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CHEMICAL ENGINEERING TRANSACTIONS
VOL. 74, 2019
A publication of
The Italian Association
of Chemical Engineering
Online at www.cetjournal.it
Guest Editors: Sauro Pierucci, Jiří Jaromír Klemeš, Laura Piazza
Copyright © 2019, AIDIC Servizi S.r.l.
ISBN 978-88-95608-71-6; ISSN 2283-9216
In Vitro Degradation of Gelatin/Carboxymethylcellulose
Scaffolds for Skin Tissue Regeneration
Fasai Wiwatwongwana*, Prayoon Surin
Department of Advanced Manufacturing Technology, Faculty of Engineering, Pathumwan Institute of Technology, 833
Rama 1 Road, Wangmai, Pathumwan, Bangkok 10330, Thailand
fasaiw227@gmail.com
The field of tissue engineering has grown in response to many medical needs for tissue replacement. For the
skin replacement, there have to develop the different types of tissue engineered skin substitutes. The artificial
scaffold have to be a biodegradable material with appropriate degradation rate for skin regeneration. In this
study, gelatin blended with carboxymethylcellulose (CMC) scaffold was fabricated via freeze drying method.
The various gelatin and CMC ratios were 100/0, 90/10, 80/20, 70/30 and 60/40, respectively. In vitro
degradation of the scaffold was done by degradation in lysozyme in PBS buffer within desired time. The
swelling ratio of the scaffolds was done by immersing in PBS buffer for 2 h at 37oC. The results revealed that
the gelatin/CMC (80:20) scaffold showed the highest value of swelling ratio and have an appropriate
degradation rate with completely degraded after 36 h. Moreover, the gelatin/CMC (90:10) also showed good
degradation rate and high value of swelling ratio. However, the gelatin/CMC (60:40) scaffold occurred fast
degradation rate with the lowest value of swelling ratio. The in vitro degradation result was consistency with
the result of swelling ratio. This could imply the best condition for scaffold fabrication from biological and
physical analysis which might suitable for skin tissue engineering applications.
1. Introduction
Tissue engineering is an interdisciplinary field which can be referred to biomaterials development that
combining scaffold, cells and biologically active molecules into functional tissues. For example porous scaffold
for improving, maintaining and restoring the function of skin tissues and organs. Skin loss of patient can cause
from different ways such as accidents, burns, ulcers and diseases. The scaffold, skin replacement, has been
widely used and recently available. It have to be biocompatible and biodegradable material with non-toxicity.
The scaffold functions have to prevent infection, accelerate the wound healing and generate the skin tissues
(Ma 2004 and Ma & Elisseeff 2006). Moreover, it can be allowed skin around the wound to reproduce a new
regeneration of skin tissues in a suitable conditions. However, the presently available scaffolds are expensive
due to its components. Therefore, the cheaper scaffold which has the same functions is the propose of this
research. The scaffold fabrication have to be designed for supporting the recovery mechanism of skin
functions. The applications of tissue engineering is the main propose of the scaffold design. Its components
can be used from the naturally derived or chemically synthesized in the compositions (Hollinger 2012).
The important functions of the scaffold are the appropriate degradation properties and suitable physical
properties which have to be perfectly fit the rate of skin tissue regeneration that would need around 2 weeks to
be completed. Moreover, their mechanical properties, ease of processing and biochemical activity are
prominent in the design of tissue engineering materials that elicit a specific and desired biological response
(Fisher et al. 2007).
Nanofibrous structure such as aerogel-based scaffolds can be used in tissue engineering applications. There
have a study of bi-component natural aerogels Alginate-Gelatin (AG) and Chitosan-Gelatin (CS/G) produced
by Supercritical gel drying. The results revealed that both A/G and CS/G mixtures formed uniform gels and
Supercritical gel drying provided nanostructured morphology. The porosity values higher that 80% of all the
aerogels. CS/G aerogels crosslinked with Glutaraldehyde (GTA) showed improved in thermal behavior
DOI: 10.3303/CET1974260
Paper Received: 10 June 2018; Revised: 2 October 2018; Accepted: 3 February 2019
Please cite this article as: Wiwatwongwana F., Surin P., 2019, In Vitro Degradation of Gelatin/carboxymethylcellulose Scaffolds for Skin Tissue
Regeneration, Chemical Engineering Transactions, 74, 1555-1560 DOI:10.3303/CET1974260
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(Baldino et al. 2015). Other scaffolds such as small diameter vascular grafts can be used to treat peripheral
vascular pathologies and ischemic heart diseases. The small grafts made of poly (ε-caprolactone) (PCL) and
poly (glycerol sebacat) (PGS) by electrospinning technique. Their surface was modified by dynamic coating of
gelatin at 37 oC for 1 h followed by UV-irradiation to reduce water permeability.The results showed that gelatin
could improve the properties of PCL:PGS electrospun grafts by decreasing in the water permeability and did
not affect the 3D structure and pore interconnectivity of the scaffolds (Ferrari et al. 2017).
There have been studied in vitro degradation of polycaprolactone and polycaprolactone/gelatin nanofibrous
scaffold. The polycaprolactone (PCL) and polycaprolactone/gelatin (PCL/Ge) 70:30 nanofibrous scaffolds
were fabricated using electrospinning technique and compared on in vitro degradation rate to determine a
more suitable scaffold for skin tissue engineering application. The result showed that both PCL and PCL/Ge
(70:30) nanofibrous scaffolds were degraded after 8th week. The degradation rate of PCL nanofibrous scaffold
was slower and did not has obvious morphological changes. PCL nanofibrous scaffold with slow degradation
rate was more suitable to be used for long-term implantation and long-term drug delivery carrier while PCL/Ge
(70:30) nanofibrous scaffold with faster degradation rate might have the potential to be used for skin tissue
engineering application (Mim & Sultana 2017).
Moreover, there have been studied the degradation of a collagen-chondroitin-6-sulfate matrix by collagenase
and chondroitinase. Type I collgen-chondroitin-6-sulfate (collgen-GAG) scaffolds, produced by freeze-drying
techniques. The objective was to evaluate changes in the microstructure and mechanical properties of
selected collagen-GAG scaffolds as they degrade in an in vitro model system. Carbodiimide-cross-linked
matrices were found to have a higher cross-link density, a higher compressive stiffness and a greater
resistance to collagenase and chondroitinase, compared to non-cross-linked controls and matrices that were
cross-linked by the dehydrothermal process (Pek et al. 2004). The functional behavior of novel poly(ε-
caprolactone) (PCL) membranes functionalized with reduced graphene oxide (rGO) nanoplatelets under
simulated in vitro culture conditions (phosphate buffer solution (PBS) at 37 ◦C) during 1 year, in order to
elucidate their applicability as scaffolds for in vitro neural regeneration. After 1 year of hydrolytic degradation,
the pH was barely affected, and the rGO nanoplatelets mainly remained in the membranes which envisaged
low cytotoxic effect. The degradation rate of the membranes herein reported would perfectly fit the rate of
neural tissue regeneration that would need around 1 month to be completed. On the other hand, the presence
of rGO nanomaterials accelerated the loss of mechanical stability of the membranes. However, it is envisioned
that the gradual degradation of the PCL/rGO membranes could facilitate cells infiltration, interconnectivity, and
tissue formation (Gonzalez et al. 2018).
Among the most commonly utilized material is natural ECM (extracellular matrix)-based polymer such as
collagen. However, due to high cost of collagen this research aimed to use gelatin which is a denatured
structure of collagen and has been used for medical application as a biomaterial and as an additive or gel
capsules in drugs (Olsen et al. 2003). There have many researches approved for in vitro biocompatible test for
gelatin with fibroblast cells. The results of those research fields showed that gelatin scaffolds could be able to
maintain cells with good affinity and proliferation after 14 days of culturing without any signs of biodegradation
(Lee et al. 2005). CMC was used as a hydrogel for wound dressing, a scaffold for various tissue engineering
applications and an injectable material for bone augmentation (Biswal & Singh 2004 and Capitani et al. 2000).
Various treatments such as dehydrothermal treatment and chemical treatment have been used for
strengthening the scaffold structure (Haugh et al. 2011). Therefore, using gelatin blended with CMC as a
scaffold for skin implantation could be improved in physical and biological properties of the scaffold for skin
tissue engineering. This scaffold could be used instead of collagen which is currently undergoing as skin
substitute in the tissue engineering. The current study focused on the scaffolds made from gelatin blended
with CMC in various ratios. Degradation and swelling properties of the gelatin/CMC scaffolds were evaluated
in comparison to pure gelatin scaffold.
2. Materials and methods
2.1 Gelatin-CMC scaffold preparation
Type A gelatin was purchased from BIO BASIC INC, Canada. It was a reagent grade and derived from pork
skin with bloom number of 240-270 and pH 4.5-5.5 at 25 oC. Its viscosity was 3.5-4.5 cps and moisture less
than 12.0%. Carboxymethylcellulose sodium salt (CMC) was purchased from Sigma-Aldrich, St. Louis, MO,
USA. It was medium viscosity with 400-800 cps in a 2% aqueous solution at 25 oC. The gelatin solution was
prepared by mixing gelatin powder with DI water at 0.8 wt% (w/w) then leaved it at room temperature for 1
hour before stirred it at 50°C for another 1 hour. The CMC solution was prepared by mixing CMC powder with
DI water at 0.8 wt% (w/w) and then stirred it at 70 °C for 30 minutes. The gelatin solution was mixed with CMC
solution in various ratios which were 100/0, 90/10, 80/20, 70/30 and 60/40, respectively and stirred it at 50 °C
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for 15 minutes of each condition. Finally, the solutions were pipetted into 24-well culture plate with volume of 1
ml per well and freezed them for 24 hours at -20 °C. The sample was then placed in a Lyophilizer (Freeze-Dry
Machine) at -50 °C for 24 hours to produce a gelatin/CMC scaffold with porous structure and put all
gelatin/CMC scaffolds in a humid controlled container.
The scaffold was then cross-linked using a physical process of dehydrothermal treatment (DHT). It was
exposed to 140 °C under vacuum for 48 h (Wiwatwongwana & Promma 2015). The diagram of gelatin/CMC
scaffold preparation was shown in Figure 1.
Figure 1: Schematic diagram of 0.8 wt% (w/w) gelatin/CMC scaffold preparation.
2.2 Degradation of Gelatin-CMC scaffold
The enzyme lysozyme was purchased from BIO BASIC INC, Canada. It was purified from chicken egg white
into a lyophilized powder. It was white powder with moisture less than 6.0%. Its molecular mass was 14,307
Da and isoelectric point (pi) of 11.35. The enzyme was active over a broad pH range (6.0-9.0). This research
used this enzyme for gelatin/CMC scaffold degradation. An enzyme concentration was reported in enzyme
units (u) per ml. The selected concentration of enzyme was 31.2 u/ml (0.1 mg/ml PBS) (Pek et al. 2004). The
gelatin/CMC scaffold with various conditions were placed in 24-well culture plate and the enzyme solution was
added with volume of 2 ml per well. Then, it was incubated in oven at 37
oC. After 0.5, 1, 1.5, 3, 9, 15, 24, 36
and 48 h, the scaffolds were removed and rinsed with DI water to remove the enzyme. Then, dried the
scaffolds and weighed it. The weight remain of the scaffold after degradation was calculated by the following
equation (1):
Weight remain (%) = 100 W W
W
100 (1)
Where: W is initial weight of the scaffold
W is final weight of the scaffold
2.3 Determination of swelling ratio
The swelling ratio of gelatin/CMC scaffolds was investigated. The gelatin/CMC scaffold in all conditions were
placed in a PBS buffer at 37 oC for 2 h to swell and denature the gelatin and CMC. PBS buffer within the pores
was expelled by pressing the swollen scaffolds between sheets of kimwipe tissue paper with placed the
scaffold on each side of paper for 5 s. The scaffold was weighted and the weight was recorded as wet weight.
The swelling ratio of gelatin/CMC scaffold was calculated by the following equation (2) (Pek et al. 2004):
Swelling ratio =
W W
W
(2)
Where: W is initial weight of the scaffold
W is wet weight of the scaffold
3. Results
3.1 In vitro biodegradation of gelatin/CMC scaffolds
The gelatin/CMC scaffolds was shown in Figure 2. The appearance and average size of G100T (Figure 2(a))
was similar to G82T (Figure 2(b)). The average diameter and height of all gelatin/CMC conditions were similar
compared to pure gelatin scaffold with average diameter was from 11.71±0.22 mm to 13.92±0.14 mm and
average height was from 4.28±0.09 mm to 5.26±0.25 mm as shown in Table 1. The G73T showed the lowest
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value in both diameter and height. All gelatin/CMC scaffolds in various conditions were compared on in vitro
degradation by lysozyme. In this study, gelatin/CMC scaffolds with all conditions were incubated in lysozyme
in PBS solution at 37 oC for 0.5, 1, 1.5, 3, 9, 15, 24, 36 and 48 h. The results showed that all gelatin/CMC
conditions were completely degraded after 36 hours as illustrated in Figure 3. The degradation of the
scaffolds which added the CMC in a condition of G91T, G82T and G73T showed the appropriate degradation
rate with completely degraded after 36 hours. While, pure gelatin scaffold (G100T) and gelatin/CMC scaffold
in a condition of G64T were completely degraded after 9 hours. At the initial time of degradation (Table 2),
scaffold G100T occurred the weakest in structure with weight remain of 18.34% at degradation time of 0.5
hour while scaffold G64T showed the weakest structure with weight remain of 10.56% at degradation time of
3 hours. The other scaffolds (G91T, G82T and G73T) showed the stronger in structure with can remain their
weight of 27.50%, 31.67% and 38.18%, respectively after degraded in lysozyme for 3 hours.
Figure 2: 0.8 wt% (w/w) of gelatin/CMC scaffolds (a) G100T and (b) G82T.
Table 1: Average diameter and height of 0.8 wt% (w/w) gelatin/CMC scaffolds (n=5).
Scaffolds Average diameter (mm) Average height (mm)
G100T 13.88±0.33 4.96±0.29
G91T
G82T
G73T
G64T
13.92±0.14
13.17±0.27
11.71±0.22
13.91±0.17
5.26±0.25
5.10±0.26
4.28±0.09
4.94±0.23
Figure 3: In vitro degradation behaviour of Figure 4: Swelling ratio of 0.8 wt% (w/w)
0.8 wt% (w/w) gelatin/CMC scaffolds. gelatin/CMC scaffolds (n=5) (*significant different
p<0.05 relative to G100T).
Table 2: Weight remain (%) at initial time (0.5, 1, 1.5, and 3 hours) of 0.8 wt% (w/w) gelatin/CMC scaffolds.
Scaffolds
Degradation time (hour)
0.5 1 1.5 3
G100T 18.34 15.75 14.87 14.58
G91T
G82T
G73T
G64T
93.87
94.63
95.54
96.05
79.77
88.23
91.18
78.22
74.91
66.96
85.28
77.11
31.67
27.50
38.18
10.56
* *
*
(a) (b)
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3.2 Swelling ratio of gelatin/CMC scaffolds
Figure 4 illustrated the swelling ratio of all gelatin/CMC scaffolds. In overall, the swelling ratio of all scaffold
conditions was from 10.55±1.26 to 34.96±1.81 as shown in Table 3. According to the figure 4, the
composition of gelatin and CMC at ratio 80:20 showed the highest value of water absorption compared to pure
gelatin scaffold (18.82±0.83) with significant different. Increasing CMC resulted in collapsed structure. The
G64T scaffold showed the lowest of swelling ratio with significant different compared to pure gelatin scaffold.
The average swelling ratios of G82T and G64T were 34.96±1.81 and 10.55±1.26, respectively. The result of
swelling ratio was consistency with the result of in vitro degradation in lysozyme. The G82T scaffold occurred
the best value of swelling ratio and have an appropriate degradation rate. The G64T scaffold occurred the
lowest value of swelling ratio with fast degradation rate.
Table 3: Swelling ratio of 0.8 wt% (w/w) gelatin/CMC scaffolds (n=5).
Scaffolds Average swelling ratio SD
G100T 18.82 0.83
G91T
G82T
G73T
G64T
33.36
34.96
16.83
10.55
1.27
1.81
2.28
1.26
4. Discussion
This results reported in this study concluded that increasing CMC into gelatin scaffolds at an appropriate
content could be improved in biological and physical properties of scaffolds. The gelatin blended with CMC
scaffolds at ratio of 80:20 (G82T) showed the highest value of the swelling ratio with significant different
compared to pure gelatin scaffold. G82T scaffold also showed good degradation rate in lysozyme solution with
could remain their structure and degraded completely after 36 hours that might suitable for skin regeneration.
However, the scaffold G100T and G64T showed weak in its structure with gave a low value of swelling ratio
and fast degradation rate with completely degraded after 9 hours which was not suitable for skin regeneration.
This results suggested that improving gelatin scaffold by blending with CMC at the ratio of 80:20 and using
thermal crosslinking technique might be applied for tissue engineering applications. Moreover, it could be
suggested that CMC could be used as a scaffold strengthening at appropriate content. The degradation of the
scaffolds would be increased if the CMC content was too much.
5. Conclusion
The scaffolds which made from gelatin blended with CMC in various conditions were studied in this research.
In vitro degradation of all scaffold conditions was investigated by immersing in lysozyme in PBS solution at the
desired time. The degradation results showed the best condition of gelatin/CMC ratio at 80:20 with completely
degraded after 36 hours and showed the highest value of swelling ratio. It was found that too much of CMC
content decreased in material strengthening. Increasing of CMC content at an appropriate content could be
improved in biological and physical properties of scaffold. From the results, G82T could be used to design for
skin tissue engineering applications.
Acknowledgments
This research conducted under the support of Department of Advanced Manufacturing Technology and
Department of Manufacturing Engineering, Faculty of Engineering, Pathumwan Institute of Technology.
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