Materials Sciences and Applications, 2013, 4, 458-470
doi:10.4236/msa.2013.48056 Published Online August 2013 (http://www.scirp.org/journal/msa)
Study on Chitosan-Lactate Sponges with Oriented Pores as
Potential Wound Dressing
Chen Lai1,2, Yi Chen3, Shujiang Zhang3
1Biomedical Engineering Research Center, Beijing University Shenzhen Institution, Shenzhen, China; 2Sichuan University, Chengdu
China; 3The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China.
Received May 21st, 2013; revised June 26th, 2013; accepted July 8th, 2013
Copyright © 2013 Chen Lai et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The motive of this work was to provide an inexpensive potential wound dressing using chitosan lactate (LCH) which
was synthesized by the grafting lactic acid onto the amino groups in chitosan (CH) without a catalyst. The XRD and 13C
NMR results demonstrated that the grafting by lactic acid took place at C2 site in CH, leading to the destruction of the
regularity of the packing in the original CH chains and formation of the amorphous CH salts. The unique device was
developed in our experiments which could yield an approximately vertical thermal gradient, forming the uniformly ver-
tical pores in LCH sponges. TEM images revealed that both TBA and LCH concentration affected the micro-structure
of the sponges, although they worked via different mechanisms. In the water suction experiments, the capillary coeffi-
cient Ks was introduced to evaluate the structure-function relationship. The positive or negative influence of LCH, TBA
and porosity on Ks clearly stood out when their relationships were plotted graphically. The in vitro biocompatibility of
LCH sponges was evaluated. The results obtained indicated that LCH sponges exhibited bio-safety at lower concentra-
tion (25%) during short time (1 day). However, highly concentrated extraction showed a serious toxic effect on both
HSF and HaCaT cells. The release kinetics for hydrophilic and hydrophobic drugs with different formulation sponges
was determined in in vitro release experiments. The contribution of the drug diffusion, matrix erosion and microstruc-
ture of porous materials must be taken into account on the release mechanism. The method and the structure described
in present paper provided a starting point for the design and fabrication of a family of chitosan derivatives based porous
materials with potentially broad applicability
Keywords: Chitosan-Lactate; Sponges; Wound Dressing; Modification
1. Introduction
Chitosan is obtained from the partial deacetylation of
chitin which is the main component of the exoskeleton of
crustaceans, composing of 2-amino-2-deoxy-D-glucose
units linked through β-(1→4) bonds. It is also crystalline
and shows polymorphism depending on its physical state.
Chitosan is a positively charged molecule and soluble in
diluted acidic aquous solution. Because of its unique
property like antimicrobial activity [1], hemostasis func-
tion [2], biodegradability [3], chitosan has attracted sci-
entific interest for various types of biomedical applica-
tions. By different method, chitosan can be easily proc-
essed into membrane, scaffold, sponge, powder and hy-
drogel. Numerous works have been done on the modifi-
cation of chitosan, leading to various derivatives with
improved properties. Among these derivatives, chitosan
lactate (LCH) is very popular for its great biocompatibil-
ity, antibacteria, low toxicity and simple preparation. The
ranking of cytotoxicity among these derivatives is chito-
san hydrochloride > chitosan glutamate > chitosan lactate
[4]. Meanwhile, the ranking of biocompatible was estab-
lished by various laboratories to be methylpyrrolidone
chitosan > chitosan lactate > glycol chitosan > chitosan
glutamate > chitosan hydrochloride [5]. Due to the cati-
onic nature of CH, LCH can be obtained through simple
and green approaches without catalysis. Some research-
ers’ studies suggest that LCH is a safe and efficient gene
carrier [6]. It also has been used to develop CPC (cal-
cium phosphate cement) composites with higher strength
and increased strain before failure [7]. Controlling the
structure of LCH can be done by drying processes in
suitable molds. Being produced into porous structures for
use in tissue regeneration and drug control release is one
of its most promising features. During the freezing proc-

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 459
ess ice crystals nucleate and grow along the lines of
thermal gradients. Ice removal by lyophilization gener-
ates a porous material. Thereby pore orientation and
morphology can be directed by controlling the geometry
of the temperature gradients during freezing. Some ex-
cipients have the same effects such as tertiary butyl al-
cohol (TBA) which is a widely used co-solvent and acts
as a mass-transfer accelerator in lyophilization processes.
It can have great effect on the crystal habit of ice [8] with
very low toxicity [9]. It is miscible with water in any
proportion and could be removed rapidly and completely
by freeze-drying because of its high vapor pressure and
high melting point.
In recent years, wound dressing based on chitosan and
its derivatives have been commercially available [4,10].
Chitosan-based materials can provide a non-protein ma-
trix for 3D tissue growth and activate macrophages for
tumoricidal activity. Previous studies have shown that
chitosan-based materials can effectively accelerate wo-
und healing and prevent scar-forming by initiating fibro-
blast proliferation and collagen deposition [5,11]. Wound
repair is a complex process involving many different cell
types. Among them, HaCaT (human immortal keratino-
cytes) and HSF (Human skin fibro-blasts) could present a
useful model for the study of potential cytotoxic. Ha-
CaT cells are of dermal keratinocyte origin. They are
immortalized but not tumorigenic and constitute a direct
comparison with normal human epidermal kerationcytes.
During the initial inflammatory phase fibroblasts start to
enter the wound where they synthesize and later remodel
new extracellular matrix. An ideal dressing should offer
moist environment at the wound surface, allow gaseous
exchange, prevent from microorganisms, absorb exudates
and be removed without pain. Despite numerous ad-
vantage and unique properties, chitosan and some deri-
vates have a very slow degradation rate, and its mole-
cules degrade in an uncontrollable manner. So it eventu-
ally had to be removed from the wound, leading to me-
chanical damage of newly regenerated tissue and causing
pain or discomfort to the patients. Furthermore, adequate
ventilation of wound site is very important to avoid
wound contamination with microorganisms and accumu-
lation of CO2. However, chitosan based materials have
good barrier properties towards oxygen, nitrogen, carbon
dioxide and air [12], which hampers their use for wound
dressing. Due to the unique structural features of sponges
which are soft and flexible with interconnected micro-
pores, they have good fluid absorption capability. If there
were unidirectional channel in the sponge, the air and
vapor permeability would have been strengthened. One
might further hypothesize that the capillarity phenome-
non occurring as a consequence of unidirectional channel
might allow more rapid absorption of exudates from the
wound.
However, only a few research articles have been re-
ported about LCH with very limited information. In this
paper, we present a LCH sponge with unidirectional
channel, carrying antibiotics to enhance their antibacte-
rial activity. Although homo- and copolymers based on
Poly lactic acid (PLA) have been widely used in modi-
fying the CH, it exhibits many problems, such as the
residues of the catalyst and other organic solvents.
Therefore, lactic acid is considered instead of PLA in our
experiments. We developed an effective device to obtain
steep temperature gradient to form vertical channels in
the sponges. For the improved antimicrobial activities,
the drug control release mechanism was investigated
using penicillin and erythromycin as model drug. The
main objective of this research is to discuss the chemical
and physical properties, drug release ability and cytotox-
icity of LCH sponges for the potential application as in-
expensive wound dressing.
2. Materials and Methods
2.1. Materials
Lactic acid aqueous solution [85% (w/w)], chitosan (Mw
~389,000 and 92% deacetylated), and tertiary butyl al-
cohol (TBA) were all purchased from Sigma Aldrich Co.
(Milwaukee, USA), and were used without further pu-
rification. Human skin fibroblasts (HSF) and human im-
mortal keratinocytes (Hacat) were purchased from Kun-
ming Cell Bank of Chinese Academy of Sciences. Peni-
cillin and erythromycin were gifted from Guangzhou
Medical College.
2.2. Preparation of LCH Sponge
0.8 g chitosan and 0.5 mL lactate acid aqueous solution
was dispersed in 40 mL deionized water. The mixture
was stirred overnight at 70˚C, and a clear viscous, light
yellow aqueous solution was obtained. The reaction
mixture was subjected to dialysis (seamless cellulose
tubing with MWCO-14,000 and pore size-50 Å) to re-
move excess lactic acid for 3 days. The dialysis medium
was exchanged with fresh water several times. The mix-
ture solution was placed within a refrigerator to freeze at
−78˚C for 24 hours. The frozen hydrogel was lyophilized
within a freeze-dryer for 48 hours. The light yellow and
fluffy powders of chitosan lactate (LCH) were fabricated.
As shown in Figure 1, LCH solution (3% - 10% w/w)
was mixed with TBA (3% - 10% w/w) by stirring till
uniform solution was formed. The mixture was added to
the freezing device as shown in Figure 1. The device
consisted of two parts: a solution container and a coolant
reservoir. The massive copper block in the coolant res-
ervoir acted as the thermal conductor and was continu-
ously cooled by liquid nitrogen (−196˚C). The solution
container side is made of polytetrafluoroethylene (PTFE)

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 460
freezing
lyophilization
soaked in liquid
nitrogen
(a)
(b)
(c)
(d)
(e)
Figure 1. Scheme for preparation process of LCH sponge.
(a) LCH solution; (b) Picture of the solution container. The
massive copper block in the coolant reservoir acted as the
thermal conductor and was continuously cooled by liquid
nitrogen. The solution container side is made of PTFE and
surrounded by PUR; (c) Illustration of the vertical tem-
perature controlled device; (d) Picture of a frozen sample
placed in a liquid nitrogen bath during the freezing process;
(e)The sponge with a size of 2 cm × 1 cm.
and surrounded by polyurethane (PUR) which is the ex-
cellent heat preservation materials. The top of the so-
lution container is exposed to the atmosphere at room
temperature. This device yields an approximately vertical
thermal gradient, leading to the vertical growth of ice
crystal. Freezing was accomplished by immersing the
device in liquid nitrogen bath. The samples were then
lyophilized until dry. After lyophilization, the sponges
were further dried at 37˚C in vacuum drying oven for 24
hours to allow the solvent to be completely evaporated.
The round-shaped sponges were stored in desiccators for
future use.
2.3. Contact Angle Measurement and Water
Absorption Studies
Static contact angles of LCH sponges were measured
with a contact angle analyzer (OCA 20 Dataphysics,
German) using the sessile drop technique. The meas-
urements were carried out at room temperature in air
with deionized water as the probe liquid. The average
contact angles of three points at different sites of samples
were measured. The water adsorbing rate of sponges
were determined with direct optical images and video by
a camera. Data were analyzed using SCA 20 software.
2.4. Morphological Characterization and
Analysis
Morphological characterization was conducted using scan-
ning electron microscopy (SEM, 30 XLFEG, Philips,
The Netherlands). The samples were prepared by break-
ing them in liquid nitrogen. Fragments were adhered on a
cupreous stub by double-faced adhesive tape and coated
with gold and further analyzed.
2.5. NMR Spectroscopy and X-Ray Diffraction
Carbon-13 spectra were recorded on a Bruker DRX-400
spectrometer (operating frequency of 400 MHz). Sam-
ples were analysed in D2O solution at 60˚C in 5 mm o.d.
tubes. DDS was used as external standard. 13C NMR
were recorded using 90˚ pulse, 15,000 Hz spectral width,
8000 data points, 0.54 s acquisition time, 3 s relaxation
delay.
X-ray diffraction (XRD) analysis was performed on an
X-ray diffractometer (XRD) (X Pert’PRO, PANalytical,
The Netherlands) using a flat camera and 40 keV Cukα
(k = 0.15418 nm) radiation. The samples were scanned
from 10˚ to 90˚ with a scan speed of 10˚/min, respec-
tively.
2.6. Porosity Determination of Scaffolds
Porosity of sponge was determined following the method
described in the reference [13]. Briefly, ethanol was used
as the liquid phase and kept at 25˚C. A bottle filled with
ethanol was weighed (W1). Then a sponge sample weight-
ing WS was immersed into the bottle and weighed (W2)
is the density of ethanol at 25˚C. The size of the cylin-
drical scaffold including radius (R) and height (H) was
measured. The porosity (P) was calculated using the
equation as follows:



2
12
1π
s
PWWW RH 

 

(1)
2.7. Cells Culture and Seeding
HSF and HaCat cells were cultured based on the same pr
Cedure. Two kinds of cells were cultured respectively in
DMEM (Sigma-Aldrich, USA)/10% (v/v) FBS (GIB-
COFBS-10099-141, Germany), together with supple-
ments of 2.5 mM L-glutamine. The medium was re-
placed once in every 3 days and the cultures were main-
tained at 37˚C in a wet atmosphere containing 5% CO2.
When the cells reached 80% - 90% confluence, they
were trypsinized with 0.25% trypsin containing 0.53 mM
EDTA (Invitrogen, USA) prior to being used in the ex-
periments.
In all cytoxicity tests performed, culture medium with
0.64% phenol and standard culture medium were used as
positive and negative controls, respectively. Phenol is

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 461
known to have a strong cytotoxic effect leading to exten-
sive cell death, and is commonly used as a positive con-
trol for cell death.
To assess the short-term cytotoxicity of the developed
sponges, the GB/T16886.7 standard test method was
used. The fabricated sponges were sterilized using gam-
ma irradiation from Co60 sources with 25kGy. Sponges
were extracted for 24 hours at 37˚C, using standard cul-
ture medium as the extracting fluid which was constant
and equal to 10 mL. Cells were seeded in 96 wells plates
(n = 10), at a density of 3000 cells per well. Cell re-
sponse was evaluated after 1 day, 2 days, 4 days and 7
days of incubation time at 37˚C, in humidified atmos-
phere containing 5% CO2. Because LCH is water soluble
salt of chitosan, immersion in aqueous solution for a long
period may result in sponge degradation. Hydrolysis might
lead to changes of porous structure of the LCH sponge,
therefore the different micro-structure of sponges does not
have great effect on the extraction liquid cyctotoxicity.
2.8. Quantification of Viable Cells (MTT Assay)
Using MTT (3-(4,5-Dimethylthiazo-a-yl)-2,5-diphenylte-
trazoluim bromide) to indirectly reflect viable cell num-
bers has been widely applied which is based on the fact
that metabolically active cells interact with a tetrasolium
salt in an MTT reagent to produce a soluble formazan
dye. In experiments, MTT was dissolved in PBS at a
concentration of 5 mg/ml, sterilized by filtration, and a
volume of 20 μl added to each well. The solution was
then transferred to a cuvette and placed in a Thermos-
pectronic Genesis10 UV-vis spectrophotometer, from which
the absorbance at 570 nm was measured. The viability of
the cells cultured with fresh SFM was used as control. The
relative cell viability (%) was calculated according to the
following equation based on absorbance at 570 nm:


100%
sb
relative cellviabilityODOD(2)
2.9. In Vitro Release Studies
In vitro drug release studies were performed using the
USP rotating-basket dissolution apparatus (RC 806,
Tianda, China). The receptor medium consisted of 500
ml of PBS at pH 7.4. In order to simulate the one-dimen-
sional drug release from the sponges as clinic skin wound
dressing, the sponges were constrained in a glass mi-
cro-breaker to guarantee that only a single open face with
the effective transport area of 2.54 cm2 could contact
PBS solution. The sample-carrying micro-breaker was
placed in the basket and lower into the PBS solution. The
baskets were kept in a thermostated water bath at 37˚C
with temperature fluctuation ±0.1˚C and were rotated at
100 rpm to eliminate the boundary layer effect. In the
controlled release studies, penicillin and erythromycin
were chosen as hydrophilic and hydrophobic drug mod-
els, respectively. And the drug loading achieved the drug
saturated solubility in the LCH sponges. The amount of
penicillin and erythromycin was assayed spectropho-
tometrically at 213 nm and 241 nm, respectively, using
UV spectrophotometer (UV-1800, Shimadzu, Japan). At
the sampling time points, a 100 µL sample was removed
from the release medium and an equivalent volume of
PBS solution was replaced to maintain constant volume.
In the experiments with erythromycin, 0.25 mol/l NaOH
solution was added into the sample as coloring reagent
(1:1 volume ratio) before the spectrophotometer meas-
urements. All studies were performed in triplicate.
For the purpose of comparison, similar experiments
were performed in a sealed aluminum vessel. The vessel
was then immersed in liquid nitrogen and stored for 24
hours in a freezer at −78˚C. Subsequently, they were ly-
ophilized in freeze-dryer for 48 hours.
3. Results and Discussion
3.1. Characterization of LCH sponges
CH and LCH were characterized in the solution state by
13C NMR (as shown in Figure 2). As the most notable
feature, C = O 13C NMR signal of the LCH appears as
triplets, corresponding to the –COOH in the structure. The
signals of C4, C5, C3, C6 are only slight changed by the
formation of COOH, demonstrating that the modification
mainly happened on the amino group of C2 as shown in
Figure 3. C1 NMR signal of LCH is a singlet and the
C2 signal of it also appears as one peak, suggesting that
LCH presents 2-fold helical structure [14]. In contrast, the
C1 of CH is split into a number of sharp peaks, and the C2
is split into a doublet with the separation of which is 1.64
ppm (see Table 1). Moreover, the signal of C1 at 99.0
ppm are strongly shifted by the modification. This finding
Figure 2. 13C NMR spectra of LCH and CH.

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 462
Figure 3. Lactic acid grafted chitosan.
Table 1. 13C chemical shifts of LCH and CH (ppm from TMS).
C1 C4 C5 C3 C6 C2 C = O CH3
CH
99.0,
95.6,
94.1,
90.0
78.2
76.3 73.2 71.4 69.5 62.2 57.62 55.98 178.33 22.46
LCH 105.2 78.9
77.4 72.6 70.5 69.2 62.5 58.3
184.7
181.2
178.5
22.9
19.3
suggests that a drastic conformational change of CH
might occur during the modification with lactic acid. The
NMR spectra are generally insensitive to the change of
crystal structure, as a complementary means XRD can
further support the NMR results. As shown in Figure 4,
the crystallinity of the LCH is much lower than that of
the CH. The peaks having lattice angel 2θ = 10.3˚ and
19.8˚ correspond to the equatorial (200) and (020) which
are typical fingerprints of semi-crystalline CH. In the
LCH diffractogram distinct peak with maxima occurring
at the angles 2θ = 20.4˚ and a slight peak at 2θ = 7.4˚
appear. But the peak at 2θ = 26.4˚ disappears. Upon
transformation of the CH into the form of LCH, the
crystalline structure changes. The grafting by lactic acid
takes place at C2 which is inside the crystallites, result-
ing in the destruction of the regularity of the packing in
the original CH chains and formation of the amorphous
CH salts. LCH has a more amorphous structure than CH,
thus shortening the degradation time of wound dressing.
As far as water solubility and biodegradation are con-
cerned, LCH is found to be superior to CH. Control of
sponge pore morphology is critical for controlling cellu-
lar colonization rates and organization within an engi-
neering tissue. Figure 5 shows the typical SEM images
of the sponges prepared in the sealed aluminum vessel
and the vertical temperature controlled device, respec-
tively.
From the images shown above, it is obvious that the
freezing process can have a dramatic effect on the micro-
structure of sponges. The freezing in the sealed alumi-

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 463
Figure 4. XRD patterns of LCH and CH.
num vessel generate highly connected pores ranging
from 15 to 60 nm. In contrast, the samples prepared in
special device have vertical oriented channels. This to-
tally different pore structure is most likely the result of
the difference in ice growth condition. Under the freezing
condition, the solution would be separated into pure ice
phase and solvent phase. After ices sublimate during the
freeze-drying process, the porous structure duplicated the
ice crystal morphology. For the freezing process in alu-
minum vessel with high thermal conductivity, fast freez-
ing resulted in small ice crystals formation. Freezing
started from the metal wall of the device to the center of
the solution, this inhomogeneous temperature gradient in
the system led to the crystals growth towards all direc-
tions. The samples solidified under space-dependent free-
zing conditions resulted in an inhomogeneous pore stru-
cture as shown in Figure 5(a). When solidification oper-
ated in the unidirectional freezing devices, ice could
grow under a relatively stable temperature gradient. Dur-
ing solidification, the sample was laterally surrounded by a
PTFE frame; thus, the isothermal levels within the sam-
ple ran parallel to the copper plate. That might result in
homogeneous ice-crystal morphology. The pores were
directed vertically within the sponge, almost parallel to
the heat flow imprinted during the freezing process.
While investigating the effects of TBA on the micro-
structure of sponges, we note that the smooth channel
walls appeared significantly more textured with TBA
concentration increasing from 3% to 20%. A number of
lyophilization technologies based on the use of TBA
have suggested [6,15] that at lower concentration of TBA
(3%) there were enough water molecules to surround
TBA molecules, forming stable clathrate hydrates in
which water molecules bonded to form an ice-like cage
[6].
Water under this condition tended to behave like ice
crystal which has highly ordered structure. This produces
the elongate, oriented, uniform ice crystals which grow
Figure 5. SEM images of LCH sponges prepared under
different condition. (a) In sealed aluminum vessel by im-
mersion in liquid nitrogen; (b) In vertical temperature con-
trolled device with TBA.
parallel to the thermal gradient. The micrographs of Fig-
ure 6(b) also demonstrate two levels of porosity at the
concentration of 5% TBA. The elongate, oriented micro-
tubule with thicker wall can be observed. On the other
side, the surfaces of microtubule walls exhibited many
small pores which are arranged in parallel arrays along
ridges or grooves in the surface. This two-phase structure
is most likely the result of the difference in ice nucleation
conditions in the system. With the increasing concentra-
tion of TBA, because no enough water molecules left to
surround the TBA molecule, there was more opportunity
for TBA molecules to self-associate and form dimmers
that were connected by hydrogen bonds. These low-den-
sity water clusters could not produce ice-like crystals due
to their lack of long-range order. Hence, many smaller
amorphous ice grains formed at the wall of microtubule
like Figure 6(b). The fact that many of smaller ice grains

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 464
(a) (b)
(c) (d)
Figure 6. Dependence of cross-section of spongy microstructure on concentration (w/w) of TBA in solution frozen in the setup
with vertical temperature gradient by contact with liquid nitrogen: (a) 3%; (b) 5%; (c) 15%; (d) 20%.
formed at the higher concentration was in agreement
with observations reported elsewhere [16,17]. As the
increasing of TBA concentration (15% and 20%), the
morphology of amorphous ice tended to be irregular,
suggesting that TBA could act as effective ice crystal
growth modifiers and exert a strong influence on the ex-
ternal morphology and /or crystalline structure of ice.
Many small irregular holes were formed, with pleat-sha-
ped tracing of microscopic ice formation as shown in
Figures 6(c) and (d). The micromorphologies of channel
and even the holes in the walls of the channels inherently
affect both the mechanical and solubility properties of
the sponges.
3.2. The Properties as Wound Dressing
3.2.1. Water Absorption Behavior
The absorption of excessive interstitial fluid in the
wound bed is critical to wound healing. Excessive mois-
ture inhibits cell proliferation leading to tissue saturation
and wound break down. The sponges prepared in our
experiment are soft and elastic with uniform vertical
pores in which capillarity phenomenon often occurs.
Capillarity capacity is not often reported for sponge ma-
terials, however, it is a useful parameter to understand
the structure-function relationship and the future devel-
opment of LCH sponges in wound dressing. For various
classes of porous material, including paper, powders,
rocks and soil, previous studied have shown that the wa-
ter uptake can be expressed as well-known Washburn
Equation [18]
1
2
mKst

 (3)
where m is the water absorbed by a porous solid per unit
of surface area, t means the elapsed time, Ks is defined as
the capillary coefficient characterizing the quantification
of the capillary suction. The equation predicts a linear
relation between m and t1/2 with a slope Ks.
The suction behavior of each sample is illustrated in
Figure 7(a), where the liquid mass absorbed per unit
area (m) is plotted versus the square root of the elapsed

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 465
(a) (b)
(c) (d)
Figure 7. The water adsorption behavior of samples with various formulations. (a) Water absorption rate of various samples;
&: 3%TBA, 3%LCH; Ο: 3%TBA, 5%LCH; Ω: 3%TBA, 7%LCH; ●: 3%TBA, 10%LCH; #: 5%TBA, 3%LCH; *:
5%TBA, 5%LCH; ♦: 5%TBA, 7%LCH; ♣: 5%TBA, 10%LCH; ∆: 10%TBA, 3%LCH; 10%TBA, 5%LCH; ◊: 10%TBA,
7%LCH; ♥: 10%TBA, 10%LCH; (b) Effect of TBA concentration on Ks; ■: 3%LCH; ●: 5%LCH; ▲: 7%LCH; ▼L:
10%LCH; (c) Effect of LCH concentration on Ks. ▲: 10%TBA; ●: 5%TBA; ■: 3%TBA; (d) Effect of porosity on Ks.
time (t). The samples prepared with lower LCH or TBA
concentration present steep slope, indicating the rapid
water uptake. The effects of LCH and TBA concentration
on the Ks are shown in Figures 7(b) and (c). From the
results given above, it is obvious that an increase in LCH
concentration results in a decrease of Ks. In contrast to
LCH tendency, as the concentration of TBA increases,
the Ks also increases. The fluctuation of Ks versus TBA
concentration seems to be much flatter compared to those
of LCH. Therefore, we believed that the LCH concentra-
tion plays a much more influential role than TBA con-
centration on the suction capacity of the sponges. Several
researchers have found evidence that a less dense porous
material is able to absorb a higher liquid amount for the
same porosity [19]. Although TBA can alter ice crystal
growth and hence affect the ice morphology, the change
of TBA concentration does not lead to dramatic change
in solution viscosity. LCH concentration makes a sub-
stantial contribution to the compactness in sponges by
influencing the viscosity of the solution. The samples
made from high viscosity often exhibit higher strength
and less elastic in present experiments. Therefore, the
fluctuation in LCH concentration leads to a dramatic
effect on suction capacity. Generally speaking, the suc-
tion capacity in porous materials (Ks) has a first corre-
spondence with their pore size and amount.To further
assess the relationship between Ks and porosity, their
correlation is presented in Figure 7(d). A positive corre-
lation of Ks with porosity seems to exist though with a
quite low statistical significance (r2 = 0.5299), corrobo-
rating that the suction capacity does not depend on the
porosity completely in present study. The same porosity

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 466
corresponds to quite different value of Ks especially at
higher porosity. One possible reason for these results lies
in the influence of other parameter on the capillary coef-
ficient as expressed:

1
2
0
Ks Cr    (4)
where C is a constant corresponding to the liquid, repre-
sents the effective porosity (the open porosity actually
accessible by the liquid), r
0 is the median pore size,
means the pore tortuosity of the solid which is a non-
dimensional parameter. One classic definition of tortu-
ousity is often given by Bear [20]. In case of parallel
cylindrical capillaries which are perpendicularly arranged
with regard to the bed of a liquid it is about 1. The chan-
nels in our experiments are vertical cylindrical capillaries,
but the holes in the channel walls make the fluid route
more tortuous than theoretical condition. Such fluid
movement in present capillary system is hard to reconcile
with vertical movement in ideal parallel cylindrical cap-
illary. It is likely that many factors are at play in deter-
mining the tortuosity including the LCH, TBA concen-
tration and the freezing behavior, resulting in a more
complex dependence of Ks on the porosity. Their rela-
tionship therefore deviates from linear relationship. How-
ever, the capillary behavior of the sponge described in
present paper is rather complicated and may deserve a
further study.
3.2.2. Cell Viability
Figure 8 shows the viability of HaCaT and HSF cell
after being cultured with the extraction medium, pre-
pared by immersing sponges in it. The effects of LCH on
two kinds of cell viability are found to be time and con-
centration dependent. At lower concentration extraction
(<25%), LCH time-dependently inhibited HSF cell pro-
liferation by approximately 88% (2 days) and 86% (4
days). With increasing concentration (50% - 100%), the
incubation time influenced cell viability only marginally.
The results suggest that the higher concentration extrac-
tion medium from LCH sponges is harmful for the HSF
cells. A dramatic increase of cytotoxicity on HaCaT cells
by the increasing LCH concentration is also observed. At
25% LCH extraction time-dependently inhibited HaCaT
cell proliferation by approximately 89% (2 days) and
94% (4 days). Higher concentration led to a significant
decrease in viable cells, but it seemed that the 50% LCH
extraction was much safer for HaCaT cells than HSF
cells. As shown, the viability of the HaCaT cells cultured
in the 50% LCH extraction medium is 93% after 1 day
incubation, while only 50% HSF cells viability is ob-
tained by the same treatment. According to the United
States pharmacopoeia about toxicity classification [21],
50% concentration of LCH extraction shows cytotoxicity
(a)
(b)
Figure 8. Indirect cytotoxicity evaluation of LCH sponges
based on the viability of HSF cells (a) and HaCaT cells (b)
*p < 0.05, **p < 0.01, ***p < 0.001: significant deviations from
the control at the respective extraction concentration and
incubation time.
grade of 0 - 1 for the HaCaT cells. By treatment with
100% LCH extraction, only approximately 15% cells
showed viable phenomena by MTT assay.
Chitosan and its derivatives exhibit outstanding bioac-
tive properties, whereas they have been reported to pos-
sess distinct toxic effects, as red blood cell lysis or cell
growth inhibition [22]. In this experiment, LCH sponges
were prepared for the potential use of wound dressing, so
HaCaT and HSF were used as referenced cell lines to
evaluate their potential cytotoxic effects. The results ob-
tained indicate that LCH sponges exhibit bio-safety at
lower concentration (25%) during short time (1 day). The
sponges prepared in our experiment are even less toxic
than chitosan 1130 which has high molecular weight [23].
However, highly concentrated extraction shows a serious
toxic effect on both HSF and HaCaT cells. HSF cells are
much more sensitive to materials toxicity than HaCaT

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 467
cells. This is probably due to the differentiated nature of
these two cells. Cationic polymers have been known to
be cytotoxic materials, however the mechanism has not
been fully understood. There is a general acceptance that
the interaction between the positively charged chitosan
polymers and the negatively charged cell membrane de-
stabilizes the cells structure, resulting in the rupture of
cell membrane followed by cell death [24,25]. In our
experiment, we can believe that the cytotoxic effect is
partly originated by the increased acidity caused by the
degradation of the lactate-modified chitosan sponges.
As we mentioned above that the sponges prepared in
our experiment are a potential skin wound dressing. Usu-
ally, there is only one side of sponges will exposure to a
skin wound in clinic application which is different from
our cytotoxic test. In the case of immersion in extraction
medium, the sponges hydrolyzed much faster than clini-
cal application condition, resulting in significant increas-
ing in concentration and cyctotoxicity. Once LCH wound
dressing is used clinically, the dramatic dependence of
cyctotoxicity on the time and concentration will level
off.
3.2.3. In Vitro Release Studies
In our research, the sponges are made of hydrophilic chi-
tosan salts, the controlled release in this kind of system
must take into account three components: matrix erosion,
drug diffusion and the structure of the porous microen-
viroment [26]. So drug release is controlled by two proc-
esses: erosion of the matrix and diffusion of the drug. As
many classical drug release model, the pores in the ma-
trix are classified into accessible and isolated region [27].
In our experiments, for simplification purpose, the pore
which drug molecule located can be idealized as a cylin-
der. In Figures 9(a) and (b), we show release curves for
penicillin and erythromycin in sponges with different
formulation, representing the effects of the drug proper-
ties and matrix microstructure on the release behavior
from bulk-degrading polymer. The figures allow for a
direct visualization that the drug release increases with
the increasing of TBA amount in the matrix. As we dis-
used above, when was below 20%, increasing TBA led to
larger pore in sponges which was much more accessible
to solvent. In hydrophilic drug system, Initial burst re-
lease is not observed, and penicillin is released entirely
within 80 hours. The drug release in 10% TBA system
presents linear release curve indicating a quasi zero-order
kinetics. The curves in penicillin release do not exhibit
square root of time profile, suggesting the release behav-
ior is governed by both diffusion and erosion process.
Penicillin is hydrophilic drug which can form homoge-
nous system with LCH solution, so the release of drug
from the LCH sponges must be accompanied with matrix
erosion. As shown in Figure 10(a), penicillin causes
extensive wrinkling in the internal surface of the pore but
without noticeable aggregate, demonstrating the good
dispersion of drugs in matrix. At the same time, dissolu-
tion of matrix structure can be accelerated by the release
of drug from the sponge network because the release of
drug leaves an imperfection on the network, thereby
weakening the microstructure of matrix. Compared to
erythromycin system, penicillin exhibits faster release
characteristics due to the association of the faster erosion
of the matrix and drug dissolution.
Exponential Functions were used to the experimental
data of in vitro release of erythromycin from sponges
with various formulations. Good correlation coefficients
(R2 = 0.99 - 0.97) were obtained for each case. As hy-
drophobic drug, erythromycin failed to solute in the LCH
solution, causing efficient phase separation in the poly-
mer solution consequently. Unlike penicillin which is
trapped within the polymer uniformly, erythromycin was
adsorbed on the surface of matrix. As shown in Figure
10(b), drug solids aggregate and form circular shape on
the surface of internal pores. These could responsible for
the initial burst release of erythromycin (Figures 9(c)
and (d)). Due to this kind of morphological characteris-
tics, the drug release has very limited influence on the
matrix microstructure. The erythromycin/LCH sponges
are much more compact and stiffer than those of penicil-
lin. In the experiments, we observed that the permeation
of the PBS solution in this kind of matrix only led to a
limited erosion. For the 5% LCH drug release system, the
release curves display typical diffusion-controlled release
curves, indicating that the diffusion is the predominant
mechanism of drug release. For the 7% LCH drug release
system, the action of erosion is no longer negligible and
the curve profiles polynomial function characteristics.
The LCH solution with concentration of 7% is too vis-
cous to dissolve the erythromycin, leading to low drug-
loading in matrix. The results of these simulations lead
us to believe that the balance between diffusion and ero-
sion will title towards erosion under this condition.
4. Conclusions
Due to the presence of two functional groups chitosan
was modified by lactic acid without using catalysts in our
present experiments. The crystallinity of LCH decreases
after modification because the side chains replace the
–NH2 groups in CH and destroy the regularity of packing
between CH chains. As a complementary means to XRD,
13C NMR demonstrates that modification mainly hap-
pened on the amino group of C2 in CH structure. The
results of 13C NMR suggest that a drastic conformational
change of CH might occur during the modification with
lactic acid, which agrees well with the XRD findings.
TEM images show that different types of freezing be-
havior and TBA concentration lead to entirely different

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 468
(a) (b)
(c) (d)
Figure 9. Cumulative release of penicillin and erythromycin from the sponges with various formulations. (a) penicillin from
the 5% LCH; (b) penicillin from the 7% LCH;(c) erythromycin from the 5% LCH; (d) erythromycin from the 7% LCH. The
dots represent data points and the solid lines are the corresponding release curves computed from the model.
micro-structure of sponges in our experiment. TBA does
not exhibit a considerable inhibition effect on the ice
crystallization, but it shows considerable influence on the
crystal growth morphologies, possibly due to the pres-
ence of the specific weak hydrogen binding between
TBA and water molecular.
As a potential wound dressing, the properties of water
absorption behavior, cell viability and in vitro release are
evaluated. Capillarity capacity parameter, Ks, is intro-
duced for the understanding of the structure-function rela-
tionship and the future development of LCH sponges in
wound dressing. The water absorption depends on the
LCH concentration more dramatically at higher TBA.
Indirect cyctotoxicity evaluation using HSF and Ha-
CaT cells indicates that the effect of LCH sponges on
cyctotoxicity is time and concentration dependent. The
results obtained indicate that LCH sponges exhibit bio-
safety at lower concentration (25%) during short time (1
day) and much safer for HaCaT than HSF. However,
highly concentrated extraction shows a serious toxic ef-
fect on both HSF and HaCaT cells using the soaking
method in our experiments.
The in vitro release characteristic of hydrophilic and
hydrophobic drug from the LCH sponges confirms that
both the drug molecules diffusion and matrix erosion
play important roles during the drug release. The in vitro
release study of penicillin showed no burst effect, while
the in vitro release study of erythromycin showed a clear
burst effect with an initial fast release phase followed by
a sustained release phase. Due to the hydrophilic proper-
ties, penicillin can accelerate the degradation of biode-
gradable carrier. As a result, the hydrophobic drug ery-
thromycin release was significantly more prolonged than
penicillin.
Wound healing is indeed a very complex process invo-
lving many complex steps as: the inflammatory phase,
the proliferative phase and the remodel phase. In the pa-
per, we just present a promising candidate in wound

Study on Chitosan-Lactate Sponges with Oriented Pores as Potential Wound Dressing 469
(a) (b)
Figure 10. The internal surface of the sponge pores with penicillin (a) and erythromycin (b).
dressing. Further investigation such as animal and clini-
cal test still needed to evaluate this kind of dressing in
the future.
5. Acknowledgements
This research was supported by Major State Basic Re-
search Development Program (973 Project No. 2012CB
933603), Science and technology research Foundation of
Shenzhen Bureau of science and technology & informa-
tion (JC200903170498A).
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