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DEVELOPMENT OF SCAFFOLDS BASED ON CHITOSAN, TYPE I
COLLAGEN AND HYALURONIC ACID AS BIOMATERIAL FOR
THREE-DIMENSIONAL CELL CULTURE
Lucas Rabello1, Vicente Trindade2, Ênio Oliveira3, Daniela L. Fabrino3
1Bioprocess Engineering Undergrad student, Federal University of São João del Rei, Ouro Branco (MG), Brasil 2 Metallurgical Engineering Department, Federal University of Ouro Preto, Ouro Preto (MG), Brasil
3Chemistry, Biotechnology and Bioprocess Engineering Department, Federal University of São João del Rei,
Ouro Branco (MG), Brasil
E-mail: [email protected]
Abstract. Biomaterials are used in contact with living tissue and can be implanted to replace or repair tissues
that have been severely damaged. For this purpose, these materials can be used as scaffolds that seek to produce
a microenvironment in an ex vivo field of cultivation, contributing to the progress of regenerative medicine.
Among the biomaterials used in the production of the scaffolds, some biopolymers stand out such as chitosan,
collagen and hyaluronic acid. In this way, this work had as objectives to produce scaffolds through the
combination of these three biopolymers and to analyze its morphology as well as its influence on a VERO cell
culture. Through the lyophilization of the polymer solutions produced, we obtained scaffolds with pores between
10 and 100 μm in diameter. In addition, it was evidenced through optical microscopy that the VERO cell line
cultured on these scaffolds was not rejected. Although quantitative cell viability testing is still required, our
results point to this triad of biopolymers as a promising biomaterial in tissue engineering.
Keywords: three-dimensional cell culture, scaffolds, chitosan, collagen, hyaluronic acid
1. INTRODUCTION
Cell culture emerged at the beginning of the XX century as a method to study cell
behavior free from the uncountable variants on a living organism (FRESHNEY, 2016).
However, conventional cell culture, performed on hard and flat surfaces as culture T-flasks
and Petri dishes may cause cytoskeleton remodeling and cell flattening. These alterations may
lead to nucleus distortion and alter gene expression and protein synthesis (VERGANI et al.,
2004; THOMAS et al., 2002). Therefore, two dimension cell culture (2D) provides a non-
natural condition which leads to the development of faulty cells from the physiologically
point of view (SUN et al. 2006). Attempting to minimize the damage caused to cell
population in vitro three-dimension cell (3D) culture was developed.
The 3D cell culture made possible the production of artificial tissues and organs in
laboratories and the advent of the tissue engineering technology which is devoted to the
development of solutions to provide the substitution of implants, prosthesis and grafts used by
the regenerative medicine (TAVARES, 2011). Therefore, it is important the development of
techniques used on the 3D cell cultures such as the scaffolds, to allow anatomical complex
structures to be artificially synthesized. In this regard, this work offers a helping hand with a
careful literature review and practical assays that contribute to the development of these
structures.
2. LITERARTURE REVIEW
Scaffolds
Scaffolds are porous structures used to induce 3D cell cultures; the grown cells are able to
migrate among their fibers and attach to them forming 3D structures (BRESLIN &
O’DROSCOLL, 2013).
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The desired characteristics on a scaffold include water retention ability, tenacity to hold
the cells in a stretched position; porosity so that the 3D arrangement is possible,
biodegradability to make holes to the budding cells and connectivity to allow oxygen and
nutrient flux (DUTTA, R. & DUTTA, A., 2009).
In addition to providing an appropriate physical structure for cell adherence, the chemical
properties of the scaffolds are also important, as it is known that the adhesion links happen in
a particular way depending on the material that constitutes the scaffolds and through these
links different gene expression that leads to phenotypic alterations of the cell population (KIM
et al., 1999). Thus, the extracellular matrix (ECM) has an important role in the control of cell
growth and differentiation (SOUZA & PINHAL, 2011).
Also, the production of scaffolds can be made with different materials such as the
biopolymers.
Biopolymers
Biopolymers are macromolecules such as polysaccharides, proteins, nucleic acids and
lipids produced by living organisms (IUPAC Gold Book). These biomaterials have the
advantages to be abundant, low cost, similar to ECM, biodegradable with nontoxic products
generated in this process, as well as being biocompatible. Due to these factors, their use is
abundant in several sectors of the biomedical industry and in the scaffolds production (PIRES
et al., 2015). Among these biopolymers the type I collagen, chitosan and hyaluronic acid
(HA) are in a privileged position regarding 3D cell culture due to their particular
characteristics.
Chitosan
Chitosan is a biopolymer formed by monomeric units of N-acetil-D-glucosamine e D-
glucosamine; a highly organized crystalline structure, insoluble in water medium and most of
organic solvents (LARANJEIRA & FÁVERE, 2009).
The main properties of chitosan that lead to its high usage as a biomaterial in 3D related
research are due to its non-toxicity properties, biocompatibility e biodegradability (KIM et al.,
2001), notwithstanding its great flexibility which allows its usage as biomaterial to produce
films, gels and membranes (LARANJEIRA & FÁVERE, 2009). In addition, scaffolds that
incorporate chitosan in their constitution turn up more resistant mechanically when compared
to others (TSAI et al., 2013).
On a neutral pH the chitosan acquires a positive global charge by the protonation of its
amino groups. This property has a great importance to 3D cell culture as it gives this molecule
the ability to make links with glycosaminoglycans, proteoglycans and other negatively
charged molecules in an electrostatic way (SHANDY & SHARMA, 1990). This phenomenon
favors the retention of signaling molecules as growing factors providing better growing
condition to the cell population (LARANJEIRA & FÁVERE, 2009).
Collagen
The collagen is a fibrous protein of the ECM, it is produced by the connective tissue cells
and a variety of cells (ALBERTS, 2010). This protein has structural functions such as being
responsible for forming complexes with glycosaminoglycans which are important in the
retention of signaling molecules and growing factors (SOUZA & PINHAL, 2011).
When dissolved in an acid solution, the collagen aggregates in fibrils with fluted cross
patterns forming highly organized scaffolds with a substantial variety of usages on the 3D cell
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culture (PACAK et al., 2011). However, this biomaterial has the disadvantage of having fast
biodegradability and low stiffiness (ANGELEA et al., 2004). These problems are serious but
can be minimized by using crosslinker agents or by association with other materials during
the production of scaffolds. As an example, the chitosan which interacts with the collagen by
hydrogen bonds allowing the production of hybrid scaffolds with complementary capabilities
on the 3D structures (RAMASAMY & SHANMUGAN, 2014).
Hyaluronic acid
The hyaluronic acid is the simplest of the GAGs, present in the ECM and constituted of a
regular repeated sequence of up to 25 thousands of non-sulfated disaccharides units, found in
variable quantities in all fluids and tissues of adult animals. The hyaluronic acid provides
resistance against compression forces on the tissues and joints, often serving to create free
spaces to where cells can migrate (ALBERTS, 2010).
It has been widely used on scaffolds production due to its biocompatibility and large
capacity to incorporate a wide range of signaling molecules (LAM et al., 2013). It can bind to
proteoglycans and form structures capable to retain signaling molecules (SOUZA &
PINHAL, 2011). Lastly, by being negatively charged at pH near the neutral point, this
molecule attracts cations to its structure and these molecules bring with themselves a large
amount of water turning the HA a highly hydrated molecule (LAM et al., 2013).
Moreover, the addition of HA on the scaffolds has been associated to the increase of cell
proliferation and migration through signaling pathways unleashed by its association to CD44
and RHAMM receptors (ZHU et al., 2006).
It is interesting notice that the cited biopolimers have different properties that when
combined increase the success chance of a 3D cell culture. Therefore, a variety of works that
use these biomaterials at different mixings, in order to provide the best culture conditions, is
found in the literature. (ANGELEA et al., 2004; ZHU et al., 2014; MAHMOUD &
SALAMA, 2016; SIONKOWSKA et al., 2016).
3. OBJECTIVES
This work had as a main objective to produce scaffolds by combining chitosan,
hyaluronic acid and type I collagen to evaluate their morphology and influences on a cell
culture.
4. MATERIAIS AND METHODS
Production of scaffolds
The scaffolds production was done in collaboration with Prof. Dr. Ênio Nazaré de
Oliveira Júnior, and used low molecular weight chitosan extracted from Pandalus borealis
shrimp (92% of desacetylation - Primex ehf), type I collagen from rat tail (4 mg/ml, SIGMA-
ALDRICH) and HA from cockscomb (SIGMA-ALDRICH), used in different mass
proportions (3:0:0; 0:3:0; 0:0:3; 0.05:2.95:0; 0:2,73:0.27; 2.5:0:0.5; 1:1:1 milligrams of
chitosan, type I collagen and HA respectively).
Stock solutions were then prepared (concentration of 0.3% m/v) using 0.5M of acetic acid
as a solvent, from which aliquots were taken to produce the scaffolds at the above cited
proportions.
The polymers solution volumes, corresponding to the defined mass shown above, were
spilled on 24 well culture plates and then frozen. After complete freezing, the solution was
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lyophilized for 4 hours, at -50ºC and 125 μmHg of pressure. Lastly, the scaffolds were
immersed in hydrated ethyl alcohol (70% v/v) for 30 minutes to be cleaned up (asepsis), then
extensively washed with phosphate saline buffer (PBS 1x) to remove the residual alcohol
and/or acid groups.
.
Scaffolds Scanning Electron Microscopy (SEM) analysis
The analysis of the scaffolds surface was done in partnership with Prof Vicente Trindade
PhD, from the Federal University of Ouro Preto, at the Inspect S50 (FEI Company/EDS:
Quantax da Brucker).
Cell Culture Preparation
The VERO cell line was used to perform all the experiments and a master and a work
bank were created to guarantee its maintenance during the whole work. From the work bank
cell were grown in T-25, with DMEM (SIGMA D0822), 10% fetal bovine serum (FBS)
(SIGMA F7524) and 1% of antibiotics (10 mg penicillin; 10 mg streptomycin; 25 μg
amphotericin B per ml – SIGMA A5955)
The cells were then expanded to T-75 flasks until they achieved 70% of confluence. At
this point, the culture was trypsinized (gibco 25200-056, 0.05% work solution). The cell
counting was performed and the volume was adjusted to achieve a 2.5∙104 cell/mL work
suspension. From this suspension, 2 mL were taken (5∙104 cells) and added to each scaffold
which were put in the CO2 incubator at 37ºC for 48 hours.
Cell Viability Analysis
The cell viability was carried out using trypan blue dye 0.4% (SIGMA-ALDRICH). The
culture medium was removed and the scaffolds washed with PBS to remove dead or non-
adherent cells. Then, the adherent cells were treated with trypsin and this solution was mixed
with culture medium plus SFB 10% to inhibit trypsin action. The cells were stained with
Trypan blue and cell count of viable cells was performed. The same procedures were done in
a 2D cell culture as a control set.
5. RESULTS AND DISCUSSION
Scaffolds Production
Once it was stipulated the optimal proportions among the polymers to produce the
scaffolds through a review of the literature, the solutions were frozen and lyophilized
following the protocols reviewed (MATSIKO et al., 2012).The structures obtained were
sponge like, the so called scaffolds.
Under macroscopic analysis it was not possible to verify if they were actually porous as a
scaffold should be. Moreover, the lyophilization of HA solution did not lead to the formation
of any solid structure. This result highlights the fact that although this polymer possesses
different interesting properties to 3D cell culture, such as its hydration and retaining of
signaling molecules abilities, its use as a scaffold is difficult due to its short living time and
lack of mechanical resistance when in an aqueous solution (COLLINS & BIRKINSHAW,
2013). Despite these characteristics, the use of this polymer on scaffolds production should
not be discouraged because different techniques of chemical modifications in its polymeric
chain have been described in the literature to promote its cross linking with other polymers,
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in order to provide better stability of this polymer (COLLINS & BIRKINSHAW, 2013). This
way one could use its beneficial properties as referred by Sanad et al. (2017), Weinstein-
Oppenheimer et al. (2017), Hu et al. (2017) e Raia et al. (2017).
Yet, through the macroscopic analysis of the scaffolds, it was observed that those which
had chitosan in their constitution presented themselves as more robust when handled. This
characteristic corroborates the literature that points out that chitosan is a resistant biomaterial
when compared to other biopolymers such as the collagen (TSAI et al., 2013).
Scaffolds Scanning Electron Microscopy (SEM) analysis
Once the macroscopic analyses were not enough to evaluate the porosity of the produced
structures, the SEM was chosen as a tool to evaluate this characteristic. Using this technique,
it was possible to show that the produced scaffolds were porous as can be seen on the figures
1 and 2.
This result was promising since one of the objectives of this work was the production of
porous 3D structures suitable to the 3D cell culture technique. The presence of pores on these
structures is important to induce this kind of culture as they mimic the ECM of the original
tissues and allow the in vitro 3D rearrangement of the cells (DUTTA, R. & DUTTA, A.,
2009).
Figure 1: SEM of the chitosan (A and B) and chitosan-hyaluronic acid (C and D) scaffolds. The left images (A
and C) provide a general view of the scaffolds surfaces (scale bar 3 mm) and the ones on the right side (B and
D) provide a more detailed view of the pores morphology on these biomaterials (scale bar 500 m).
A B
C D
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Figure 2: SEM of the collagen type I-hyaluronic acid (A and B) and chitosan-collagen type I-hyaluronic acid (C
and D) scaffolds. The left images (A and C) provide a general view of the scaffolds surfaces (scale bar 3 mm in A
and 500µm in C) and the ones on the right side (B and D) provide a more detailed view of the pores morphology
on these biomaterials (scale bar 1mm in B and 3mm in D).
The microscopy of the scaffolds C and CQ (figure 3) show images that led to
misinterpretation, once they exhibited a dense and compact film at first view. This result
would not be good once what a 3D cell culture needs is a porous structure. However
Versteegden et al. (2017) showed that collagen scaffolds may present dense walls with a
porous interior. This way, we believe that these structures C and CQ should be re-evaluated
by SEM but on a transversal section to check this morphology.
Figure 3: SEM of the type I collagen structures (A) and type I collagen chitosan (B), scale bar 500 m.
A B
A B
C D
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After confirmation of the production of 3D porous scaffolds, on the other polymer
mixings, the pore size analysis was conducted and the results can be seen at table 1
Table 1: Pore size found on the scaffolds by scanning electron microscopy
(values of media and standard error)
Scaffold composition Pore size (µm)
Chitosan-hyaluronic acid 94 (±36)
Collagen-chitosan Didn´t show pores at SEM
Collagen-hyaluronic acid 70 (±39)
Chitosan-collagen-hyaluronic acid 41 (±15)
The produced material on this work presented pores between 10 and 100 μm. The
scaffold comprised by chitosan, collagen and HA polymers in equal proportion was the one
which presented more homogeneous pore sizes, 41 (±15), as predicted by the exam of media
and standard deviation. Figure 4 shows the illustrative images of the pores used in this
calculation.
Figure 4: SEM highlighting the pore sizes with emphasis in the sizes of the collagen scaffolds typo I-hyaluronic
acid (A), chitosan-hyaluronic acid (B) and chitosan-collagen typo I-hyaluronic acid (C).
Regarding the 3D cell culture scenario the pores of the scaffolds have an important role in
the maintenance of the 3D structure as well as an important influence on their adhesion,
migration and cell proliferation (VAN TIENEN et al., 2002). In relation to this influence,
A B
C
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factor such as cell type, pore sizes and chemical composition of the scaffolds rule the positive
aspects of the cell scaffolds interactions (O’BRIEN et al., 2005). For instance, in scaffolds of
silicon nitride, endothelial cells adhere mainly to structures with pores smaller than 80 μm
whether fibroblasts prefer scaffolds with pore sizes larger than 90 μm (SALEM et al., 2001).
However, in poly lactic acid scaffolds smooth muscle cells can attach to pores with diameter
ranging from 63 to 150 μm and fibroblasts to pore ranging from 38 to 150 μm. (ZELTINGER
et al.,2001).
Generally, the majority of cell types show preferences by linking to structures with pores
that have bigger sizes than their own characteristic size, favoring the interactions with
adjacent cells which link to one another forming a support structure (O’BRIEN et al., 2005).
This way, the pores of the scaffolds can vary from 3 to 1000 μm diameter depending on the
application of the scaffold (SHIM et al., 2017).
Considering all those factors, it is observed that the scaffolds produced on this work
presented pores with suitable size to perform 3D cell culture; however, data about size and
uniformity of the pores per se are not enough to predict which of the produced scaffolds is the
most suitable for 3D cell culture. Thus, it is necessary to perform tests on the cell population
viability and its quantification when in contact with these biomaterials.
The cell interaction with the biopolymers was observed by optical microscopy showing
that the VERO cell line did not reject these biomaterials, as seen in figure 5. However, it was
not possible to accomplish the cell ablation from the scaffolds in order to perform tests on the
cell population viability and its quantification. The reason why the trypsin protocol did not
work remains unsolved and, in the near future, other techniques such as MTT assay or
quantification of fluorescent labeled genetic material will be carried out as suggested by ZHU
et al., 2013.
Figure 5: optical microscopy of VERO cells in the type I collagen scaffold in magnification of 100x (A) and 400x
(B)
6. CONCLUSIONS
It was possible to point out the readiness and efficiency of porous scaffolds production by
lyophilization and that the chitosan, type I collagen and HA polymers association at equal
proportions, contributed with the formation of structures containing more homogeneous sized
pores when compared to the other mixings tested, not to mention that the chitosan contributed
to the hardiness of these structures.
VERO cells A B
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It was also observed that pure HA scaffolds have special needs of chemical treatment or
association with other biomaterials to increase their stability and resistance.
Lastly, it was evidenced by optical microscopy that the VERO cells did not reject the
biomaterial. Notwithstanding, it is still required to perform tests on the cell population
viability and its quantification, our data points out that the triad of biomaterial here studied, in
an equitable proportion, as a promising biomaterial on the 3D cell culture field.
Acknowledgments
The authors are thankful to Professors Ênio Oliveira, PhD and Vicente Trindade, PhD
for co-advising and SEM performance respectively; to Samille Henriques for her helpful hand
with the experiments; to the Program of Tutoring Education (PET) and to the Ministry of
Education (MEC) for the grant with which this work was done.
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14° Congresso da Sociedade Latino Americana de Biomateriais, Orgãos Artificiais e Engenharia de Tecidos - SLABO5ª Edição do Workshop de Biomateriais, Engenharia de Tecidos e Orgãos Artificiais - OBI
20 a 24 de Agosto de 2017 - Maresias - SP - Brasil
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