here may be easily operated to fit such small bone defects.

1. Introduction GAGs were reported previously [10]. However, materials based on

Glycosaminoglycans (GAGs) are polyanionic linear polysaccharides
with a repeating disaccharide units structure. GAGs are components of
various structural and connective tissues such as skin, cartilage, and
cornea [1]. Each tissue produces GAGs with specific polymeric chains
binded with proteins, enzymes, or ions [2–4]. The connective tissues of
marine organisms are rich in sulfated glycosaminoglycans [5,6]. The
isolation of GAGs from marine organisms, e.g. the Aetobatus narinari
[7], Cyclopterus lumpus [8], Anodonta cygnea [9], or Salmo salar [10] has
already been reported.
Glycosaminoglycans can be identified by the spectrophotometric
assay in the absorption spectrum of 1,9-dimethylmethylene blue (DMB)
[11,12]. The DMB complex can be stabilized by the use of formate
buffer. Another dye type for GAGs identification is Alcian blue [13].
However, the DMB use is a more sensitive method than Alcian blue use
[14].
The obtainment of scaffolds based on collagen with glycosami-
noglycans has already been reported [15–17]. Such materials present
appropriate biocompatibility and are not cytotoxic. GAGs are also
added to chitosan-based materials [18,19]. The obtained composites
were characterized as biocompatible and bioresorbable. Moreover, the
appropriate biological properties of chitosan/collagen scaffolds with
chitosan, or their mixture, exhibit low stability in aqueous environment
and need to be cross-linked to improve their properties.
Tannic acid is a glucose and gallic acid polyphenol. It can be applied
to scaffolds as a cross-linker due to the hydrogen and electrostatic in-
teractions formation [20]. Tannic acid addition to the polymeric scaf-
folds results in the obtainment of materials biocompatible and stable in
aqueous conditions [21,22].
The aim of the study was to obtain scaffolds based on chitosan and
collagen, glycosaminoglycans-enriched ones, cross-linked by tannic
acid. The novel materials were characterized by physicochemical
methods as well as an in vitro experiment. Such scaffolds can find a
potential clinical application to fill small bone or cartilage defects.
2. Materials and methods
2.1. Sample preparation
Collagen was isolated from rat tail tendons in our laboratory.
Chitosan (DD = 77%, Mv = 5.4 × 105 g/mol) and tannic acid were
purchased from Sigma-Aldrich Company (Germany). Polymers were
dissolved in 0.1M acetic acid at 1 wt.% concentration each. The pro-
cedure of glycosaminoglycans (GAGs) isolation was according to the

∗
Corresponding author.
E-mail address: beatakaczmarek8@gmail.com (B. Kaczmarek).

https://doi.org/10.1016/j.polymertesting.2017.11.026
Received 5 September 2017; Received in revised form 21 November 2017; Accepted 25 November 2017
Available online 27 November 2017
0142-9418/ © 2017 Elsevier Ltd. All rights reserved.
B. Kaczmarek et al.
procedure reported previously and the presence of hyaluronic acid and
chondroitin sulfate in the isolated GAGs mixture was noticed by the
spectrophotometric method [10]. The GAGs solution at 1% concentra-
tion in distilled water was prepared.
2.2. Scaffolds preparation
Chitosan and collagen solutions were mixed in the 50/50 wt ratio
[23]. During stirring, 1 and 5 wt.% of the isolated glycosaminoglycans
solution was added. Then, tannic acid was added as a cross-linker in the
20% weight ratio based on the previous studies [20,22]. The obtained
solutions were frozen and lyophilizated at −55 °C and 5Pa (ALPHA 1-
2LD plus, CHRIST, Germany) for 24 h. As a result, 3D porous structures
(scaffolds) were obtained.
2.3. Scaffolds porosity and density
The density and porosity of scaffolds were measured by isopropanol
displacement, because it does not wet the sample [24]. Samples were
put into the known volume of isopropanol (V1). After 5 min the change
in volume of isopropanol-impregnated scaffold was measured. The
sample was removed from the solution and again the difference in
isopropanol volume was determined. The density of the porous sample
(d) was calculated as follows:
W 2.7.1. Collagenase
d=
V2 − V3 (1)
where W is sample weight, V2 is the total volume of isopropanol and
isopropanol-impregnated scaffold, V3 is the isopropanol volume after
sample removing. The porosity (P) of the scaffold was calculated using
the equation:
V1 − V3
P=
V2 − V3 (2)
where V2, V3 as above, V1 is the initial volume of isopropanol.
2.4. Scanning electron microscope
The morphology of the samples was studied using Scanning Electron
Microscope (SEM) (LEO Electron Microscopy Ltd, England). Scaffolds
were frozen in liquid nitrogen for 3 min and gentry cut with a razor
scalpel for the interior structure observation. Samples were covered by
gold and scanning electron microscope images were made with re-
solution 500 μm.
For the cells observation on the scaffold the samples were dried in
the CO2 critical point dryer, attached to the microscope adhesive
holders and covered with a gold layer. The SEM images were taken
under JEOL JSM5410 scanning electron microscope (JEOL, Tokyo,
Japan).
2.5. Swelling properties and liquid uptake
Swelling behavior was measured by immersing the scaffolds in a
phosphate-buffer saline (PBS) solution (pH = 7.4) for 2, 24, 48, 72 h.
After each period of time, the immersed materials were gently dried and
weighed. The swelling ratios were then calculated using equation (3)
[22]:
mS (t ) − mS (0)
swelling [%] = ∗100%
mS (0) (3)
where ms(t) is the weight of the scaffolds after immersion in PBS for the
period of time and ms(0) is the weight before immersion.
Liquid uptake is the percentage liquid content after the scaffolds
immersion. It is related with the scaffolds weight changes. Scaffolds are
placed in the determined PBS mass (m0) and are then removed without
squeezing. The rest of the PBS solution was weighed (mt) and the liquid
164
Polymer Testing 65 (2018) 163–168
uptake was calculated [22]:
mt − m 0
Liquid uptake = ∗100%
m0 (4)
2.6. Mechanical testing
Mechanical properties were measured by mechanical testing ma-
chine (Z.05, Zwick/Roell, Germany) for each kind of sample. Scaffolds
were immersed in PBS for 2, 4, 24, 48 and 72 h. After those periods of
time samples were put between two discs and compressed.
Measurement was carried out in PBS solution (pH = 7.4). The com-
pressive modulus is a Young modulus for the compression process,
where it determines the stiffness of an elastic composite. It was calcu-
lated from the slope of the stress-strain curves in the linear region
(strain from 2 to 5%). The compressive stress is the force on the surface
area which has to be applied to compress the scaffold to 20% of its
height [22]. For each kind of composite at least ten samples were tested
and the standard deviation was calculated.
2.7. Enzymatic degradation
For the enzymatic degradation study, the scaffolds with a de-
termined mass were immersed in 1 ml of 0.1M Tris-HCl (pH = 7.4)
containing 50 mM CaCl2 and incubated in 37 °C for 30 min. 1 ml of
0.1M Tris-HCl containing 50 units of collagenase from Clostridium his-
tolyticum, Type I (Sigma-Aldrich, Poland) was added to the solution.
The scaffolds were incubated at 37 °C for 1 h. The reaction was stopped
by the addition of 0.2 ml 0.25M EDTA. The samples were rinsed with
distilled water and immersed in methanol for 3 h. Then, they were
rinsed with distilled water again, frozen, and lyophilized. The percen-
tage of weight loss was determined by the calculation of mass difference
between scaffolds before and after degradation [24].
2.7.2. Hyaluronidase
The samples with the known weight were immersed in PBS and
placed at 37 °C for 30 min. 1 ml of PBS containing 50 units of hya-
luronidase from bovine testes (Sigma-Aldrich, Germany) was added to
the solution and incubated for 1 h. Then, the samples were rinsed with
distilled water three times and immersed in methanol for 3 h. They
were rinsed with distilled water again, frozen, and lyophilized. The
percentage of weight loss was calculated [25].
2.7.3. In vitro tests
The in vitro testes were carried out with the procedure published
previously [17,22]. Scaffolds (0.5 cm height, 0.12 mm diameter) were
soaked in 70% EtOH (water solution) and washed in sterile phosphate
buffer solution (PBS; pH = 7.4). For the preliminary assessment of
scaffolds suitability for cell growth, human osteosarcoma cell line SaOS-
2 was used [26]. These cells are often the first choice to assess scaffolds
biocompatibility, especially if they are aimed at bone tissue engineering
application [27,28]. 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. Cell-seeded scaf-
folds were examined with the CellTiter96Aqueous One Solution Cell
Proliferation Assay (MTS, Promega, Poland). MTS solution was diluted
10x in phenol-free alpha-MEM and 400 μl aliquots were added per well
per scaffold. The absorbance at 490 nm was measured after 30 min
incubation at 37 °C in the dark [27,28]. Results were expressed as %
change in cell viability compared to results obtained for unmodified
scaffolds.
B. Kaczmarek et al. Polymer Testing 65 (2018) 163–168

Table 1
The density (d) and porosity (P) of scaffolds based on chitosan, collagen and their mixture
enriched by glycosaminoglycans cross-linked by tannic acid addition.

Specimen d [mg/cm3] P [%]

CTS+1%GAG 15.1 ± 1.3 96.2 ± 0.7

CTS+5%GAG 17.4 ± 0.9 97.3 ± 0.5
Coll+1%GAG 7.9 ± 0.8 92.7 ± 0.8
Coll+5%GAG 8.2 ± 0.7 93.4 ± 0.6
CTS/Coll+1%GAG 13.9 ± 0.9 96.9 ± 0.7
CTS/Coll+5%GAG 14.3 ± 0.3 97.1 ± 0.2

3. Results and discussion

3.1. Scaffolds porosity and density

Fig. 2. Swelling behavior of different scaffolds cross-linked by tannic acid based on

The porosity and density of the cross-linked scaffolds based on
chitosan, collagen, and their mixture enriched by glycosaminoglycans
addition are listed in Table 1.
Each scaffold shows the porosity above 90%, which is necessary
when considering the material for tissue engineering applications [29].
The density and porosity increase together with the increasing amount
of glycosaminoglycans added to the sample; however, the changes are
not significant. It is related with the increasing number of hydrogen
interactions between chitosan and collagen as well as glycosami-
noglycans. It causes the structure stabilization and the scaffolds density
and porosity increase. The components content in the scaffold influ-
ences its density and porosity parameters.
3.2. Scanning electron microscope
Scanning electron microscope (SEM) images of chitosan, collagen
and their mixture with 5% glycosaminoglycans addition cross-linked by
tannic acid are shown in Fig. 1.
Each type of scaffold presents a porous structure with inter-
connected pores. Such type of pores is necessary to allow for the nu-
trients flow inside the scaffold and, as a result, cells proliferation within
the whole scaffold volume. Changes in the pores diameter are not sig-
nifically dependent on the sample content. The average pore size for
scaffolds based on chitosan with 5% GAGs addition is 150 μm
( ± 33 μm), and based on collagen with 5%GAGs is 183 μm ( ± 57 μm).
chitosan, collagen, and their mixture with 1 and 5% glycosaminoglycans addition after
immersion in PBS for 2, 24, 48, and 72 h.
The pore size of scaffold based on chitosan/collagen mixture is 180 μm
( ± 29 μm) for 1% GAGs addition and 208 μm ( ± 41 μm) for 5% GAGs
content.
3.3. Swelling properties and liquid uptake
The scaffolds activity after their immersion in PBS was detected by
swelling and liquid uptake measurement (Figs. 2 and 3). Swelling is
related with the increasing scaffolds mass after immersion in the liquid.
The PBS uptake is related with the presence of interconnected pores in
the scaffolds structure and the solution flow inside the sample.
All the scaffolds are highly swellable, which is related with the
presence of hydrophilic groups in the polymeric chains. The highest
percentage of swelling was noticed for the collagen-based scaffolds;
lower swelling behavior was found for the samples based on chitosan
and collagen mixture, and the lowest for samples based on chitosan.
The initial swelling is highest due to the liquid flow inside the scaffold.
Then, the percentage swelling decreases with an increasing immersion
time. There are no significant changes in the liquid uptake values be-
tween the scaffolds; however, for collagen with 5% glycosaminogly-
cans, high decrease in liquid uptake is observed. It can be related with
the high swelling behavior of this scaffold type and result from the

Fig. 1. SEM images of scaffolds cross-linked by tannic

acid addition based on a) CTS+5%GAG b) Coll
+5%GAG c) CTS/Coll+1%GAG d) CTS/Coll
+5%GAG.

165
B. Kaczmarek et al. Polymer Testing 65 (2018) 163–168

Table 2
The percentage of weight loss after the enzymatic degradation by collagenase of scaffolds
based on chitosan, collagen and their mixture with 1 and 5% glycosaminoglycans addi-
tion cross-linked by tannic acid.

Specimen Δm [mg] weight change [%]

CTS+1%GAG 0.7 ± 0.7 3.6 ± 0.4

CTS+5%GAG 0.8 ± 0.2 4.0 ± 0.6
Coll+1%GAG 1.4 ± 0.3 3.5 ± 0.7
Coll+5%GAG 1.2 ± 0.2 6.8 ± 0.3
CTS/Coll+1%GAG 1.8 ± 0.7 5.9 ± 0.6
CTS/Coll+5%GAG 1.7 ± 0.5 5.7 ± 0.4

Table 3
The percentage of weight loss after the enzymatic degradation by hyaluronidase of
scaffolds based on chitosan, collagen and their mixture with 1 and 5% glycosaminogly-
Fig. 3. Liquid uptake of PBS solution to the scaffolds cross-linked by tannic acid based on cans addition cross-linked by tannic acid.
chitosan, collagen, and their mixture with 1 and 5% glycosaminoglycans addition after
Specimen Δm [mg] weight change [%]
immersion in PBS for 2, 24, 48, and 72 h.
CTS+1%GAG 12.4 ± 1.1 41.3 ± 1.7
CTS+5%GAG 11.9 ± 0.8 40.9 ± 1.2
Coll+1%GAG 6.7 ± 0.5 10.7 ± 0.7
Coll+5%GAG 5.8 ± 0.4 10.5 ± 1.3
CTS/Coll+1%GAG 6.9 ± 0.7 25.6 ± 0.8
CTS/Coll+5%GAG 6.4 ± 0.3 24.2 ± 0.5

Fig. 4. Compressive modulus (Emod) of scaffolds cross-linked by tannic acid based on

chitosan, collagen, and their mixture with 1 and 5% glycosaminoglycans addition, cross-
linked by tannic acid.

Fig. 6. SaOS-2 viability (mean ± standard deviation bars) *p < 0.05 vs. CTS+1%GAG;
#p < 0.05 vs. Coll+1%GAG, Student t-test.

Fig. 5. Maximum compressive force (Fmax) of scaffolds based on chitosan, collagen, and
their mixture with 1 and 5% glycosaminoglycans addition, cross-linked by tannic acid.

closing of its pores.

All the scaffolds kept the cylindrical shape and did not dissolve after
immersion in PBS. Previous studies showed that scaffolds without cross-
linking are not stable in aqueous environment and a cross-linker ad-
dition is necessary to improve their stability [22].
Fig. 7. SEM image of scaffold based on chitosan/collagen with 5% glycosaminoglycans
addition cross-linked by tannic acid (magnification 1 × 1500).
3.4. Mechanical testing

The compressive modulus (Emod) (Fig. 4) and maximum

166
B. Kaczmarek et al.
compressive force (Fmax) (Fig. 5) were determined for scaffolds cross-
linked by tannic acid enriched with glycosaminoglycans.
The scaffolds immersion before measurement allows imitating the
conditions inside the body. All the cross-linked scaffolds were in the
cylindrical shape before and after immersion which allowed for the
mechanical testing. Initial measurements showed the highest mechan-
ical parameters of the scaffolds based on chitosan and chitosan/col-
lagen mixture. However, after 48 and 72 h, swelling was advanced and
the compressive modulus and maximum compressive force decreased.
After immersion for a period longer than 72 h, mechanical parameters
did not change significantly (results not showed). The use of the chit-
osan and collagen mixture improves the material stability which was
observed as the lowest difference before the first and the last mea-
surement. The amount of glycosaminoglycans did not significantly in-
fluence the scaffolds mechanical properties.
3.5. Enzymatic degradation
3.5.1. Collagenase
The percentage of weight loss as a result of enzymatic scaffolds
degradation by collagenase is listed in Table 2. The results showed the
weight change in the range of 3.5–6.8%. The highest weight loss was
noticed for the collagen scaffold with 5% glycosaminoglycans addition.
The addition of chitosan to collagen improves the material stability
against collagenase.
3.5.2. Hyaluronidase
The results of percentage weight loss after the enzymatic degrada-
tion by hyaluronidase are shown in Table 3. The enzymatic degradation
by hyaluronidase resulted in scaffolds weight changes higher than in
the case of collagenase. The changes are in the range of 10.5% for
collagen, 25% for chitosan/collagen, and 41% for chitosan. All the
scaffolds are sensitive to the enzymatic degradation by hyaluronidase;
however, the presence of chitosan improves this sensitivity. The content
of glycosaminoglycans does not significantly influence the obtained
results.
3.5.3. In vitro tests
The viability of SaOS-2 cells on each scaffold type are shown in
Fig. 6. CTS+5% GAG presented higher cell viability in comparison to
the same scaffolds with 1% GAG or two Coll-based scaffolds with the
tested GAG concentration. Interestingly, both of the tested CTS/Coll
scaffolds showed higher cell viability compared to single-component
scaffolds. Moreover, 5% GAG addition to such scaffolds increased cell
viability compared to similar scaffolds with 1%GAG. Good bio-
compatibility of the chitosan/collagen scaffolds with glycosaminogly-
cans without cross-linking by tannic acid were previously presented by
our group [10]. Based on these and previous results, it can be concluded
that tannic acid seems to improve the scaffolds biocompatibility [22].
Moreover, an increasing amount of glycosaminoglycans in CTS/Coll
scaffolds results in an increased cells viability. The SEM image of
scaffold based on chitosan/collagen mixture with 5% glycosaminogly-
cans addition cross-linked by tannic acid is shown in Fig. 7. It was
noticed that cells attached to the scaffold surface and the extracellular
matrix formation was initiated.
4. Conclusion
The biocompatible, porous scaffolds cross-linked by tannic acid,
based on chitosan, collagen, and glycosaminoglycans-enriched ones
were obtained. The results showed that the use of chitosan and collagen
mixture improves the scaffolds physicochemical properties and cells
viability. Moreover, the glycosaminoglycans amount did not sig-
nificantly influence the materials properties. However, a higher GAGs
content enhances the Coll/CTS scaffolds biological properties. All the
scaffolds are enzymatically degraded; however, hyaluronidase caused
167
Polymer Testing 65 (2018) 163–168
their higher weight loss than collagenase. These studies indicate that
scaffolds based on chitosan, collagen, and glycosaminoglycans cross-
linked by tannic acid display properties suitable for tissue engineering
applications.
Acknowledgement
Financial support from the National Science Centre (NCN, Poland)
Grant No UMO-2015/19/N/ST8/02176 (BK) is gratefully acknowl-
edged. This work was supported in part by the National Center of
Science (NCN, Poland) grant No UMO-2016/21/B/NZ5/00217 (AMO).
References
[1] W.D. Comper, T.C. Laurent, Physiological function of connective tissue poly-
saccharides, Physiol. Rev. 58 (1978) 255–315.
[2] D.R. Coombe, Biological implications of glycosaminoglycans interactions with
haemopoietic cytokines, Immunol. Cell Biol. 86 (2008) 598–607.
[3] V. Vreys, G. David, Mammalian heparanase: what is the message? J. Cell. Mol. Med.
11 (2007) 427–452.
[4] X. Xu, Y. Dai, Heparin: an intervenor in cell communication, J. Cell. Mol. Med. 14
(2010) 175–180.
[5] E.O. Kozlowski, M.S.G. Pavao, L. Borsig, Ascidin dermatan sulfates attenuate me-
tastasis, inflammation and thrombosis by inhibition of P-selectin, J. Thromb.
Haemost. 9 (2011) 1807–1815.
[6] S. Mizumoto, D. Fingmoon, K. Sugahara, Interaction of chondroitin sulfate and
dermatan sulfate from various biological sources with heparin-binding growth
factors and cytokines, Glycoconj. J. 30 (2013) 619–632.
[7] G. Sadhasivam, A. Muthuvel, A. Pachaiyappan, B. Thangavel, Isolation and char-
acterization of hyaluronic acid from the liver of marine stingray Aerobatusnatinari,
Int. J. Biol. Macromol. 54 (2013) 84–89.
[8] C.G. Panagos, D. Thomson, C. Moss, C.D. Bavington, H.G. Olafsson, D. Uhrin,
Characterization of hyaluronic acid and chondroitin/dermatan sulfate from the
lumpsucker fish, C. Lumpus, Carbohyd. Polym. 106 (2014) 25–33.
[9] M. Lopes-Lima, I. Ribeiro, R.A. Pinto, J. Machado, Isolation, purification and
characterization of glycosaminoglycans in the fluids of the mollusk
Anodontacygnea, Comp. Biochem. Physiol. A 141 (2005) 319–326.
[10] B. Kaczmarek, A. Sionkowska, K. Łukowicz, A.M. Osyczka, The cells viability study
on the composites of chitosan and collagen with glycosaminoglycans isolated from
fish skin, Mat. Lett. 206 (2017) 166–168.
[11] K.B. Taylor, G.M. Jeffree, A new basic metachromatic dye, 1:9-dimethyl methylene
blue, Histochem. J. 1 (1969) 199–204.
[12] J.E. Stone, N. Akhtar, S. Botchway, C.A. Pennock, Interactions of 1,9-di-
methylmethylene blue with glycosaminoglycans, Ann. Clin. Biochem. 31 (1994)
147–152.
[13] W. Gold, A simple spectrophotometric method for estimating glycosaminoglycan
concentrations, Anal. Biochem. 99 (1979) 183–191.
[14] R.W. Ferndale, C.A. Sayers, A.J. Barrett, A direct spectrophotometric microassay for
sulfated glycosaminoglycans in cartilage cultures, Connect. Tissue Res. 9 (1982)
247–248.
[15] B.A. Harley, J.H. Leung, E.C. Silva, L.J. Gibson, Mechanical characterization of
collagen-glycosaminoglycans scaffolds, Acta. Biomater. 3 (2007) 463–474.
[16] E. Farrell, F.J. O'Brien, P. Doyle, J. Fischer, I. Yannas, B.A. Harley, B. O'Connell,
P.J. Prendergast, V.A. Campvell, A collagen-glycosaminoglycan scaffold supports
adult rat mesenchymal stem cell differentiation along osteogenic and chondrogenic
routes, Tissue Eng. 12 (2006) 459–468.
[17] B. Kaczmarek, A. Sionkowska, A.M. Osyczka, Collagen-based scaffolds enriched
with glycosaminoglycans isolated from skin of Salmo salar fish, Polym. Test. 62
(2017) 132–136.
[18] A. Denuziere, D. Ferrier, A. Domard, Interactions between chitosan and glycosa-
minoglycans (chondroitin sulfate and hyaluronic acid): physicochemical and bio-
logical studies, Ann. Pharm. Fr. 58 (2000) 47–53.
[19] F.-Y. Fan, C.-C. Chiu, C.-L. Tseng, H.-S. Lee, Y.-N. Pan, K.-C. Yang,
Glycosaminoglycan/chitosan hydrogel for matrix-associated autologous chon-
drocyte implantation: an in vitro study, J. Med. Biol. Eng. 34 (2014) 211–217.
[20] A. Sionkowska, B. Kaczmarek, K. Lewandowska, Modification of collagen and
chitosan mixtures by the addition of tannic acid, J. Mol. Liq. 199 (2014) 318–323.
[21] V. Natarajan, N. Krithica, B. Madhan, P.K. Sehgal, Preparation and properties of
tannic acid cross-linked collagen scaffold and its application in wound healing, J.
Biomed. Mat. Res. B. Appl. Biomater. 5 (2013) 560–567.
[22] B. Kaczmarek, A. Sionkowska, A.M. Osyczka, The comparison of physic-chemical
properties of chitosan/collagen/hyaluronic acid composites with nano-hydro-
xyapatite cross-linked by dialdehyde starch and tannic acid, Polym. Test. 62 (2017)
171–176.
[23] K. Lewandowska, A. Sionkowska, S. Grabska, B. Kaczmarek, M. Michalska, The
miscibility of collagen/hyaluronic acid/chitosan blends investigated in dilute so-
lutions and solids, J. Mol. Liq. 220 (2016) 726–730.
[24] J. Kozłowska, A. Sionkowska, Effects of different crosslinking methods on the
properties of collagen-calcium phosphate composite materials, Int. J. Biol.
Macromol. 74 (2015) 397–403.
[25] B.F. Ticar, Z. Rohmah, S.I. Mussatto, J.M. Lim, S. Park, B.D. Choi, Hyaluronidase-
B. Kaczmarek et al.
inhibitory activities of glycosaminoglycans from Liparis tessellatus eggs, Carbohyd.
Polym. 161 (2017) 16–20.
[26] S.B. Rodan, Y. Imai, M.A. Thiede, G. Wesolowski, D. Thompson, Z. Bar-Shavit,
S. Shull, K. Mann, G.A. Rodan, Characterization of a human osteosarcoma cell line
(SaOS-2) with osteoblastic properties, Cancer Res. 47 (1987) 4961–4966.
[27] J. Filipowska, G.C. Reilly, A.M. Osyczka, A single short session of media perfusion
induces osteogenesis in hBMSCs cultured in porous scaffolds, dependent on cell
differentiation stage, Biotechnol. Bioeng. 113 (2016) 1814–1824.
168
Polymer Testing 65 (2018) 163–168
[28] J. Filipowska, J. Pawlik, K. Cholewa-Kowalska, G. Tylko, E. Pamula,
L. Niedzwiedzki, M. Szuta, M. Laczka, A.M. Osyczka, Incorporation of sol–gel
bioactive glass into PLGA improves mechanical properties and bioactivity of com-
posite scaffolds and results in their osteoinductive properties, Biomed. Mat. 9
(2014) 065001.
[29] V. Karageorgiou, D. Kaplan, Porosity and 3D biomaterial scaffolds and osteogenesis,
Biomaterials 26 (2005) 5474–5491.