Porous Architecture and Permeability Evaluation of Silk

Fibroin Scaffold from Direct Dissolution Salt Leaching

Method

Hermawan Judawisastra1*, Febri Ramdani Nugraha1, Untung Ari Wibowo1

1
Department of Material Science and Engineering, Faculty of Mechanical and
Aerospace Engineering, Institut Teknologi Bandung, Jl. Ganesa 10, Bandung 40132,
Indonesia

hermawan.judawisastra@material.itb.ac.id

Abstract. Tissue engineering scaffold is used to facilitate and support cell/tissue growth.
Fabricating of silk fibroin scaffold using the direct dissolution salt leaching method has been
successfully done, significantly reduced processing time than the traditional method. Cells
attachment, cells growth, cells migration, and diffusion of nutrients and waste was affected by
architecture and permeability of scaffold. In this study three-dimensional analysis on porous
architecture and permeability evaluation of direct dissolution salt leaching scaffolds were
evaluated. Scaffolds were fabricated from different NaCl particle size 158 μm, 250 μm, 378
μm and 503 μm at silk fibroin concentration 12% w/v. Scaffold architecture was evaluated
using microcomputed tomography (micro-CT) method. Scaffold permeability was measured
using falling head method. This research has successfully revealed the effects of NaCl particle
size to porous architecture and permeability of silk fibroin scaffold resulting from direct
dissolution salt leaching method, which is important for cells attachment, cell proliferation, cell
differentiation, tissue formation, and scaffold mechanical properties.

Keyword: Micro-CT, Permeability, Porous architecture, Salt leaching, Scaffold

1. Introduction
Tissue engineering aims to regenerate damaged tissues, by developing biological substitutes that
restore, maintain or improve tissue function. For successful tissue engineering, three main components
that are required include scaffold, cells and growth factors. The scaffold is a three-dimensional (3D)
structure is used to facilitate cell/tissue growth and the transport of nutrients and wastes while
degrading gradually itself. Scaffold essentially act as a template for tissue formation and are typically
seeded with cells and growth factors. These cell-seeded scaffolds are either cultured in vitro to
synthesize tissues which can then be implanted into an injured site, or are implanted directly into the
injured site, using the body’s own systems, where regeneration of tissues or organs is induced in vivo
[1–3]. Materials scaffold have to be non-toxic, sterile, biodegradable and biocompatible [4]. Polymer
materials widely used for tissue engineering because easy to control biodegradability and
processability. The naturally derived polymers include proteins of natural extracellular matrices such
as collagen and glycosaminoglycan, alginic acid, chitosan, and polypeptides, etc. [5]. Silk fibroin
polymers from silkworms (e.g., Bombyx mori) consist of repetitive protein sequences and provide
structural roles in cocoon formation [6]. Fibroin silk shows biocompatibility, high mechanical
properties, and degradation products are less toxic and there is no adverse immune response in the host
system [7,8]. Judawisastra (2016) has been successfully developed silk fibroin scaffold using the direct
dissolution salt leaching method that significantly reduces processing time and temperature compared
to conventional methods. This method combines the direct dissolution method using CaCl2-formic
acid mixture to produce silk fibroin solution and salt leaching fabrication to produce porous silk fibroin
scaffold [9].

Cell growth, cell proliferation, cell differentiation and mechanical properties of scaffold are influenced
by scaffold architecture, including pore size, scaffold porosity, scaffold wall thickness, pore
interconnectivity, and scaffold specific surface area, and scaffold permeability [10–13]. Pore
architecture of a scaffold has a direct impact on cell attachment, cell penetration and migration during
cell seeding, the diffusion of oxygen, nutrients, and waste products, and provides a 3-dimensional (3D)
microenvironment inducing cell assembly and differentiation [10,11,14,15]. Mechanical properties
greatly affected by scaffold wall thickness [12]. The scaffold permeability influencing tissue formation,
by the diffusion of oxigen and nutrients, waste removal and cell migration into the scaffold [13,16].
Scaffold architecture from salt leaching fabrication can be varied by varying salt particle size, the salt
particle dissolved to result in a porous scaffold [12]. Scaffold architecture can affect each other. Pore
size can affect on scaffold porosity, scaffold wall thickness, pore interconnectivity, and scaffold
specific surface area [10,12,17,18]. The scaffold permeability is a function of porosity, pore
architecture, and the pore connectivity [16].

Scaffold architecture can be performed by two-dimensional (2D) and three-dimensional (3D) image
analysis. 2D evaluation can be obtained from scanning electron microscopy (SEM) examination results
which provide a measure only in a 2D image. This evaluation required destructive and tedious sample
preparation, and does not allow the measurement of scaffold specific surface area, the pore volume,
and connectivity parameters. 3D evaluation can be obtained by means of X-ray microcomputed
tomography (micro-CT) scan. This technique is non-destructive and can be used to measure scaffold
specific surface area, pore volume, porosity, pore size, pore interconnectivity [13,19]. Evaluation 3D
architecture of natural and synthetic scaffolds using micro-CT has been widely doing [7,11,12,19–26].
C. Correia, et al, and S. Lin-Gibson, et al, has evaluated 3D porous architecture of silk fibroin and
EBPADMA scaffolds respectively, resulting from traditional salt leaching method using micro-CT
[7,12]. Scaffold permeability of Tri-calcium phosphate (TCP) scaffold has been conducted and showed
the influence pore size, porosity, pore connectivity, and surface area [17]. However, evaluation of 3D
architecture of silk fibroin scaffold from the direct dissolution salt leaching method including the
correlation with scaffold permeability has not been done yet. In this research, evaluation of 3D porous
architecture of silk fibroin scaffold from the direct dissolution salt leaching method including the
correlation with scaffold permeability will be carried out. Effect of NaCl particle size variation to 3D
porous architecture and permeability of scaffold will be also evaluated.

2. Material and method

2.1. Material and preparation of the silk fibroin scaffold
Bombyx mori silkworm cocoons was from CV. Wisata Ilmu Sutera, Bandung, Indonesia. Sodium
bicarbonate, sodium chloride, 70% ethanol, and demineralized water were from PT Bratachem,
Bandung, Indonesia. Calcium chloride and formic acid were from Merck Millipore, Germany.
The silk fibroin scaffold fabrication was started by degummed the silk cocon twice in 0.5 wt% NaHCO3
for 1 hour to remove the sericin, rinsed with demineralized water and then dried in a fume hood
overnight. The degummed silk was weighed and directly dissolved in 8 wt% CaCl 2-formic acid then
mixed using magnetic stirrer homogenized for 3 hours in room temperature to produce solution silk
fibroin with concentration 12% w/v, then poured and mixed into a 3.5 cm diameter mold containing
NaCl particle. The NaCl particle size was manually sieved and varied in 158 ± 47 μm, 250 ± 58 μm,
378 ± 42 μm, 503 ± 31 μm. NaCl-silk mixture was fixed at ratio gr-mass to ml-volume 5:1. NaCl-silk
mixture was placed at RT overnight to evaporate the solvent and to become NaCl-silk block. The NaCl-
silk block was immersed in 70% ethanol for 1 hour, followed by immersing into the demineralized
water for 3 days to remove NaCl particle by replacing the water every 6 hours [9].

2.2. Characterization of the silk fibroin scaffold

Silk fibroin scaffold architecture evaluation
Scaffold three-dimensional (3D) architecture was evaluated using a Bruker high-resolution micro-CT
Skyscan 1173 scanner (Skyscan, Kontich, Belgium) with following parameter: pixel size of 11,044
µm, integration time of 1.3 s., X-ray source at 35 keV and 190 µA, rotation range of 1800, and rotation
step of 0.2. Data sets were reconstructed using standardized cone-beam reconstruction software
(NRecon v1.7.1, SkyScan). The output format is 2240 x 2240 bitmap images. Two-dimensional (2D)
or 'sliced' bitmap images from micro-CT were evaluated using CTAn (CT Analyser, v1.16.4.1,
SkyScan) software. The scaffold architecture analysis doing by choosing an appropriate region of
Interest (ROI) on a single cross-section image and integrating of all the ROIs across all the selected
image levels to the volume of interest (VOI). Then the image is segmented into binary images with a
threshold of 5–255 (grey values). Despeckle process was carried out to reduce noise dot/single dot
object. Binary images were used to build three-dimensional models.
To evaluate scaffold architecture using CTAn software, five parameters were measured by means of
different 3D analysis tools: pore size was measured using the Structure Separation algorithm, scaffold
porosity was determined using the Porosity algorithm, scaffold wall thickness was calculated using the
Structure Thickness algorithm, pore interconnectivity was measured using the ROI shrink-wrap
algorithm, and scaffold specific surface area was measured using the Object surface / Volume Ratio
algorithm, see Table 1.

Table 1. Scaffold architecture: Description and 3D analysis tools.

Scaffold
No. Description and 3D Analysis Tools
architecture
1 Pore size [21]. Pore size is defined as average the contiguous pore space (black voxels)
in three (X, Y, Z)
3D Analysis CTAn: Structure Separation
2 Scaffold Total porosity is the volume of all volume pore as a percent of the total
porosity [27]. VOI volume
3D Analysis CTAn: Porosity
3 Scaffold wall Scaffold wall thickness defined as the diameter of the largest sphere on
thickness [27]. solid of the scaffold
3D Analysis CTAn: Structure Thickness
4 Pore Pore interconnectivity defined the accessible pore space for a virtual
interconnectivity object, with a diameter equal to minimum connection size. The difference
[25]. between the original VOI and the VOI after Shrink-Wrap corresponds to
the pore space that is accessible.

Pore interconnectivity (%)

Volume VOI − Volume VOI 𝑆ℎ𝑟𝑖𝑛𝑘 − 𝑤𝑟𝑎𝑝
=( ) 𝑥 100%
Volume VOI − Volume solid 𝑠𝑐𝑎𝑓𝑓𝑜𝑙𝑑
The ROI shrink-wrap using stretch over hole less than 4 voxel based on
the smallest protein that stimulates osteogenesis [28].
5 The scaffold The specific surface area defined the surface area of the interface between
specific surface material and void divide by the apparent volume of the scaffold
area [11]. 3D Analysis CTAn: Object surface / Volume Ratio
 Scaffold permeability measurement
The scaffold specific permeability (k ) was measured using a falling head method, see Equation (1)
[29].
µ
𝑘=𝐾 (1)
𝜌𝑔
where ρ is the density of demineralized water (0.998 Mg m-3), µ is dynamic viscosity demineralization
water (8.9 x 10-4 Pa s), and g is gravity acceleration (9.8 m s-2), K is hydraulic conductivity. This is a
scalar quantity which measures how easily a fluid is transported through a tortuous void space [29].
The hydraulic conductivity (K) is calculated using Equation (2) [29].
𝑎 𝐿 𝐻1
𝐾= 𝑙𝑛 (2)
𝐴 𝑡 𝐻2
The initial head difference H1 is recorded at t = t0, then time required for water in the standpipe to drop
from the upper to the lower level H2 is recorded (t). α is the cross-sectional area of the standpipe, A is
the cross-sectional area of the sample of scaffold, L is the scaffold thickness.
Three specimens on each sample were used to determine the scaffold permeability.

3. Result and discussion

3.1. The scaffold 3D model in varying NaCl particle size
Figure 1 shows the scaffolds 3D model resulting from different NaCl particle size. It can be easily
observed that the increase of NaCl particle size generates larger pore size. Scaffold resulting from NaCl
particle size 158 μm shows the highest number of the pore whilst the largest NaCL particle size 503
μm results in the lowest number of the pore. Scaffolds were highly porous and the pores are
interconnected. This scaffold morphology confirms with silk fibroin scaffold resulted from literature
[22].

Figure 1. 3D model of silk fibroin scaffolds with different NaCl particle sizes:
(a) 158 μm, (b) 250 μm, (c) 378 μm, (d) 503 μm.
3.2. Scaffold architecture results from different NaCl particle size
Figure 2 shows scaffold architecture measurement resulted from different particle NaCl size: Mean
pore size, scaffold porosity, scaffold wall thickness, pore interconnectivity, and the scaffold specific
surface area. The use of larger NaCl particle size generates larger pore size, higher scaffold porosity,
higher scaffold wall thickness, higher pore interconnectivity, but decrease the scaffold specific surface
area.
200 88
Mean pore size (µm)

Scaffold porosity (%)

86
150 84
82
80
100
78
76
50 74
72
0 70
158 250 378 503 158 250 378 503
NaCl particle size (µm) NaCl particle size (µm)

(a) (b)
Scaffold wall thickness

Pore interconnectivity
60 105
50 100
95
40
(µm)

90
(%)

30
85
20
80
10 75
0 70
158 250 378 503 158 250 378 503
NaCl particle size (µm) NaCl particle size (µm)

(c) (d)
Scaffold specific surface

0.16
0.14
0.12
area (1/m)

0.10
0.08
0.06
0.04
0.02
0.00
158 250 378 503
NaCl particle size (µm)

(e)
Figure 2. Scaffold architecture measurement results (a) Mean pore size, (b) Scaffold porosity, (c)
Scaffold wall thickness, (d) Pore interconnectivity, and (e) The scaffold specific surface area.

Pore size evaluation

Figure 2(a) shows the mean scaffold pore size resulting from different NaCl particle sizes. The use of
larger NaCl particle size generates higher scaffold pore size. During salt leaching fabrication, the pore
size of the scaffold follows the size of the salt used, when the salt dissolves in the salt leaching process,
it produces a porous structure in the scaffold [3,12]. Increasing NaCl particle size from 158 to 503 µm
resulted in higher pore size from 97.72 - 158.35 µm. The pore size more than ~5 µm is required to
produce scaffold that meets the requirement for mammalian/human tissue reconstructions [30]. The
pore size measurement results are smaller than the correspond NaCl particle size used. The use of
larger NaCl particle size resulted in larger difference between the particle size and pore size measured.
This phenomena is related with the incorporation of the incomplete pores at the volume of interest
surface during 3D analysis and was also obtained in other 3D analysis of silk fibroin scaffolds [12,22].

Scaffold porosity evaluation

Figure 2(b) shows the scaffold porosity measurement resulting from different NaCl particle sizes. The
use of larger NaCl particle size generates higher scaffold porosity. Increasing NaCl particle size
generates higher pore size which caused larger scaffold pore volume, see Figure 3. The large scaffold
pore volume will result in high scaffold porosity [10]. The increase in NaCl particle size used from
158 to 503 µm resulted in increasing scaffold porosity from 77.61 to 84.08%, indicating highly porous
scaffolds. Highly porous scaffold, above 79% porosity, is suitable for tissue engineering scaffold
[22,30].
Scaffold with high porosity (82%) was obtained in the scaffold produced from small NaCl particle size
158 µm, see Figure 2(b). The use of small particle size 158 µm did not produce low porosity scaffold.
Scaffold porosity was determined by pore size and number of pore [10,12]. Figure 1(a) shows scaffold
from NaCl particle size 158 µm has the most pore number. This indicates the high porosity in this
scaffold was caused by domination of the high pore number than that of small pore size.
4.1
4
Scaffold pore volume

3.9
( x 10^8 µm^3)

3.8
3.7
3.6
3.5
3.4
3.3
3.2
3.1
158 250 378 503
NaCl particle size (µm) Figure 3. Scaffold pore volume resulted from
different NaCl particle size.

Scaffold wall thickness evaluation

Figure 2(c) shows the scaffold wall thickness resulted from different NaCl particle sizes. The
increasing of NaCl particle size generates thicker scaffold wall thickness. The larger NaCl particle size
from 158 to 503 µm resulted in the thicker scaffold wall thickness from 35.08 - 50.63 µm. The pore
size is directly related to the wall thickness for scaffolds with a near identical porosity. As the pore size
decreases, a larger number of pores must exist to maintain a constant porosity, therefore, the average
wall thickness must also decrease [12].The use of NaCl particle size from 158 to 503 µm increased
scaffold pore size up to 62%, see 3.2.1, whilst increased scaffold porosity only 8%, see Table 2.
Therefore the thicker scaffold wall thickness was dominated by increasing scaffold pore size as the
scaffold porosity was maintained identic.

Table 2. Scaffold porosity resulted from different NaCl particle size.

NaCl particle size (µm)

158 250 378 503
Scaffold porosity (%) 82.64 77.61 80.61 84.08

Pore interconnectivity evaluation

Figure 2(d) shows pore interconnectivity resulted from different NaCl particle sizes. The use of larger
NaCl particle size increased pore interconnectivity. The use of NaCl particle size from 158 to 503 µm
resulted in increasing pore interconnectivity from 92.72 to 97.78%, indicating high pore
interconnectivity scaffolds. Highly pore interconnectivity scaffold, above 90% porosity, is suitable for
tissue engineering scaffold [22,30]. As mentioned in 3.2.2, increasing NaCl particle size generates
higher scaffold porosity. Pore interconnectivity increases along with porosity, see Figure 4. The higher
porosity results in greater pore volume, creating higher interconnected pores [17].
100
Pore interconnectivity (%)

95

Porosity (%)
90
Porosirty (%)

Pore interconnectivity
85 (%)

80

75

70
158 250 378 503
NaCl particle size (µm)

Figure 4. Increasing pore interconnectivity along with porosity resulting from the use
of larger NaCl particle size.

The scaffold specific surface area evaluation

Figure 2(e) shows the scaffold specific surface area resulted from different NaCl particle sizes. The
use of larger NaCl particle size from 158 to 503 µm decreases the scaffold specific surface area from
0.12 to 0.077 m-1. This is in accordance with research of Fergal J. O'Brien's et al. [18] which developed
the relationship of the scaffolds specific surface area with the average pore size (d), see Equation (3).
The use of larger NaCl particle size generates higher scaffold pore size. Increasing scaffold pore size
decreases the scaffolds specific surface area.

0.718
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑠𝑢𝑟𝑓𝑎𝑐𝑒 𝑎𝑟𝑒𝑎 = (3 )
𝑑

3.3. The scaffold specific permeability evaluation

Specific permeability results of fibroin scaffold resulting from different NaCl particle sizes is shown
in Figure 5. The use of larger NaCl particle size results in higher scaffold specific permeability. The
use of larger NaCl particle size from 158 to 503 µm increases scaffold specific permeability from 0.62
x 10-11 to 2.09 x 10-11 m2. As mentioned in point 3.2, increasing the NaCl particle size generates larger
pore size, higher scaffold porosity, higher pore interconnectivity, and decreases scaffold specific
surface area. Higher scaffold porosity and pore interconnectivity will provide more space for fluid to
flow and result in higher specific permeability, see Figure 6 [17]. Low specific surface area means
low friction on the surface of the scaffold. Lower specific surface area generates lower impede of fluid
to flow and consequently results in high specific permeability, see Figure 7 [31].
 30
Scaffold specific permeability

25
( x 10^-12 m^2)

20

15

10

0
158 250 378 503
NaCl particle size (µm) Figure 5. The scaffold specific
permeability in varying NaCl particle size.

100 25

Porosity (%)

Scaffold specific permeability

Pore interconnectivity (%)

95
20
Pore interconnectivity (%)

( x 10^-12 m^2)
90
Porosity (%)

15 Scaffold specific permeability ( x

85 10^-12 m^2)

10
80

5
75

70 0
158 250 378 503
NaCl particle size (µm)

Figure 6. The increase of scaffold specific permeability along with scaffold porosity and pore
interconnectivity resulting from the use of larger NaCl particle size.

0.14 25
Scaffold specific surface area (1/m)

0.12
Scaffold specific permeability

20

0.10 Scaffold specific surface area (1/m)

( x 10^-12 m^2)

15 Scaffold specific permeability ( x

0.08
10^-12 m^2)
0.06
10

0.04

5
0.02

0.00 0
158 250 378 503
NaCl particle size (µm)

Figure 7. Correlation of the scaffold specific surface area and the scaffold specific permeability at
different NaCl particle size.
4. Conclusion
Three dimensional analysis on porous architecture and permeability evaluation of silk fibroin scaffold
from direct dissolution salt leaching method have been conducted. Evaluation of scaffold architecture
showed the use of larger NaCl particle size increased the scaffold pore size. The larger pore size
resulted in higher scaffold porosity, thicker scaffold wall thickness, higher pore interconnectivity but
lower scaffold specific surface area. The use of larger NaCl particle size resulted in higher scaffold
specific permeability. Higher scaffold porosity and pore interconnectivity provide more space for fluid
to flow. Lower specific surface area generates low friction on the surface of the scaffold and lower
impede of fluid to flow. This research has successfully revealed the effects of NaCl particle size to
porous architecture and permeability of silk fibroin scaffold resulting from direct dissolution salt
leaching method, which is important for cells attachment, cell growth, cell proliferation, cell
differentiation, tissue formation, and scaffold mechanical properties.

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