International Journal of Biological Macromolecules 92 (2016) 645653

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Characterization of gelatin/chitosan scaffold blended with aloe vera

and snail mucus for biomedical purpose
Daniel Enrique Lpez Angulo , Paulo Jos do Amaral Sobral
University of So Paulo. Depto de Eng. de Alimentos FZEA USP, Av. Duque de Caxias Norte, 225, CEP, 13635-900 Pirassununga, (SP), Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Biologically active scaffolds used in tissue engineering and regenerative medicine have been generating
Received 19 May 2016 promising results in skin replacement. The present study aims to test the hypothesis that the incorpo-
Received in revised form 6 July 2016 ration of Aloe vera and snail mucus into scaffolds based on gelatin and chitosan could improve their
Accepted 7 July 2016
structure, composition and biodegradability, with a potential effect on bioactivity. Homogeneous pore
Available online 21 July 2016
diameter as well as pore walls in the composite scaffold could be seen in the SEM image. The pores in
the scaffolds were interconnected and their sizes ranged from 93 to 296 m. The addition of Aloe vera
Keywords:
and snail mucus enlarged the mean pore size with increased porosity and caused changes in the pore
Biocompatible scaffolds
Tissue regeneration
architecture. The FTIR analysis has shown good afnity and interaction between the matrix and the Aloe,
Interconnected matrix which may decrease water-binding sites, so this fact hindered the water absorption capacity of the mate-
rial. The mechanical properties could explain the highest swelling capacity of the snail scaffold, because
the high percentage of elongation could facilitate the entry of liquid in it, generating a matrix with plenty
of uid retention. The real innovation in the present work could be the use of these substances (Aloe and
snail mucus) for tissue engineering.
2016 Elsevier B.V. All rights reserved.

1. Introduction A scaffold should be biocompatible, non-antigenic, biodegradable

Acute trauma, genetic skin disorders and chronic wounds may
result in skin loss, with burns and scalds being major contributors
to rapid, extensive, deep (i.e. full thickness) wounds with substan-
tial areas of skin damage, frequently without the possibility of
skin regeneration [1]. These often cannot be successfully treated
with common routine surgical skin grafting techniques [2,3]. The
conventional therapies used to repair skin defects include auto-
graft, allograft and xenograft. Although often there are limitations
regarding donor sites and high risk of immune rejection and disease
transmission [4,5].
In recent years, biologically active scaffolds used in tissue
engineering and regenerative medicine have been generating
promising results in skin replacement and cartilage regeneration.
Many kinds of synthetic and natural polymer materials have been
used as scaffolds. Their function is to provide mechanical support
and to allow the adhesion and proliferation of the cells embedded
on the scaffold or the cells migrating from a surrounding tissue [6].
Corresponding author.
E-mail addresses: daniel.lopez@usp.br, daniel.lopezan@usach.cl
(D.E. Lpez Angulo).
and have a three-dimensional structure with adequate porosity
and pore size [7,8]. Gelatin is a biodegradable and biocompatible
polymer and is able to make polyion complexes. Due to these prop-
erties, gelatin is commonly used in drug and cell delivery for tissue
engineering applications targeting several tissues such as bone,
cartilage and skin [9,10].
Among the numerous materials that have been evaluated for
scaffold fabrication, in tissue engineering, chitosan is considered
promising, attributing to its non-toxicity excellent biocompatibility
and biodegradability [11]. Chitosan is a polysaccharide-type biolog-
ical polymer composed of glucosamine and N-acetyl glucosamine
linked with 14 glucosidic linkage. Chitosan is generally pro-
duced by alkaline deacetylation of chitin. The N-acetyl glucosamine
in chitosan is a structural component, also found in the gly-
cosaminoglycans. This suggests that chitosan may be able to
interact with growth factors, receptors, and adhesion proteins
[12,13]. This polymer is found to be degraded by enzymes in the
human body, and the degradation products are non-toxic [14].
In the context of incorporating new biomaterials that have
regenerative and stimulating properties of cell growth, snail mucus
has been used in medicine from ancient times for pain relief, for
the treatment of burn injuries, other injuries and various diseases
[15]. Research on the secretions of the snail (Helix aspersa) have

http://dx.doi.org/10.1016/j.ijbiomac.2016.07.029
0141-8130/ 2016 Elsevier B.V. All rights reserved.
646 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653
conrmed that the mucus contains an unusual combination of
natural ingredients with benecial and therapeutic qualities for
human skin, including allantoin and glycolic acid [16,17]. Accord-
ing to the USA Food and Drug Administration (FDA), allantoin is a
safe and effective active compound for skin protection at a dosage
range of 0.52.0% [18]. Glycolic acid is the most widely used alpha-
hydroxy acid for skin care cosmetic products and for the treatment
of skin diseases including actinic keratosis, hyperkeratosis and seb-
orrheic eczema [19].
Aloe vera (Aloe barbadensis Miller) is a member of the Liliaceae
family. The parenchymatic tissue of Aloe vera leaves (gel) contains
over 9899% water and more than 60% of the dry matter (DM) is
made up of polysaccharides [20]. Aloe vera gel is not only a hydrat-
ing and moisturizing agent for the skin, but it has also been reported
to possess immunomodulatory, anti-inammatory, anti-allergic,
antibacteric and wound and burn healing properties [2123]. There
is a consistent number of reports regarding biologically active gly-
coproteins from Aloe vera [24]. Lately, Choi et al. [25] reported the
proliferation and wound healing effect of a 5.5 kDa glycoprotein.
This glycoprotein was isolated by activity-guided sequential frac-
tionation from Aloe vera gel and was found to enhance keratinocyte
proliferation.
Pore size, scaffold porosity, and the general pore structure
all have signicant effects upon tissue structure and inltration
into material constructs [26]. Therefore, Gelatin/Chitosan/Aloe vera
(GCA) and Gelatin/Chitosan/Snail mucus (GCS)-based scaffold poly-
mers may be combined to obtain a variety of biocompatible
products for tissue engineering. Besides, there is no information
related to their physical properties and skin generation properties.
Porous chitosan/gelatin scaffold can be prepared via polyelectrolyte
complex formation, freeze-drying, and post-crosslinking with glu-
taraldehyde. Glutaraldehyde is the most widely used crosslinker
agent because it is easily available, inexpensive and capable of
accomplishing the crosslinking in a relatively short time. Although
other crosslinking agents could be used to reduce cytotoxicity, they
cannot match glutaraldehyde in collagen stabilization. Although
glutaraldehyde toxicity poses a risk for biocompatibility, it has also
been shown that this can be reduced by decreasing the concen-
tration of glutaraldehyde in the solution that generates the vapor,
and/or by an efcient removal of unreacted glutaraldehyde left in
the material after the crosslinking treatment [27].
Hence, this study aims to test the hypothesis that the incor-
poration of Aloe vera and snail mucus into scaffolds based on
gelatin and chitosan could improve their structure, composition
and biodegradability, with a potential effect on bioactivity, thereby
marking this new biomaterial as a prospective scaffold candidate
for tissue engineering. Finally, it is important to note that the real
innovation could be the use of these substances (Aloe vera and snail
mucus) for tissue engineering.
2. Material and methods
2.1. Materials
The macromolecules used for scaffold production were: pigskin
gelatin (molecular weight 5.2 104 Da; bloom 260; moisture
content = 9.98%), supplied by Gelnex South America (It, Santa Cata-
rina, Brazil), and chitosan (derived from crab shell with minimum
deacetylation degree of 85%, MW: 2 105 ), obtained from Sigma
(St. Louis, MO, USA). Acetic acid (Sigma), PBS (Sigma Aldrich) and
glycerol (Synth) were all chemical reagents of analytical grade. Aloe
vera pure extract (A) was obtained by Mundo Aloe Vera Pica (Chile),
snail mucus (S) was purchased from Lacofar LTDA (Chile).
2.2. Biochemical characterization
The concentration of the amino acids present in pigskin gelatin
was determined by high-performance liquid chromatography
(HPLC) following the methodology described by Daz et al. [28].
2.3. Fabrication of porous scaffolds
A blend of gelatin (G) and chitosan (CH) was prepared by thor-
ough mixing of gelatin (2%) and chitosan (1%) (control) solution
in 0.05 M acetic acid in the ratio of 1:1 and stirred with a mag-
netic bar at 50 C for 2 h. Similarly, Aloe vera (A) and snail mucus
(S) blended G-CH composite was prepared by mixing different
weighted quantities of A and S with the G-CH solution to obtain a
nal concentration of 0.07% (A1, S1, with low Aloe and snail mucus
concentration), 0.15% (A2, S2, with high Aloe and snail mucus
concentration) (w/v), and of both (AS 1 and AS 2, low and high
concentration, respectively). Glycerol was added in 0.3%. The solu-
tion blend was then poured into a glass petri dish and left for 2 h on
liquid nitrogen system chamber (190 C), prior to freeze-drying,
at 58 C, for 18 h. The sponge obtained was cross-linked inside
desiccators containing glutaraldehyde (10%) in 200 ml 90% ethanol,
during 2 h. Then, the sponge was immersed on NaOH solution (1%)
and washed with distilled water two times, and treated with 5%
(w/v) NaBH4 solution. The scaffolds, again after thorough washing
with distilled water, were frozen at 190 C, and freeze-dried in
vacuum for 18 h.
2.4. Microstructural observation
The morphology of the freeze-dried composite scaffolds was
observed by using Scanning Electron Microscope (Hitachi TM 3000,
Japan). The sample was placed on double-sided carbon tape in a vac-
uum chamber, prior to measurement. The pore sizes of the scaffolds
were measured using image visualization software (Image J 1.45s,
NIH Image, USA). The values were expressed as mean standard
deviation.
2.5. Scaffold porosity
The porosity of scaffolds was measured by liquid displacement
method, at 25 C. Absolute ethanol was used as a displacement liq-
uid since it can easily penetrate the scaffolds and would not induce
shrinking or swelling. The specimens, pycnometer and ethanol,
were kept at 25 C for 1 h before testing. A pycnometer lled with
ethanol was weighed (W1). A scaffold specimen of known weight
(WS) was immersed into the bottle, and then the bottle was sub-
merged slowly until all the air in the scaffold was removed; lastly
the pycnometer was relled with ethanol and weighed (W2). The
scaffold saturated with ethanol was removed from the pycnometer
and then the pycnometer was weighed (W3) [29]. Then, the volume
of the scaffold (VS) specimen was calculated using Eq. (1), while the
total volume of the pores (VP) was calculated using Eq. (2).
W 1 W 2 + WS
VS = (1)

W 2 W 3 WS
VP = (2)

Then the porosity of the specimen was calculated using Eq. (3).
VP W 2 W 3 WS
= = (3)
(VP + VS) W1 W3
 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653 647

2.6. Swelling property (water binding capacity) Table 1

Amino acid contents in the pigskin gelatin of this study (g/100 g).

The swelling property of the gelatin scaffolds was exam-
ined according to [30]. The scaffolds were placed in a 0.4 mg/ml
phosphate-buffered saline solution (PBS), pH 7.4, at 37 C, for 1,
2, 7, 14, 21 and 28 days. Subsequently, swelling was calculated
according to initial dry and nal wet weight of the samples, and
expressed as%. Data from each sample were calculated using three
measurements.
2.7. Mechanical properties
The tensile and compression properties of the composite scaf-
folds were determined using a Texture Analyzer icon TA.XT2i
(Stable microsystems) instrument, at room temperature (22 C).
Briey, for tensile properties, freeze-dried samples, of 2.5 mm
thickness, were cut into the size of 5 cm 1 cm. The gauge length
between the two grips was set at 40 mm and the speed of testing
was set at 5 mm/min. The values of tensile strength, elongation at
break (EAB) and Youngs modulus were determined from the stress-
strain curves. The results were expressed as mean standard
deviation (n = 10).
The compression tests were performed on the scaffolds cylin-
ders (25 mm in diameter and 20 mm in thickness). Samples were
compressed up to 50% of their initial thickness, under a load cell of
10 kn at the displacement rate of 1 mm min1 . Compressive mod-
ules of the scaffolds were determined by the average slope of the
stressstrain curve over the strain range of 05%. Data from each
sample were calculated using ten measurements.
2.8. Fourier transform infrared spectroscope
The Fourier transform infrared spectroscopy (FTIR) spectra of
scaffolds were performed using a FTIR Spectrophotometer (Perkin
Elmer Spectrum one, Germany) in the wavenumber range of
4003500 cm1 with resolution of 4 cm1 . 16 scans were per-
formed. The FTIR spectra were taken in absorbance mode.
2.9. X-ray diffraction
XRD patterns of the gelatin-based scaffold were recorded over
a 2 range of 560 , using an X-ray diffractometer (Rigaku, model
Miniex600, Japan), with a step angle of 2 C/min.
2.10. In vitro biodegradation
The biodegradation study of the scaffolds was carried out
in vitro by incubating the scaffolds in PBS bath with pH 7.4 with
a concentration of 0.4 mg/ml of lysozyme which was close to the
concentration of lysozyme in typical wound exudation uid with
inammation [31], in an incubation dish, and kept at 37 C. At a pre-
determined time interval, samples were taken from the medium,
washed with distilled water and freeze-dried. The degradable ratio
(D, %) was calculated by a weight loss using Eq. (4).
Wo Wt
D= 100%
Wo
Where, WO denotes the original dry/wet weight, while Wt is the
dry/wet weight at time t. Each biodegradation experiment was
repeated three times and the average value was taken as the per-
centage biodegradation.
2.11. Statistical analysis
Statistical analysis and interpretation of all results were done
by using the software GraphPad Prism version 6.0 for Windows
Amino acid g/100 g
Ala 8.2
Arg 11.2
Asp 5.8
Gly 23.3
Iso 1.2
Leu 2.7
Glu 9.4
Lys 3.5
Meth 0.5
Fen 1.6
Tyr 0.6
Tre 1.6
Pro 13.7
Hypro 11.1
Val 2.4
Hist 0.9
Ser 3.1
(GraphPad Software, San Diego, California, USA). One-way ANOVA
with Fischer LSD posttest was performed to obtain the variance
between and within all treatments. P-values less than 0.05 denoted
statistical signicance.
3. Results and discussion
The amino acid composition was expressed as g of amino
acid residues/100 g of gelatin (Table 1). Pigskin gelatin had higher
amount of glycine (Gly), followed by proline (Pro) and arginine
(Arg), corresponding to 23.3, 13.7 and 11.1% of the total of amino
acids, respectively, agreeing with results found in the Ref. [32],
which reported a Pro and Arg content of 15.1 and 11.1 g/100 g,
respectively. It is important to analyze the presence of Pro and
Hyp because the correlation between these amino acid content in
gelatin can affect its melting temperature [33]. Moreover, the rela-
tionship between the denaturation temperatures and the amino
acid content (Pro + Hyp) has been established for vertebrate and
invertebrate species in relation to the mechanism of stabilization of
the triple helix by the pyrrolidine residues [33]; it has been reported
Pro and Hyp content of 13.9 and 11.1 (g/100 g), respectively [32]. In
addition, Chiou et al. [34] it was reported similar result for pigskin
gelatins amino acid, around 22.9% for proline (Pro) and hydrox-
yproline (Hyp). Similar results have been reported by Eastoe et al.
[35], where Pro, Hyp, Arg and Gly content was 13.2, 9.1, 4.9 and
33.0 (g/100 g), respectively.
It is also important to highlight the presence of lysine (3.5%),
since glutaraldehyde has the ability to react with the functional
group of the residue of this amino acid, crosslinking adjacent
peptides. This fact is important for improving the mechanical prop-
erties of the scaffold formed after the freeze-drying process.
3.1. SEM micrograph analysis of composite scaffolds
SEM was used to study the scaffold porous microstructure and
morphology. Homogeneous pore diameter as well as pore walls
(4) thickness in the composite scaffold can be seen in the SEM image.
The pores in the scaffolds (Fig. 1) were interconnected and their
sizes ranged from 93 to 296 m for crosslinked composite scaf-
folds, the mean pore diameter was between 119 38 m and
207 61 m (Table 2). Scaffold porosity, pore size, and the overall
pore structure all have important effects upon tissue formation and
inltration into biomaterial constructs. Interconnecting pores facil-
itate the loading of cells into scaffold materials while the increased
internal surface area provides sites for attachment and spreading.
The effects of pore size on tissue regeneration has been empha-
sized by experiments demonstrating optimum pore size of 5 m
648 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653

Fig. 1. Scanning electron micrographs of the freeze-dried gelatin-chitosan based A and S scaffolds: A1 and A2 are low and high A concentration, S1 and S2 are low and high
S concentration, AS 1 and AS 2 contain A blended with S in low concentration and A blended with S in high concentration. (see nomenclature under each photo).

for neovascularization [36], 515 m for broblast ingrowth [37],
20 m for the ingrowth of hepatocytes [38], 200350 m for
osteoconduction [39], and 20125 m for regeneration of adult
mammalian skin [40]. Pore interconnectivity is also critical to
ensure that all cells are within 200 m from blood supply in order
to provide for mass transfer of oxygen and nutrients [38,41]. There-
fore the material reported here could be used for a wide range of
different cellular activities as for example; angiogenesis of mul-
tilayered agent-based model simulation/in vivo rat implantation,
cell inltration of primary rat osteoblasts, chondrogenesis of rabbit
marrow stromal cells, proliferation of human foreskin broblasts
and smooth muscle cell differentiation of dog bone marrow stem
cells. However, it should be noted that cell differentiation is also
dependent on the cell type, scaffold material, and fabrication con-
ditions [4247].
The observation revealed that the addition of Aloe vera and
snail mucus increased the mean pore size and porosity and caused
some changes in the pore architecture. Besides pore size and its
uniformity, porosity also determine the mechanical properties and
the water retention property, thus the scaffold can easily absorb
the culture medium to facilitate the cells for the migration, adher-
ence and proliferation in the porous structure. Aloe vera has been
reported to contain several biologically active and nutritionally
important compounds such as glycoproteins, saccharides, vitamins
and antioxidants. These compounds, potentially, by virtue of their
water-soluble/absorbing nature, readily attract cells in the medium
and promote their activities on the scaffold.
The porosity of all Aloe vera and snail mucus scaffolds formula-
tions was higher than 90% (Table 2), which suggested all scaffolds
were with good porosity. Taking into account the results of the SEM,
it could be suggested that all formulations were with good pore size
and porosity, which indicated that the sponge scaffold meets the
basic requirements as tissue engineering material.
3.2. Mechanical properties
The uniaxial tensile and compressive data of the control and
composite scaffold blended with 0.07% and 0.15% (w/v) of Aloe vera
and snail mucus scaffolds are presented in Table 3. The control scaf-
fold exhibited maximum tensile strength (0.18 0.043 MPa), which
was due to the H bond/ionic interaction between the functional
groups OH, COOH and NH2 of gelatin and the OH and NH2
 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653 649

Table 2
Control and Aloe vera/snail mucus scaffolds morphological parameters.

Treatments1 Pore size (m) S.D. Porosity (%) S.D. Pore size range (m)

Control 119.21 38.16 80.2a 0.19 93248

A1 187.95 40.17 96.7b 0.07 162281
S1 190.95 37.82 98.3b 0.01 162230
A2 207.03 61.46 94.3b 0.04 162265
S2 204.51 49.21 93.8b 0.03 159291
AS 1 199.45 54.34 95.5b 0.02 162248
AS 2 201.53 59.99 94.0b 0.02 162296
1
A1 and A2 are low and high A concentration, S1 and S2 are low and high S concentration, AS 1 and AS 2 contain A blended with S in low concentration and A blended
with S in high concentration. S.D: Standard deviation. Different letters indicate signicant differences (p < 0.05).

Table 3
Mechanical properties1 of gelatin/chitosan scaffolds blended with Aloe vera and snail mucus.

Treatments2 Y.M. (MPa) S.D. T.S. (MPa) S.D. EAB (%) S.D. Compressive Mod. (MPa)
a a a
Control 4.99 1.03 0.18 0.04 3.96 0.01 0.04a
A1 0.85bd 0.23 0.05bc 0.02 7.62a 0.01 0.05a
S1 0.64b 0.15 0.08b 0.01 13.16b 0.01 0.03a
A2 0.89be 0.36 0.06b 0.02 8.34a 0.01 0.02ca
S2 0.23bc 0.04 0.06b 0.02 23.52c 0.04 0.10b
AS 1 0.71bf 0.21 0.04bc 0.01 7.22a 0.01 0.12bd
AS 2 1.65gb 0.68 0.11bd 0.04 6.92a 0.01 0.02ac
1
Y.M.: Youngs modulus, T.S.: Tensile strength, EAB: elongation at break. S.D.: standard deviation.
2
A1 and A2 are low and high A concentration, S1 and S2 are low and high S concentration, AS 1 and AS 2 contain A blended with S in low concentration and A blended
with S in high concentration. Different letters indicate signicant differences (p < 0.05).

groups of chitosan. The tensile properties of the control scaffold are
comparable with the corresponding data presented by the other
researchers, such as Enrione et al. [6], which reported for frozen
and freeze-dried gelatin/chitosan/hialuronic acid scaffold a tensile
strength between 0.240.44 MPa. A similar result was also obtained
previously by Mao et al. [48], for freeze-dried gelatin-chitosan scaf-
fold crosslinking with glutaraldehyde, where the tensile strength
was 0.45 MPa. Kakkar et al. [49] reported a tensile strength for
freeze-dried gelatin-chitosan scaffold of 0.107 0.83 MPa. Liu et al.
[50] and Tseng et al. [51] reported tensile modulus of about
0.0900.1 MPa and the EAB of about 20% and 12% for the colla-
gen/chitosan and gelatin/chitosan scaffolds designed for skin tissue
engineering.
In this study, the average EAB of the control scaffold was 3.96%
(Table 3). It has been reported that the EAB of commercial gelatin
scaffold was 4.70% [52], which was slightly higher than our obser-
vation. Those scaffold prepared with snail mucus had signicantly
lower Young modulus and higher EAB, this may be due to the inter-
ference of its their functional groups (C O, COOH). It seemed that
the kind of polymer and its ionic nature did modify the interaction
between the molecules and the resultant elasticity.
Compressive properties of gelatin/chitosan scaffolds are of
interest in the study of cell-mediated contraction of the scaffolds
[53]. In previous reports, the compressive moduli of gelatin-
chitosan-based scaffolds designed for skin tissue engineering were
around 0.0220.034 MPa [50]. Thein-Han et al. [54] reported a
compression modulus of gelatin/chitosan scaffold, ratio 2:1, of
0.072 MPa.
amide I can be seen in the range of 16331639 cm1 , amide II near
15461551 cm1 and amide III in the range of 12391241 cm1 .
Therefore, the spectrum conrms the presence of -helix with -
turns with characteristic absorption band designating the peptide
bonds (CONH) for amides I, II and III. The peak of amide A is due to
stretching vibration of N H bonds. The peak of amide I is caused by
stretching vibration of C O bonds. N H bending and C H stretch-
ing vibration are responsible for amide II band, and the amide III
peak is made up of C N stretching vibration. Other bands observed
at 1451 cm1 are due to COO groups.
When the control spectrum is compared with the scaffold con-
taining A and S, it was observed that the peaks corresponding to
the amino group and hydroxyl group have become broader and
deeper. This indicates a possibility that S and A have interacted with
the free amino and the hydroxyl group of chitosan and gelatin. It
was observed absorption band at 2920 cm1 due to the symmet-
rical and asymmetrical C H stretching of the CH2 groups, which
is attributed to the pyranose ring. This band is also characteristic
of the presence of aliphatic (CH) groups in these compounds. The
absorption band at 1240 cm1 is due to the stretching vibrations
of C O groups of esters and phenols, which are more sharper in
the spectra containing A and S. The absorption peak at 1079 cm1
is due to the C O stretching of polysaccharides of Aloe vera, which
was absent in the control spectrum. The peaks around 1404 cm1
in the spectrum correspond to carboxyl COOH stretching bands.
All these spectra show evidence of intermolecular interactions and
good molecular compatibility between gelatin and chitosan, and
between the other components.

3.3. FTIR analysis 3.4. X-ray diffraction

FTIR analysis is an important tool to determine the chemical
changes at molecular level. The width and intensity of spectral
bands as well as the position of peaks are all sensitive to chem-
ical changes and to conformations of macromolecules [55]. The
FTIR spectra of the control, Aloe vera and snail mucus compos-
ite scaffold are shown in Fig. 2. The typical band assignments
for raw gelatin reported in the literature [56,57] are: amide A
(N H stretching vibration) found at around 3300 cm1 , whereas
X-ray diffractograms of gelatin/chitosan blended with Aloe vera
and snail mucus composite lms are shown in Fig. 3. All of these
spectra showed the main crystal diffraction peak around 2 = 20
in the XRD pattern. The XRD results suggested that there were
good compatibility and interaction between gelatin and chitosan
molecules in the scaffold. When Aloe and snail mucus components
were added in higher rates in the G-CH scaffold, the peak intensity
was reduced. This result indicate the decreasing of crystallinity of
650 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653

Fig. 2. FTIR spectra of lyophilized gelatin-chitosan blended with Aloe vera and snail mucus, (a) 0.07% and (b) 0.15%. A1 and A2 are low and high A concentration, S1 and S2
are low and high S concentration, AS 1 and AS 2 contain A blended with S in low concentration and A blended with S in high concentration.

the scaffold. This was because of the incorporation of amorphous
Aloe and snail into the matrix network. The little decrease in the
crystallinity of chitosan/gelatin scaffolds is due to the hydrogen
bonding between gelatin, which leads to their good compatibility
[58,59]. Moreover, other peaks appear around 2 = 29 , 36 , 39 ,
44 , 48 , 49 and 58 , which may originate from new crystals gen-
erated in the complex.
3.5. Swelling behavior
The water retention ability of the G-CH-based scaffold, which
is an essential attribute of wound dressing, was investigated. The
swelling pattern for the control scaffold does not change markedly
during the time of study, reaching a high absorption capacity of
about 600% (Fig. 4). Peng et al. [60], also reported a high swelling
capacity of crosslinked chitosan/gelatin composite. They suggested
this high water retention property might be attributed to the
hydrophilic nature of chitosan and gelatin. A high water uptake is
benecial to maintain the shape of scaffold and transfer nutrients
and wastes. By incorporating A and S in lower percentage, a signi-
cant increase in the percentage of swelling from 14 days to the end
of the storage period was observed. Certainly, these added com-
ponents generate a change in the structure of the scaffold, where
the polysaccharides provided by Aloe vera and snail increase the
porosity of the material allowing greater ease of ingress of water,
which was previously observed by SEM.
The incorporation of Aloe in high proportion interferes with the
process of liquid absorption in the material, limiting its swelling
 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653 651

70,000 main enzyme in wound exudation uid that could hydrolyze the
(1 4) glycosidic linkage in chitosan [62], Aloe vera [63] and snail
60,000
AS 1
mucus [64]. Fig. 5 shows the dynamic degradation of the scaffolds
50,000 after incubation in PBS and lysozyme solution at different times.
AS 2
The graphic shows a tendency of an increase in the percentage of
40,000
Intensity

A2
degradation of scaffolds during the 28 days of incubation. In gen-
30,000 A1 eral, no signicant differences were observed in the percentage of
Control degradation within each of the evaluated days, notwithstanding
20,000
the percentage degradation in the early days of incubation is sig-
S2

10,000
0 70 and 80%, proving thus its biodegradability in a similar body uid
0 20 40 60
2 (degree)
Fig. 3. XRD images of gelatin/chitosan scaffolds blended with Aloe vera and snail
mucus. A1 and A2 are low and high A concentration, S1 and S2 are low and high S
concentration, AS 1 and AS 2 contain A blended with S in low concentration and A
blended with S in high concentration.
between 300 and 450%, which is signicantly lower than in the
material without Aloe. The FTIR analysis has shown that a good
afnity and interaction between the matrix and the Aloe scaffold
may decrease water-binding sites, so this fact hindered the water
absorption capacity of the material. Finally, the mechanical prop-
erties could explain the highest swelling capacity of the S2 scaffold,
because the high percentage of elongation could facilitate the entry
of liquid in S2, generating a matrix with plenty of uid retention.
3.6. In vitro biodegradation
Controllable biodegradation rate plays crucial roles in regulat-
ing cell proliferation and tissue regeneration [61]. Lysozyme is the
nicantly lower in relation to the end of the period. After 28 days
S1
of incubation, the wound-healing scaffold was degraded between
80
environment. All G-CH-based scaffolds were degraded at similar
fasted rate, this might have happened because the larger pore helps
enzyme diffusion, leading to rapid degradation as seen in all kinds
of scaffold.
Although these in vitro data demonstrate the sensitivity of the
scaffold to different degradation mechanisms, an in vivo study
would be necessary to capture the effect of the relevance between
the matrix and the Aloe and snail mucus concentrations and bioac-
tivity.
4. Conclusions
In conclusion, we have obtained and characterized a biocompos-
ite scaffold possessing lysozyme sensitivity, exhibiting attractive
mechanical properties for soft tissue applications by combining of
natural and biocompatible biopolymers with regenerating prop-
erties. Due to the promising miscibility between gelatin-chitosan
and Aloe/snail mucus molecules, scaffolds with homogeneous net-
works and interconnected porous structures can be achieved after

Swelling study 0.07% Swelling study 0.15 %

1200 1200

1000 1000

800 800 Control

Swelling %
Swelling %

Control S2
600 600
S1 A2
400 A1 400 AS 2
AS 1
200 200

0
Incubaon period (days)
Incubaon period (days)

Fig. 4. Swelling behavior of gelatin/chitosan scaffolds blended with Aloe vera and snail mucus with concentration of 0.07% and 0.15%. A1 and A2 are low and high A
concentration, S1 and S2 are low and high S concentration, AS 1 and AS 2 contain A blended with S in low concentration and A blended with S in high concentration.

In vitro biodegradaon 0.07 % In vitro biodegradaon 0.15 %

100 100
90 90
80 80
70 70
Control control
Degradaon (%)

Degradaon (%)

60 60
S1 S2
50 50
40 A2
A1 40
30 AS 1 30 AS 2
20 20
10 10
0
2 4 6 8 28
Time (Days) Time (Days)

Fig. 5. In vitro biodegradation of gelatin/chitosan scaffolds blended with Aloe vera and snail mucus with concentration of 0.07% and 0.15%. A1 and A2 are low and high A
concentration, S1 and S2 are low and high S concentration, AS 1 and AS 2 contain A blended with S in low concentration and A blended with S in high concentration.
652 D.E. Lpez Angulo, P.J. do Amaral Sobral / International Journal of Biological Macromolecules 92 (2016) 645653
crosslinking and freeze-drying. These new scaffolds have improved
exibility and water absorption capacity compared to G-CH scaf-
folds. The in vitro degradation study revealed that the materials can
be almost completely hydrolyzed by PBS and lysozyme solutions,
which indicates their excellent and controllable degradability, with
a potential effect also on the cell response. This scaffold type may
prove to be useful as a vehicle to facilitate tissue development
in vitro prior to transplantation.
Conicts of interest
For this investigation the authors have no any actual or potential
conict of interest that could inappropriately inuence their work.
Funding
The authors would like to thank at the Brazilian National Council
for Scientic and Technological Development (CNPq) and TWAS,
the academy of sciences for the developing world, for the research
grants (190232/2014-5 CNPq/TWAS-FR: 3240279900).
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