Improvement of biomaterials used in tissue engineering by an ageing treatment

ORIGINAL PAPER

Improvement of biomaterials used in tissue engineeringby an ageing treatment

Cristian A. Acevedo • Paulo Dıaz-Calderon •

Javier Enrione • Marıa J. Caneo • Camila F. Palacios •

Caroline Weinstein-Oppenheimer • Donald I. Brown

Received: 17 May 2014 / Accepted: 26 October 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract Biomaterials based on crosslinked sponges of

biopolymers have been extensively used as scaffolds to

culture mammal cells. It is well known that single bio-

polymers show significant change over time due to a phe-

nomenon called physical ageing. In this research, it was

verified that scaffolds used for skin tissue engineering (based

on gelatin, chitosan and hyaluronic acid) express an ageing-

like phenomenon. Treatments based on ageing of scaffolds

improve the behavior of skin-cells for tissue engineering

purposes. Physical ageing of dry scaffolds was studied by

differential scanning calorimetry and was modeled with

ageing kinetic equations. In addition, the physical properties

of wet scaffolds also changed with the ageing treatments.

Scaffolds were aged up to 3 weeks, and then skin-cells

(fibroblasts) were seeded on them. Results indicated that

adhesion, migration, viability, proliferation and spreading of

the skin-cells were affected by the scaffold ageing. The best

performance was obtained with a 2-week aged scaffold

(under cell culture conditions). The cell viability inside the

scaffold was increased from 60 % (scaffold without ageing

treatment) to 80 %. It is concluded that biopolymeric scaf-

folds can be modified by means of an ageing treatment,

which changes the behavior of the cells seeded on them. The

ageing treatment under cell culture conditions might become

a bioprocess to improve the scaffolds used for tissue engi-

neering and regenerative medicine.

Keywords Physical ageing � Scaffolds � Tissue

engineering

Introduction

The preparation of tissue engineering systems is not an

easy process. First, it is necessary to have an adequate

material called scaffold, which carries the mammal cells

into a bioreactor and allows the tridimensional culture

expansion. The system formed by cells attached on a

scaffold is a complex biosystem where many bioprocesses

occur to transform the tridimensional culture in a tissue

engineering device. The selection of an adequate scaffold

and its treatment are critical steps in the design of tissue

engineering systems to support the cellular component.

Scaffolds based on crosslinked sponges of gelatin (Ge) and

chitosan (Ch) are extensively used for tissue engineering

applications. Ge/Ch-scaffolds have been used as biomaterials

to regenerate skin [1], bones [2] and cartilages [3]. The fact

that Ge/Ch-scaffolds are successful for tissue engineering

C. A. Acevedo (&)

Centro de Biotecnologıa, Universidad Tecnica Federico Santa

Marıa, Avenida Espana 1680, Valparaıso, Chile

e-mail: [email protected]

P. Dıaz-Calderon � J. Enrione

Biopolymer Research and Engineering Lab, Escuela de

Nutricion y Dietetica, Facultad de Medicina, Universidad de los

Andes, Monsenor Alvaro del Portillo 12455, Las Condes,

Santiago, Chile

M. J. Caneo � C. F. Palacios � D. I. Brown

Laboratorio de Biologıa de la Reproduccion y del Desarrollo,

Instituto de Biologıa, Facultad de Ciencias, Universidad de

Valparaıso, Avenida Gran Bretana 1111, Valparaıso, Chile

M. J. Caneo � C. F. Palacios

Escuela de Tecnologıa Medica, Facultad de Medicina,

Universidad de Valparaıso, Alcalde Sergio Prieto Nieto 452,

Vina del Mar, Chile

C. Weinstein-Oppenheimer

Escuela de Quımica y Farmacia, Facultad de Farmacia,

Universidad de Valparaıso, Avenida Gran Bretana 1093,

Valparaıso, Chile

123

Bioprocess Biosyst Eng

DOI 10.1007/s00449-014-1319-x

applications can be explained by two reasons: (a) gelatin

contains an Arg-Gly-Asp (RGD)-like sequence which pro-

motes cell adhesion and migration [4]; and (b) gelatin is

blended with chitosan to improve its physical properties

because Ge/Ch-copolymers form polyelectrolyte complexes

[5]. Moreover, modifications of Ge/Ch-sponges with hyalu-

ronic acid (Ha) produce Ge/Ch/Ha-scaffolds that mimic the

extracellular matrix allowing the growth of skin-cells [6].

It has been described that ageing of biomaterials is a

problem in the pharmaceutical industry, because it can limit

the shelf-life of pharmaceutical devices [7]. Polymers and

biopolymers show significant change over time due to the

non equilibrium condition of the amorphous fraction. Phe-

nomena associated to physical ageing have been studied

extensively in synthetic polymers, some pharmaceutical

polymers [7] and a few scaffolds [8]. Physical ageing is of

significant importance at both the scientific and technolog-

ical levels, because it leads to changes to the properties of

the material, these include hardening, densification,

increased brittleness and decreased permeability [9, 10]. In

pharmaceutical polymers, this phenomenon strongly influ-

ences the drug diffusion and consequently alters biological

phenomena [7]. Information about shelf-life or ageing of

biopolymer scaffolds is scarce in the scientific literature.

It has been described that single biopolymer systems based

on gelatin or chitosan express physical ageing [9, 11]. Nev-

ertheless, the Ge/Ch/Ha-sponges are complex systems, which

are not easy to characterize. Most critically, the Ge/Ch/Ha-

scaffolds are sponges covalently crosslinked, of which there is

little information describing the physicochemical properties

and how that affects the cell behavior [12, 13].

It is well known that cells have the ability to interact

with extracellular matrix. Specifically skin-cells sense

change of collagen-fibrils [14], and probably may sense

changes of collagen-derived protein as gelatin. If the

scaffolds components change with ageing, it is likely that

this can affect the behavior of the cells seeded on them.

Since gelatin and chitosan express physical ageing, we

hypothesized that Ge/Ch/Ha-scaffolds also express struc-

tural changes caused by ageing, and the cells cultured on

them would be able to sense these changes.

In this work, we verified that Ge/Ch/Ha-scaffolds express

an ageing-like phenomenon and that this modified the skin-

cells behavior. This finding opens an alternative to investi-

gate novel strategies to improve tissue engineering matrixes.

Materials and methods

Preparation of Ge/Ch/Ha-scaffolds

Gelatin (from bovine, bloom value 200, grade Ph Eur BP

NF) was purchased from Merck (Germany), chitosan (from

crab shells, 88 % deacetylated, 120 kDa, food grade) was

purchased from Quitoquimica (Chile) and hyaluronic acid

(medical grade, 980 kDa) was purchased from Lifecore

(USA). EDC (1-ethyl-(3,3-dimethyl-aminopropyl)-carbo-

diimide), MES (2-morpholine-ethane sulfonic acid) and

NHS (N-hydroxysuccinimide) were purchased from

Sigma–Aldrich (USA). The scaffolds were prepared using

the method described by Liu et al. [6] with minimum

modifications as described below.

A solution of Ge/Ch/Ha was prepared mixing gelatin

(1.0 %) with chitosan (2 % in 1 % acetic acid) and hyal-

uronic acid (0.01 %) at 50 �C in proportions of 7:2:1

(Ge:Ch:Ha). The solution was poured into a Petri dish

adjusting the volume to obtain a height of 3 mm. The Ge/

Ch/Ha solution was cooled at 4 �C, frozen at -80 �C,

immersed in liquid nitrogen and lyophilized using a freeze

dry system (Labconco, USA). Then, the dry Ge/Ch/Ha-

sponge was crosslinked by the use of a solution composed

by EDC (30 mM), MES (50 mM) and NHS (8 mM), using

ethanol 90 % as solvent. The resultant crosslinked matrix

was then washed with ethanol, frozen and lyophilized. The

Ge/Ch/Ha-scaffolds were stored with silica gel until

experimentation.

Skin-cells

The skin-cells were obtained from rat dermis (strain cpr

100). Rats were anesthetized with ketamine/xylazine (5 and

2 mg per 100 g of body weight, respectively), shaved and

disinfected with a povidone-iodine solution. A biopsy of

1 cm2 was taken from the dorsal area of the animal.

The biopsy was washed with saline phosphate-buffered

(PBS) containing penicillin (100 U/mL) and streptomycin

(100 lg/mL), cut in 1-mm pieces and incubated for 3 h at

37 �C in collagenase solution (2 mg/mL). The epidermis

layer and visible fat were discarded. Then, the cells were

recovered by centrifugation and cultured in DMEM (Gib-

co-Invitrogen, USA) with 10 % fetal bovine serum (FBS)

in 25 cm2 T-flasks under standard cell culture condition

(37 �C and 5 % CO2).

Histochemistry and immunohistochemistry

The scaffolds were fixed in Bouin’s solution for 24 h,

dehydrated and embedded in Paraplast-Plus (Sigma, USA).

Then, the scaffolds were cut completely using a microtome

(Leica, Germany). Five micrometer-thick serial sections

were obtained and mounted on silane-coated microscope

slides, deparaffinized and rehydrated.

The cell distribution and morphology were analyzed

using a trichrome stain (Hematoxylin/Erythrosine B–

Orange G/Methyl blue) [5, 13]. The first section of each

series (15 sections) was stained and analyzed.

Bioprocess Biosyst Eng

123

Selected sections of each series were immunostained

with anti-TGF-b antibody (1:100 dilution, rabbit poly-

clonal, ab66043; Abcam, UK). After use of primary anti-

body, sections were incubated with biotinylated anti-rabbit

IgG (1:500 dilution) and processed using a commercial

peroxidase-ABC kit (Vector, USA), stained with DAB as

chromogen and counterstained using Harris Hematoxylin.

Viable biomass measurement

Resazurin assay was used to measure viable biomass [15].

Viable biomass inside and outside the scaffold was mea-

sured to estimate adhesion and migration of cells seeded

onto the scaffold, respectively.

The cells or scaffolds with cells were incubated in

24-wells culture plate with 200 ll of fresh culture medium

and 200 ll of resazurin solution (50 lM; Sigma–Aldrich,

USA) for 4 h at 37 �C. The viable biomass was estimated

by resorufin production, which was measured by fluores-

cence (excitation at 544 nm and emission at 590 nm) with

a plate reader (Appliskan Thermo Scientific, USA). For

each experiment, a calibration of a known viable cell

number was made (cells were counted in a Neubauer

chamber with the viability dye trypan blue).

Differential scanning calorimetry (DSC)

Thermal properties were analyzed using a differential

scanning calorimeter (DSC, Mettler Toledo, Switzerland).

Prior to the analysis, all samples were equilibrated (con-

stant weight) in a chamber with 75 % relative humidity

(using NaCl saturated solution). Then, a sample of 15 mg

was hermetically sealed in an aluminum pan of 100 lL.

The thermal scanning conditions were: scan 1) heating

from -20 �C to 130 �C at 10 �C/min, holding at 130 �C

for 1 min; and scan 2) cooling from 130 �C to -20 �C,

holding at -20 �C for 5 min and reheating to 130 �C at

10 �C/min. The DSC was previously calibrated using

indium as a standard (melting at 156.6 ± 0.3 �C and

enthalpy of 28.45 ± 0.6 J/g) and an empty pan was used as

reference.

Physical properties

Bulk-density was estimated as the ratio between the dry

mass and the total volume occupied by the scaffold. The

volume as a whole was measured with a micrometer

(Mitutoyo, Japan) recording the width, length and thickness

of the scaffolds in at least five different positions.

Porosity of the scaffold was measured using gas pic-

nometry (pycnometer MVP 1305, Micrometrics, USA)

using helium as carrying gas.

Prior to bulk-density and porosity measurements the wet

scaffolds were lyophilized as described before.

Mathematical modeling

To model changes associated with ageing (U) as a function

of time (t), the Kohlrausch–William–Watts (KWW) equa-

tion was applied to experimental data [16]:

U ¼ exp � t

T

� �B� �

where T is the characteristic time of ageing and B is a

dimensionless coefficient (measure of non-exponentiality).

The changes of enthalpy relaxation (DH) associated to

physical ageing and other properties were represented by

their dimensionless functions:

U ¼ X � X1ð ÞX0 � X1ð Þ

where X is the property measured, X0 is the initial value

and X? is the value at equilibrium.

Experimental values were fitted to the KWW equation

using a non-linear least square method (Newton’s method)

with the tool Solver for Microsoft Excel. The quality of the

fitting was estimated using the correlation coefficient

(r) between experimental and calculated value.

Statistical methods

Basic statistical analyses to compare among different kind

of samples (e.g. scaffolds treated with different ageing

time) were made using analysis of variance (ANOVA,

p \ 0.05). In addition, comparisons between groups of

samples were made using the Tukey test.

Results and discussion

Biological ageing of the tissue engineering system

Ageing of biological systems based on cell cultured into

scaffolds could be beneficial for tissue engineering pur-

poses. In our model of skin-cells culture on Ge/Ch/Ha-

scaffold, short-time ageing produces beneficial effects,

including cellular migration, development of cell clusters

(colonies) and release of growth factors [13].

In Fig. 1 the behavior of skin-cells seeded into scaffolds

at 1 week and after 2 weeks can be seen. Figure 1a shows

that the cell distribution inside the scaffold changes with

short time of ageing, improving cell homogeneity after

2 weeks. Figure 1b shows single cells typically observed at

1 week, but Fig. 1c shows that with time cells form clus-

ters and they express TGF-b.

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123

These results show that a whole biological system

changes significantly over time, and these changes are

beneficial in terms of tissue engineering. If the scaffold is

considered totally inert, the mentioned changes could be

associated only with cell processes. But, our hypothesis is

that the scaffolds express ageing changes affecting the cell

behavior. To verify this hypothesis, the expression of

putative phenomena associated with physical ageing was

studied in Ge/Ch/Ha-scaffolds.

Physical ageing of dry scaffold

An initial experiment based on DSC was made to verify

expression of phenomena associated whit ageing in dry Ge/

Ch/Ha-scaffolds (Fig. 2a). The first DSC-scan shows two

thermal transitions: (a) a glass transition temperature (Tg)

with an inflection point close to 37.4 �C (onset 31.9 �C,

endpoint 39.8 �C and heat capacity variation of 148.0 J/

kg �C); and (b) an endotherm with onset at 72.4 �C (peak

85.9 �C, endpoint 99.2 �C and enthalpy of 10.6 J/g). The

endotherm observed in the first scan indicates the presence

of an ordered-like fraction in the scaffold, which could be

associated with the melting of gelatin [5, 12, 13]. The

second DSC-scan showed a completely amorphous struc-

ture indicating the irreversibility of the endothermic tran-

sitions previously discussed.

A spontaneous endothermic event on the thermogram

was observed when the amorphous material in glassy state

was stored (see Fig. 2a). The latter strongly indicated that

the scaffold structure changes with ageing time. In poly-

mers and biopolymers (e.g. gelatin) this phenomenon

associated with physical ageing is often called enthalpy

relaxation or structural relaxation [9, 10].

Figure 2b shows the change in excess of enthalpy of

aged scaffolds. Experimental data of Fig. 2b fitted well to

KWW equation, classically used to model the physical

ageing kinetics [16]. The KWW equation has been used to

model the enthalpy relaxation process of biopolymers as

carbohydrates [17] and proteins [10]. This equation is a

stretched exponential function, where the parameter

B (0 B B B 1) is a measure of its non-exponentiality. If the

parameter B is close to 1, it corresponds to a single

relaxation time with exponential behavior. The estimated

value of B was lower than 1 (B = 0.58), indicating a

Fig. 1 Behavior of skin-cells seeded into scaffolds. a Cell counting

and distribution after 1 and 2 weeks of seeded. b Single cells at

1 week (stained with the thrichrome stain, bar 50 lm). c Cell cluster

after 2 weeks showing positive immunostaining for TFG-b (counter-

stained with hematoxylin, bar 50 lm)

Fig. 2 Thermophysical behavior of dry scaffold. a Thermograms of

scaffolds in semicrystalline state (original material) and amorphous

state aged 24 h at 20 �C. b Enthalpy relaxation (DH) kinetics of

amorphous material and its fitting using KWW equation. Coefficients

fitted were, DH? = 2.64 [J/g], T = 9.69 [h] and B = 0.58

(dimensionless)

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distribution of relaxation time rather than a single relaxa-

tion time. In complex biopolymers systems such as gela-

tinized starches the B values reported are between 0.2 and

0.4 [17]. In addition, the fitting of KWW equation pro-

duced an excess in enthalpy at equilibrium (DH?) of 2.64

[J/g], which is a value similar to values reported for pure

gelatin (2.4 [J/g]) [10].

As far as we know this phenomenon has not been

described in complex systems such as copolymers cova-

lently crosslinked with EDC (like our system). So our

results suggest the occurrence of this relaxation-like phe-

nomenon, which could have an important effect in the

scaffold properties.

Ageing of wet scaffold

Results described before shows that scaffolds can change in

glassy state. However, when the scaffold is used for tissue

engineering applications, it is immersed in a bioreactor

with culture medium at physiological temperature (37 �C)

taken a rubbery state (eliminating in theory all thermal

history of the material). To study if the wet scaffold

(rubbery state) under cell culture conditions is affected by

ageing, we kept the scaffold immersed in medium (DMEM

with 10 % FBS) at 37 �C for 15 days (360 h).

The results shown in Fig. 3 described that porosity and

bulk-density change over time. Porosity decreased linearly

in the time studied (p \ 0.05; ANOVA), but bulk-density

kinetic had a behavior similar to KWW equation. In terms

of tissue engineering, this phenomenon could be affecting

the cell behavior, because it has been reported that porosity

reduction in Ge/Ch/Ha-scaffolds improves the cell growth

and expression of growth factors [12].

Figure 4a shows that the glass transition changes over

ageing time. This kinetic can be modeled by the KWW

equation which describes the ageing phenomenon. Addi-

tionally, the Tg was increased and the heat capacity vari-

ation decreased (Fig. 4b). Yoshioka et al. [8] obtained

changes of Tg in PLGA-scaffolds aged in PBS at 37 �C.

These authors reported decreasing of Tg (second scan) by

the degradation of the polymer and reduction of the

molecular weight. Nevertheless, we obtained an increase of

the Tg (first scan) in contrast to the decrease of heat

Fig. 3 Physical behavior of wet scaffold. Prior to measure porosity

and bulk-density, the wet scaffolds were lyophilized. a Porosity

kinetic. b Bulk-density (q) kinetic and its fitting using KWW

equation. Coefficients fitted were, q? = 50.23 [kg/m3], T = 349.84

[h] and B = 0.56 (dimensionless)

Fig. 4 Glass transition kinetic. Wet scaffolds were sampled and

lyophilized, and then equilibrated at 75 % relative humidity. a Glass

transition temperature (Tg) and its fitting using KWW equation.

Coefficients fitted were, Tg? = 88.98 [�C], T = 341.54 [h] and

B = 0.69 (dimensionless). b Glass transition temperature (Tg) and

heat capacity variation (DCp) relationship in a semi-log plot

Bioprocess Biosyst Eng

123

capacity, suggesting an increase of the crystalline fraction.

The latter has been informed in PLLA-scaffolds aged in

alkaline medium at 37 �C [18], where the rise of Tg was

associated to increase of crystallinity.

Our results show that physical and thermophysical

properties of the scaffold change with ageing. It is well

known that changes in extracellular matrix affect the cell

behavior. In our previous work [13], it was reported that

different gelatin content modify the Tg and porosity of Ge/

Ch/Ha-scaffolds, and that these modifications improved the

cell growth. Then a question arises, if a treatment based on

ageing of the scaffold could also modify the behavior of

cells seeded on them.

Effect of ageing treatment on cell affinity

A key issue in tissue engineering is the interaction between

the biomaterial or scaffold and the living cells. Cell

attachment and migration in the scaffold are relevant for

building an implant system, which mimic a tissue and

therefore is useful for tissue engineering applications [19].

To estimate the affinity between cells and the aged scaf-

folds, cell adhesion and migration outside the scaffold were

studied.

A set of aged scaffolds with different ageing time was

prepared. For that, the scaffolds (without cells) were kept

in cell culture medium (DMEM with 10 % FBS) at 37 �C

for 1, 2 and 3 weeks. Then, skin-cells were seeded on the

aged scaffolds to study their affinity.

Figure 5a shows the cell adhesion. Scaffold not aged has

a cell adhesion close to 39 %. Similar values have been

reported for this kind of scaffolds [6, 13]. However, when

the scaffold is aged, the adhesion decreases to reach a value

close to 25 %. It is very likely that the ageing affects some

structuring properties of the scaffolds, and which might

influence the behavior of the cells in contact with them.

Figure 5b shows that ageing affects the migration out-

side the scaffold (p \ 0.05; ANOVA). The scaffold not

aged has low migration rate in comparison with scaffold

aged for 2 and 3 weeks (p \ 0.05; Tukey test). In terms of

tissue engineering this effect could be beneficial, because

in early stage it is necessary to have cells growing within

the scaffold. But in a long term, when the scaffold is

implanted on patients, it is necessary that the cells can

migrate outside the scaffold for tissue healing or repair.

These results suggest that the scaffold is a non-inert

matrix and that ageing phenomenon affects the cell

behavior.

Effect of ageing treatment on physiological cell

behavior

The physiological behavior (viability and proliferation) of

cells cultured onto scaffolds with different ageing time (1,

2 and 3 weeks) was analyzed using histological techniques.

Cells were seeded on aged scaffolds and after 1 week of

culture they were fixed to prepare the histological analysis.

Figure 6 shows cells observed (single cells, clusters of

cells, pyknotic cells and mitotic figures).

Figure 7 shows physiological rates (viability and mitotic

rate) in three zones of the scaffolds. Viability of cells

cultured onto aged scaffold is higher than non-aged scaf-

folds (Fig. 7a). All zones of aged scaffolds had at least

80 % of viability. However, in the middle and bottom

zones of the not aged scaffolds displayed viabilities close

to 60 %. This is an important finding since cell viability

within the whole thickness of the scaffold has been

described as a relevant success factor for medical devices

in tissue engineering [19, 20].

Figure 7b shows the mitotic rate, indicating that the

scaffold aged for 2 weeks is the most proliferative

Fig. 5 Affinity between cells and aged scaffolds. Cells were seeded

on scaffolds with different ageing levels. Scaffolds with cells

(2 9 104 cells/cm2) were incubated in a 24-wells culture plate.

a Cell adhesion. Adhesion is the rate between the number of cells

initially seeded onto the scaffold and viable cells attached after 24 h.

b Cell migration. Migration rate is a relative value of viable biomass

outside the scaffold (onto plastic well bottom) at 48 h, and it was

arbitrary considered a value of 100 % for not aged scaffolds. Asterisk

symbols (* and **) show not significant differences (p [ 0.05; Tukey

test)

Bioprocess Biosyst Eng

123

promoting matrix. The mitotic rate in the intermediate zone

of scaffolds aged for 2 weeks was 0.53 %. This value is

slightly lower than mitotic rates reported to fetal monolayer

cultures. For instance, a mitotic rate of mouse fetal skin

fibroblasts was reported as close to 1.3 % [21]. Prolifera-

tion within the scaffold is another success factor for tissue

engineering devices [19], indicating that the ageing bio-

process is improving the scaffold performance probably

influencing cell signaling through chemical and physical

stimuli.

These results suggest that the biological properties of the

scaffold could be improved using the ageing phenomena as

a bioprocess.

Effect of ageing treatment on cell distribution

The importance of cell spreading within a scaffold has been

recognized in the tissue engineering field for optimizing the

scaffold properties [19]. Figure 8 shows the cell distribu-

tion inside the scaffolds. Figure 8a indicates strongly that

the higher biomass is obtained with a scaffold aged for

2 weeks. In addition, the scaffold with 2 weeks of ageing

has a homogeneous distribution of cells inside the scaffold,

which again shows that the 2 weeks ageing bioprocess

improved the native scaffold.

Figure 8b shows the distribution of aggregated cells in

different zones of the scaffold. We observed that the

scaffold aged for 2 weeks has the higher presence of cell

cluster and the best distribution of them. Cell aggregation

is a very important factor in tissue engineering because it

allows cell–cell interaction and it is a qualitative evidence

of cell growth. It has been reported that cell clusters of

skin-cells immobilized in fibrin-scaffolds appear after the

third day of seeded [22]. This could be inducing a micro-

environment favorable for the synthesis of growth factors,

such as TGF-b [23], which are recognized as relevant

determinants of tissue regeneration. Our results show that

the cell clusters inside the aged scaffolds expressed TGF-b(Fig. 6e), but single cells into non-aged scaffold did not

express TGF-b (Fig. 6f). This is a significant finding since

TGF-b family members have been reported to improve

tissue healing [24] and to participate in wound healing

Fig. 6 Photomicrographs of

histological sections from

scaffolds seeded with cells.

Sections stained with the

thrichrome stain. a Single cells

(bar 50 lm). b Cluster of cells

(bar 50 lm). c Pyknotic cells

(bar 25 lm). d Polar view of a

cell in metaphase (bar 25 lm).

e Cell clusters into aged

scaffolds with positive

immunostaining for TGF-b(counterstained with

hematoxylin) (bar 50 lm).

f Cell into non-aged scaffold

with negative immunostaining

for TGF-b (bar 25 lm)

Bioprocess Biosyst Eng

123

through the stimulation of macrophage recruitment, re-

epithelization and wound contracture [25].

These results strongly suggest that the behavior of cells is

modified by the ageing of the scaffold. There could be an

optimal degree of ageing to cultivate cells for tissue engi-

neering. In our model, we obtained the best condition with

2 weeks of ageing, originating an improved construct, as

compared with the starting material. This exhibits the main

features for tissue formation on an artificial substrate such as

attachment, cell viability, proliferation and migration.

Conclusions

Polymeric scaffolds based on crosslinked Ge/Ch/Ha-

sponges express a phenomenon related with enthalpy

relaxation. Additionally, the physical properties of wet

scaffolds change with time. Treatments based on ageing

allow the design of modified scaffolds.

The cell behavior is affected by the ageing time of the

scaffold. Biological responses of the cell culture as cell

adhesion, viability and proliferation changed with the

ageing of the scaffold. In our experimental model, the best

condition was obtained with a 2 weeks aged scaffold under

cell culture conditions.

Finally, we concluded that the scaffold is not an inert

matrix, and its ageing affects the behavior of cells seeded

on them. The scaffolds used for tissue engineering could be

improved using an ageing treatment under cell culture

conditions. Overall, the bioprocess described herein can be

proposed to build wound and tissue healing medical

devices.

Acknowledgments The authors wish to thank CONICYT from

Chile by FONDECYT Grant 1120166.

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