Accepted Manuscript Title: Dynamic Mechanical and Swelling Properties of Maleated Hyaluronic Acid Hydrogels Author: Hai Lin Jun Liu Kai Zhang Yujiang Fan Xingdong Zhang PII: DOI: Reference:
S0144-8617(15)00078-8 http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.047 CARP 9635
To appear in: Received date: Revised date: Accepted date:
23-7-2014 10-12-2014 15-1-2015
Please cite this article as: Lin, H., Liu, J., Zhang, K., Fan, Y., and Zhang, X.,Dynamic Mechanical and Swelling Properties of Maleated Hyaluronic Acid Hydrogels, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.01.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
1 2
·
A novel hyaluronic acid modification method was developed.
3
·
The degree of substitution of the novel derivative, maleated hyaluronic acid (MaHA) is much higher than that of the methacrylated hyaluronic acid (MeHA)
5
reported in the literature. ·
moduli than those of MeHA.
7
9
·
The crosslinking density and hydrophilicity of the introduced groups on HA
us
8
The photopolymerized hydrogels of MaHA have higher compressive storage
cr
6
ip t
4
molecule affect the swelling behavior of hydrogels.
Ac ce p
te
d
M
an
10
1
Page 1 of 37
10 11
Dynamic Mechanical and Swelling Properties of
13
Maleated Hyaluronic Acid Hydrogels
ip t
12
cr
14
Hai Lin*, Jun Liu, Kai Zhang, Yujiang Fan, Xingdong Zhang*
16
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
17
*Corresponding Authors:
18
Hai Lin: [email protected], Phone: (86)28-85417078
19
Xingdong Zhang: [email protected], Phone: (86)28-85412757
an
M Ac ce p
te
d
20 21
us
15
2
Page 2 of 37
21 Abstract
23
A series of maleated hyaluronan (MaHA) are developed by modification with maleic anhydride.
24
The degrees of substitution (DS) of MaHA vary between 7% and 75%. The DS of MaHA is both
25
higher and wider than methacrylated HA derivatives (MeHA) reported in the literature. MaHA
26
hydrogels are then prepared by photopolymerization and their dynamic mechanical and swelling
27
properties of the hydrogels are investigated. The results showed that MaHA hydrogels with
28
moderate DS (25%, 50% and 65%) have higher storage modulus and lower equilibrium swelling
29
ratios than those with either low or high DS (7%, 15% and 75%). Theoretical analyses also
30
suggest a similar pattern among hydrogels with different DS. The results confirm that the
31
increased cross-linking density enhances the strength of hydrogels. Meanwhile, the hydrophilicity
32
of introduced groups during modification and the degree of incomplete crosslinking reaction
33
might have negative impact on the mechanical and swelling properties of MaHA hydrogels.
34
Keywords
36 37
cr
us
an
M
d
te
Ac ce p
35
ip t
22
Hydrogels, hyaluronic acid, mechanical property, swelling kinetics, photopolymerization
3
Page 3 of 37
37 38
1. Introduction Hyaluronic acid (HA) or hyaluronan plays a key structural role for aggrecan assembly,
40
although it forms a smaller part of the extracellular matrix than collagen in most tissues or organs.
41
HA also shows significant advantages of water reservation capacity. Furthermore, HA is built up
42
by repeating disaccharide units composed of N-acetyl-D-glucosamine and D-glucuronic acid. The
43
regular structure of HA provides the same composition regardless of the materials source, and
44
therefore it is non-allergenic. In recent decades, research on HA, HA derivatives and HA-based
45
materials has brought a lot of attention from both basic science and applied clinical applications,
46
which includes drug delivery system (Lim, Cho, Lee & Kim, 2012; Petersen et al., 2013), tissue
47
engineering (Kim, Mauck & Burdick, 2011; Park, Choi, Hu & Lee, 2013; Yu, Cao, Zeng, Zhang &
48
Chen, 2013), plastic filling (Fakhari & Berkland, 2013; Yeom et al., 2010), wound dressing
49
(Hanjaya-Putra et al., 2013; Kirk et al., 2013; Niiyama & Kuroyanagi, 2014) and bio-printing
50
(Murphy, Skardal & Atala, 2013; Pescosolido et al., 2011). Researchers have also reviewed the
52 53 54 55
cr
us
an
M
d
te
Ac ce p
51
ip t
39
different aspects of HA and HA-based biomaterials (Burdick & Prestwich, 2011; Collins & Birkinshaw, 2013; Khan & Ahmad, 2013; Schante, Zuber, Herlin & Vandamme, 2011; Xu, Jha, Harrington, Farach-Carson & Jia, 2012). The hydroxyl groups of HA are typically the main targets of modification because the
carboxyl groups of HA are thought to be related to the bio-synthesis process of extracellular
56
matrix (Christner, Brown & Dziewiatkowski, 1977). Among the published modification methods
57
on hydroxyl groups of HA, esterifications with alkyl succinic anhydrides or methacrylic anhydride
58
are the most well-known methods (Kenne et al., 2013; Khan & Ahmad, 2013). The as-obtained
59
methacrylated HA (MeHA) is especially suitable for photopolymerization which makes the MeHA 4
Page 4 of 37
popular for further biomaterials and tissue engineering applications. Although MeHA is
61
extensively used in the biomaterials field, its preparation process and the final product are not
62
flawless. The MeHA reaction conditions cannot be controlled easily. The degree of substitution
63
(DS) of MeHA is low and its range of DS is narrow too. To some extent the inadequate
64
esterification is one of the main reasons leading to the unsatisfied mechanical properties of the
65
MeHA hydrogels.
us
cr
ip t
60
In this article, we investigate a novel Sodium Hyaluronate (HAs) modification method which
67
yields in HAs derivative with higher DS and improved properties. Furthermore, hydrogels were
68
prepared by obtained HAs derivatives and their mechanical properties and swelling kinetics were
69
characterized.
70
2. Experimental
M
an
66
71
2.1
72
Sodium Hyaluronate (HAs) was purchased from Bloomage Freda Biopharm Co. Ltd.,
73
(Qingdao, Shandong, China). The viscosity-average molecular weight (M) of HAs was tested by
75 76 77
d te
Ac ce p
74
Materials
the capillary viscometry method, identifying a M of 1.0-1.1×103 kDa. Photoinitiator Irgacure 2959 and methacrylic anhydride (MAA) were purchased from Sigma.
Analytical-grade maleic anhydride (MA) and anhydrous ethyl alcohol were purchased from Kelong Chemical Co. Ltd. (Chengdu, China) and were used without further treatments.
78
Analytical-grade formamide was dried by anhydrous magnesium sulfate and redistillation before
79
use.
80
2.2
81
2.2.1 Modification of HAs
Methods
5
Page 5 of 37
The HAs was modified to install photoactive polymerizable groups as following: 1.0g HAs
83
was added to 80ml dry formamide. The solution was heated at 50oC to obtain a homogeneous
84
solution under intense stirring. Maleic anhydride was dissolved in 20ml dry formamide and then
85
added to the HAs solution. The reaction was subsequently proceeded for 5h before cooling to
86
room temperature. The cooled solution was further precipitated in cold anhydrous ethyl alcohol
87
under stirring. The precipitant was purified through repeating washing with anhydrous ethyl
88
alcohol and centrifugal separation. The washed and centrifuged product was decanted and then
89
dissolved in distilled water. The solution was neutralized with 2N sodium hydroxide solution. The
90
HAs derivative was finally dialyzed against deionized water, lyophilized and stored at -20 oC
91
(MaHA).
M
an
us
cr
ip t
82
In order to better understand the modification efficiency, we also prepared a methacrylated
93
HA derivative (MeHA) as a control based on the methods reported in the literature (Bian et al.,
94
2013; Bian, Zhai, Zhang, Mauck & Burdick, 2012). Briefly, methacrylic anhydride (MAA, Sigma),
95
which is 10-fold excess to the primary hydroxyl groups in HAs, is reacted with HAs in an aqueous
97 98 99 100
te
Ac ce p
96
d
92
solution at 4oC. The reaction pH was kept in between 8.0 and 9.0 by adjusting with 5N NaOH. The same reaction time of 5h was picked in order to compare with the MaHA. The product was precipitated in anhydrous ethyl alcohol, dialyzed and finally lyophilized (MeHA). The as-prepared MaHA and MeHA specimens are listed in Table 1. The specimens are named
by their reactants (e.g., Me or Ma), and the degrees of substitution (DS).
101
2.2.2 Characterization of modified HAs
102
2.2.2.1
103
1
1
H Nuclear Magnetic Resonance (NMR)
H-NMR spectroscopy was used to verify the esterification and to enable the quantitative 6
Page 6 of 37
104
calculation of the DS. The HAs derivatives (both MeHA and MaHA) were dissolved in D2O with
105
a concentration of 8-10 mg/mL. The spectra were recorded using a Bruker AVIII 400 HD nuclear
106
magnetic resonance spectrometer (Swiss). 2.2.2.2
108
ATR FT-IR was applied to identify the successful modification (-unsaturated ester/acid).
109
The HA derivatives (MeHA & MaHA) were scanned from 400 cm-1 to 4000 cm-1 with a resolution
110
of 2 cm-1 by using Thermo Fisher Nicolet IS10 (USA). The original HA powder was also scanned
111
under the same condition and served as control.
ip t
107
an
us
cr
Attenuated Total Reflect Fourier Transform Infrared Spectroscopy (ATR FT-IR)
2.2.3 Preparation of HAs hydrogels
113
A 0.1% (w/v) Irgacure 2959 solution (Sigma) was prepared. MaHA was added to the initiator
114
solution with a concentration of 20 mg/mL. After thorough dissolution of MaHA under stirring,
115
100L of the solution was pipetted to plastic cylindrical molds (custom-made, with the internal
116
diameter of 8mm and the height of 2mm). The filled molds were sealed by cover glasses on both
117
ends. The photocrosslinked hydrogels were prepared by exposing the solution to UV light
119 120 121
d
te
Ac ce p
118
M
112
(OmniCure S1500 (USA), 365nm, ~16 mW/cm2) for 60 seconds at each end. 2.2.4 Characterization of HAs hydrogels 2.2.4.1
Dynamic Mechanical Analysis (DMA)
The DMA of compression tests were performed on NETZSCH DMA 242C equipped with a
122
controller of TASC 414/4 (NETZSCH, German). Both frequencies of 1.0 Hz and 10 Hz were
123
applied, which simulated the normal and the limit of physiological stride frequency. The tests were
124
carried out at constant temperature of 25oC. The testing parameters were set as amplitude of 20m,
125
the preload force of 0.001N and the force track of 120%. Three cylindrical samples with a 7
Page 7 of 37
diameter of 8mm and a height of 2mm were used for each modification condition. The results
127
were analyzed by NETZSCH Proteus program, and statistical analysis was performed by student t
128
test on the average E’ and E’’ which were calculated according to the data exported with a regular
129
time interval.
ip t
126
130
2.2.4.2
131
The dry weights of hydrogels (w0) can be calculated according to the solution concentrations
132
and volumes. After soaking in 100mM PBS (pH = 7.2) at 37oC at regular time intervals, the buffer
133
on surface of hydrogels was carefully absorbed and the gels weighed (wt) to determine the
134
swelling ratio (Q) which is defined as (wt-w0)/w0.
an
us
cr
Swelling tests
2.2.5 Kinetics and structure analysis
136
2.2.5.1 Kinetics analysis
137
The swelling of the cross-linked hydrogels is commonly described to follow the first-order
140 141 142
d
te
139
kinetics (Schott, 1992a, b), which can be expressed as following equation (Equation 1).
(1)
Ac ce p
138
M
135
Where, Qt means the swelling ratio at time t, and Qe stands for the swelling ratio when the hydrogels reach equilibrium. The Equation 1 becomes to Equation 2 after integration. (2)
143
The Equation 2 is derived based on Fick’s laws which only apply under the following
144
conditions (Schott, 1992a, b). The samples should have a large aspect ratio, i.e., the surface of the
145
sample is much larger than its thickness (H). Moreover, both H and diffusion coefficient (D)
146
should remain constants during the swelling process. 8
Page 8 of 37
147 148
The Fickian equation has another expression shown as Equation 3.(Jovanovic & Adnadjevic, 2013; Schott, 1992a)
149
ip t
150
(3)
Where D is diffusion coefficient, t is the diffusion time and H is the thickness of the hydrogel.
With enough diffusion time, the Equation 3 can be approximately simplified as Equation 4.
152
(4)
153
Compare Equation 4 with Equation 2, the constant D can be calculated from the slope of the
For the entire swelling process, the Scott’s second-order equation is obeyed.
156
159
160 161
162
d
After integration between the limits t(0, t) and Qt(0, Qe), and make A=1/kQe2 and B=1/Qe,
te
158
(5)
then the Equation 5 is transferred to Equation 6.
Ac ce p
157
us
an
155
linear fit curve.
M
154
cr
151
(6)
Where A and B are coefficients with physical meanings. At the very beginning of the swelling, that is t → 0, then
and thus yields:
(7)
163
The intercept A is the reciprocal of the initial swelling rate, corresponding to the stage when
164
(1) the solvent has permeated the entire hydrogel and (2) before the strain on the network begins
165
to retard swelling.
166
On the contrary, at a long time,
, we then deduce that B=1/Qt, which means B is the 9
Page 9 of 37
167
reciprocal of equilibrium swelling ratio. 2.2.5.2 Structure analysis
169
As mentioned above, the network structure of the MaHA hydrogels is one of the driving
170
forces of swelling kinetics. Therefore, a number of analyses based on swelling experiments were
171
conducted to investigate important parameters used to characterize the network structure of the
172
hydrogels, including the polymer volume fraction in the swollen state (v2,s), the molecular weight
173
of the polymer chain between two neighboring crosslinking points (
174
mesh size (ξ).
cr
us
an
176
The relationship between
and v2,s was developed and described by Peppas et al. as
following (Buckley & Martin, 1962; Peppas, Hilt, Khademhosseini & Langer, 2006):
177
Where
179
(18 cm3/g for water);
182 183 184 185
is the molar volume of solvent
te
is the number average molecular weight of HA,
is the specific volume of the dry HA (0.8137 cm3/g for HA) (Leach,
Ac ce p
181
d
(8)
178
180
), and the corresponding
M
175
ip t
168
Bivens, Patrick & Schmidt, 2003);
is the Flory-Huggins interaction parameter between
polymer-solvent (0.439 for HA) (Ottenbrite & Kim, 2000); and
is the polymer volume
fraction in the relaxed state, which is defined as the state of a polymer immediately after crosslinking but before swelling in a solvent. The effective crosslink density
is calculated by the Equation 9 (Leach, Bivens, Patrick &
Schmidt, 2003).
186
(9)
187
The hydrogel mesh size ξ which is defined as the average distance between crosslinks in the 10
Page 10 of 37
188
hydrogel can be determined theoretically. (Meybodi, Imani & Mohammad, 2013)
189
(10)
190
Where
191
state. For HA, the root-mean-square end-to-end distance was reported in literature as following
192
(Leach, Bivens, Patrick & Schmidt, 2003):
(11)
us
193
cr
ip t
is the root-mean-square distance between two adjacent crosslinks in the solvent-free
Where n is the number of disaccharide repeat units for HA with a given molecular weight. Since
195
the molecular weight of the repeat unit is 379.32 g/mol, the equation can be transformed as:
an
194
196
199 200 201 202 203
M
by
d
Equation 10 and replacing
gives Equation 13.
te
198
Therefore, the mesh size of HA hydrogels can be calculated by substitution of Equation 12 in
(13)
Ac ce p
197
(12)
3. Results and Discussion
The well-known mechanism for the formation of MeHA by hyaluronic acid and methacrylic
anhydride in an aqueous environment has been thoroughly investigated before (Burdick, Chung, Jia, Randolph & Langer, 2005; Smeds, Pfister-Serres, Hatchell & Grinstaff, 1999). The MaHA
204
reaction between HAs and maleic anhydride in organic solvent follows a similar mechanism
205
shown in Figure 1. In anhydrous solvent, esterification reaction can easily occur between the
206
primary hydroxyl group at C-6 of the Glucosamine and anhydride through a ring opening
207
mechanism. Recently, Vasi et al. reported an identical HA modification gained in a two step 11
Page 11 of 37
synthesis (Vasi, Popa, Butnaru, Dodi & Verestiuc, 2014). By comparison, the sodium hyaluronan
209
used as raw materials in our described preparation did not require an ion exchange into the acidic
210
form to obtain solubility in a polar organic solvent. Consequently, our modification process
211
enables a shorter and quicker access to yield a maleated hyaluronan derivative. Nonetheless, the
212
HAs derivatives obtained by routes are consistent judged by NMR as well as FT-IR analysis (see
213
section 3.1&3.2).
us
1
cr
ip t
208
214
3.1
215
As shown in Figure 2, the spectrum of MeHA displays two peaks at approximately 5.6 and 6.0
216
ppm, which corresponds to the introduced methacrylate moieties (Smeds & Grinstaff, 2001;
217
Smeds, Pfister-Serres, Hatchell & Grinstaff, 1999). Figure 2 also shows that the grafted maleic
218
ester in MaHA present peaks at 5.9 and 6.5ppm which is slightly different from the data shown in
219
literature (6.1 and 6.7ppm, in CD3SOCD3) (Vasi, Popa, Butnaru, Dodi & Verestiuc, 2014). In
220
agreement with Vasi’s study, the appearance of peak at 8.1 ppm is related to the proton from
221
COOH group. The peak at 6.2 ppm is not expected in MaHA structure, and it possible relates to
223 224 225
an
M
d
te
Ac ce p
222
H NMR
the protons of maleic acid which was hydrolyzed from maleic anhydride and attached to the MaHA molecule by hydrogen bonds. According to the integral area of protons, only 0.50%-2.19% double bonds in high DS MaHA derivatives are contributed by attaching maleic acid. The broadened peaks between 3.2 and 3.8 ppm are referred to the protons of the pyranose rings (Leach,
226
Bivens, Patrick & Schmidt, 2003). Furthermore, the peak at 1.9ppm belongs to the protons of
227
methyl (-CH3) in N-acetyl group and served as the reference peak to calculate the DS. According
228
to the integral area of protons of unsaturated bonds and methyl, the DS in MeHA is around 3.33%
229
and the DS of MaHA samples ranges from 7.13% to 75.47%, correspondingly. 12
Page 12 of 37
The variation of DS of MeHA could be altered by the amount of methacrylic anhydride,
231
reaction time and the solution pH (Burdick, Chung, Jia, Randolph & Langer, 2005). Our study of
232
MeHA suggests that merely 3.33% functionalization were achieved under the reaction conditions
233
of 10-fold MAA and reaction time of 5h. Even with a different set of parameters (e.g., 20-fold
234
MAA, reaction time of 24h and accurate control over pH), we can only obtain the MeHA
235
derivative with a DS around 20%. However, the DS of MeHA reported in the literature is less than
236
29% (Bian et al., 2013). The hydrolysis of MAA in water and the sensitivity of pH and
237
temperature of this reaction make it very difficult to ideally control over the DS of MeHA.
238
However, the reaction between HAs and maleic anhydride in anhydrous organic solvent can fully
239
control over the DS of MaHA by reaction conditions. As a result, we achieved a variety of DS for
240
MaHA that shown in both Table I and Figure 2.
M
an
us
cr
ip t
230
3.2
242
The ATR FT-IR spectra of HAs and HAs derivatives (MeHA and MaHA) with different DS
243
are shown in Figure 3. With the increase of DS in MaHA, new peaks were identified according to
245 246 247
te
Ac ce p
244
ATR FT-IR
d
241
the obtained DS. The peaks at 1719 cm-1, 1576 cm-1 and the shoulder peak between these peaks were related to the -unsaturated ester/acid (-CO-C=C-CO-) of the modified HAs (Haxaire, Marechal, Milas & Rinaudo, 2003). The synergistic effect of the carbonyl groups, the olefin and its conjugation of the -system at its sp2-hybrids contribute to the band development of the
248
modified MaHA. Compared with the spectra of MaHA samples, the spectrum of MeHA shows
249
both a slight shift and intensity difference at the peak around 1610 cm-1 , because MeHA has a
250
different double conjugated group (CH2=C(C)-CO-). The results of ATR FT-IR are consistent with
251
the data in literature (Vasi, Popa, Butnaru, Dodi & Verestiuc, 2014) and confirm that the functional 13
Page 13 of 37
252
molecules are branched on the polysaccharide chain, and the DS is affected by the feed ratios in
253
MaHA.
254
3.3
255
The average moduli of different MaHA hydrogels were calculated and shown in Figure 4.
256
Most of the samples have storage moduli larger than 200kPa, and the 25MaHA hydrogels have the
257
largest E’, around 290kPa. Previous research of our group showed that the ternary hydrogels
258
which prepared by collagen, chondroitin sulfate and hyaluronic acid had a compressive modulus
259
around 45–54 kPa (Guo et al., 2012; Zhang et al., 2011). According to other published data, the
260
MeHA hydrogels have the modulus range between 3.5 and 53.6 kPa (Bian et al., 2013), depending
261
on the hydrogel concentration and curing time. Therefore, the MaHA hydrogels we developed
262
have a much higher mechanical property than the MeHA hydrogels reported in literature. In
263
addition, the range of the storage modulus might be another important aspect, because the
264
mechanical property differences in materials might cause significantly different responses in
265
biological system, such as stem cell differentiation and bioactive molecule secretion (Choi et al.,
267 268 269
ip t
cr
us
an
M
d
te
Ac ce p
266
Dynamic Mechanical Analysis
2013; Kim, Khetan, Baker, Chen & Burdick, 2013; Marklein, Soranno & Burdick, 2012). The difference of storage modulus among hydrogel samples developed in this study and those reported in the literature is more than 100 kPa. In addition, the range of storage modulus of hydrogels development in our study can be even more significant at higher concentration and longer curing
270
time. Currently, the hydrogels developed for cartilage tissue engineering typically have relatively
271
weaker mechanical properties than those of natural cartilage: Young’s modulus 0.5-60 kPa (Bian
272
et al., 2013) to 0.699 MPa (human fetal) (Callahan, Ganios, Childers, Weiner & Becker, 2013), or
273
compressive modulus ≈2 to over 100 kPa (Burdick & Prestwich, 2011) to 2.78MPa (rabbit) 14
Page 14 of 37
(Zhang et al., 2013). In addition, the range of modulus variation for those hydrogels is quite
275
narrow too (Marklein & Burdick, 2010; Toh, Lim, Kurisawa & Spector, 2012). Our ongoing study
276
is investigating how these MaHA hydrogels with higher mechanical property perform in vitro and
277
in vivo.
ip t
274
The DMA results also suggest that the E’ do not always increase along with the increase of DS.
279
Meanwhile, as expected, the loss moduli of the MaHA hydrogels are much lower than their
280
storage moduli, suggesting that the elastic properties of those hydrogels are more pronounced than
281
their viscous properties. The statistical analyses on storage moduli show that significant difference
282
exists between each group, except for 50MaHA and 60MaHA. Generally speaking, if the double
283
bonds are fully reacted during the photopolymerization, the more double bonds grafted on the
284
HAs, the higher the storage moduli of the formed hydrogels. Since the storage moduli of
285
hydrogels do not increase with the DS when the DS is quite high (50% or more) (see figure 4), we
286
deduced that not all the unsaturated bonds are cross-linked under current reaction conditions. As a
287
result, the MaHA with a suitable DS will achieve optimized mechanical property. Otherwise, a
289 290 291
us
an
M
d
te
Ac ce p
288
cr
278
complex construct composed of HA and other reactive compositions, which can react with more of the double bonds, may further improve the mechanical property of the hydrogel. 3.4
Hydrogel swelling kinetics analysis
A gravimetric method was applied to investigate the swelling process of newly developed
292
MaHA hydrogels. Swelling ratios of different MaHA hydrogels are shown as the symbols in
293
Figure 5. To have a better understanding of the swelling behavior, we try to fit the process with the
294
first order exponential decay equation:
295
equations can simulate the swelling process very well with coefficients of determination (R2) are
The lines in Figure 5 indicate that the
15
Page 15 of 37
296
higher than 0.9850 (see table 2). The parameters of the equations are shown as Table 2. The swelling capacity of all hydrogel samples increases with time. All MaHA hydrogels
298
show the similar pattern of reaching their equilibrium swelling states after a certain period of time.
299
With the help of simulation equations, the equilibrium swelling ratios (Qe) of different hydrogels
300
can be calculated, ranges between 104 and 154. Compared with literature data, the swelling ratios
301
have a wide range due to the difference in preparation methods. In our previous work,
302
interpenetrating ternary hydrogels have swelling ratios of 4-14 g sol/g gel (Guo et al., 2012; Zhang
303
et al., 2011). The 25MaHA samples have the smallest Qe. 50MaHA and 65MaHA samples have
304
equivalent Qe values, which are slightly higher than that of 25MaHA. As the spline connected line
305
of Qe in Figure 6, the Qe decreases with the increase of the DS up to around 35%. The decrease of
306
Qe at the earlier stage is caused by the increase of the degree of crosslinking when the DS is
307
climbing. This trend is consistent with reported other chemically crosslinked hydrogels (Kenne et
308
al., 2013; Sivakumaran, Maitland, Oszustowicz & Hoare, 2013). However, the swelling ratios
309
raise slowly between 35% and 75% DS. It is assumed that unsaturated bonds introduced into the
311 312 313
cr
us
an
M
d
te
Ac ce p
310
ip t
297
HAs molecule cannot react completely by photopolymerization under insufficient exposure time. Hence, the highly hydrophilic carboxyl groups grafted on the polymer chain yield in an enhanced water retaining capacity inside the network after more carboxyl groups are introduced. Therefore, the positive effects of hydrophilicity and negative effects due to crosslinking are balanced at an
314
appropriate DS, which eventually leads to the minimum value of swelling ratio at equilibrium. As
315
a result, a medium DS (25%-50% in this study) is necessary for the appropriate swelling of
316
hydrogels.
317
The simulation curves and equations can also derive the time to achieve dynamic equilibrium. 16
Page 16 of 37
The time for hydrogels to reach 80% and 90% of the theoretical swelling ratio is listed in Table 2.
319
Hydrogels prepared by 50MaHA have the shortest time of 5.24 hrs and 8.26 hrs, respectively,
320
what indicates that the hydrogels need to be soaked in the buffer for at least 10h to reach their
321
swollen state.
ip t
318
According to the Equation 2, the linear fit could describe the swelling process. However, in
323
our case, only the first 12 hours swelling process can be fitted by the Equation 2, as the data of
324
subsequent times deviated from the prior theoretical calculation. The fitting lines are shown as
325
Figure 7. The deviation might be caused by factors such as the change of the hydrogels thickness,
326
and the variation of diffusion coefficient during the swelling process. Thus, the first-order kinetics
327
may be only suitable at the early stage of swelling and cannot predict or describe the entire
328
swelling process (Figure 7, fitting lines are made up to 12h only).
M
an
us
cr
322
However, based on the excellent fit of the swelling data up to 12 hrs, the parameters of linear
330
fit lines, together with their diffusion coefficients are calculated according to Equation 4, and
331
listed in Table 3. The theoretical calculated data of the diffusion coefficients show a similar trend
333 334 335
te
Ac ce p
332
d
329
with the storage modulus of hydrogels described in the previous section. With the increase of cross-linking at early stage, the hydrophilic groups on HAs molecules are immobilized which leads to an easier situation for the diffusion of solvent. With the increase of DS, the branched groups cannot be fully reacted under the curing conditions. Then, the introduced free hydrophilic
336
side groups hinder the diffusion of water molecule. According to the data of hydrogels prepared by
337
MeHA in literature (Bian et al., 2013), the MeHA hydrogels with an increasing concentration or
338
UV exposure time have a diffusion coefficient of 3.8-6.3×10-7 cm2/s. The decrease of diffusivity
339
was explained by the increasing of crosslinking density (Bian et al., 2013). In comparison to 17
Page 17 of 37
MeHA, the MaHA hydrogels have an equivalent diffusion coefficient which is 4.44-6.21 ×10-7
341
cm2/s. For MaHA hydrogels, not only the crosslinking density will affect the diffusivity, but also
342
the hydrophobic and hydrophilic properties of the introduced functionalities affect the diffusion of
343
water and the solvatisation with it.
ip t
340
To describe the entire swelling process, the second-order equation is applied. Figure 8 shows
345
the swelling kinetics of the hydrogels plotted according to the transferred second-order kinetics
346
equation. The fitting parameters are shown in Table 4. According to the fitting lines in Figure 8
347
and theoretically calculated parameters in Table 4, the initial swelling rate (Qi) and the equilibrium
348
swelling ratio (Qe’) can be calculated. The results indicate that the Qe’s of hydrogels have a
349
similar pattern compared to the results of simulation equations described above: the MaHA
350
hydrogels with the lowest DS have the largest swelling ratio, and the samples with moderate DS
351
(25% to 65%) have similar swelling ratios. Schott proposed that the swelling rate is proportional
352
to two factors (Schott, 1992a). The first factor is the unrealized swelling percentage. The second
353
factor is the internal specific boundary area. This area encloses all the sites that have not interacted
355 356 357
us
an
M
d
te
Ac ce p
354
cr
344
with water nor swollen at a given time, but the sites will be hydrated and swell in due course. In our case, the amorphous domains in MaHA hydrogels are expanded and the resultant stress on the crystalline domains increases during the swelling process. However, the stressed crosslinking crystalline domains can resist further swelling. Thus, the swelling rate of MaHA hydrogels is
358
proportional to their relative swelling capacity. The MaHA hydrogels with higher percentages of
359
crystalline domains should have higher unrealized swelling capacities and initial swelling rates as
360
compared to the rest hydrogels with either lower or higher DS. As shown in Table 4, 50MaHA
361
hydrogels have the highest initial swelling rate. With the combined effects of crosslinking and 18
Page 18 of 37
hydrophilicity, the swelling behavior of hydrogels has a similar trend with their mechanical
363
properties described in 3.3.
364
3.5 Hydrogel analysis
365
Based on the previous analysis of the swelling behavior of MaHA hydrogels, the theoretical
366
calculated structural parameters are listed in Table 5 according to Qe and Qe’, respectively. The
369 370
cr
showed a trend of decrease between 7MaHA and 65MaHA, and then increase at
75MaHA. The
us
368
and
changed inversely with the effect of crosslinking on
and . The range of
was 298-434nm or 336-480nm when calculating with Qe or Qe’, respectively. These results
an
367
ip t
362
show a similar interval and good permeability.
Swelling ratios vary with the amounts of introduced functional groups on HAs and the curing
372
conditions, both of which decide the degree of cross-linking and hydrophilicity in hydrogels. In
373
our study, the molecular weights between crosslinks (
374
mesh sizes ( ) of MaHA hydrogels are calculated by using Qe or Qe’. These parameters of
375
hydrogels are different due to their difference in the inner structure and chemical compositions.
377 378 379
d
te
), effective crosslink densities ( ) and
Ac ce p
376
M
371
When the DS was lower than 50%, the degree of cross-linking affects more on the inner structure of hydrogels than the hydrophilicity does. When the DS was higher than 50%, the influence of hydrophilicity dominated the inner structure of hydrogels over the degree of crosslinking. 4. Conclusions
380
The maleated hyaluronic acid (MaHA) was prepared, and its degree of substitution (DS) was
381
controlled by adjusted reaction conditions. ATR FT-IR as well as NMR results indicate the
382
successful introduction of maleic ester. Additionally, 1H NMR results showed that the DS ranges
383
between 7% and 75%. Compared with the DS of MeHA in the literature, the MaHA yielded in a 19
Page 19 of 37
higher and wider DS. Furthermore, the modification reaction can be easily controlled and it is
385
shorter and more efficient to attain high DS maleated hyaluronan than literature report. Following
386
the preparation of MaHA hydrogels by photopolymerization, their dynamic mechanical properties
387
and swelling behavior were studied. Dynamic mechanical analyses showed that the 25MaHA
388
hydrogel has the highest storage modulus (~ 290kPa). The change of hydrogel’s storage modulus
389
indicates that the mechanical property of hydrogels is determined not only by the density of
390
cross-linking but also the hydrophilicity of the introduced groups during modification. Swelling
391
studies and theoretical calculations also confirmed that both cross-linking and hydrophilicity
392
affects the swelling properties of the hydrogels. Calculations with both fitting and swelling
393
kinetics equations were performed. Although the calculated results are based on several
394
assumptions and might deviate from the data obtained by analytical devices, they are meaningful
395
for inter-comparisons among samples and inspiring the product improvements. The hydrogels of
396
25, 50 and 65MaHA have similar equilibrium swelling ratios (Qe) that range between 104 and 124.
397
The Qe of 25, 50, 65MaHA hydrogels are lower than those of hydrogels of 7, 15 and 75MaHA,
399 400 401
cr
us
an
M
d
te
Ac ce p
398
ip t
384
which range between 125 and 173. Meanwhile, the diffusion coefficients (D) of 25, 50 and 65MaHA hydrogels are higher than those of 7, 15 and 75MaHA hydrogels. The analysis of the swelling structure suggests that the MaHA hydrogels with low Qe and high D have lower molecular weight between crosslinks (
) and smaller mesh size ( ). The structural characteristics
402
of MaHA hydrogels are caused by the increase of cross-linking density and hydrophilicity. Future
403
studies will report the biocompatibility studies following ISO 10993, and in vitro and in vivo
404
performance of the MaHA hydrogels for cartilage tissue engineering applications.
405 20
Page 20 of 37
406
Acknowledgements This study was financially supported by the National Key Technology Research and
408
Development Program (2012BAI42G00), the National Science Foundation for Young Scientists of
409
China (51403134), the Application Technology Research and Demonstration Projects of Hainan
410
Province China (SQ2014ZDXM0294) and the Foundation of Jiangsu Collaborative Innovation
411
Center of Biomedical Functional Materials, China.
cr us
References
Bian, L., Hou, C., Tous, E., Rai, R., Mauck, R.L., & Burdick, J.A. (2013). The influence of hyaluronic acid
an
hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy. Biomaterials, 34(2), 413-421.
Bian, L., Zhai, D.Y., Zhang, E.C., Mauck, R.L., & Burdick, J.A. (2012). Dynamic Compressive Loading Enhances Cartilage Matrix Synthesis and Distribution and Suppresses Hypertrophy in hMSC-Laden
M
Hyaluronic Acid Hydrogels. Tissue Engineering Part A, 18(7-8), 715-724.
Buckley, D.J., & Martin, B. (1962). The swelling of polymer systems in solvents. II. Mathematics of diffusion. Journal of Polymer Science Part a-Polymer Chemistry, 56(163), 175-188.
d
Burdick, J.A., Chung, C., Jia, X., Randolph, M.A., & Langer, R. (2005). Controlled Degradation and 386-391.
te
Mechanical Behavior of Photopolymerized Hyaluronic Acid Networks. Biomacromolecules, 6(1), Burdick, J.A., & Prestwich, G.D. (2011). Hyaluronic Acid Hydrogels for Biomedical Applications. Advanced Healthcare Materials(23), H41-H56.
Callahan, L.A.S., Ganios, A.M., Childers, E.P., Weiner, S.D., & Becker, M.L. (2013). Primary human chondrocyte extracellular matrix formation and phenotype maintenance using RGD-derivatized PEGDM hydrogels possessing a continuous Young’s modulus gradient. Acta Biomaterialia, 9, 6095-6104.
Ac ce p
412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443
ip t
407
Choi, J., Choi, B., Park, S.H., Pai, K., Li, T., Min, B.H., & Park, S. (2013). Mechanical Stimulation by Ultrasound Enhances Chondrogenic Differentiation of Mesenchymal Stem Cells in a Fibrin-Hyaluronic Acid Hydrogel. Artificial Organs, 37(7), 648-655. Christner, J.E., Brown, M.L., & Dziewiatkowski, D.D. (1977). Interaction of Cartilage Proteoglycans with Hyaluronic Acid The role of the hyaluronic acid carboxyl groups. Biochem. J.(167), 711-716. Collins, M.N., & Birkinshaw, C. (2013). Hyaluronic acid based scaffolds for tissue engineering-A review. Carbohydrate Polymers, 92(2), 1262-1279. Fakhari, A., & Berkland, C. (2013). Applications and emerging trends of hyaluronic acid in tissue engineering, as a dermal filler and in osteoarthritis treatment. Acta Biomaterialia, 9(7), 7081-7092. Guo, Y., Yuan, T., Xiao, Z., Tang, P., Xiao, Y., Fan, Y., & Zhang, X. (2012). Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering. Journal of Materials Science-Materials in Medicine, 23(9), 2267-2279. Hanjaya-Putra, D., Shen, Y.I., Wilson, A., Fox-Talbot, K., Khetan, S., Burdick, J.A., Steenbergen, C., & 21
Page 21 of 37
Gerecht, S. (2013). Integration and Regression of Implanted Engineered Human Vascular Networks During Deep Wound Healing. Stem Cells Translational Medicine, 2(4), 297-306. Haxaire, K., Marechal, Y., Milas, M., & Rinaudo, M. (2003). Hydration of Polysaccharide Hyaluronan Observed by IR Spectrometry. I. Preliminary Experiments and Band Assignments. Biopolymers, 72, 10-20. Jovanovic, J., & Adnadjevic, B. (2013). The Effect of Primary Structural Parameters of Poly(methacrylic
ip t
acid) Xerogels on the Kinetics of Swelling. Journal of Applied Polymer Science, 127(5), 3550-3559.
Kenne, L., Gohil, S., Nilsson, E.M., Karlsson, A., Ericsson, D., Kenne, A.H., & Nord, L.I. (2013).
Modification and cross-linking parameters in hyaluronic acid hydrogels-Definitions and analytical
cr
methods. Carbohydrate Polymers, 91(1), 410-418.
Khan, F., & Ahmad, S.R. (2013). Polysaccharides and Their Derivatives for Versatile Tissue Engineering Application. Macromolecular Bioscience, 13(4), 395-421.
us
Kim, I.L., Khetan, S., Baker, B.M., Chen, C.S., & Burdick, J.A. (2013). Fibrous hyaluronic acid hydrogels that direct MSC chondrogenesis through mechanical and adhesive cues. Biomaterials(34), 5571-5580. Kim, I.L., Mauck, R.L., & Burdick, J.A. (2011). Hydrogel design for cartilage tissue engineering: A case
an
study with hyaluronic acid. Biomaterials, 32(34), 8771-8782.
Kirk, J.F., Ritter, G., Finger, I., Sankar, D., Reddy, J.D., Talton, J.D., Nataraj, C., Narisawa, S., Millan, J.L., & Cobb,
R.R.
(2013).
Mechanical
and
biocompatible
characterization
of
a
cross-linked
collagen-hyaluronic acid wound dressing. Biomatter, 3(4).
M
Leach, J.B., Bivens, K.A., Patrick, C.W., & Schmidt, C. (2003). Photocrosslinked Hyaluronic Acid Hydrogels: Natural, Biodegradable Tissue Engineering Scaffolds. Biotechnology and Bioengineering, 82(5), 578-589.
d
Lim, H.J., Cho, E.C., Lee, J.A., & Kim, J. (2012). A novel approach for the use of hyaluronic acid-based hydrogel nanoparticles as effective carriers for transdermal delivery systems. Colloids and Surfaces
te
a-Physicochemical and Engineering Aspects, 402, 80-87. Marklein, R.A., & Burdick, J.A. (2010). Spatially controlled hydrogel mechanics to modulate stem cell interactions. Soft Matter, 6(1), 136-143.
Marklein, R.A., Soranno, D.E., & Burdick, J.A. (2012). Magnitude and presentation of mechanical
Ac ce p
444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487
signals influence adult stem cell behavior in 3-dimensional macroporous hydrogels. Soft Matter, 8(31), 8113-8120.
Meybodi, Z.E., Imani, M., & Mohammad, A. (2013). Kinetics of dextran crosslinking by epichlorohydrin: A rheometry and equilibrium swelling study. Carbohydrate Polymers(92), 1792-1798. Murphy, S.V., Skardal, A., & Atala, A. (2013). Evaluation of hydrogels for bio-printing applications. Journal of Biomedical Materials Research Part A, 101A(1), 272-284. Niiyama, H., & Kuroyanagi, Y. (2014). Development of novel wound dressing composed of hyaluronic acid and collagen sponge containing epidermal growth factor and vitamin C derivative. J Artif Organs, 17(1), 81-87. Ottenbrite, R.M., & Kim, S.W. (2000). Polymeric Drugs and Drug Delivery Systems. CRC Press. Park, H., Choi, B., Hu, J., & Lee, M. (2013). Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering. Acta Biomaterialia, 9(1), 4779-4786. Peppas, N.A., Hilt, J.Z., Khademhosseini, A., & Langer, R. (2006). Hydrogels in BIology and Medicine: From Molecular Principles to Bionanotechnology. Advanced Materials(18), 1345-1360. Pescosolido, L., Schuurman, W., Malda, J., Matricardi, P., Alhaique, F., Coviello, T., van Weeren, P. R., Dhert, W.J.A., Hennink, W.E., & Vermonden, T. (2011). Hyaluronic Acid and Dextran-Based Semi-IPN 22
Page 22 of 37
523 524
Hydrogels as Biomaterials for Bioprinting. Biomacromolecules, 12(5), 1831-1838. Petersen, S., Kaule, S., Teske, M., Minrath, I., Schmitz, K.P., & Sternberg, K. (2013). Development and In Vitro Characterization of Hyaluronic Acid-Based Coatings for Implant-Associated Local Drug Delivery Systems. Journal of Chemistry. Schante, C.E., Zuber, G., Herlin, C., & Vandamme, T.F. (2011). Chemical modifications of hyaluronic acid Polymers(85), 469-489.
ip t
for the synthesis of derivatives for a broad range of biomedical applications. Carbohydrate Schott, H. (1992a). Kinetics of Swelling of Polymer and Their Gels. Journal of Pharmaceutical Sciences, 81(5), 467-470.
cr
Schott, H. (1992b). Swelling Kinetics of Polymer. J. Macromol. Sci-Phys., B31(1), 1-9.
Sivakumaran, D., Maitland, D., Oszustowicz, T., & Hoare, T. (2013). Tuning drug release from smart microgel-hydrogel composites via cross-linking. Journal of Colloid and Interface Science, 392, 422-430.
us
Smeds, K.A., & Grinstaff, M.W. (2001). Photocrosslinkable polysaccharides for in situ hydrogel formation. Journal of Biomedical Materials Research, 54, 115-121.
Smeds, K.A., Pfister-Serres, A., Hatchell, D.L., & Grinstaff, M.W. (1999). Synthesis of a novel
an
polysaccharide hydrogel. J.M.S. Pure Appl. Chem., A36(7&8), 981-989.
Toh, W.S., Lim, T.C., Kurisawa, M., & Spector, M. (2012). Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomaterials, 33(15), 3835-3845.
M
Ana-Maria Vasi, Marcel Ionel Popa, Maria Butnaru, Gianina Dodi, & Verestiuc, L. (2014). Chemical functionalization of hyaluronic acid for drug delivery applications. Materials Science & Engineering C(38), 177-185.
d
Xu, X., Jha, A.K., Harrington, D.A., Farach-Carson, M.C., & Jia, X. (2012). Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter, 8(12), 3280-3294.
te
Yeom, J., Bhang, S.H., Kim, B.S., Seo, M.S., Hwang, E.J., Cho, I.H., Park, J.K., & Hahn, S.K. (2010). Effect of Cross-Linking Reagents for Hyaluronic Acid Hydrogel Dermal Fillers on Tissue Augmentation and Regeneration. Bioconjugate Chemistry, 21(2), 240-247. Yu, F., Cao, X., Zeng, L., Zhang, Q., & Chen, X. (2013). An interpenetrating HA/G/CS biomimic hydrogel
Ac ce p
488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522
via Diels-Alder click chemistry for cartilage tissue engineering. Carbohydrate Polymers, 97(1), 188-195.
Zhang, K., Zhang, Y., Yan, S., Gong, L., Wang, J., Chen, X., Cui L., & Yin J. (2013). Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(L-glutamic acid) and chitosan. Acta Biomaterialia, 9, 7276-7288.
Zhang, L., Li, K., Xiao, W., Zheng, L., Xiao, Y., Fan, H., & Zhang, X. (2011). Preparation of collagen-chondroitin sulfate-hyaluronic acid hybrid hydrogel scaffolds and cell compatibility in vitro. Carbohydrate Polymers, 84(1), 118-125.
23
Page 23 of 37
Figure Captions Figure 1 The reaction scheme between HAs and maleic anhydride.
ip t
Figure 2 The 1H NMR spectra of MeHA and MaHA. Number 1&2 stand for the protons in unsaturated bond in MaHA. Number 1’ stand for the protons in unsaturated bond in MeHA. Number 3 stands for the methyl protons of N-acetyl groups in HAs molecule. The degree of substitution (DS) is calculated according to the integral area of 1&2 and 3 or 1’ and 3.
us
cr
Figure 3 The ATR FT-IR spectra of HAs and HAs derivatives. In MaHA spectra, the bands at 1719 cm-1 to 1576 cm-1 are featured by C=O, C=C and their conjugation effect. These featured bands are the main difference in the spectra of original HAs and MeHA.
an
Figure 4 The storage and loss modulus of MaHA hydrogels. Except for 50MaHA and 65MaHA, significant difference exists between each group of MaHA hydrogels.
M
Figure 5 The swelling ratios of MaHA hydrogels at time interval and the first order exponential decay equation (ExpDec1) fitting curves. The symbols stand for the swelling ratios of different MaHA hydrogels. The lines are fitted according to ExpDec1.
d
Figure 6 The spline connected line of swelling ratio at equilibrium of MaHA hydrogels. It is calculated based on the ExpDec1 fitting results of swelling ratio.
te
Figure 7 Data plotted according to the first-order kinetics (Equation 2, section 2.2.5.1) and the fitting lines. Only the early stage of the swelling process can be fitted. The deviation might be caused by change of the hydrogels thickness and the variation of diffusion coefficient during the swelling process. The diffusion coefficients (D) are calculated accordingly.
Ac ce p
524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555
Figure 8 Data plotted according to the second-order kinetics (Equation 6, section 2.2.5.1) and the fitting lines. The initial swelling rate (Qi) and the equilibrium swelling ratio (Qe’) are calculated based on the parameters of the fitting lines.
24
Page 24 of 37
O
O HO
ONa O OH
O
O O
+
Dry Formamide
NH CH3
O
50oC
O
O HO
ONa O
OH
O
n
O
HO OH
O O
O O
NH CH3
n
te
d
M
an
us
cr
Figure 1 The reaction scheme between HAs and maleic anhydride.
Ac ce p
555 556 557
OH
O
OH
ip t
O
25
Page 25 of 37
M
te
d
Figure 2 The 1H NMR spectra of MeHA and MaHA. Number 1&2 stand for the protons in unsaturated bond in MaHA. Number 1’ stand for the protons in unsaturated bond in MeHA. Number 3 stands for the methyl protons of N-acetyl groups in HAs molecule. The degree of substitution (DS) is calculated according to the integral area of 1&2 and 3 or 1’ and 3.
Ac ce p
558 559 560 561 562 563
an
us
cr
ip t
557
26
Page 26 of 37
cr
ip t
563
te
d
M
an
Figure 3 The ATR FT-IR spectra of HAs and HAs derivatives. In MaHA spectra, the bands at 1719 cm-1 to 1576 cm-1 are featured by C=O, C=C and their conjugation effect. These featured bands are the main difference in the spectra of original HAs and MeHA.
Ac ce p
565 566 567 568
us
564
27
Page 27 of 37
568 350
300
*
* *
*
07MaHA
15MaHA
E' E''
*
ip t
200
150
cr
E' & E''(kPa)
250
us
100
50
25MaHA
50MaHA
65MaHA
75MaHA
MaHA hydrogels
te
d
M
Figure 4 The storage and loss modulus of MaHA hydrogels. Except for 50MaHA and 65MaHA, significant difference exists between each group of MaHA hydrogels.
Ac ce p
569 570 571 572
an
0
28
Page 28 of 37
572 160
ip t
120
100
cr
80
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75HA-MA ExpDec1 Fit of Swelling Ratio
60
us
Swelling Ratio (g Sol/g Gel)
140
40
0
10
15
20
25
30
Time (Hrs)
te
d
M
Figure 5 The swelling ratios of MaHA hydrogels at time interval and the first order exponential decay equation (ExpDec1) fitting curves. The symbols stand for the swelling ratios of different MaHA hydrogels. The lines are fitted according to ExpDec1.
Ac ce p
573 574 575 576 577
5
an
20
29
Page 29 of 37
577 Spline Line of Qe
150
ip t
140 130
cr
120 110 100 90 80 10
20
30
50
60
70
80
DS (%)
578
te
d
M
Figure 6 The spline connected line of swelling ratio at equilibrium of MaHA hydrogels. It is calculated based on the ExpDec1 fitting results of swelling ratio.
Ac ce p
579 580 581
40
an
0
us
Swelling Ratio at equilibrium (Qe)
160
30
Page 30 of 37
581
5
3
cr
ln(Qe/Qe-Q)
4
ip t
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA Linear Fit of ln(Qe/Qe-Q)
6
2
us
1
0
10
15
20
25
30
Time (hrs)
te
d
M
Figure 7 Data plotted according to the first-order kinetics (Equation 2, section 2.2.5.1) and the fitting lines. Only the early stage of the swelling process can be fitted. The deviation might be caused by change of the hydrogels thickness and the variation of diffusion coefficient during the swelling process. The diffusion coefficients (D) are calculated accordingly.
Ac ce p
582 583 584 585 586 587
5
an
0
31
Page 31 of 37
587 0.32 0.28
0.20
cr
0.16 0.12 0.08
us
t/Q (g Gel/g Sol hrs)
0.24
ip t
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA Linear Fit of t/Q
0.04
0
10
15
20
25
30
Time (hrs)
te
d
M
Figure 8 Data plotted according to the second-order kinetics (Equation 6, section 2.2.5.1) and the fitting lines. The initial swelling rate (Qi) and the equilibrium swelling ratio (Qe’) are calculated based on the parameters of the fitting lines.
Ac ce p
588 589 590 591 592
5
an
0.00
32
Page 32 of 37
Table 1 MaHA and MeHA Specimens with various DS 03MeHA 07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
10 5 7.5 10 12.5 15 20
pH
DS
4 50 50 50 50 50 50
8.0-9.0 / / / / / /
3.33% 7.13% 14.71% 25.92% 50.14% 66.15% 75.47%
* The reaction time was 5 hrs for all cases.
us an M d te Ac ce p
593 594
MAA MA MA MA MA MA MA
Temp (oC)
ip t
Specimen Reactant Dosage*
cr
592
33
Page 33 of 37
Table 2 Swelling Parameters of Simulation Equations of MaHA Hydrogels Based on Increasing DS y0
A
t
R2
T80% hrs
T90% hrs
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
154.92±4.27 149.93±1.88 104.35±4.23 106.26±2.79 106.15±4.84 125.93±1.47
-115.07±4.68 -106.09±1.83 -68.56±4.48 -71.01±2.40 -70.19±4.73 -88.23±1.92
7.08±0.49 8.94±0.28 4.84±0.69 4.34±0.41 5.81±0.76 6.76±0.44
0.9902 0.9986 0.9850 0.9936 0.9879 0.9963
9.30 11.30 5.77 5.24 6.95 8.48
14.21 17.50 9.13 8.26 10.98 13.17
Ac ce p
te
d
M
an
us
597 598 599
ip t
Sample
cr
594 595 596
34
Page 34 of 37
601
Table 3 The Parameters of Fitting Lines According to Equation 2* and Diffusion Coefficients (D) Slope
Intercept
R2
D (cm2/s)×10 -7
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
0.1128±0.0066 0.0974±0.0021 0.1698±0.0083 0.1837±0.0103 0.1673±0.0045 0.1376±0.0088
0.3744±0.0472 0.3696±0.0148 0.4964±0.0597 0.5216±0.0745 0.4253±0.0324 0.4655±0.0633
0.9801 0.9973 0.9859 0.9813 0.9957 0.9761
4.46 4.44 6.14 5.74 6.21 5.11
ip t
Sample
cr
599 600
*Equation 2:
Ac ce p
te
d
M
an
us
602 603
35
Page 35 of 37
B×103
A×102
R2
Qi
Qe’
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
5.76±0.19 5.86±0.18 8.30±0.15 8.59±0.12 8.03±0.13 7.30±0.13
2.23±0.27 2.76±0.29 2.26±0.23 1.76±0.19 2.48±0.21 1.95±0.20
0.9922 0.9924 0.9975 0.9984 0.9978 0.9976
44.80 36.23 44.27 56.92 40.27 51.20
173.61 170.65 120.48 116.41 124.53 136.99
*Equation 6:
ip t
Sample
cr
606
Table 4 The parameters of Fit Lines According to Equation 6* and Swelling Ratios at Initial (Qi) and Equilibrium (Qe’)
us
603 604 605
Ac ce p
te
d
M
an
607
36
Page 36 of 37
607 608
Table 5 The Structure Parameters of MaHA Hydrogels
(mol/cm3)
(g/mol) 1.86×105 1.67×105 1.15×105 1.19×105 1.19×105 1.45×105
(nm)
6.59×10-6 7.38×10-6 1.07×10-5 1.04×10-5 1.03×10-5 8.47×10-6
434 406 298 305 306 357
2.12×105 1.95×105 1.41×105 1.35×105 1.49×105 1.62×105
(mol/cm3) 5.79×10-6 6.30×10-6 8.71×10-6 9.08×10-6 8.23×10-6 7.57×10-6
(nm) 480 457 347 336 361 388
Ac ce p
te
d
M
an
us
609 610
(g/mol)
ip t
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
Qe’
cr
Qe Sample
37
Page 37 of 37
S0144-8617(15)00078-8 http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.047 CARP 9635
To appear in: Received date: Revised date: Accepted date:
23-7-2014 10-12-2014 15-1-2015
Please cite this article as: Lin, H., Liu, J., Zhang, K., Fan, Y., and Zhang, X.,Dynamic Mechanical and Swelling Properties of Maleated Hyaluronic Acid Hydrogels, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.01.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
1 2
·
A novel hyaluronic acid modification method was developed.
3
·
The degree of substitution of the novel derivative, maleated hyaluronic acid (MaHA) is much higher than that of the methacrylated hyaluronic acid (MeHA)
5
reported in the literature. ·
moduli than those of MeHA.
7
9
·
The crosslinking density and hydrophilicity of the introduced groups on HA
us
8
The photopolymerized hydrogels of MaHA have higher compressive storage
cr
6
ip t
4
molecule affect the swelling behavior of hydrogels.
Ac ce p
te
d
M
an
10
1
Page 1 of 37
10 11
Dynamic Mechanical and Swelling Properties of
13
Maleated Hyaluronic Acid Hydrogels
ip t
12
cr
14
Hai Lin*, Jun Liu, Kai Zhang, Yujiang Fan, Xingdong Zhang*
16
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
17
*Corresponding Authors:
18
Hai Lin: [email protected], Phone: (86)28-85417078
19
Xingdong Zhang: [email protected], Phone: (86)28-85412757
an
M Ac ce p
te
d
20 21
us
15
2
Page 2 of 37
21 Abstract
23
A series of maleated hyaluronan (MaHA) are developed by modification with maleic anhydride.
24
The degrees of substitution (DS) of MaHA vary between 7% and 75%. The DS of MaHA is both
25
higher and wider than methacrylated HA derivatives (MeHA) reported in the literature. MaHA
26
hydrogels are then prepared by photopolymerization and their dynamic mechanical and swelling
27
properties of the hydrogels are investigated. The results showed that MaHA hydrogels with
28
moderate DS (25%, 50% and 65%) have higher storage modulus and lower equilibrium swelling
29
ratios than those with either low or high DS (7%, 15% and 75%). Theoretical analyses also
30
suggest a similar pattern among hydrogels with different DS. The results confirm that the
31
increased cross-linking density enhances the strength of hydrogels. Meanwhile, the hydrophilicity
32
of introduced groups during modification and the degree of incomplete crosslinking reaction
33
might have negative impact on the mechanical and swelling properties of MaHA hydrogels.
34
Keywords
36 37
cr
us
an
M
d
te
Ac ce p
35
ip t
22
Hydrogels, hyaluronic acid, mechanical property, swelling kinetics, photopolymerization
3
Page 3 of 37
37 38
1. Introduction Hyaluronic acid (HA) or hyaluronan plays a key structural role for aggrecan assembly,
40
although it forms a smaller part of the extracellular matrix than collagen in most tissues or organs.
41
HA also shows significant advantages of water reservation capacity. Furthermore, HA is built up
42
by repeating disaccharide units composed of N-acetyl-D-glucosamine and D-glucuronic acid. The
43
regular structure of HA provides the same composition regardless of the materials source, and
44
therefore it is non-allergenic. In recent decades, research on HA, HA derivatives and HA-based
45
materials has brought a lot of attention from both basic science and applied clinical applications,
46
which includes drug delivery system (Lim, Cho, Lee & Kim, 2012; Petersen et al., 2013), tissue
47
engineering (Kim, Mauck & Burdick, 2011; Park, Choi, Hu & Lee, 2013; Yu, Cao, Zeng, Zhang &
48
Chen, 2013), plastic filling (Fakhari & Berkland, 2013; Yeom et al., 2010), wound dressing
49
(Hanjaya-Putra et al., 2013; Kirk et al., 2013; Niiyama & Kuroyanagi, 2014) and bio-printing
50
(Murphy, Skardal & Atala, 2013; Pescosolido et al., 2011). Researchers have also reviewed the
52 53 54 55
cr
us
an
M
d
te
Ac ce p
51
ip t
39
different aspects of HA and HA-based biomaterials (Burdick & Prestwich, 2011; Collins & Birkinshaw, 2013; Khan & Ahmad, 2013; Schante, Zuber, Herlin & Vandamme, 2011; Xu, Jha, Harrington, Farach-Carson & Jia, 2012). The hydroxyl groups of HA are typically the main targets of modification because the
carboxyl groups of HA are thought to be related to the bio-synthesis process of extracellular
56
matrix (Christner, Brown & Dziewiatkowski, 1977). Among the published modification methods
57
on hydroxyl groups of HA, esterifications with alkyl succinic anhydrides or methacrylic anhydride
58
are the most well-known methods (Kenne et al., 2013; Khan & Ahmad, 2013). The as-obtained
59
methacrylated HA (MeHA) is especially suitable for photopolymerization which makes the MeHA 4
Page 4 of 37
popular for further biomaterials and tissue engineering applications. Although MeHA is
61
extensively used in the biomaterials field, its preparation process and the final product are not
62
flawless. The MeHA reaction conditions cannot be controlled easily. The degree of substitution
63
(DS) of MeHA is low and its range of DS is narrow too. To some extent the inadequate
64
esterification is one of the main reasons leading to the unsatisfied mechanical properties of the
65
MeHA hydrogels.
us
cr
ip t
60
In this article, we investigate a novel Sodium Hyaluronate (HAs) modification method which
67
yields in HAs derivative with higher DS and improved properties. Furthermore, hydrogels were
68
prepared by obtained HAs derivatives and their mechanical properties and swelling kinetics were
69
characterized.
70
2. Experimental
M
an
66
71
2.1
72
Sodium Hyaluronate (HAs) was purchased from Bloomage Freda Biopharm Co. Ltd.,
73
(Qingdao, Shandong, China). The viscosity-average molecular weight (M) of HAs was tested by
75 76 77
d te
Ac ce p
74
Materials
the capillary viscometry method, identifying a M of 1.0-1.1×103 kDa. Photoinitiator Irgacure 2959 and methacrylic anhydride (MAA) were purchased from Sigma.
Analytical-grade maleic anhydride (MA) and anhydrous ethyl alcohol were purchased from Kelong Chemical Co. Ltd. (Chengdu, China) and were used without further treatments.
78
Analytical-grade formamide was dried by anhydrous magnesium sulfate and redistillation before
79
use.
80
2.2
81
2.2.1 Modification of HAs
Methods
5
Page 5 of 37
The HAs was modified to install photoactive polymerizable groups as following: 1.0g HAs
83
was added to 80ml dry formamide. The solution was heated at 50oC to obtain a homogeneous
84
solution under intense stirring. Maleic anhydride was dissolved in 20ml dry formamide and then
85
added to the HAs solution. The reaction was subsequently proceeded for 5h before cooling to
86
room temperature. The cooled solution was further precipitated in cold anhydrous ethyl alcohol
87
under stirring. The precipitant was purified through repeating washing with anhydrous ethyl
88
alcohol and centrifugal separation. The washed and centrifuged product was decanted and then
89
dissolved in distilled water. The solution was neutralized with 2N sodium hydroxide solution. The
90
HAs derivative was finally dialyzed against deionized water, lyophilized and stored at -20 oC
91
(MaHA).
M
an
us
cr
ip t
82
In order to better understand the modification efficiency, we also prepared a methacrylated
93
HA derivative (MeHA) as a control based on the methods reported in the literature (Bian et al.,
94
2013; Bian, Zhai, Zhang, Mauck & Burdick, 2012). Briefly, methacrylic anhydride (MAA, Sigma),
95
which is 10-fold excess to the primary hydroxyl groups in HAs, is reacted with HAs in an aqueous
97 98 99 100
te
Ac ce p
96
d
92
solution at 4oC. The reaction pH was kept in between 8.0 and 9.0 by adjusting with 5N NaOH. The same reaction time of 5h was picked in order to compare with the MaHA. The product was precipitated in anhydrous ethyl alcohol, dialyzed and finally lyophilized (MeHA). The as-prepared MaHA and MeHA specimens are listed in Table 1. The specimens are named
by their reactants (e.g., Me or Ma), and the degrees of substitution (DS).
101
2.2.2 Characterization of modified HAs
102
2.2.2.1
103
1
1
H Nuclear Magnetic Resonance (NMR)
H-NMR spectroscopy was used to verify the esterification and to enable the quantitative 6
Page 6 of 37
104
calculation of the DS. The HAs derivatives (both MeHA and MaHA) were dissolved in D2O with
105
a concentration of 8-10 mg/mL. The spectra were recorded using a Bruker AVIII 400 HD nuclear
106
magnetic resonance spectrometer (Swiss). 2.2.2.2
108
ATR FT-IR was applied to identify the successful modification (-unsaturated ester/acid).
109
The HA derivatives (MeHA & MaHA) were scanned from 400 cm-1 to 4000 cm-1 with a resolution
110
of 2 cm-1 by using Thermo Fisher Nicolet IS10 (USA). The original HA powder was also scanned
111
under the same condition and served as control.
ip t
107
an
us
cr
Attenuated Total Reflect Fourier Transform Infrared Spectroscopy (ATR FT-IR)
2.2.3 Preparation of HAs hydrogels
113
A 0.1% (w/v) Irgacure 2959 solution (Sigma) was prepared. MaHA was added to the initiator
114
solution with a concentration of 20 mg/mL. After thorough dissolution of MaHA under stirring,
115
100L of the solution was pipetted to plastic cylindrical molds (custom-made, with the internal
116
diameter of 8mm and the height of 2mm). The filled molds were sealed by cover glasses on both
117
ends. The photocrosslinked hydrogels were prepared by exposing the solution to UV light
119 120 121
d
te
Ac ce p
118
M
112
(OmniCure S1500 (USA), 365nm, ~16 mW/cm2) for 60 seconds at each end. 2.2.4 Characterization of HAs hydrogels 2.2.4.1
Dynamic Mechanical Analysis (DMA)
The DMA of compression tests were performed on NETZSCH DMA 242C equipped with a
122
controller of TASC 414/4 (NETZSCH, German). Both frequencies of 1.0 Hz and 10 Hz were
123
applied, which simulated the normal and the limit of physiological stride frequency. The tests were
124
carried out at constant temperature of 25oC. The testing parameters were set as amplitude of 20m,
125
the preload force of 0.001N and the force track of 120%. Three cylindrical samples with a 7
Page 7 of 37
diameter of 8mm and a height of 2mm were used for each modification condition. The results
127
were analyzed by NETZSCH Proteus program, and statistical analysis was performed by student t
128
test on the average E’ and E’’ which were calculated according to the data exported with a regular
129
time interval.
ip t
126
130
2.2.4.2
131
The dry weights of hydrogels (w0) can be calculated according to the solution concentrations
132
and volumes. After soaking in 100mM PBS (pH = 7.2) at 37oC at regular time intervals, the buffer
133
on surface of hydrogels was carefully absorbed and the gels weighed (wt) to determine the
134
swelling ratio (Q) which is defined as (wt-w0)/w0.
an
us
cr
Swelling tests
2.2.5 Kinetics and structure analysis
136
2.2.5.1 Kinetics analysis
137
The swelling of the cross-linked hydrogels is commonly described to follow the first-order
140 141 142
d
te
139
kinetics (Schott, 1992a, b), which can be expressed as following equation (Equation 1).
(1)
Ac ce p
138
M
135
Where, Qt means the swelling ratio at time t, and Qe stands for the swelling ratio when the hydrogels reach equilibrium. The Equation 1 becomes to Equation 2 after integration. (2)
143
The Equation 2 is derived based on Fick’s laws which only apply under the following
144
conditions (Schott, 1992a, b). The samples should have a large aspect ratio, i.e., the surface of the
145
sample is much larger than its thickness (H). Moreover, both H and diffusion coefficient (D)
146
should remain constants during the swelling process. 8
Page 8 of 37
147 148
The Fickian equation has another expression shown as Equation 3.(Jovanovic & Adnadjevic, 2013; Schott, 1992a)
149
ip t
150
(3)
Where D is diffusion coefficient, t is the diffusion time and H is the thickness of the hydrogel.
With enough diffusion time, the Equation 3 can be approximately simplified as Equation 4.
152
(4)
153
Compare Equation 4 with Equation 2, the constant D can be calculated from the slope of the
For the entire swelling process, the Scott’s second-order equation is obeyed.
156
159
160 161
162
d
After integration between the limits t(0, t) and Qt(0, Qe), and make A=1/kQe2 and B=1/Qe,
te
158
(5)
then the Equation 5 is transferred to Equation 6.
Ac ce p
157
us
an
155
linear fit curve.
M
154
cr
151
(6)
Where A and B are coefficients with physical meanings. At the very beginning of the swelling, that is t → 0, then
and thus yields:
(7)
163
The intercept A is the reciprocal of the initial swelling rate, corresponding to the stage when
164
(1) the solvent has permeated the entire hydrogel and (2) before the strain on the network begins
165
to retard swelling.
166
On the contrary, at a long time,
, we then deduce that B=1/Qt, which means B is the 9
Page 9 of 37
167
reciprocal of equilibrium swelling ratio. 2.2.5.2 Structure analysis
169
As mentioned above, the network structure of the MaHA hydrogels is one of the driving
170
forces of swelling kinetics. Therefore, a number of analyses based on swelling experiments were
171
conducted to investigate important parameters used to characterize the network structure of the
172
hydrogels, including the polymer volume fraction in the swollen state (v2,s), the molecular weight
173
of the polymer chain between two neighboring crosslinking points (
174
mesh size (ξ).
cr
us
an
176
The relationship between
and v2,s was developed and described by Peppas et al. as
following (Buckley & Martin, 1962; Peppas, Hilt, Khademhosseini & Langer, 2006):
177
Where
179
(18 cm3/g for water);
182 183 184 185
is the molar volume of solvent
te
is the number average molecular weight of HA,
is the specific volume of the dry HA (0.8137 cm3/g for HA) (Leach,
Ac ce p
181
d
(8)
178
180
), and the corresponding
M
175
ip t
168
Bivens, Patrick & Schmidt, 2003);
is the Flory-Huggins interaction parameter between
polymer-solvent (0.439 for HA) (Ottenbrite & Kim, 2000); and
is the polymer volume
fraction in the relaxed state, which is defined as the state of a polymer immediately after crosslinking but before swelling in a solvent. The effective crosslink density
is calculated by the Equation 9 (Leach, Bivens, Patrick &
Schmidt, 2003).
186
(9)
187
The hydrogel mesh size ξ which is defined as the average distance between crosslinks in the 10
Page 10 of 37
188
hydrogel can be determined theoretically. (Meybodi, Imani & Mohammad, 2013)
189
(10)
190
Where
191
state. For HA, the root-mean-square end-to-end distance was reported in literature as following
192
(Leach, Bivens, Patrick & Schmidt, 2003):
(11)
us
193
cr
ip t
is the root-mean-square distance between two adjacent crosslinks in the solvent-free
Where n is the number of disaccharide repeat units for HA with a given molecular weight. Since
195
the molecular weight of the repeat unit is 379.32 g/mol, the equation can be transformed as:
an
194
196
199 200 201 202 203
M
by
d
Equation 10 and replacing
gives Equation 13.
te
198
Therefore, the mesh size of HA hydrogels can be calculated by substitution of Equation 12 in
(13)
Ac ce p
197
(12)
3. Results and Discussion
The well-known mechanism for the formation of MeHA by hyaluronic acid and methacrylic
anhydride in an aqueous environment has been thoroughly investigated before (Burdick, Chung, Jia, Randolph & Langer, 2005; Smeds, Pfister-Serres, Hatchell & Grinstaff, 1999). The MaHA
204
reaction between HAs and maleic anhydride in organic solvent follows a similar mechanism
205
shown in Figure 1. In anhydrous solvent, esterification reaction can easily occur between the
206
primary hydroxyl group at C-6 of the Glucosamine and anhydride through a ring opening
207
mechanism. Recently, Vasi et al. reported an identical HA modification gained in a two step 11
Page 11 of 37
synthesis (Vasi, Popa, Butnaru, Dodi & Verestiuc, 2014). By comparison, the sodium hyaluronan
209
used as raw materials in our described preparation did not require an ion exchange into the acidic
210
form to obtain solubility in a polar organic solvent. Consequently, our modification process
211
enables a shorter and quicker access to yield a maleated hyaluronan derivative. Nonetheless, the
212
HAs derivatives obtained by routes are consistent judged by NMR as well as FT-IR analysis (see
213
section 3.1&3.2).
us
1
cr
ip t
208
214
3.1
215
As shown in Figure 2, the spectrum of MeHA displays two peaks at approximately 5.6 and 6.0
216
ppm, which corresponds to the introduced methacrylate moieties (Smeds & Grinstaff, 2001;
217
Smeds, Pfister-Serres, Hatchell & Grinstaff, 1999). Figure 2 also shows that the grafted maleic
218
ester in MaHA present peaks at 5.9 and 6.5ppm which is slightly different from the data shown in
219
literature (6.1 and 6.7ppm, in CD3SOCD3) (Vasi, Popa, Butnaru, Dodi & Verestiuc, 2014). In
220
agreement with Vasi’s study, the appearance of peak at 8.1 ppm is related to the proton from
221
COOH group. The peak at 6.2 ppm is not expected in MaHA structure, and it possible relates to
223 224 225
an
M
d
te
Ac ce p
222
H NMR
the protons of maleic acid which was hydrolyzed from maleic anhydride and attached to the MaHA molecule by hydrogen bonds. According to the integral area of protons, only 0.50%-2.19% double bonds in high DS MaHA derivatives are contributed by attaching maleic acid. The broadened peaks between 3.2 and 3.8 ppm are referred to the protons of the pyranose rings (Leach,
226
Bivens, Patrick & Schmidt, 2003). Furthermore, the peak at 1.9ppm belongs to the protons of
227
methyl (-CH3) in N-acetyl group and served as the reference peak to calculate the DS. According
228
to the integral area of protons of unsaturated bonds and methyl, the DS in MeHA is around 3.33%
229
and the DS of MaHA samples ranges from 7.13% to 75.47%, correspondingly. 12
Page 12 of 37
The variation of DS of MeHA could be altered by the amount of methacrylic anhydride,
231
reaction time and the solution pH (Burdick, Chung, Jia, Randolph & Langer, 2005). Our study of
232
MeHA suggests that merely 3.33% functionalization were achieved under the reaction conditions
233
of 10-fold MAA and reaction time of 5h. Even with a different set of parameters (e.g., 20-fold
234
MAA, reaction time of 24h and accurate control over pH), we can only obtain the MeHA
235
derivative with a DS around 20%. However, the DS of MeHA reported in the literature is less than
236
29% (Bian et al., 2013). The hydrolysis of MAA in water and the sensitivity of pH and
237
temperature of this reaction make it very difficult to ideally control over the DS of MeHA.
238
However, the reaction between HAs and maleic anhydride in anhydrous organic solvent can fully
239
control over the DS of MaHA by reaction conditions. As a result, we achieved a variety of DS for
240
MaHA that shown in both Table I and Figure 2.
M
an
us
cr
ip t
230
3.2
242
The ATR FT-IR spectra of HAs and HAs derivatives (MeHA and MaHA) with different DS
243
are shown in Figure 3. With the increase of DS in MaHA, new peaks were identified according to
245 246 247
te
Ac ce p
244
ATR FT-IR
d
241
the obtained DS. The peaks at 1719 cm-1, 1576 cm-1 and the shoulder peak between these peaks were related to the -unsaturated ester/acid (-CO-C=C-CO-) of the modified HAs (Haxaire, Marechal, Milas & Rinaudo, 2003). The synergistic effect of the carbonyl groups, the olefin and its conjugation of the -system at its sp2-hybrids contribute to the band development of the
248
modified MaHA. Compared with the spectra of MaHA samples, the spectrum of MeHA shows
249
both a slight shift and intensity difference at the peak around 1610 cm-1 , because MeHA has a
250
different double conjugated group (CH2=C(C)-CO-). The results of ATR FT-IR are consistent with
251
the data in literature (Vasi, Popa, Butnaru, Dodi & Verestiuc, 2014) and confirm that the functional 13
Page 13 of 37
252
molecules are branched on the polysaccharide chain, and the DS is affected by the feed ratios in
253
MaHA.
254
3.3
255
The average moduli of different MaHA hydrogels were calculated and shown in Figure 4.
256
Most of the samples have storage moduli larger than 200kPa, and the 25MaHA hydrogels have the
257
largest E’, around 290kPa. Previous research of our group showed that the ternary hydrogels
258
which prepared by collagen, chondroitin sulfate and hyaluronic acid had a compressive modulus
259
around 45–54 kPa (Guo et al., 2012; Zhang et al., 2011). According to other published data, the
260
MeHA hydrogels have the modulus range between 3.5 and 53.6 kPa (Bian et al., 2013), depending
261
on the hydrogel concentration and curing time. Therefore, the MaHA hydrogels we developed
262
have a much higher mechanical property than the MeHA hydrogels reported in literature. In
263
addition, the range of the storage modulus might be another important aspect, because the
264
mechanical property differences in materials might cause significantly different responses in
265
biological system, such as stem cell differentiation and bioactive molecule secretion (Choi et al.,
267 268 269
ip t
cr
us
an
M
d
te
Ac ce p
266
Dynamic Mechanical Analysis
2013; Kim, Khetan, Baker, Chen & Burdick, 2013; Marklein, Soranno & Burdick, 2012). The difference of storage modulus among hydrogel samples developed in this study and those reported in the literature is more than 100 kPa. In addition, the range of storage modulus of hydrogels development in our study can be even more significant at higher concentration and longer curing
270
time. Currently, the hydrogels developed for cartilage tissue engineering typically have relatively
271
weaker mechanical properties than those of natural cartilage: Young’s modulus 0.5-60 kPa (Bian
272
et al., 2013) to 0.699 MPa (human fetal) (Callahan, Ganios, Childers, Weiner & Becker, 2013), or
273
compressive modulus ≈2 to over 100 kPa (Burdick & Prestwich, 2011) to 2.78MPa (rabbit) 14
Page 14 of 37
(Zhang et al., 2013). In addition, the range of modulus variation for those hydrogels is quite
275
narrow too (Marklein & Burdick, 2010; Toh, Lim, Kurisawa & Spector, 2012). Our ongoing study
276
is investigating how these MaHA hydrogels with higher mechanical property perform in vitro and
277
in vivo.
ip t
274
The DMA results also suggest that the E’ do not always increase along with the increase of DS.
279
Meanwhile, as expected, the loss moduli of the MaHA hydrogels are much lower than their
280
storage moduli, suggesting that the elastic properties of those hydrogels are more pronounced than
281
their viscous properties. The statistical analyses on storage moduli show that significant difference
282
exists between each group, except for 50MaHA and 60MaHA. Generally speaking, if the double
283
bonds are fully reacted during the photopolymerization, the more double bonds grafted on the
284
HAs, the higher the storage moduli of the formed hydrogels. Since the storage moduli of
285
hydrogels do not increase with the DS when the DS is quite high (50% or more) (see figure 4), we
286
deduced that not all the unsaturated bonds are cross-linked under current reaction conditions. As a
287
result, the MaHA with a suitable DS will achieve optimized mechanical property. Otherwise, a
289 290 291
us
an
M
d
te
Ac ce p
288
cr
278
complex construct composed of HA and other reactive compositions, which can react with more of the double bonds, may further improve the mechanical property of the hydrogel. 3.4
Hydrogel swelling kinetics analysis
A gravimetric method was applied to investigate the swelling process of newly developed
292
MaHA hydrogels. Swelling ratios of different MaHA hydrogels are shown as the symbols in
293
Figure 5. To have a better understanding of the swelling behavior, we try to fit the process with the
294
first order exponential decay equation:
295
equations can simulate the swelling process very well with coefficients of determination (R2) are
The lines in Figure 5 indicate that the
15
Page 15 of 37
296
higher than 0.9850 (see table 2). The parameters of the equations are shown as Table 2. The swelling capacity of all hydrogel samples increases with time. All MaHA hydrogels
298
show the similar pattern of reaching their equilibrium swelling states after a certain period of time.
299
With the help of simulation equations, the equilibrium swelling ratios (Qe) of different hydrogels
300
can be calculated, ranges between 104 and 154. Compared with literature data, the swelling ratios
301
have a wide range due to the difference in preparation methods. In our previous work,
302
interpenetrating ternary hydrogels have swelling ratios of 4-14 g sol/g gel (Guo et al., 2012; Zhang
303
et al., 2011). The 25MaHA samples have the smallest Qe. 50MaHA and 65MaHA samples have
304
equivalent Qe values, which are slightly higher than that of 25MaHA. As the spline connected line
305
of Qe in Figure 6, the Qe decreases with the increase of the DS up to around 35%. The decrease of
306
Qe at the earlier stage is caused by the increase of the degree of crosslinking when the DS is
307
climbing. This trend is consistent with reported other chemically crosslinked hydrogels (Kenne et
308
al., 2013; Sivakumaran, Maitland, Oszustowicz & Hoare, 2013). However, the swelling ratios
309
raise slowly between 35% and 75% DS. It is assumed that unsaturated bonds introduced into the
311 312 313
cr
us
an
M
d
te
Ac ce p
310
ip t
297
HAs molecule cannot react completely by photopolymerization under insufficient exposure time. Hence, the highly hydrophilic carboxyl groups grafted on the polymer chain yield in an enhanced water retaining capacity inside the network after more carboxyl groups are introduced. Therefore, the positive effects of hydrophilicity and negative effects due to crosslinking are balanced at an
314
appropriate DS, which eventually leads to the minimum value of swelling ratio at equilibrium. As
315
a result, a medium DS (25%-50% in this study) is necessary for the appropriate swelling of
316
hydrogels.
317
The simulation curves and equations can also derive the time to achieve dynamic equilibrium. 16
Page 16 of 37
The time for hydrogels to reach 80% and 90% of the theoretical swelling ratio is listed in Table 2.
319
Hydrogels prepared by 50MaHA have the shortest time of 5.24 hrs and 8.26 hrs, respectively,
320
what indicates that the hydrogels need to be soaked in the buffer for at least 10h to reach their
321
swollen state.
ip t
318
According to the Equation 2, the linear fit could describe the swelling process. However, in
323
our case, only the first 12 hours swelling process can be fitted by the Equation 2, as the data of
324
subsequent times deviated from the prior theoretical calculation. The fitting lines are shown as
325
Figure 7. The deviation might be caused by factors such as the change of the hydrogels thickness,
326
and the variation of diffusion coefficient during the swelling process. Thus, the first-order kinetics
327
may be only suitable at the early stage of swelling and cannot predict or describe the entire
328
swelling process (Figure 7, fitting lines are made up to 12h only).
M
an
us
cr
322
However, based on the excellent fit of the swelling data up to 12 hrs, the parameters of linear
330
fit lines, together with their diffusion coefficients are calculated according to Equation 4, and
331
listed in Table 3. The theoretical calculated data of the diffusion coefficients show a similar trend
333 334 335
te
Ac ce p
332
d
329
with the storage modulus of hydrogels described in the previous section. With the increase of cross-linking at early stage, the hydrophilic groups on HAs molecules are immobilized which leads to an easier situation for the diffusion of solvent. With the increase of DS, the branched groups cannot be fully reacted under the curing conditions. Then, the introduced free hydrophilic
336
side groups hinder the diffusion of water molecule. According to the data of hydrogels prepared by
337
MeHA in literature (Bian et al., 2013), the MeHA hydrogels with an increasing concentration or
338
UV exposure time have a diffusion coefficient of 3.8-6.3×10-7 cm2/s. The decrease of diffusivity
339
was explained by the increasing of crosslinking density (Bian et al., 2013). In comparison to 17
Page 17 of 37
MeHA, the MaHA hydrogels have an equivalent diffusion coefficient which is 4.44-6.21 ×10-7
341
cm2/s. For MaHA hydrogels, not only the crosslinking density will affect the diffusivity, but also
342
the hydrophobic and hydrophilic properties of the introduced functionalities affect the diffusion of
343
water and the solvatisation with it.
ip t
340
To describe the entire swelling process, the second-order equation is applied. Figure 8 shows
345
the swelling kinetics of the hydrogels plotted according to the transferred second-order kinetics
346
equation. The fitting parameters are shown in Table 4. According to the fitting lines in Figure 8
347
and theoretically calculated parameters in Table 4, the initial swelling rate (Qi) and the equilibrium
348
swelling ratio (Qe’) can be calculated. The results indicate that the Qe’s of hydrogels have a
349
similar pattern compared to the results of simulation equations described above: the MaHA
350
hydrogels with the lowest DS have the largest swelling ratio, and the samples with moderate DS
351
(25% to 65%) have similar swelling ratios. Schott proposed that the swelling rate is proportional
352
to two factors (Schott, 1992a). The first factor is the unrealized swelling percentage. The second
353
factor is the internal specific boundary area. This area encloses all the sites that have not interacted
355 356 357
us
an
M
d
te
Ac ce p
354
cr
344
with water nor swollen at a given time, but the sites will be hydrated and swell in due course. In our case, the amorphous domains in MaHA hydrogels are expanded and the resultant stress on the crystalline domains increases during the swelling process. However, the stressed crosslinking crystalline domains can resist further swelling. Thus, the swelling rate of MaHA hydrogels is
358
proportional to their relative swelling capacity. The MaHA hydrogels with higher percentages of
359
crystalline domains should have higher unrealized swelling capacities and initial swelling rates as
360
compared to the rest hydrogels with either lower or higher DS. As shown in Table 4, 50MaHA
361
hydrogels have the highest initial swelling rate. With the combined effects of crosslinking and 18
Page 18 of 37
hydrophilicity, the swelling behavior of hydrogels has a similar trend with their mechanical
363
properties described in 3.3.
364
3.5 Hydrogel analysis
365
Based on the previous analysis of the swelling behavior of MaHA hydrogels, the theoretical
366
calculated structural parameters are listed in Table 5 according to Qe and Qe’, respectively. The
369 370
cr
showed a trend of decrease between 7MaHA and 65MaHA, and then increase at
75MaHA. The
us
368
and
changed inversely with the effect of crosslinking on
and . The range of
was 298-434nm or 336-480nm when calculating with Qe or Qe’, respectively. These results
an
367
ip t
362
show a similar interval and good permeability.
Swelling ratios vary with the amounts of introduced functional groups on HAs and the curing
372
conditions, both of which decide the degree of cross-linking and hydrophilicity in hydrogels. In
373
our study, the molecular weights between crosslinks (
374
mesh sizes ( ) of MaHA hydrogels are calculated by using Qe or Qe’. These parameters of
375
hydrogels are different due to their difference in the inner structure and chemical compositions.
377 378 379
d
te
), effective crosslink densities ( ) and
Ac ce p
376
M
371
When the DS was lower than 50%, the degree of cross-linking affects more on the inner structure of hydrogels than the hydrophilicity does. When the DS was higher than 50%, the influence of hydrophilicity dominated the inner structure of hydrogels over the degree of crosslinking. 4. Conclusions
380
The maleated hyaluronic acid (MaHA) was prepared, and its degree of substitution (DS) was
381
controlled by adjusted reaction conditions. ATR FT-IR as well as NMR results indicate the
382
successful introduction of maleic ester. Additionally, 1H NMR results showed that the DS ranges
383
between 7% and 75%. Compared with the DS of MeHA in the literature, the MaHA yielded in a 19
Page 19 of 37
higher and wider DS. Furthermore, the modification reaction can be easily controlled and it is
385
shorter and more efficient to attain high DS maleated hyaluronan than literature report. Following
386
the preparation of MaHA hydrogels by photopolymerization, their dynamic mechanical properties
387
and swelling behavior were studied. Dynamic mechanical analyses showed that the 25MaHA
388
hydrogel has the highest storage modulus (~ 290kPa). The change of hydrogel’s storage modulus
389
indicates that the mechanical property of hydrogels is determined not only by the density of
390
cross-linking but also the hydrophilicity of the introduced groups during modification. Swelling
391
studies and theoretical calculations also confirmed that both cross-linking and hydrophilicity
392
affects the swelling properties of the hydrogels. Calculations with both fitting and swelling
393
kinetics equations were performed. Although the calculated results are based on several
394
assumptions and might deviate from the data obtained by analytical devices, they are meaningful
395
for inter-comparisons among samples and inspiring the product improvements. The hydrogels of
396
25, 50 and 65MaHA have similar equilibrium swelling ratios (Qe) that range between 104 and 124.
397
The Qe of 25, 50, 65MaHA hydrogels are lower than those of hydrogels of 7, 15 and 75MaHA,
399 400 401
cr
us
an
M
d
te
Ac ce p
398
ip t
384
which range between 125 and 173. Meanwhile, the diffusion coefficients (D) of 25, 50 and 65MaHA hydrogels are higher than those of 7, 15 and 75MaHA hydrogels. The analysis of the swelling structure suggests that the MaHA hydrogels with low Qe and high D have lower molecular weight between crosslinks (
) and smaller mesh size ( ). The structural characteristics
402
of MaHA hydrogels are caused by the increase of cross-linking density and hydrophilicity. Future
403
studies will report the biocompatibility studies following ISO 10993, and in vitro and in vivo
404
performance of the MaHA hydrogels for cartilage tissue engineering applications.
405 20
Page 20 of 37
406
Acknowledgements This study was financially supported by the National Key Technology Research and
408
Development Program (2012BAI42G00), the National Science Foundation for Young Scientists of
409
China (51403134), the Application Technology Research and Demonstration Projects of Hainan
410
Province China (SQ2014ZDXM0294) and the Foundation of Jiangsu Collaborative Innovation
411
Center of Biomedical Functional Materials, China.
cr us
References
Bian, L., Hou, C., Tous, E., Rai, R., Mauck, R.L., & Burdick, J.A. (2013). The influence of hyaluronic acid
an
hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy. Biomaterials, 34(2), 413-421.
Bian, L., Zhai, D.Y., Zhang, E.C., Mauck, R.L., & Burdick, J.A. (2012). Dynamic Compressive Loading Enhances Cartilage Matrix Synthesis and Distribution and Suppresses Hypertrophy in hMSC-Laden
M
Hyaluronic Acid Hydrogels. Tissue Engineering Part A, 18(7-8), 715-724.
Buckley, D.J., & Martin, B. (1962). The swelling of polymer systems in solvents. II. Mathematics of diffusion. Journal of Polymer Science Part a-Polymer Chemistry, 56(163), 175-188.
d
Burdick, J.A., Chung, C., Jia, X., Randolph, M.A., & Langer, R. (2005). Controlled Degradation and 386-391.
te
Mechanical Behavior of Photopolymerized Hyaluronic Acid Networks. Biomacromolecules, 6(1), Burdick, J.A., & Prestwich, G.D. (2011). Hyaluronic Acid Hydrogels for Biomedical Applications. Advanced Healthcare Materials(23), H41-H56.
Callahan, L.A.S., Ganios, A.M., Childers, E.P., Weiner, S.D., & Becker, M.L. (2013). Primary human chondrocyte extracellular matrix formation and phenotype maintenance using RGD-derivatized PEGDM hydrogels possessing a continuous Young’s modulus gradient. Acta Biomaterialia, 9, 6095-6104.
Ac ce p
412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443
ip t
407
Choi, J., Choi, B., Park, S.H., Pai, K., Li, T., Min, B.H., & Park, S. (2013). Mechanical Stimulation by Ultrasound Enhances Chondrogenic Differentiation of Mesenchymal Stem Cells in a Fibrin-Hyaluronic Acid Hydrogel. Artificial Organs, 37(7), 648-655. Christner, J.E., Brown, M.L., & Dziewiatkowski, D.D. (1977). Interaction of Cartilage Proteoglycans with Hyaluronic Acid The role of the hyaluronic acid carboxyl groups. Biochem. J.(167), 711-716. Collins, M.N., & Birkinshaw, C. (2013). Hyaluronic acid based scaffolds for tissue engineering-A review. Carbohydrate Polymers, 92(2), 1262-1279. Fakhari, A., & Berkland, C. (2013). Applications and emerging trends of hyaluronic acid in tissue engineering, as a dermal filler and in osteoarthritis treatment. Acta Biomaterialia, 9(7), 7081-7092. Guo, Y., Yuan, T., Xiao, Z., Tang, P., Xiao, Y., Fan, Y., & Zhang, X. (2012). Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering. Journal of Materials Science-Materials in Medicine, 23(9), 2267-2279. Hanjaya-Putra, D., Shen, Y.I., Wilson, A., Fox-Talbot, K., Khetan, S., Burdick, J.A., Steenbergen, C., & 21
Page 21 of 37
Gerecht, S. (2013). Integration and Regression of Implanted Engineered Human Vascular Networks During Deep Wound Healing. Stem Cells Translational Medicine, 2(4), 297-306. Haxaire, K., Marechal, Y., Milas, M., & Rinaudo, M. (2003). Hydration of Polysaccharide Hyaluronan Observed by IR Spectrometry. I. Preliminary Experiments and Band Assignments. Biopolymers, 72, 10-20. Jovanovic, J., & Adnadjevic, B. (2013). The Effect of Primary Structural Parameters of Poly(methacrylic
ip t
acid) Xerogels on the Kinetics of Swelling. Journal of Applied Polymer Science, 127(5), 3550-3559.
Kenne, L., Gohil, S., Nilsson, E.M., Karlsson, A., Ericsson, D., Kenne, A.H., & Nord, L.I. (2013).
Modification and cross-linking parameters in hyaluronic acid hydrogels-Definitions and analytical
cr
methods. Carbohydrate Polymers, 91(1), 410-418.
Khan, F., & Ahmad, S.R. (2013). Polysaccharides and Their Derivatives for Versatile Tissue Engineering Application. Macromolecular Bioscience, 13(4), 395-421.
us
Kim, I.L., Khetan, S., Baker, B.M., Chen, C.S., & Burdick, J.A. (2013). Fibrous hyaluronic acid hydrogels that direct MSC chondrogenesis through mechanical and adhesive cues. Biomaterials(34), 5571-5580. Kim, I.L., Mauck, R.L., & Burdick, J.A. (2011). Hydrogel design for cartilage tissue engineering: A case
an
study with hyaluronic acid. Biomaterials, 32(34), 8771-8782.
Kirk, J.F., Ritter, G., Finger, I., Sankar, D., Reddy, J.D., Talton, J.D., Nataraj, C., Narisawa, S., Millan, J.L., & Cobb,
R.R.
(2013).
Mechanical
and
biocompatible
characterization
of
a
cross-linked
collagen-hyaluronic acid wound dressing. Biomatter, 3(4).
M
Leach, J.B., Bivens, K.A., Patrick, C.W., & Schmidt, C. (2003). Photocrosslinked Hyaluronic Acid Hydrogels: Natural, Biodegradable Tissue Engineering Scaffolds. Biotechnology and Bioengineering, 82(5), 578-589.
d
Lim, H.J., Cho, E.C., Lee, J.A., & Kim, J. (2012). A novel approach for the use of hyaluronic acid-based hydrogel nanoparticles as effective carriers for transdermal delivery systems. Colloids and Surfaces
te
a-Physicochemical and Engineering Aspects, 402, 80-87. Marklein, R.A., & Burdick, J.A. (2010). Spatially controlled hydrogel mechanics to modulate stem cell interactions. Soft Matter, 6(1), 136-143.
Marklein, R.A., Soranno, D.E., & Burdick, J.A. (2012). Magnitude and presentation of mechanical
Ac ce p
444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487
signals influence adult stem cell behavior in 3-dimensional macroporous hydrogels. Soft Matter, 8(31), 8113-8120.
Meybodi, Z.E., Imani, M., & Mohammad, A. (2013). Kinetics of dextran crosslinking by epichlorohydrin: A rheometry and equilibrium swelling study. Carbohydrate Polymers(92), 1792-1798. Murphy, S.V., Skardal, A., & Atala, A. (2013). Evaluation of hydrogels for bio-printing applications. Journal of Biomedical Materials Research Part A, 101A(1), 272-284. Niiyama, H., & Kuroyanagi, Y. (2014). Development of novel wound dressing composed of hyaluronic acid and collagen sponge containing epidermal growth factor and vitamin C derivative. J Artif Organs, 17(1), 81-87. Ottenbrite, R.M., & Kim, S.W. (2000). Polymeric Drugs and Drug Delivery Systems. CRC Press. Park, H., Choi, B., Hu, J., & Lee, M. (2013). Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering. Acta Biomaterialia, 9(1), 4779-4786. Peppas, N.A., Hilt, J.Z., Khademhosseini, A., & Langer, R. (2006). Hydrogels in BIology and Medicine: From Molecular Principles to Bionanotechnology. Advanced Materials(18), 1345-1360. Pescosolido, L., Schuurman, W., Malda, J., Matricardi, P., Alhaique, F., Coviello, T., van Weeren, P. R., Dhert, W.J.A., Hennink, W.E., & Vermonden, T. (2011). Hyaluronic Acid and Dextran-Based Semi-IPN 22
Page 22 of 37
523 524
Hydrogels as Biomaterials for Bioprinting. Biomacromolecules, 12(5), 1831-1838. Petersen, S., Kaule, S., Teske, M., Minrath, I., Schmitz, K.P., & Sternberg, K. (2013). Development and In Vitro Characterization of Hyaluronic Acid-Based Coatings for Implant-Associated Local Drug Delivery Systems. Journal of Chemistry. Schante, C.E., Zuber, G., Herlin, C., & Vandamme, T.F. (2011). Chemical modifications of hyaluronic acid Polymers(85), 469-489.
ip t
for the synthesis of derivatives for a broad range of biomedical applications. Carbohydrate Schott, H. (1992a). Kinetics of Swelling of Polymer and Their Gels. Journal of Pharmaceutical Sciences, 81(5), 467-470.
cr
Schott, H. (1992b). Swelling Kinetics of Polymer. J. Macromol. Sci-Phys., B31(1), 1-9.
Sivakumaran, D., Maitland, D., Oszustowicz, T., & Hoare, T. (2013). Tuning drug release from smart microgel-hydrogel composites via cross-linking. Journal of Colloid and Interface Science, 392, 422-430.
us
Smeds, K.A., & Grinstaff, M.W. (2001). Photocrosslinkable polysaccharides for in situ hydrogel formation. Journal of Biomedical Materials Research, 54, 115-121.
Smeds, K.A., Pfister-Serres, A., Hatchell, D.L., & Grinstaff, M.W. (1999). Synthesis of a novel
an
polysaccharide hydrogel. J.M.S. Pure Appl. Chem., A36(7&8), 981-989.
Toh, W.S., Lim, T.C., Kurisawa, M., & Spector, M. (2012). Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomaterials, 33(15), 3835-3845.
M
Ana-Maria Vasi, Marcel Ionel Popa, Maria Butnaru, Gianina Dodi, & Verestiuc, L. (2014). Chemical functionalization of hyaluronic acid for drug delivery applications. Materials Science & Engineering C(38), 177-185.
d
Xu, X., Jha, A.K., Harrington, D.A., Farach-Carson, M.C., & Jia, X. (2012). Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter, 8(12), 3280-3294.
te
Yeom, J., Bhang, S.H., Kim, B.S., Seo, M.S., Hwang, E.J., Cho, I.H., Park, J.K., & Hahn, S.K. (2010). Effect of Cross-Linking Reagents for Hyaluronic Acid Hydrogel Dermal Fillers on Tissue Augmentation and Regeneration. Bioconjugate Chemistry, 21(2), 240-247. Yu, F., Cao, X., Zeng, L., Zhang, Q., & Chen, X. (2013). An interpenetrating HA/G/CS biomimic hydrogel
Ac ce p
488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522
via Diels-Alder click chemistry for cartilage tissue engineering. Carbohydrate Polymers, 97(1), 188-195.
Zhang, K., Zhang, Y., Yan, S., Gong, L., Wang, J., Chen, X., Cui L., & Yin J. (2013). Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(L-glutamic acid) and chitosan. Acta Biomaterialia, 9, 7276-7288.
Zhang, L., Li, K., Xiao, W., Zheng, L., Xiao, Y., Fan, H., & Zhang, X. (2011). Preparation of collagen-chondroitin sulfate-hyaluronic acid hybrid hydrogel scaffolds and cell compatibility in vitro. Carbohydrate Polymers, 84(1), 118-125.
23
Page 23 of 37
Figure Captions Figure 1 The reaction scheme between HAs and maleic anhydride.
ip t
Figure 2 The 1H NMR spectra of MeHA and MaHA. Number 1&2 stand for the protons in unsaturated bond in MaHA. Number 1’ stand for the protons in unsaturated bond in MeHA. Number 3 stands for the methyl protons of N-acetyl groups in HAs molecule. The degree of substitution (DS) is calculated according to the integral area of 1&2 and 3 or 1’ and 3.
us
cr
Figure 3 The ATR FT-IR spectra of HAs and HAs derivatives. In MaHA spectra, the bands at 1719 cm-1 to 1576 cm-1 are featured by C=O, C=C and their conjugation effect. These featured bands are the main difference in the spectra of original HAs and MeHA.
an
Figure 4 The storage and loss modulus of MaHA hydrogels. Except for 50MaHA and 65MaHA, significant difference exists between each group of MaHA hydrogels.
M
Figure 5 The swelling ratios of MaHA hydrogels at time interval and the first order exponential decay equation (ExpDec1) fitting curves. The symbols stand for the swelling ratios of different MaHA hydrogels. The lines are fitted according to ExpDec1.
d
Figure 6 The spline connected line of swelling ratio at equilibrium of MaHA hydrogels. It is calculated based on the ExpDec1 fitting results of swelling ratio.
te
Figure 7 Data plotted according to the first-order kinetics (Equation 2, section 2.2.5.1) and the fitting lines. Only the early stage of the swelling process can be fitted. The deviation might be caused by change of the hydrogels thickness and the variation of diffusion coefficient during the swelling process. The diffusion coefficients (D) are calculated accordingly.
Ac ce p
524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555
Figure 8 Data plotted according to the second-order kinetics (Equation 6, section 2.2.5.1) and the fitting lines. The initial swelling rate (Qi) and the equilibrium swelling ratio (Qe’) are calculated based on the parameters of the fitting lines.
24
Page 24 of 37
O
O HO
ONa O OH
O
O O
+
Dry Formamide
NH CH3
O
50oC
O
O HO
ONa O
OH
O
n
O
HO OH
O O
O O
NH CH3
n
te
d
M
an
us
cr
Figure 1 The reaction scheme between HAs and maleic anhydride.
Ac ce p
555 556 557
OH
O
OH
ip t
O
25
Page 25 of 37
M
te
d
Figure 2 The 1H NMR spectra of MeHA and MaHA. Number 1&2 stand for the protons in unsaturated bond in MaHA. Number 1’ stand for the protons in unsaturated bond in MeHA. Number 3 stands for the methyl protons of N-acetyl groups in HAs molecule. The degree of substitution (DS) is calculated according to the integral area of 1&2 and 3 or 1’ and 3.
Ac ce p
558 559 560 561 562 563
an
us
cr
ip t
557
26
Page 26 of 37
cr
ip t
563
te
d
M
an
Figure 3 The ATR FT-IR spectra of HAs and HAs derivatives. In MaHA spectra, the bands at 1719 cm-1 to 1576 cm-1 are featured by C=O, C=C and their conjugation effect. These featured bands are the main difference in the spectra of original HAs and MeHA.
Ac ce p
565 566 567 568
us
564
27
Page 27 of 37
568 350
300
*
* *
*
07MaHA
15MaHA
E' E''
*
ip t
200
150
cr
E' & E''(kPa)
250
us
100
50
25MaHA
50MaHA
65MaHA
75MaHA
MaHA hydrogels
te
d
M
Figure 4 The storage and loss modulus of MaHA hydrogels. Except for 50MaHA and 65MaHA, significant difference exists between each group of MaHA hydrogels.
Ac ce p
569 570 571 572
an
0
28
Page 28 of 37
572 160
ip t
120
100
cr
80
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75HA-MA ExpDec1 Fit of Swelling Ratio
60
us
Swelling Ratio (g Sol/g Gel)
140
40
0
10
15
20
25
30
Time (Hrs)
te
d
M
Figure 5 The swelling ratios of MaHA hydrogels at time interval and the first order exponential decay equation (ExpDec1) fitting curves. The symbols stand for the swelling ratios of different MaHA hydrogels. The lines are fitted according to ExpDec1.
Ac ce p
573 574 575 576 577
5
an
20
29
Page 29 of 37
577 Spline Line of Qe
150
ip t
140 130
cr
120 110 100 90 80 10
20
30
50
60
70
80
DS (%)
578
te
d
M
Figure 6 The spline connected line of swelling ratio at equilibrium of MaHA hydrogels. It is calculated based on the ExpDec1 fitting results of swelling ratio.
Ac ce p
579 580 581
40
an
0
us
Swelling Ratio at equilibrium (Qe)
160
30
Page 30 of 37
581
5
3
cr
ln(Qe/Qe-Q)
4
ip t
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA Linear Fit of ln(Qe/Qe-Q)
6
2
us
1
0
10
15
20
25
30
Time (hrs)
te
d
M
Figure 7 Data plotted according to the first-order kinetics (Equation 2, section 2.2.5.1) and the fitting lines. Only the early stage of the swelling process can be fitted. The deviation might be caused by change of the hydrogels thickness and the variation of diffusion coefficient during the swelling process. The diffusion coefficients (D) are calculated accordingly.
Ac ce p
582 583 584 585 586 587
5
an
0
31
Page 31 of 37
587 0.32 0.28
0.20
cr
0.16 0.12 0.08
us
t/Q (g Gel/g Sol hrs)
0.24
ip t
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA Linear Fit of t/Q
0.04
0
10
15
20
25
30
Time (hrs)
te
d
M
Figure 8 Data plotted according to the second-order kinetics (Equation 6, section 2.2.5.1) and the fitting lines. The initial swelling rate (Qi) and the equilibrium swelling ratio (Qe’) are calculated based on the parameters of the fitting lines.
Ac ce p
588 589 590 591 592
5
an
0.00
32
Page 32 of 37
Table 1 MaHA and MeHA Specimens with various DS 03MeHA 07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
10 5 7.5 10 12.5 15 20
pH
DS
4 50 50 50 50 50 50
8.0-9.0 / / / / / /
3.33% 7.13% 14.71% 25.92% 50.14% 66.15% 75.47%
* The reaction time was 5 hrs for all cases.
us an M d te Ac ce p
593 594
MAA MA MA MA MA MA MA
Temp (oC)
ip t
Specimen Reactant Dosage*
cr
592
33
Page 33 of 37
Table 2 Swelling Parameters of Simulation Equations of MaHA Hydrogels Based on Increasing DS y0
A
t
R2
T80% hrs
T90% hrs
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
154.92±4.27 149.93±1.88 104.35±4.23 106.26±2.79 106.15±4.84 125.93±1.47
-115.07±4.68 -106.09±1.83 -68.56±4.48 -71.01±2.40 -70.19±4.73 -88.23±1.92
7.08±0.49 8.94±0.28 4.84±0.69 4.34±0.41 5.81±0.76 6.76±0.44
0.9902 0.9986 0.9850 0.9936 0.9879 0.9963
9.30 11.30 5.77 5.24 6.95 8.48
14.21 17.50 9.13 8.26 10.98 13.17
Ac ce p
te
d
M
an
us
597 598 599
ip t
Sample
cr
594 595 596
34
Page 34 of 37
601
Table 3 The Parameters of Fitting Lines According to Equation 2* and Diffusion Coefficients (D) Slope
Intercept
R2
D (cm2/s)×10 -7
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
0.1128±0.0066 0.0974±0.0021 0.1698±0.0083 0.1837±0.0103 0.1673±0.0045 0.1376±0.0088
0.3744±0.0472 0.3696±0.0148 0.4964±0.0597 0.5216±0.0745 0.4253±0.0324 0.4655±0.0633
0.9801 0.9973 0.9859 0.9813 0.9957 0.9761
4.46 4.44 6.14 5.74 6.21 5.11
ip t
Sample
cr
599 600
*Equation 2:
Ac ce p
te
d
M
an
us
602 603
35
Page 35 of 37
B×103
A×102
R2
Qi
Qe’
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
5.76±0.19 5.86±0.18 8.30±0.15 8.59±0.12 8.03±0.13 7.30±0.13
2.23±0.27 2.76±0.29 2.26±0.23 1.76±0.19 2.48±0.21 1.95±0.20
0.9922 0.9924 0.9975 0.9984 0.9978 0.9976
44.80 36.23 44.27 56.92 40.27 51.20
173.61 170.65 120.48 116.41 124.53 136.99
*Equation 6:
ip t
Sample
cr
606
Table 4 The parameters of Fit Lines According to Equation 6* and Swelling Ratios at Initial (Qi) and Equilibrium (Qe’)
us
603 604 605
Ac ce p
te
d
M
an
607
36
Page 36 of 37
607 608
Table 5 The Structure Parameters of MaHA Hydrogels
(mol/cm3)
(g/mol) 1.86×105 1.67×105 1.15×105 1.19×105 1.19×105 1.45×105
(nm)
6.59×10-6 7.38×10-6 1.07×10-5 1.04×10-5 1.03×10-5 8.47×10-6
434 406 298 305 306 357
2.12×105 1.95×105 1.41×105 1.35×105 1.49×105 1.62×105
(mol/cm3) 5.79×10-6 6.30×10-6 8.71×10-6 9.08×10-6 8.23×10-6 7.57×10-6
(nm) 480 457 347 336 361 388
Ac ce p
te
d
M
an
us
609 610
(g/mol)
ip t
07MaHA 15MaHA 25MaHA 50MaHA 65MaHA 75MaHA
Qe’
cr
Qe Sample
37
Page 37 of 37
