Journal of Biomaterials and Nanobiotechnology, 2011, 2, 216-225
doi:10.4236/jbnb.2011.23027 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
Biodegradable Polysaccharide Gels for Skin
Scaffolds
Stephen J. Juris1, Anja Mueller1, Brian T. L. Smith1, Samantha Johnston1, Robert Walker1,
Robert D. Kross2
1Central Michigan University, Mt. Pleasant, USA; 2Kross Link Labs, Bellmore, USA.
Received March 2nd, 2011; revised May 2nd 2011; accepted June 10th, 2011.
ABSTRACT
A variety of skin substitutes are used in the treatment of full-thickness burns. Substitutes made from skin can harbor
latent viruses, and artificial skin grafts can heal with extensive scarring, failing to regenerate structures such as glands,
nerves, and hair follicles. Biodegradable and biocompatible hydrogels, however, rarely mimic the strength of the epi-
dermis. Therefore, novel and practical skin scaffold materials remain to be developed. Polysaccharides form hydrogels
with predicted inherent biocompatibility. This paper describes the preparation and biocompatibility of unique hydrogel
skin scaffolds from plant-extracted polysaccharide mixtures of specific sources, types, and molecular weight fractions.
These hydrogels have a range of mechanical and degradation properties with the potential to fulfill the multiple, di-
verse functions of artificial skin, including protection, compatibility with different cell types, biodegradation, and re-
lease of needed signals for cell growth and wound healing.
Keywords: Hydrogel, Polysaccharides, Skin Scaffold
1. Introduction
The skin is the largest organ in the body and serves many
important functions, such as protection against infection,
immune surveillance, perception of touch, and tempera-
ture regulation [1,2]. Skin contains two layers: the strong
epidermis, which is composed mainly of keratinocytes
that form the thick protective layer with hair follicles and
glands, and the dermis, which contains many different
cell types, including collagen-producing fibroblasts, blood
vessel-forming endothelial cells, motor and sensory neu-
rons, and immunoregulatory cells.
When skin is superficially damaged the wound is rap-
idly repaired. However, when the injury destroys both
epidermis and dermis, patients often die from infection or
loss of plasma [3]. If patients survive with the help of skin
grafts, skin often heals with the formation of scar tissue
without the regeneration of some cell types [4-7], in-
cluding hair follicles and sebaceous glands [8]. Thus, an
improved scaffold should release cytokines to promote
healing and cell differentiation. Also, artificial skin often
contains animal material, such as collagen, which may
contain infectious material. Due to these problems, there
is a need for improved methods and materials for fabri-
cating artificial skin [9,10]. Unfortunately, there are
limited biocompatible and biodegradable materials avai-
lable. Hydrogels have been used as an alternate artificial
skin, because good hydration is the most important ex-
ternal factor responsible for optimal wound healing [11-
14], as water is necessary for transport of nutrients to the
growing cells. Hydrogels, however, are commonly not
physically strong enough to mimic the tough, thin dermis
[9].
The polymer most often used in skin scaffold synthesis
is a polyester based on lactic acid (PLA) and glycolic acid
(PGA) [15-19]. However, these polyesters have been
shown to degrade to their monomeric components, re-
sulting in high local acidity that can destroy proteins
[20-22]. Thus, a material that avoids acidity and is capa-
ble of delivering bioactive molecules would be more
useful.
Polysaccharides are biopolymers with predicted bio-
compatibility, and many polysaccharides form physical
hydrogels [23]. There are also a variety of polysaccha-
rides that form helical structures and thus are stronger
materials. In this study, we used four different gel-
forming polysaccharides: ι-carrageenan, κ-carrageenan,
xanthan gum, and konjac gum [24-26]. The two carra-
geenans and xanthan form strong double helical confor-

Biodegradable Polysaccharide Gels for Skin Scaffolds 217
mations with specific cations present [27,28], and konjac
forms a partially-ordered single helix with the right car-
bocation [29]. Xanthan and konjac gum are mostly used
as thickeners, as are the carrageenans, which are also
known to form H-bond-stabilizing reversible gels [23].
Gel formation is a two-step process induced by specific
counterions; first the double helices aggregate, then the
aggregates combine at a certain combination of tempera-
ture and concentration, stiffening the gel. Konjac and xan-
than gum gel formation seems to be dependent mostly on
ionic strength (which also drives helix formation), con-
centration, and temperature [24,25].
Polysaccharides may indeed be a useful material as a
skin scaffold with the capability of delivering molecules
involved in wound healing; some polysaccharides even
improve wound healing [9,30]. Mostly natural polysac-
charides have been investigated as biomaterials, such as
chitosan [5,7,31], cellulose [32], collagen [33,34], and
hyaluronic acid [35-37]. Chitin/poly(lactic acid-co-gly-
colic acid) (PGLA) blends [38] and starch-based blends
[39,40] have been reported for protein drug delivery and
tissue engineering scaffolds, respectively. A crosslinked
hyaluronic acid hydrogel has been loaded with bioactive
cytokines and successfully induced angiogenesis [36].
This paper reports the preparation of hydrogels made
from plant polysaccharides with different mechanical
strengths and activities. Mixtures of these extracted poly-
saccharides have already been used successfully in the
development of a skin patch for drug delivery [41].
Developed polysaccharide skin scaffolds are required
to be biocompatible, promote healing, and mimic the
strength of dermis and epidermis in order to prevent fur-
ther tissue damage. This skin scaffold will be made of
two layers, a dense, strong top layer for keratinocytes
mimicking the epidermis, and a porous, softer layer for
fibroblasts, mimicking the dermis. This paper reports the
testing of biocompatibility and strength of polysaccha-
rides and polysaccharide hydrogels synthesized from
plant extracts, as well as their method of preparation.
2. Materials and Methods
2.1. Materials
EMD Chemicals: HPLC grade water, 30% hydrogen
peroxide; Fisher Scientific: potassium phosphate mono-
basic and dibasic, potassium chloride (KCl), calcium
lactate (C6H10CaO6), hydrogen chloride (HCl); Sigma:
Triton X-100, Bovine Serum Albumin (BSA); TIC Gums:
κ-carrageenan (Colloid 710 H Powder), ι-carrageenan
(Colloid 881 M Powder), Xanthan (Ticaxan Rapid Pow-
der), Konjac (Konjac High Viscosity), Ticagel (121-AGF
Powder); Arch Personal Care Produces (ARCH): Cosmo-
cil preservative; Aldrich: sodium benzoate (NaC6H5CO2),
N-methyl pyrrolidone, glucose (C6H12O6), glycerin
(C3H5(OH)3); Hyclone: Heat inactivated fetal bovine se-
rum; American Type Culture Collection: MeWo human
fibroblast cell line; Invitrogen: Minimum Essential Me-
dia (MEM), L-glutamine, penicillin/streptomycin, Tryp-
sin/EDTA; Roche Biochemicals: formazan dye WST-1.
Water was purified through a Barnstead E-pure filter
system and collected at 18 MOhm.
2.2. Instrumentation
1) UV spectra were obtained on an Agilent 8453 UV/Vis
Spectrometer utilizing Agilent ChemStation software. 2)
Gel permeation chromatography was conducted on a
Waters Breeze system consisting of a Waters 1515 iso-
cratic pump, 717+ autosampler, and 2414 refractive in-
dex detector, a column heater, and with Breeze software,
with Fluka polyethylene glycol (PEG) standards on two
Tosohaas TSK-Gel 7.5 mm × 30 cm columns in series,
with deionized ultra-filtered (DIUF) water as solvent at
1.0 mL/min and 30˚C. 3) IR spectroscopy was performed
with KCl pellets on a Nicolet Magna IR 560 spectrome-
ter. 4) Differential scanning calorimetry (DSC) was car-
ried out at a heating rate of 10˚C/min using a TA Instru-
ments Inc. model 2100 with a 2910 DSC cell at 50
mL/min. 5) Thermal gravimetric analysis (TGA) meas-
urements used a TA Instruments 2950 with a Thermal
Analyst 2100 control unit and a 10˚C/min heating rate in
nitrogen atmosphere. 6) A TAQ800 Dynamic Mechani-
cal Analyzer (DMA) was used for mechanical analysis at
a strain ramp from 0.01% - 0.4%. The modulus was cal-
culated from the slope of the linear region of a stress vs.
strain curve. 7) Cell viability, as measured by hydrolysis
of the formazan dye WST-1, was measured using a Spec-
tramax microplate spectrometer. Measurements were
taken at 450 nm, and the 650 nm reference absorbance
was subtracted from all samples before subtracting media
reading from experimental samples.
2.3. Preparation of the Hydrogels
Hydrogels were prepared following Dr. Kross’ patent
[41]. To prepare the “original” hydrogel, konjac gum
(0.168 g), xanthan gum (0.112 g), κ-carrageenan (0.08 g),
and ι-carrageenan (0.16 g) were initially dispersed in
N-methyl pyrrolidone (1.2 g), and the mixture was stirred
into water (17.09 g). Potassium chloride (0.08 g), cal-
cium lactate (0.16 g), sodium benzoate (0.02 g), and
Cosmocil CQ (0.02 g) were added, and the mixture was
stirred until all dissolved. Glucose (0.90 g) and malic
acid (0.05 g) were mixed initially and then stirred in
slowly until the solution became more viscous. The solu-
tion was weighed, heated to 85˚C to initiate gelling, and
reweighed. The water lost in heating was re-added to the

Biodegradable Polysaccharide Gels for Skin Scaffolds 218
mixture, which was again heated to 85˚C. The gel was
then poured into five separate Petri dishes (5 cm diameter)
in 2 g quantities and allowed to cool and gel at 4˚C. Gels
produced had a thickness of about 0.9 mm, which re-
duced up to 0.3 mm over time as water was lost. The
variability between the different compositions was +/–
0.15 mm.
The preparation of the “alternative” hydrogel also fol-
lowed the patent [41]. Ticagel 121-AGF Powder (0.5 g),
sodium benzoate (0.02 g) and Cosmocil CQ (0.02 g)
were added to preweighed glycerin (1.0 g) and stirred.
DIUF water (18.46 g) was added to the beaker and the
mixture stirred until thickened by heating, as above, and
the liquid then similarly poured and allowed to gel during
cooling.
Variations of the original composition included: 150%
konjac, elimination of KCl, xanthan, one of the carra-
geenans, or Cosmocil. When the release of Bovine Serum
Albumin (BSA) from the gel was to be measured, BSA
was added directly to the pre-gel mixture before heating.
Variations of the alternate composition included reducing
the amount of water. The method for the respective
preparations of these mixtures was exactly the same as
above.
2.4. Determination of Relative Hydrogen
Bonding Strength
The broad –OH absorption peak, present on the spectra at
3000 - 3500 cm–1, was used to quantify the relative av-
erage strength of H-bonding present in the various gels.
The calculation involves the initial vertical subdivision of
the –OH peak into ~50 cm–1 increments. The heights of
each of these increments from the base of the spectrum to
the peak were then measured, in millimeters, and totaled.
This sum divided by 100 became the division factor. Be-
ginning at the higher frequency end of the peak, each
increment was numbered and the sum then divided by the
division factor resulting in a weighted average. This total
number was reflective of the degree of overall H-bonding
strength. Higher values (lower frequency vibrations) in-
dicate less relative hydrogen bonding strength. If the
sample contains –NH bonds as well, only samples with
the same amount of –OH and –NH peaks can be com-
pared with each other.
2.5. Cell Culture
The human fibroblast cell line MeWo was cultured in
MEM supplemented with 10% heat-inactivated fetal bo-
vine serum, 2 mM L-glutamine, 100 U/ml penicillin, and
100 μg/mL streptomycin. Cells were incubated at 37˚C in
5% CO2. Cells were passaged using 0.25% Trypsin/
EDTA and centrifuged for 5 minutes at 200 x g.
2.6. Cell Viability Assay
Individual components of prepared polysaccharide skin
scaffolds, except konjac gum and Ticagel (due to insolu-
bility), were tested for toxicity with human fibroblasts by
incubating the individual components with 5 × 104 MeWo
cells for either 3 or 7 days. Hydrogels were also tested by
cutting formed hydrogels to the diameter of a 96-well
plate and incubating them with 5 × 104 MeWo cells for 7
days. The formazan dye WST-1 was used to measure the
metabolic activity of cells per manufacturer’s instruc-
tions. Samples were read at both 450 nm (sample reading)
and 650 nm (reference). Samples were tested on at least
three replicates and the error reported as the standard
deviation of the mean of each sample.
2.7. Primary Skin Irritation in Rabbits
The Primary Skin Irritation study followed the Organiza-
tion for Economic Cooperation and Development (OECD)
Guidelines for the Testing of Chemicals, Test No. 404
[42], and was performed by Eurofins PSL (725 Cranbury
Road, East Brunswick, NJ 08816) [43]. NIH guidelines
for the care and use of laboratory animals (NIH Publica-
tion #85-23 Rev. 1985) have been observed. An original
gel was prepared under sterile conditions and addition-
ally sterilized with ethanol and stored under moist condi-
tions.
In short, three naïve, male New Zealand albino rabbits
(young adults) were kept in controlled conditions for 13
days. A 0.5 g, 1 in2 piece of the original hydrogel was
applied to one 6-cm2 intact dose site on each animal. In-
dividual dose sites were scored according to the Draize
scoring system [44] at approximately 30 - 60 min, 24, 48,
and 72 h, and at 7, 10, and 14 days after patch removal.
The resulting Primary Dermal Irritation Index (PDII) was
classified as follows: 0, non-irritating; >0 - 2.0, slightly
irritating; 2.1 - 5.0, moderately irritating; >5.0, severely
irritating. The animals were observed for signs of gross
toxicity and behavioral changes at least once daily during
the test period.
2.8. Assessment of the Ready Biodegradability of
the Original Hydrogel with the Closed Bottle
Test
The assessment of the Ready Biodegradability of the
original hydrogel was determined via a standard test for
biodegradability [45], and was performed by Eurofins
GAB (Eutinger Str. 24, D-75223 Niefern-Öschlebronn,
Germany).
In short, closed glass bottles were filled with aerated
and inoculated test medium [46]. O2 consumption was
measured with an Oximeter and a calibrated electrode.
Sodium benzoate at 2 mg/L was used to check the activ-

Biodegradable Polysaccharide Gels for Skin Scaffolds 219
ity of the inoculum. In accordance with the regulations,
the biochemical oxygen demand (BOD) for the reference
item should be >60% of the theoretical oxygen demand
(ThOD) within 14 days.
The effluent of the municipal activated sludge plant of
Pforzheim/Germany was taken as the inoculum. The de-
gradability of the original hydrogel was determined in an
aqueous test solution at a concentration of 2 mg/L, based
on dry matter content. The solution was inoculated with a
small number of microorganisms from a mixed popula-
tion and maintained in closed bottles in the dark at a con-
stant temperature between 19.1˚C and 22.6˚C.
The hydrogel can be assumed to be inhibitory, if in a
toxicity test containing both the test item (2 mg/L) and
the reference item (2 mg/L), less than 25% degradation
occurs, based on the theoretical and chemical oxygen de-
mand (COD). The ThOD of the toxicity control was cal-
culated from the proportionate amounts of ThOD and
COD.
The test was performed in closed BOD flasks in single,
2-, and 3-fold test assays for each of the 6 measurement
dates (4, 7, 11, 14, 21, and 28 days). Ultra pure grade
water was strongly aerated for 10 min to achieve oxygen
saturation and allowed to stand for 24 h without aeration
at test temperature. The O2 content was measured at the
start of the test. Subsequently, each flask was inoculated
with 2 mL of inoculum and the volume was made up to 5
L with oxygen-saturated water. Oxygen concentrations
were measured in one vessel from each treatment group.
The exerted BOD was calculated by subtracting the
oxygen concentration (mg O2/L) of the mean initial in-
oculum blank from that of the other study groups. Then,
the measured values of the corresponding controls were
subtracted. This corrected depletion was divided by the
concentration (mg/L) of the test item, to obtain the spe-
cific BOD as mg oxygen per mg test item. The percent-
age biodegradation was calculated by dividing the spe-
cific BOD by the specific oxygen demand, calculated
from the molecular formula in accordance with the
OECD guideline (BOD = mg O2/mg hydrogel; COD =
chemical oxygen demand of the test item).
3. Results
3.1. Preparation of the Hydrogels
All gels are based on the mixture of either N-methyl
pyrrolidone, konjac gum, xanthan gum, κ-carrageenan
gum, ι-carrageenan gum, potassium chloride, calcium lac-
tate, sodium benzoate, Cosmocil CQ, glucose, and malic
acid (“Original”); or glycerin, Ticagel, sodium benzoate,
and Cosmocil CQ (“Alternate”) developed by Dr. Kross
[41]. Konjac gum is derived from the Araceae plant fam-
ily and contains 40% glucomannan gum (β1-4 man-
nose-glucose, 1.6:1 ratio, with β1-6 branching). It is used
as a thickener as a substitute for gelatin. Xanthan gum is
made by the Xanthomonas campestris bacterium. Xan-
than contains β1-4 mannose, glucose, and glucuronic
acid with β1-3 branching. Some of the sugars are also
acetylated or modified by pyruvate. Carrageenans are
water-soluble sulfated galactans with alternating β1-3-
and β1-4-bonds and some 4-linked 3,6-anhydrogalactose.
ι-carrageenan and κ-carrageenan both form gels, but dif-
fer in the amount of sulfonation: ι-carrageenan is sul-
fonated on every sugar in the chain, whereas κ-carra-
geenan is sulfonated on every second sugar. Both carra-
geenans form double helices in the presence of salt,
ι-carrageenan in the presence of Ca2+-ions, κ-carrageenan
in the presence of K+-ions. Ticagel is a mixture of carra-
geenan and carboxymethyl cellulose. Variations were
made by removing components or by reducing or increas-
ing the amount of components.
Cosmocil and sodium benzoate are antimicrobials.
N-methyl pyrrolidone, glucose, and glycerin are added to
regulate the viscosity. Malic acid is added for pH ad-
justment. Glucose has also additional protein-protective
properties.
3.2. Characterization of Physical, Thermal, and
Mechanical Properties
IR spectroscopy was used to analyze the relative amount
of hydrogen bonding (the higher the value, the lower the
average relative strength of hydrogen bonding). This me-
thod is based on the frequency shift in the OH stretching
peak resulting from hydrogen bonding [47]. The integra-
tion across the peak reveals a shift to more or less average
relative hydrogen bonding strength if the same type of
materials (i.e. the same amount of ratio and amount of
functional groups) are compared. Differential scanning
calorimetry (DSC) was used to measure the glass transi-
tion temperature (Tg) and melting point (Mp). Thermal
gravimetric analysis (TGA) was used to determine the
thermal stability. Discrete melting points in the DSC data
indicate the presence of crystalline regions in the other-
wise amorphous hydrogels. In general, the TGA graphs
show continuous loss of material with increasing heat,
but all show distinct rapid loss steps with the percent of
gel lost in that step (Tables 1-3). These data indicate that
there are distinct regions in the gel that are phase- sepa-
rated, suggesting that the gel is not homogeneous. The
weight of the gels was determined over time to measure
water loss. Dynamic mechanical analysis (DMA) was
used to determine the mechanical strengths of the pre-
pared hydrogels. Initial results are summarized in Tables
1-3.
The original mixture showed a high Young’s modulus,
which was comparable to the modulus of contact lenses

Biodegradable Polysaccharide Gels for Skin Scaffolds 220
Table 1. Characterization of hydrogels—original with variations in konjac and xanthan gum.
Mixture H-bonding (day) TGA (˚C) Tg (˚C) Mp (˚C)Modulus in MPa (day)Poissons Ratio (day) Weight Loss in % (day)
Original 690 (d69)
52 (52%)
183 (14%) –32.2 –14.19
0.02 (d3)
0.13 (d6)
24.01 (d8)
0.04 (d6)
0.07 (d8)
34% (d6)
66% (d8)
Original without
konjac 701 (d38)
95 (63.5%)
184 (8.6%)
215 (2%)
–21.78–7.92 Sample broke (d1,2)
38.0 (d3) Sample broke Sample broke
Original 150%
konjac 901 (d1) 183 (34%) NA NA
0.014 (d1)
0.019 (d2)
Sample broke (d3)
Sample broke (d1)
0.58 (d2)
4.92 (d3)
53% (d2)
57% (d3)
Original without
xanthan 626 (d17) 47 (32.5%) 180 (61%) –32.14Not seen
0.015 (d1)
0.019 (d2)
0.020 (d3)
Sample broke (d1)
7.05 (d2)
19.61 (d3)
49% (d2)
93% (d3)
NA: Not Available.
Table 2. Characterization of hydrogels—original with variations in κ- and ι-carrageenan gum.
Mixture H-bonding (day) TGA (°C) Tg (˚C) Mp (˚C)Modulus in MPa (day)Poissons Ratio (day) Weight Loss in % (day)
Original 690 (d69)
52 (52%)
183 (14%) –32.2 –14.19
0.02 (d3)
0.13 (d6)
24.01 (d8)
0.04 (d6)
0.07 (d8)
34% (d6)
66% (d8)
Original without
-carrageenan 698 (d33) 69 (74%)
172 (10%) –32.16Not seenBroke (d1,4) 0.019 (d2) Broke Broke
Original 150%
-carrageenan 691 (d14) 186 (25.5%) NA Not seen
0.024 (d1)
0.045 (d3)
21.75 (d5)
0.30 (d3)
0.92 (d5)
23% (d3)
83% (d5)
Original without
KCl 727 (d34) 183 (27%) NA Not seen
Broke (d1)
5.51 (d2)
12.70 (d3)
0.91 (d2)
0.63 (d3)
88% (d2)
94% (d3)
Original without
-carrageenan 713 (d29) 69 (73.5%)
179 (8.5%) –32.5 Not seen
Broke (d1)
0.077 (d2)
56.21 (d3)
7.87 (d2)
5.24 (d3)
56% (d2)
82% (d3)
Original without
calcium lactate 716 (d77) 203 (5%) 224 (7.5%) NA NA 6.99 (d77) NA NA
NA: Not Available.
Table 3. Characterization of hydrogels based on the alternate mixture.
Mixture H-bonding (day) TGA (˚C) Tg (˚C) Mp (˚C) Modulus in MPa (day)
Alternate 627
175 (25%)
263 (30%) NA NA 27.68
Alternate, 90% H2O, without
Cosmocil CQ 862 (d4)
57 (35%)
201 (15%)
274 (4%)
773 (9%)
–33 NA Broke
2.46 (d14)
Alternate, 85% H2O, without
Cosmocil CQ 863 (d4)
74 (21%)
174 (11%)
250 (10%)
266 (7%)
NA NA 0.06 (d18)
0.35 (d18)
Alternate, 80% H2O, without
Cosmocil CQ 844 187 (6%) 267 (1.5%) –33.5 NA 0.01 (d11)
NA: Not Available.

Biodegradable Polysaccharide Gels for Skin Scaffolds 221
(Table 1). When konjac gum was removed from the mix-
ture, hydrogen bonding strength decreased by a small
amount, and the modulus increased by about 50%. These
gels required three days to dry at 4˚C before they could
be handled successfully. On the other hand, when the
amount of konjac gum was increased by 50%, hydrogen
bonding strength and the modulus decreased significantly.
Xanthan gum in the mixture increased hydrogen bonding
strength, but decreased the modulus significantly. These
mixtures also lost water significantly faster than the other
hydrogels described so far.
It is unexpected that increased hydrogen bonding
strength corresponds to weaker materials. These data
could possibly be explained by the amount of water pre-
sent in the mixture, such that less water leads to a higher
modulus. These data correlate well with this explanation,
as the modulus increased over time, which was accom-
panied by weight loss (and with that, water loss).
In the last three mixtures, the weight loss step that oc-
curs around 180˚C in the TGA became the major step
(Table 1). The early weight loss step (observed around
50˚C) is still present in the sample without xanthan, but
has disappeared from the other mixtures.
When either of the carrageenans was removed from
the mixture, the early weight loss step in the TGA was
seen again, and in fact the weight loss increased (Table
2). When the amount of κ-carrageenan was increased or
KCl was removed from the mixture, the higher tempera-
ture weight loss step was the only one present. When
calcium lactate was removed, this high temperature
weight loss step occurred at temperatures above 200˚C
and less weight was lost. These data indicated that this
last mixture is the most homogeneous of these gels.
The carrageenans form helices, especially with certain
ions present, such as K+ as in KCl for κ-carrageenan and
Ca2+ as in calcium lactate for ι-carrageenan. Unexpectedly,
some of the highest moduli were seen when ι-carrageenan
or the salts were removed (Table 2). These results, in
combination with the TGA data, might indicate that ho-
mogeneity increases strength, and that the thermally la-
bile phase is mechanically very stable.
Some of the samples overall also contracted signifi-
cantly when water was lost and, in extreme cases, this
effect was larger than the effect of thinning during exten-
sion, yielding unusual Poisson’s ratio values.
The alternate mixture has a similar modulus as the
original mixture (Table 3). When Cosmocil CQ, an anti-
microbial agent that reduces skin cell growth, was re-
moved, the strength of the gels was significantly reduced.
3.3. Determination of Toxicity of Hydrogel
Components and Formed Hydrogels
The biocompatibility of hydrogel components present in
the skin scaffolds was tested by incubating each compo-
nent with MeWo human fibroblasts for 3 or 7 days. The
individual components xanthan gum, κ-carrageenan, cal-
cium lactate, and sodium benzoate, all demonstrated
biocompatibility, with greater than 90% of the cells re-
maining viable after 7 days (Figure 1). ι-carageenan de-
monstrated biocompatibility after 3 days, but was toxic to
cells after 7 days (Figure 1). However, after testing the
carrageenan components with their proper counterions,
ι-carrageenan was found to be completely biocompatible
(Figure 2). When present as individual components,
N-methyl pyrrolidone, Cosmocil CQ, malic acid, and
potassium chloride were toxic after 3 days (Figure 1).
Glucose and glycerin were slightly toxic to MeWo cells,
only allowing for approximately 50% survival (Figure
1).
Since the antibacterial agent Cosmocil CQ was toxic
to MeWo cells at its use concentration, increasing con-
centrations of the antimicrobial agent sodium benzoate
were tested to determine how much could be tolerated by
fibroblasts. Whereas 0.25% sodium benzoate did not
affect cell viability, 0.5% sodium benzoate caused cell
toxicity (Figure 3).
In order to test the biocompatibility of hydrogels with
human fibroblasts, cut segments of hydrogel were incu-
–
Figure 1. Biocompatibility of individual hydrogel compo-
nents with human fibroblasts. MeWo human fibroblasts
were incubated with individual components at concentra-
tions used in the Original and Alternate hydrogel mixture
for either 3 days (black bars) or 7 days (white bars). Cell
viability was measured using the formazan dye WST-1 as
described in the Experimental section. Error bars represent
standard deviation of replicate samples.

Biodegradable Polysaccharide Gels for Skin Scaffolds 222
ι
Figure 2. Biocompatibility of carrageenan gums in the pres-
ence of their stabilizing counter ion. MeWo human fibro-
blasts were incubated for 7 days with carrageenan gums in
the presence of their counter ions at concentrations used in
the Original and Alternate hydrogel mixture. Cell viability
was measured using the formazan dye WST-1 as described
in the Experimental section. Error bars represent standard
deviation of replicate samples.
–
Figure 3. Fibroblast viability in the presence of varied con-
centrations of sodium benzoate. MeWo human fibroblasts
were incubated for 7 days in the presence of increasing con-
centrations of sodium benzoate. Cell viability was measured
using the formazan dye WST-1 as described in the Experi-
mental section. Error bars represent standard deviation of
replicate samples.
bated with MeWo human fibroblasts for 7 days. Incuba-
tion of MeWo cells with newly formed hydrogels dem-
onstrated that they were not toxic to MeWo cells (Figure
4).
Figure 4. Fibroblast viability in the presence of newly formed
hydrogels. MeWo human fibroblasts were incubated for 7
days in the presence of hydrogels cut to the diameter of a
96-well plate well. Cell viability was measured using the for-
mazan dye WST-1 as described in the Experimental section.
Error bars represent standard deviation of replicate samples.
3.4. Primary Skin Irritation in Rabbits
A primary skin irritation rabbit test was conducted to
determine the potential for original hydrogels to produce
irritation after a single topical application [43]. Individ-
ual skin irritation scores and a summary thereof were
used for calculation of Primary Dermal Irritation Index
(Table 4).
All animals appeared active and healthy during the
study [43]. Apart from the dermal irritation noted below,
there were no other signs of gross toxicity, adverse phar-
macologic effects, or abnormal behavior. No edema was
observed at any treated dose site. One hour after patch
removal, very slight erythema was noted for two of three
treated sites. The overall incidence and severity of irrita-
tion decreased with time. Both affected animals were free
of irritation by 48 hours and there was no dermal irrita-
tion noted at any site throughout the remainder of the 14-
day evaluation period. The Primary Dermal Irritation
Index for the original polysaccharide hydrogels is 0.3
[44]. Under the conditions of this study the polysaccha-
ride hydrogels are classified as slightly irritating to the
skin.
3.5. Assessment of Ready Biodegradability of the
Original Hydrogel
Degradation was determined by measuring dissolved
oxygen over a 28-day period [46] using the “Closed Bot-
tle Test”, a standard test for biodegradability by OECD
[45] and the EPA. The amount of oxygen taken up by the
microbial population during biodegradation of the test
item, corrected for uptake by the blank inoculum run in

Biodegradable Polysaccharide Gels for Skin Scaffolds 223
Table 4. Incidence and severity of skin irritation in rabbits.
Incidence of Irritation
Time after Patch
Removal Erythema Edema
Severity of
Irritation–Mean
30 - 60 minutes 2/3 0/3 0.7
24 hours 1/3 0/3 0.3
48 hours 0/3 0/3 0.0
72 hours 0/3 0/3 0.0
7 days 0/3 0/3 0.0
10 days 0/3 0/3 0.0
14 days 0/3 0/3 0.0
parallel, was expressed as a percentage of the Chemical
Oxygen Demand (COD). Sodium benzoate, at 2 mg/L
was used as a degradable reference, along with a toxicity
control with original hydrogel and 2 mg/L sodium ben-
zoate.
The COD was determined to be 2.11 mg O2/mg for the
original hydrogel, presuming that no nitrification would
occur, and based on a dry matter of 9.3% [46]. The ThOD
of the sodium benzoate was calculated to be 1.67 mg
O2/mg, and the ThOD of the mixture of sodium benzoate
plus the test item was 1.89 mg O2/mg. Biodegradation
data are shown in Table 5. In 28 days, 83.4% of the
original hydrogel was degraded.
4. Discussion
A variety of plant polysaccharide hydrogels were char-
acterized. The data showed that hydrogels can be made
with very different mechanical properties, which are
needed for the two skin scaffold layers as well as peptide
delivery particles within them. The strength, though, is
highly dependent on the drying time. Most of these hy-
drogels do not become brittle but tough upon drying; in
fact, some of the strongest biodegradable hydrogels
known are being reported. This effect is currently being
studied in detail. Both ι- and κ-carrageenan have a dou-
ble helical structure that is dependent on the presence of
the counter ion. The packing of these helices is likely the
major source of strength of these hydrogels. The data
suggest that the hydrogels are composites, with konjac
and xanthan gum as the amorphous matrix, with the car-
rageenans as fiber reinforcements. The strongest materi-
als were achieved when only k-carrageenan was used as
the fiber reinforcement. The trends will be further inves-
tigated in more detail.
The TGA data clearly demonstrate that the hydrogels
are not homogeneous, indicating that some of the carra-
geenan is present in the ordered, helical structure. The
TGA step percentages in Table 1 do not total 100% be-
Table 5. Biodegradation of the original hydrogel using the
closed bottle test.
Percent Degradation
Time
(days) Original Hydrogel Sodium
Benzoate
Toxicity
Control
4 14.5 58.7 17.8
7 28.5 64.7 40.0
11 46.2 73.4 43.7
14 56.2 69.5 62.6
21 68.1 71.7 76.4
28 83.4 85.2 86.8
cause a portion of the hydrogel volatilizes in a slow, con-
tinuous manner. These data indicate that only some of
the hydrogel is ordered and the remainder is a homoge-
neous mix. Two melting points were seen; it is assumed
that these result from “crystallized” or, more precisely,
ordered water in the hydrogel.
The DMA data show some unusually high moduli for
these hydrogels. In some cases, these are several times
higher than those for contact lenses, which are some of
the strongest known biodegradable hydrogels. The gels
were measured shortly after preparation, and then after
several days at 4˚C. In most cases, the strength increases
considerably after drying. This trend is being investi-
gated in detail; IR spectroscopy is being used to deter-
mine changes in relative hydrogen bonding strength and
for comparison with further DMA results.
Most of the individual components of the hydrogels
were found non-toxic to human fibroblasts. Although
several individual components, as single entities, were
toxic to MeWo cells, including N-methyl pyrrolidone, a
necessary component for hydrogel strength, newly
formed hydrogels containing the same concentration of
these toxic compounds were deemed biocompatible. The
toxicity of aged hydrogels is currently being investigated
because they may degrade over time and release these
toxic components. If aged hydrogels are toxic, due to
degradation and release of toxic components, omission of
individual components may aid in the prolonged bio-
compatibility of hydrogel mixtures. These data suggest
that hydrogels formed in the manner described above are
biocompatible with human fibroblasts and thus may also
be biocompatible with keratinocytes.
Skin irritation determined by the OECD standard rab-
bit test was slightly irritating during the first 24 hours,
but not irritating after that. It is possible that this initial
slight irritation results from ethanol used for sterilization.
Hydrogels were also found to be readily biodegradable
based on the OECD standard microbial test. In 28 days,

Biodegradable Polysaccharide Gels for Skin Scaffolds 224
83.4% of the original hydrogel was degraded.
Preparation of hydrogels with different mechanical
properties will allow for the development of both dermis
and epidermis mimics. The permeability, degradation
kinetics, and vascularization potential of these hydrogels,
along with the utilization of hydrogels by fibroblasts for
migration and extracellular matrix deposition, are cur-
rently being investigated.
5. Acknowledgements
We are very grateful for the donation of gums from TIC
gums and for funding from Central Michigan University.
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