development of scaffolds based on chitosan, type i ... · 5ª edição do workshop de biomateriais,...

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).

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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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



349

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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