Original article

Textile Research Journal

2018, Vol. 88(17) 1965–1978

Factors affecting the mechanical and ! The Author(s) 2017

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geometrical properties of electrostatically DOI: 10.1177/0040517517715083
journals.sagepub.com/home/trj
flocked pure chitosan fiber scaffolds

Robert Tonndorf1, Elke Gossla2, Recep Türkay Kocaman1,

Martin Kirsten1, Rolf-Dieter Hund1, Gerald Hoffmann1,
Dilbar Aibibu1, Michael Gelinsky2 and Chokri Cherif1

Abstract
The field of articular cartilage tissue engineering has developed rapidly, and chitosan has become a promising
material for scaffold fabrication. For this paper, wet-spun biocompatible chitosan filament yarns were converted
into short flock fibers and subsequently electrostatically flocked onto a chitosan substrate, resulting in a pure, highly
open, porous, and biodegradable chitosan scaffold. Analyzing the wet-spinning of chitosan revealed its advantages and
disadvantages with respect to the fabrication of the fiber-based chitosan scaffolds. The scaffolds were prepared using
varying processing parameters and were analyzed in regards to their geometrical and mechanical properties. It was
found that the pore sizes were adjustable between 65 and 310 mm, and the compressive strength was in the range
13–57 kPa.

Keywords
chitosan fiber, wet spinning, electrostatic flocking, mechanical properties, flock scaffold

The hyaline cartilage at the articular ends of bones scaffold–tissue constructs: (1) interconnected pores in
exhibits little to no intrinsic self-repair capacity in the a three-dimensional network; (2) controllable degrad-
face of damage, which may cause osteoarthritis. ation and resorption rates to match tissue growth;
Current cartilage defect treatments in clinical practice (3) surface chemistry suitable for attachment, prolifer-
are microfracture (perforation of the bone), mosaic- ation, and differentiation of cells; and (4) mechanical
plasty (transplantation of autogenous osteochondral properties similar to the tissues at the implantation
grafts from non-weight-bearing areas to the site of the site.5 Several natural and synthetic polymers have
defect), and autologous chondrocyte implantation been investigated for scaffold preparation, including
(implantation of hyaline tissue cultured from patient chitosan,6,7 which is a linear polysaccharide, a deacety-
cells).1,2 Within the last two decades the interdisciplin- lated derivative of chitin, and is isolated from crab and
ary research field of tissue engineering (TE) has devel- shrimp shells.8 It is supremely suited for TE,7,9 espe-
oped rapidly. The objective is to further improve cially for cartilage TE applications,10 as its advantages
treatments of cartilage defects,2–4 and therefore three over other biopolymers are its biodegradability
key components are under investigation: cells, signals,
and scaffolds.2,3 Scaffolds are applied as a template for 1
Institute of Textile Machinery and High Performance Material
tissue formation; for this purpose they are seeded with Technology, Technische Universität Dresden, Germany
2
cells and growth factors if required. Subsequently, Centre for Translational Bone, Joint and Soft Tissue Research, University
mechanical, chemical, and biological stimuli are utilized Hospital and Medical Faculty, Technische Universität Dresden, Germany
to guide and influence tissue formation within the scaf-
Corresponding author:
fold–tissue constructs. Robert Tonndorf, Institute of Textile Machinery and High Performance
Great efforts have been made in scaffold prepar- Material Technology, Hohe Straße 6, Dresden 01069, Germany.
ation. Hutmacher proposed the following criteria of Email: robert.tonndorf@tu-dresden.de
1966 Textile Research Journal 88(17)

into non-toxic oligosaccharides, its similarity to degradation rates and increases the number of cell
glycosaminoglycans in the extracellular matrix, and its attachment sites.25,26 The molecular weight (MW) of
hydrophilic surface, promoting cell adhesion and CHS 95/100 was between 100,000 and 250,000 g/mol,
proliferation. and for CHS 95/500 was between 200,000 and
Commonly used methods for the fabrication of two- 400,000 g/mol, as stated by the supplier. CHS 95/100
and three-dimensional scaffolds are freeze-drying, was used for fiber formation and CHS 95/500 was used
phase separation, salt leaching, cell encapsulation, gas for adhesive and substrate formation of the scaffolds.
forming, and electrospinning,11–15 as well as bubbfil The higher MW of the latter was chosen to ensure a
and centrifugal spinning.16 Likewise, textile technolo- slower degradation rate of the fiber-supporting sub-
gies are a suitable approach for a fiber-based scaffold strate, as the MW of CHS is inversely proportional to
fabrication, considering that with these technologies its degradation rate.25 Acetic acid (AcOH), sodium
structural, topographical, and mechanical properties hydroxide (NaOH), and Dulbecco’s phosphate buffered
of the constructs are widely adjustable. The most saline without calcium and magnesium (PBS) were pur-
common techniques applied for textile scaffold prepar- chased from Merck KGaA (Darmstadt, Germany).
ation are weaving, knitting, braiding, nonwoven
technologies, and electrospinning.17,18 Moreover, elec-
trostatic flocking technology has been used for the
Wet-spinning of chitosan filament yarns
preparation of three-dimensional anisotropic scaffolds, Wet-spinning of chitosan filament yarns was conducted
featuring a columnar fiber orientation to enhance the on an in-house wet-spinning pilot plant provided by
biomechanical properties under compression.19–23 Fourné Polymertechnik GmbH (Alfter, Germany) in
Gossla and colleagues recently prepared flocked scaf- accordance with Toskas and colleagues and Hild and
folds consisting of chitosan only, ensuring full bio- colleagues.27,28 The spinning dope was prepared by
degradability.24 It was found that these scaffolds were mixing at least 3 L of an aqueous solution containing
suitable for the proliferation of hMSC and Saos-2 cell 8 wt.% CHS 95/100 and 2.8 1 wt.% AcOH in deminer-
lines, hence they were proposed as a promising candi- alized water, stirring for 5–8 h in a dissolver and aging
date for articular cartilage TE applications. for 24 h. Subsequent filtration through a filter from
In this work, the possibilities and limitations of Paul GmbH & Co. KG (Steinau an der Straße,
applying wet-spun chitosan yarns in electrostatically Germany) consisting of three layers with a pore size
flocked chitosan fiber scaffolds will be shown. of 50 mm removed non-dissolved components from the
Therefore, the chitosan spinning process is character- chitosan solution.
ized regarding the drawing process and its impact on The filtered spinning dope was heated to a tempera-
the wet tensile properties of the chitosan yarns, as no ture of 60 C, extruded through a spinneret and coagu-
data are available in the literature, despite its import- lated in an aqueous alkaline bath at 50 C and a pH
ance as a parameter, especially when soaking chitosan value of 12.6. Subsequently, the yarn was continuously
fibers in an aqueous solution for TE applications. passed through three additional alkaline baths with
The following geometrical properties of prepared scaf- decreasing temperatures (50 C, 45 C, and 40 C) and
folds are examined as well: the flock fiber density, the pH values (10.6, 9.7, and 9.7). The pH of the baths
surface-area-to-volume ratio (SA:V), the porosity and was adjusted by adding NaOH. The spinneret pressure
the pore size, and the compressive strength. The was typically about 5 bar. Finally, the yarn was passed
obtained data will be used for a comparison to pure through a godet dryer with a 40 m drying path and a
three-dimensional chitosan scaffolds described in the temperature of 100 C, before being wound onto a coil.
literature. Yarn withdrawal from the baths and from the oven was
achieved by five godets and one additional godet just
before winding, resulting in a total of six drawing sec-
Experimental details tions. The drawing rate was determined by the ratio of
the take-up speed of the godets and the extrusion vel-
Materials ocity of the dope at the spinneret. The total drawing
Two chitosan (CHS) types, Chitoscience CHS 95/100 rate was the ratio between the last godet and the extru-
and CHS 95/500, were purchased from Heppe Medical sion velocity.
Chitosan GmbH (Halle, Germany), each having a Yarns with a nominal single filament diameter of 25,
degree of deacetylation (DD) greater than 92.6% and 40, and 50 mm were spun for scaffold preparation.
a viscosity of 71–150 mPa (CHS 95/100) and 351– Therefore, calculations were performed, assuming a
750 mPa (CHS 95/500), measuring a 1% CHS solution constant volumetric flow rate of chitosan (Eqs. 1.1–
in 1% acetic acid, as stated by the supplier. The types 1.6). Microsections of dry chitosan yarn samples were
were chosen for their high DD, which decreases recorded with an Axiotech 100 optical microscope by
Tonndorf et al. 1967

Zeiss (Oberkochen, Germany) and the diameter was (G, H, I and O, Q, R), the voltage (E, F, G), and the
measured and compared to the nominal diameter. fiber diameter (H, M, O) was varied. Type N and P
were prepared to achieve a high compressive strength,
hence a low voltage (30 kV) and a high fiber diameter
Mechanical analysis of chitosan yarns
(50 mm) was applied. Prior to flocking, flock fibers were
The linear mass density related tensile strength and dried at 120 C for 5 min to ensure the separability of
Young’s modulus of wet and dry chitosan yarn samples hydrophilic fibers. These fibers were then homoge-
were obtained as a function of the total drawing rate. neously distributed on the bottom electrode by means
The tensile properties of the samples were measured on of a mechanical sieve from Maag Flockmaschinen
a Z2.5 tensile tester with a 100 N load cell by Zwick GmbH. A thin layer of the chitosan adhesive (1 g of
(Ulm, Germany), based on the DIN EN ISO 2062 an aqueous solution containing 5 wt.% CHS 95/500
standard. Dry yarn samples were conditioned at 20 C and 5 wt.% AcOH) was evenly distributed on the top
and 65% relative humidity. Wet yarn samples were electrode by knife coating. By applying a voltage, the
immersed in a PBS solution for 24 h and then directly flock fibers on the bottom electrode were accelerated
tested without drying. Prior to the tensile tests, the toward the top electrode and adhered to the applied
linear mass density of the yarns was determined to cal- chitosan adhesive. During this process the temperature
culate the tensile strength in cN/tex. The gauge length was 22 C and the relative humidity was 39%.
was 250 mm and the extension rate was 250 mm/min. The as-prepared structures were thermally cured at
The values from tensile testing were analyzed with the 120 C for 15 min, whereby the adhesive was converted
software testXpert I and were averaged by means of at into a thin film and substrate, respectively. Finally, the
least 10 samples. scaffold was neutralized in a freshly prepared 0.1 M
NaOH solution (90/10 vol.% ethanol/water, 20 ml),
and subsequently immersed in 100% ethanol for 1 h
Fabrication of scaffolds and then air-dried. Ethanol-based solutions enabled
Scaffolds were prepared by electrostatic flocking as drying without substrate deformation, as alcohol dehy-
described by Gossla and colleagues.24 Flocking from drates chitosan and therefore swelling and succeeding
bottom to top was achieved using an electrostatic flock- deformation was omitted.
ing device (SPG 1000 from Maag Flockmaschinen
GmbH, Kusterdingen, Germany), which consisted of Microscopy/gravimetry-based geometrical analysis
a circular top electrode (area 26.42 cm2) and a circular
bottom electrode (area 67.93 cm2) made of aluminum
of scaffolds
alloy and set apart at a distance of 10 cm. The surface The flock fiber length in dry and in wet states as well as
of the top electrode was roughened by partial dissol- the flock fiber diameter in the wet state were determined
ution with 0.1 M NaOH and subsequently thoroughly by microscopy-based measurements. For wet fiber
rinsed with water. This procedure ensured that the dimensions, fiber samples were incubated in PBS for
adhesive film was distributed evenly on the electrode, 24 h and stained with an acid dye (Säurelicht-rot B
which would not have been possible using a non-treated from Chemie GmbH Bitterfeld Wolfen, Bitterfeld
electrode. The chitosan yarn was cut into flock fibers Wolfen, Germany). Dry and wet samples were then
with a set length of 2 mm on a cutting machine built in- distributed in glycerine and images of the fibers in lon-
house. This fiber length corresponded to the thickness gitudinal view were recorded with an Axiotech 100
of the articular cartilage.29 The flock fiber length in dry optical microscope by Zeiss. Measurements of at least
and in wet states was measured with an Axiotech 100 10 fiber samples were conducted and both dimensions
optical microscope by Zeiss. were averaged.
Various set parameters for scaffold fabrication The yield of fiber flocking and the geometrical par-
were applied, resulting in 10 differently prepared scaf- ameters of the scaffold types in dry and in wet states
fold types (Table 1), whereby the flock fiber weight were calculated using gravimetry-based measurements

Table 1. Set parameters for scaffold preparation

Scaffold type E F G H I M N O P Q R

Voltage (kV) 30 40 45 45 45 45 30 45 30 45 45
Set flock fiber mass (g) 0.5 0.5 0.25 0.5 1.0 0.5 0.5 0.5 1.0 0.25 1.0
Set flock fiber diameter (mm) 25 25 25 25 25 40 50 50 50 50 50
1968 Textile Research Journal 88(17)

from flocking and microscopically determined fiber

diameters (Eqs. 2.1–2.8). Calculated parameters were
Elemental analysis of scaffolds
the flock fiber density, the SA:V, the porosity parallel As pure chitosan scaffolds were the intended goal, the
to the plane of the chitosan film, and the pore size. For chitosan fibers and the adhesive were characterized by
pore sizes the assumption was made that the flocked their content of AcOH before and after certain washing
fibers were packed uniformly in a hexagonal packing steps, as AcOH was the only organic compound added
arrangement. For averaged values and standard devi- during the fabrication process with potentially cyto-
ation, five scaffolds of each type were investigated. toxic effects.31,32 As-spun fiber samples and the adhe-
sive, which was cured at 120 C for 5 min, were defined
as unwashed, whereas washed samples were treated
Microscopy-based geometrical analysis of scaffolds with two different washing procedures. For washing
Complementary to the gravimetric analyses, the flock procedure 1, samples were first neutralized in 0.1 M
fiber density and the distance between fibers of the NaOH for 1 h and subsequently washed in deminera-
flocked scaffolds were determined by means of micro- lized water for another 1 h. These samples served as a
scopic evaluation. To this end, samples were stained reference. For washing procedure 2, samples were neu-
with a 20 g/L Alizarin red S (Sigma) solution and sub- tralized in a freshly prepared 0.1 M NaOH ethanol-
sequently embedded in a 150 g/L gelatin (Fluka) solu- based solution (90/10 vol.% ethanol/water) for 1 h
tion. The samples were frozen in liquid nitrogen and and subsequently washed in 100% ethanol for another
stored at –80 C before cryosectioning. Cryosections 1 h, which corresponded to the actual scaffold fabrica-
were observed under a light microscope. Based on tion. For AcOH determination the weight ratio of
these sections, the fiber density was calculated as the carbon (C) to nitrogen (N) was assumed to be propor-
mean number of fibers per square millimeter. The dis- tional to the AcOH concentration within the fibers and
tance between fibers was defined as the average distance the substrate. This weight ratio was determined twice on
between the centers of a fiber and its nearest neighbors air-dried samples with a Euro EA – CHNSO Elemental
connected by an edge in a Delaunay triangulation. For Analyser from HEKAtech GmbH (Wegberg,
this purpose, representative images of the cross-sections Germany). The obtained C/N ratios were compared
were analyzed by performing a Delaunay triangulation with theoretical ratios, which were calculated based on
using the Delaunay Voronoi plug-in of the software the DD of chitosan and the amount of AcOH added.
Fiji.30 For the calculation, only fibers within 200 mm
from the margin of a detailed image at 1860 
1240 mm were considered. For each scaffold sample, Results and discussion
three cross-sections were analyzed.
Wet-spinning of chitosan filament yarns
In spinning experiments, the total drawing rates were
Mechanical analysis of scaffolds varied between 0.5 and 1.3, and for each rate several
The prepared scaffolds were compressed by a maximum hundred meters of yarn were spun. It was found that
deformation ratio of 50% over the wet fiber length. The the ratios between the drawing rates at a given godet
compressive strength as a function of the rate of (DR1. . . DR6) and the total drawing rates (DR6) were
deformation was determined on a Z2.5 tensile tester similar for all spinning experiments. The averaged
by Zwick. At least three scaffolds of each type were ratios and the corresponding coefficients of determin-
conditioned in PBS for 24 h. Then, each scaffold was ation are given in Table 2. The values from this table
cut into six square samples (15  15 mm) and swollen were calculated according to mathematical functions of
samples were submitted to compressive testing. The ini- the linear regression lines (Figure 1a). Drawing
tial load was set to 0.1 N and the compression rate was occurred primarily within the coagulation bath between
set to 5 mm/min. the spinneret and the first godet, which is represented
For the sake of clarity, the compression curves were by the drawing rate ratio DR1/DR6 having a value of
sectioned into three zones separated by a deformation 74%. About 93% of the total drawing was achieved
ratio of 12.5, 25, and 50%. For each zone the averaged after passing the second godet, and almost no drawing
maximum compressive strength was shown for differ- occurred at godets 3–6. Drawing rates substantially dif-
ently prepared scaffold types. Walther and colleagues fering from those in Table 2 were not suitable for yarn
presented how flocked structures undergo Euler buck- production as they either resulted in breaking or sag-
ling when compressed.19 Hence, the compressive ging of the water-swollen filament yarn. Hence, the
strength at different deformation ratios was compared total drawing rate was determined by exclusively con-
to Euler’s critical load, which was calculated via geo- sidering the drawing rate within the coagulation bath.
metrical parameters (Eq. 3). By applying the mathematical functions shown in
Tonndorf et al. 1969

Figure 1a, the take-up speeds of the godets are pre- literature it is not clarified whether there is a relation-
configurable according to the required total drawing ship between drawing and the tensile properties of wet
rate. For the sake of completeness, total drawing chitosan filament yarns. For the as-spun and dry chit-
rates exceeding 1.3 were not applicable as the yarn osan yarns, a linear correlation between the drawing
broke before final winding. rate and the tensile strength (Figure 2a) as well as
The single-filament diameter of the as-spun yarn was Young’s modulus (Figure 2b) was noticed. Neither cor-
a crucial parameter for scaffold preparation. The diam- relation proved to be as significant as expected, which
eters were optically measured and compared with the was attributed to the production of chitosan from nat-
calculated nominal diameter. Both values corres- ural resources and thus the chitosan raw material not
ponded, as seen in Figure 1b. Thus, it was shown that having homogeneous properties. However, an
wet-spun chitosan fibers are suitable for the prepar- increased drawing rate generally resulted in an
ation of geometrically well-defined chitosan scaffolds, increased tensile strength and Young’s modulus.
as the diameter is easily controllable. As the yarn was utilized in scaffolds, the wet tensile
In conclusion, only two set parameters, more pre- properties were of greater interest. Swollen yarn sam-
cisely the diameter of the spinneret and the first applied ples were subjected to tensile tests, revealing their ten-
drawing rate, were required as an input, making the sile properties under physiological conditions in the wet
described wet-spinning a straightforward method for state. To facilitate a wet/dry comparison, the wet and
the provision of chitosan fibers with predefined the dry tensile properties were both based upon the dry
diameters. linear mass densities. The obtained data indicated a
large drop in the tensile strength and Young’s modulus
in the wet state. The most significant changes occurred
Mechanical analysis of chitosan yarns
for highly drawn yarns. Moreover, the dependence
Drawing of chitosan yarns affects the polymer orienta- between the tensile strength and the drawing rate in
tion and therefore the tensile properties.33,34 In the the wet state was less pronounced when compared to
the dependence in the dry state, and no linear correl-
ation between the drawing rate and the wet Young’s
Table 2. Averaged ratio of drawing rate at each godet DRi to
total drawing rate DR6. Averaged ratios were determined from
modulus was noted (Figure 2). The wet modulus aver-
the slope of linear regression lines for the data points (DRi , DR6 ) aged 0.73  0.18 GPa, which was between 4% and 14%
of 26 spinning experiments; see Figure 1a of the dry modulus.
A high swelling factor of 2.4  0.2 was determined,
i 1 2 3 4 5 6 which was calculated from the ratio of the linear mass
DRi/DR6 (%) 74 93 97 98 99 100 density in wet and dry states. Water interacts strongly
Coefficient of 0.93 0.99 1 1 1 1 with the hydroxyl and amine groups of chitosan, caus-
determination ing swelling and a decrease of intermolecular forces and
hydrogen bonds, respectively, between polymer chains,

(a) 1.50 (b) 70

Godet 3
Measured filament diameter df,dry in µm

Godet 2 DR3 = 0.97·DR6

R² = 1.00 60
1.25 Godet 1
Drawing rate at godet DRi

DR2 = 0.93·DR6 50 df,dry = 1.09 d6,dry

R² = 0.99 R² = 0.89
1.00
DR1 = 0.74·DR6 40
R² = 0.93
30
0.75

20
0.50
10

0.25 0
0.25 0.50 0.75 1.00 1.25 1.50 1.75 0 10 20 30 40 50 60
Total drawing rate DR6 Nominal filament diameter d6,dry in µm

Figure 1. (a) Drawing rate at the first three godets plotted against the total drawing rate. (b) Measured filament diameter plotted
against the calculated filament diameter.
Error bars represent standard deviation for 10 samples for each nominal diameter; coefficient of determination (R2) for linear
regression.
1970 Textile Research Journal 88(17)

(a) (b)
14 18 Edry = 4.61· DR6 + 5.03
σ dry = 4.48 · DR6 + 4.41
R² = 0.60 dry R² = 0.31
dry 16
12
Tensile strength σ in cN/tex

Young's modulus E in GPa

wet 14 wet
10
12
8 10

6 σ wet = 1.42 · DR6 + 1.99 8

R² = 0.42
6
4
4
2 Ewet = -0.16 · DR6 + 0.86
2 R² = 0.05
0 0
0.25 0.5 0.75 1 1.25 1.5 0.25 0.5 0.75 1 1.25 1.5
Total drawing rate DR6 Total drawing rate DR6

Figure 2. (a) Tensile strength and (b) Young’s modulus plotted against total drawing rate.
Error bars represent standard deviation for 10 samples for each yarn. Coefficient of determination (R2) for linear regression are given.

resulting in decreased wet strength. Furthermore, orien- density, which was probably due to a decreased electro-
tation of polymer chains due to drawing is lost as well. static charge of the fibers. This circumstance in turn
Hence, the correlations between the drawing rate and implied decreased repulsing forces between incoming
the tensile strength or Young’s modulus in the wet state and adhering fibers, allowing an increased fiber packing
were small or negligible. In conclusion, the wet Young’s density. Furthermore, the thinner the flock fibers were,
modulus, which was the essential parameter for the the higher the flock fiber density became. In general, a
compressive strength of the scaffolds, was not adjust- broad range of fiber densities from 9 to 113 fibers/mm2
able by drawing. was achieved. These gravimetrically determined fiber
densities were confirmed by microscopic measurements,
which revealed densities of 73, 105, and 49 mm2 for
Geometrical analysis of scaffolds
the scaffold types H, I, and M.
For scaffold fabrication, three different flock fiber types Certain geometrical parameters in the wet state were
were used. All fibers had a nominal set fiber length of calculated based on the combination of gravimetrical
2 mm but differing set fiber diameters of 25, 40, and and microscopic measurements (Table 4): SA:V was in
50 mm. For all flock fibers the measured fiber length the range 1.8–12.8 mm. Pore sizes were in the range 65–
in dry state was less than the set fiber length, which 310 mm, and porosities were in the range 88–97%. Both
was due to the imperfection of fiber cutting. All three parameters are discussed further below. By applying
flock fiber diameters in the dry state corresponded to the Delaunay triangulation to recorded microscopic
the set diameter. The diameter in the wet state was images of the scaffolds cross-section (Figure 4), the fre-
measured on stained fiber samples (Figure 3a). An quency of discrete fiber distances, from center to center
increased fiber diameter was noticed due to swelling. between neighboring fibers, was determined. The fre-
Measured dimensions of flock fibers applied for scaf- quency distribution shifts to the left, as shown in
fold fabrication are given in Table 3. These values were Figure 4a. The mean fiber distance between neighbor-
further used for the calculation of scaffold properties. ing fibers was reduced from 140  9 (Type H) to
Ten different scaffold types with various set fabrica- 112  3 mm (Type I) as the fiber count was increased.
tion parameters having a diameter of 58 mm were The application of a larger fiber diameter (Type M)
prepared. An overview is given in Table 4. For all scaf- resulted in an increased fiber distance (145  8 mm)
folds, a dense and a homogeneous flock surface as well and in a more uneven distribution of the fibers. The
as a vertical orientation of the flock fibers were averaged values of fiber distances from microscopic
achieved (Figure 3b,c). A dry substrate weight of analysis and microscopy/gravimetry-based determined
74  4 mg, not including fibers, was measured. Not all distances are similar. Differences occur due to the real
initial fibers that were distributed onto the bottom elec- fiber distribution of the scaffolds, which is not a hex-
trode were accelerated and adhered to the adhesive; agonal arrangement as assumed for the microscopy/
as a consequence, the flocking yield was in the range gravimetry-based measured values. As the fiber flocking
55–85%. As expected, an increased initial fiber weight implemented within this work was not producing a uni-
resulted in an increased fiber density within the scaffold. form distribution of vertically aligned fibers, the pore
Decreased flocking voltages led to an increased fiber dimensions cannot be reliably determined without
Tonndorf et al. 1971

Figure 3. (a) Glycerine-embedded stained flock fibers, (b) cut scaffold sample, and (c) SEM image, bent fibers due to sample
preparation/cutting.

Table 3. Overview of microscopy-based determined flock fiber length and diameter in dry and wet states, averaged values with
standard deviation

Set flock fiber type 25 mm/2 mm 40 mm/2 mm 50 mm/2 mm 25 mm/2 mm 40 mm/2 mm 50 mm/2 mm
Dimension Length (mm) Diameter (mm)

Measurement dry 1.79  0.18 1.82  0.05 1.89  0.05 *25.1  3.2 *39.1  3.5 *50.4  1.3
Measurement wet 1.71  0.13 1.83  0.08 1.91  0.05 38.8  4.5 57.3  6.5 65.2  7.0
Dimensional changes (%) -4 1 1 55 48 29
*indicates measurements on fibers in transverse view, otherwise in longitudinal view.

Table 4. Overview of microscopy-/gravimetry-based determined geometrical parameters of various scaffold types in the wet state;
averaged values with standard deviation for five samples for each type
Scaffold type E F G H I M N O P Q R

Set flock 25 mm/ 25 mm/ 25 mm/ 25 mm/ 25 mm/ 40 mm/ 50 mm/ 50 mm/ 50 mm/ 50 mm/ 50 mm/
fiber type 2 mm 2 mm 2 mm 2 mm 2 mm 2 mm 2 mm 2 mm 2 mm 2 mm 2 mm
Fiber density 113  2 105  19 24  1 80  15 112  19 32  8 28  3 19  2 38  3 92 28  3
(fibers/mm2)
SA:V (mm) 12.8  0.2 12.0  2.2 3.0  0.1 9.2  1.8 12.7  2.1 6.4  1.5 5.7  0.5 4.2  0.4 7.8  0.63 1.8  0.5 5.8  0.5
Porosity (%) 88.4  0.2 89.2  2.0 97.1  0.1 91.7  1.6 88.6  1.9 90.2  2.3 90.7  0.8 93.1  0.7 87.3  1.0 97.1  0.8 90.6  0.8
Fiber center– 101  1 106  11 217  5.3 114  17 103  8 190  20 204  9 237  13 174  7 375  63 202  9
center
distance (mm)
Pore size (mm) 65  1 70  11 179  5.3 78  17 67  8 129  20 139  9 172  13 109  7 310  63 137  9
1972 Textile Research Journal 88(17)

(b) (c)

(a)
40
Type H
35
Type I
30
Type M
Frequency in %

25

20 (d) (e)
15

10

0
40 80 120 160 200 240 280 320 360 400 440 480
Fibre distance sc,m in µm

Figure 4. Distribution of fiber distances sc between nearest neighbors of three chitosan flock scaffolds after applying Delaunay
triangulation.
Mean of three flock scaffolds  SD. Microscopic determination was performed after Alizarin red S staining and cryosectioning (a).
Microscopic cross section of scaffold type H (b), type I (c), and type M (d). Representative image for Delaunay triangulation (e).
Scale bar ¼ 200 mm.

considering the actual distribution of the fibers. These

irregularities led to a higher pore size or fiber distance
Mechanical analysis of scaffolds
compared to the calculated values. A representative loading, unloading, and reloading
Both characteristic parameters – the pore size and curve of the scaffold type R is shown in Figure 5a,
the porosity – were proportional, whereas the pore size whereas Euler’s critical load was calculated as 10 kPa,
and the SA:V emerged as being inversely proportional based on a wet Young’s modulus of 0.30 GPa of the
to one another. In general, a high SA:V is desirable to yarn, which was calculated with respect to the swollen/
increase the surface for cell adhesion, while it also inhi- wet fiber cross-section. The lower part of the first load-
bits the nutrient supply due to small resulting pore ing curve (deformation up to 12.5%) was characterized
sizes. Therefore, one has to make a compromise as a left curvature, this may be traced back to fiber
between optimal pore size and optimal SA:V which buckling within the vertically aligned fiber assembly.
allows sufficient adhesion area while still providing effi- The loading stress strongly increased above Euler’s crit-
cient exchange of nutrients. The determined parameters ical load, which can most likely be explained by an
of this compromise do not have universal validity, increased number of fiber–fiber contacts during com-
since they need to be adjusted specifically to the pacting, as the fibers were densely packed. During
chosen scaffold material as well as the needs of the fiber compacting, free fiber bending was omitted and
applied cell types. Loh and colleagues gave an over- the slope of stress progressively increased upon further
view, wherein scaffolds examined for chondrogenesis deformation. Finally, the sharp drop of unloading
exhibited pore sizes in the range 70–860 mm and poros- curves was attributed to the slipping of adjunct fibers.
ities in the range 59–95%.11 Listed scaffolds consisted The evaluation of the obtained stress–strain curves
of different materials and were utilized for different cell with respect to the succeeding cycles indicated a very
types. For chondrogenesis within chitosan scaffolds, the small drop of the maximum compressive strength
porosity was indicated as 80% and the pore sizes were within 10 cycles. However, the loading slopes and the
in the range 70–120 mm. Most scaffold types prepared in moduli, respectively, decreased with increasing cycle
the presented paper exhibited calculated pores in the counts, whereby succeeding slopes were approaching
same range (except type G, O, and Q) and porosities a constant value. This was attributed to an irreversible
between 85 and 95%. Hence, the flocked chitosan scaf- fiber reorientation resulting in a fiber alignment differ-
folds prepared in this work are most likely suitable for ing from the vertical, which also explained the
cartilage TE applications, as stated by Gossla and col- decreased slopes of the lower part of the reloading
leagues as well, who investigated the scaffold type H curves when compared to the first loading curve.
regarding the proliferation of hMSC and Saos-2 cell Fiber reassembly is also expressed by the deformation
lines.24 energy, which is the area underneath the loading and
Tonndorf et al. 1973

(a) (b)

50 %
50

25 %
12.5 %
5
45

Deformation energy in mJ mm-1

Compressive strength in kPa

40 4 Loading
Unloading
35
Hysteresis
30 3
25
20 2
15
Euler's critical 1
10
load
5
0 0
0 10 20 30 40 50 1 2 3 4 5 6 7 8 9 10
Deformation % Cycle

Figure 5. (a) Representative loading, unloading, and reloading curve of a scaffold type R. (b) Corresponding energy of loading,
unloading, and hysteresis for each cycle.

Table 5. Compression strength and elastic modulus in kPa of various scaffold types, averaged values with standard deviation

Compression H M N O P R

Compressive strength 12.5% 21 43 82 62 81 61

25.0% 52 96 18  4 10  4 23  4 14  3
50.0% 13  4 25  10 38  7 23  8 56  6 31  5
Calculation Euler 51 82 10  1 71 14  1 10  1

the unloading curves. The difference between both buckling. Hence, the mechanical scaffold analysis
deformation energies (hysteresis) was attributed to fric- showed that the compressive strength of the scaffolds
tional or dissipated energy between fibers. As seen in can be approximated from the geometrical parameters,
Figure 5b, hysteresis was approaching a constant value even at high deformation rates.
and thus friction was approaching a constant value as Still, the mechanical strength of the uncultivated
well, which in turn was caused by the manifestation of scaffolds is approximately five times lower when com-
the reoriented fiber assembly. pared to the articular tissue, which withstands a com-
For a comparison of the different scaffold types, pressive strength of 24, 69, 112, and 168 kPa at 8, 16,
three averaged stress values, which corresponded to 24, and 32% cartilage compression.35 Nevertheless, the
the compression ratios 12.5, 25, and 50%, were mechanical strength of cultivated scaffolds is expected
chosen (Table 5 and Figure 6). The correlation between to increase, as embedded cells may form a natural
these characteristic stress values and Euler’s critical extracellular matrix, which connects the fibers within
load, which was calculated upon the geometrical par- the scaffold/tissue constructs.
ameters, was investigated. Only the first loading was
considered to ensure an analysis of the vertical fiber
Elemental analysis of scaffolds
assembly. A correlation between Euler’s critical load
and the compressive strength was ascertained Washed and unwashed fibers in addition to adhesive
(Figure 6b). As mentioned above, the experimentally samples were characterized regarding their AcOH con-
determined compressive strength increased above tent, by assuming that the concentration of AcOH was
Euler’s critical load, which certainly occurred at a proportional to the C/N ratio, which was determined
deformation of 25%. Although Euler buckling was by elemental analysis. To enable comparison of the
not applicable at such a high deformation, a correlation AcOH content at the different preparation stages of
was present. It can be presumed that fiber bending the scaffold components, the calculated and the mea-
during compacting caused this behavior. The applied sured C/N ratios were plotted (Figure 7). The calcu-
stress for fiber bending is proportional to the same lated values were referred to the source materials:
parameters that are used for the equation of Euler chitosan spinning dope, adhesive, and powder.
1974 Textile Research Journal 88(17)

(a) (b)
70
Compression 50.0 %

Experimental compressive strength

60 Compression 25.0 % y = 3.59x
R² = 0.90
Compression 12.5 %
50

40

in kPa
y = 1.53x
30
R² = 0.88

20

10 y = 0.64x
R² = 0.78
0
0 2 4 6 8 10 12 14 16 18 20
Euler's critical load based upon geometrical parameters

Figure 6. (a) Swollen scaffold sample; square with a side length of 15 mm between clamps for compressive testing. (b) Compressive
strength at a compression of 12.5, 25, and 50 % plotted against calculated Euler buckling strength corresponding to the geometrical
properties of scaffold types; error bars represent standard deviation for at least 10 samples for each data dot/scaffold type.

calculated C/N
10 for adhesive:
9.89
Weight ratio carbon/nitrogen

calculated C/N
7 for spinning
dope: 6.87
6.18
5.97
6
5.51 calculated C/N
5.21 5.30 5.21 5.28
for chitosan
5 only: 5.23
thermally washing washing as-spun washing washing
cured proc. #1 proc. #2 proc. #1 proc. #2
Powder Adhesive Fibre

Figure 7. Calculated and measured weight ratios of carbon to nitrogen for various chitosan samples.

The pure powder contained no AcOH and therefore acetate salts in the spinning baths. As expected, by
had a low calculated C/N ratio of 5.23; the spinning applying standard washing procedure 1 in alkaline
dope and the adhesive contained AcOH and thus high water, the C/N ratios of the adhesive and the fibers
C/N ratios of 6.87 and 9.89, respectively, were calcu- decreased (5.30 and 5.21, respectively) to the magnitude
lated. A high proportion of AcOH evaporated due to of the value of the pure chitosan powder (5.21), as
the thermal treatment at 120 C for 15 min. However, AcOH was neutralized and washed out. However, this
the measured C/N ratio was still 6.18, as this sample neutralization procedure resulted in the deformation of
was not washed. For the as-spun yarn, the measured the substrate. To suppress this deformation in washing
C/N ratio was 5.97, located between the calculated procedure 2, an ethanol solution was applied during
values of the powder and the spinning dope, which scaffold fabrication. Elemental analysis suggested that
was caused by an incomplete removal of AcOH and scaffold washing procedure 2 removed most AcOH
Tonndorf et al. 1975

Table 6. Electrostatic flocking in comparison to other textile technologies

Textile technology Mechanical properties Geometrical properties Applications Ref.

Weaving High mechanical Defined geometries, Nerve regeneration, Aibibu et al.18

strength low SA:V vascular graft, fascia
replacement/hernia
repair, tendon/
ligament repair
Knitting Mechanical stability Defined geometries, Vascular grafts, Aibibu et al.18
and elasticity, adapt low SA:V nerve regeneration,
to size changes dermal grafts,
tendon regeneration,
esophagus replacement,
hernia repair, small
joint reconstruction,
calvarial bone healing
Braiding High mechanical Defined geometries, Nerve regeneration, Aibibu et al.18,36
strength rope-like structures, tendon/ligament repair
low SA:V
Nonwovens Low mechanical No defined geometries Nerve regeneration, Aibibu et al.18
strength but adjustable pore cartilage engineering,
size, low SA:V tendon/ligament repair
Electrospinning Low mechanical No defined geometries Nerve regeneration, Min et al.37
strength but adjustable pore size, vascular grafts,
high SA:V bone regeneration,
skin regeneration
Electrostatic High compressive Defined geometries, Cartilage engineering This paper
flocking strength low SA:V

from the fibers (C/N ratio of 5.28) and from the adhe- reported.41 no three-dimensional and pure chitosan
sive (C/N ratio of 5.51) as well, which made this wash- structures were under investigation in recent literature.
ing procedure sufficient for the preparation of a pure Publications regarding the fabrication of mechanically
chitosan scaffold. stable three-dimensional scaffolds consisting of non-
crosslinked chitosan are rare, despite its advantage for
Comparison to pure chitosan scaffolds from minimizing the risk of cytotoxicity of residual reactants
or catalyst traces used for chitosan crosslinking.
the literature
Isotropic chitosan fiber scaffolds, having a compressive
Different textile technologies can be applied for the strength of up to 53 kPa at a deformation of 50% were
manufacture of three-dimensional scaffold structures prepared by Ucar and colleagues.42 It was highlighted
(Table 6). Making use of technologies like weaving, that these microfibrous structures had a high SA:V and
knitting, braiding, and electrostatic flocking, aniso- an interconnected porosity, but the pore sizes were not
tropic structures with a defined spatial geometry can controllable. Jana and colleagues prepared freeze-dried
be fabricated. These structures are capable of with- and pure chitosan scaffolds to ensure biocompatibil-
standing high mechanical loads when fibers are ori- ity.43 The dry scaffolds had pore sizes ranging from
ented according to the distribution of forces, whereas 100 to 500 mm and a compressive strength of up to
electrospun structures and nonwovens have isotropic 1.74 MPa, whereas wet properties were not stated.
properties. However, electrospinning is the only manu- Wang and colleagues prepared silica–chitosan hybrid
facturing technique capable of producing nanofiber scaffolds with a highly vertically oriented open porous
structures having a high SA:V. structure by combining a sol–gel process and a freeze-
In general, electrospinning of pure chitosan enables casting technique.44 Reference scaffolds prepared with
the fabrication of anisotropic and two-dimensional chitosan only had a compressive strength of about
fiber mats,37–40 making this technique suitable for the 10 kPa at a deformation of 50%. Wan and colleagues
preparation of structures that may be applied as wound produced hybrid scaffolds composed of polycaprolac-
dressings. Even though fabrication of three-dimen- tone and chitosan by a particle-leaching technique.45
sional scaffolds by electrospinning methods was If pure chitosan was applied in fabrication, the
1976 Textile Research Journal 88(17)

compressive strength was 31 kPa at a deformation of the mechanical properties of the scaffolds, which in turn
10%. The pore sizes were in the range 150–200 mm. implies that non-toxic chitosan crosslinking methods
Pourhaghgouy and Zamanian prepared unidirectional may be necessary. This is, though, only the case if
scaffolds composed of chitosan only.46 The maximum after cell culture the mechanical properties of the scaf-
compressive strength was found to be 110 kPa and the folds turn out not to be satisfactory. Hence, further
pore size was 90 mm. The chitosan scaffolds exhibited mechanical investigation after in vitro cell cultivation
foam-like structures with pores that appeared to be is needed.
hardly controllable, in some cases even completely
uncontrollable. All those scaffolds were characterized
by inter-pore openings, which were much smaller than
Equations
the actual pore sizes hindering cell infiltration and Equations for spinning:
nutrition flow. The wet compressive strengths of all
 2
those scaffolds and of the ones prepared for this work ds
were in the same range. However, further mechanical ACHS, 0 ¼ cvol  n    ð1:1Þ
2
investigation after in vitro cell cultivation is needed.
The technology of fiber flocking utilized in this work where:
has proven to be advantageous over freeze drying and ACHS, 0 ¼ flow rate area of chitosan
other methods, as the compressive strengths of up to ds ¼ nozzle diameter
57 kPa at a deformation of 50% were comparatively cvol ¼ volume concentration of chitosan in the spin-
high, while the pore and especially inter-pore sizes ning dope
were large and controllable due to the manufacturing n ¼ nozzle count.
method. Furthermore, electrostatic flocking allows easy
 2
alteration of the material or the fiber composition, if d6
needed. For instance, load-bearing fibers differing from ACHS, 6 ¼ n ð1:2Þ
2
those applied in this work are easily integrable within
the flocked structure to enhance the compressive where:
strength of the scaffold. ACHS, 6 ¼ area of chitosan yarn
d6 ¼ nominal single filament diameter.
Conclusion Vd  np  4
v0 ¼ ð1:3Þ
In the course of this work, fully resorbable scaffolds   d2s  n
manufactured by electrostatic flocking were analyzed
and an overview of the geometrical and the mechanical where:
parameters of different scaffold types was given. The v0 ¼ velocity at extrusion
scaffolds were composed of a single material, namely vd ¼ displacement volume per revolution of pump
chitosan, and featured a high compressive strength due np ¼ rotational speed of pump.
to the vertically aligned flock fibers. The applied flock-
ing technology enabled the preparation of chitosan
V_ CHS ¼ const: ¼ ACHS, 0  v0 ¼ ACHS, 6  v6 ð1:4Þ
scaffolds with high fiber density and high porosity.
The scaffolds had pore sizes of up to 310 mm and com-
pressive strength of up to 57 kPa at a deformation of where:
50%. The compressive strength of the scaffolds corre- V_ CHS ¼ volume flow chitosan
lated with easily adjustable geometrical parameters. v6 ¼ velocity of sixth/last godet.
Hence, electrostatic flocking is proposed as a technique
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
for the fabrication of tailor-made scaffolds for cartilage cvol  Vd  np
TE applications. However, the scaffold fabrication has d6 ¼ 2  ð1:5Þ
  n  v6
to be optimized in order to reduce variation in scaffold
properties, for example by technical modifications of vi
DRi ¼ , i ¼ 0...6 ð1:6Þ
the flocking device. Furthermore, a proper analysis v0
method has to be developed to determine structural
changes of the wet and swollen scaffolds during loading where:
and unloading, for example by microcomputed tomog- DRi ¼ drawing rate at a given godet
raphy. Finally, an increased Young’s modulus of the vi ¼ speed of a given godet
chitosan fibers in the wet state would be beneficial for DR6 ¼ revealed the total drawing rate.
Tonndorf et al. 1977

Equations for scaffold fabrication: where:

p ¼ pore size based on hexagonal packing.
mf, sca ¼ ms  msub ð2:1Þ Equations for Euler’s critical load:

where: 3  Eyarn  d2f, wet  fcg

mf, sca ¼ weight of fibers within scaffold E ¼ ð3Þ
256  l2f, wet
ms ¼ weight scaffold structure
msub ¼ weight of substrate.
where:
mf, sca Ewet ¼ Young’s modulus of chitosan yarn in the wet
y¼ ð2:2Þ
mf, sie state.

where: Declaration of conflicting interests

y ¼ flocking yield The authors declared no potential conflicts of interest with
msub ¼ weight of sieved fiber distributed onto respect to the research, authorship, and/or publication of this
electrode article.

4  mf, sca
fctot ¼ ð2:3Þ Funding
  d2f  lf  
The authors disclosed receipt of the following financial support
where: for the research, authorship, and/or publication of this article:
fctot ¼ flock fiber count within scaffold This work was supported by the DFG (German Research
lf ¼ measured flock fiber length (dry/wet) Foundation) at the Technische Universität Dresden (Grant
df ¼ measured flock fiber diameter (dry/wet) Numbers HU 2107/2-1, HO 1579/1-1 and GE 1133/16-1).
 ¼ chitosan density
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