Preparation, characterization, and biocompatibility evaluation of poly(Nε-acryloyl-l-lysine)/hyaluronic acid interpenetrating network hydrogels

Article abstract of DOI:10.1016/j.carbpol.2015.09.095

In the present study, poly(N_ε-acryloyl-l-lysine)/hyaluronic acid (pLysAAm/HA) interpenetrating network (IPN) hydrogels were successfully fabricated through the combination of hydrazone bond crosslinking and photo-crosslinking reactions. The HA hydrogel network was first synthesized from 3,3′-dithiodipropionate hydrazide-modified HA and polyethylene glycol dilevulinate by hydrazone bond crosslinking. The pLysAAm hydrogel network was prepared from N_ε-acryloyl-l-lysine and N,N′-bis(acryloyl)-(l)-cystine by photo-crosslinking. The resultant pLysAAm/HA hydrogels had a good shape recovery property after loading and unloading for 1.5 cycles (up to 90%) and displayed a highly porous microstructure. Their compressive moduli were at least 5 times higher than that of HA hydrogels. The pLysAAm/HA hydrogels had an equilibrium swelling ratio of up to 37.9 and displayed a glutathione-responsive degradation behavior. The results from in vitro biocompatibility evaluation with pre-osteoblasts MC3T3-E1 cells revealed that the pLysAAm/HA hydrogels could support cell viability and proliferation. Hematoxylin and eosin staining indicated that the pLysAAm/HA hydrogels allowed cell and tissue infiltration, confirming their good in vivo biocompatibility. Therefore, the novel pLysAAm/HA IPN hydrogels have great potential for bone tissue engineering applications.

Full text of DOI:10.1016/j.carbpol.2015.09.095

Accepted Manuscript
Title: Preparation, characterization and biocompatibility
evaluation of poly(N␧-acryloyl L-lysine)/hyaluronic acid
interpenetrating network hydrogels
Author: Ning Cui Junmin Qian Weijun Xu Minghui Xu Na
Zhao Ting Liu Hongjie Wang
PII:
S0144-8617(15)00960-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.095
CARP 10397
To appear in:
Received date:
Revised date:
Accepted date:
7-8-2015
23-9-2015
26-9-2015
Please cite this article as: Cui, N., Qian, J., Xu, W., Xu, M., Zhao, N.,
Liu, T., and Wang, H.,Preparation, characterization and biocompatibility
evaluation
interpenetrating
http://dx.doi.org/10.1016/j.carbpol.2015.09.095
of
poly(Nrmvarepsilon-acryloyl
network hydrogels, Carbohydrate
L-lysine)/hyaluronic
Polymers
acid
(2015),
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Preparation, characterization and biocompatibility evaluation of poly(Nɛ-acryloyl
L-lysine)/hyaluronic acid interpenetrating network hydrogels
*
Ning Cui, Junmin Qian jmqian@mail.xjtu.edu.cn, Weijun Xu, Minghui Xu, Na Zhao, Ting Liu,
Hongjie Wang
State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 710049, China
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Tel.: +86 29 82668614; fax: +86 29 82663453
Highlights
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► Poly(Nɛ-acryloyl L-lysine)/hyaluronic acid hydrogels were prepared by hydrazone bond crosslinking and photo-crosslinking.
► Poly(Nɛ-acryloyl L-lysine)/hyaluronic acid hydrogels exhibited a good shape recovery property.
► Poly(Nɛ-acryloyl L-lysine)/hyaluronic acid hydrogels showed a glutathione-responsive degradation behavior.
► Poly(Nɛ-acryloyl L-lysine)/hyaluronic acid hydrogels exhibited good in vitro and in vivo biocompatibility.
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Abstract
In the present study, poly(N -acryloyl L-lysine)/hyaluronic acid (pLysAAm/HA) interpenetrating
ɛ
network (IPN) hydrogels were successfully fabricated through the combination of hydrazone bond
crosslinking and photo-crosslinking reactions. The HA hydrogel network was first synthesized from
3,3'-dithiodipropionate hydrazide-modified HA and polyethylene glycol dilevulinate by hydrazone
bond crosslinking. The pLysAAm hydrogel network was prepared from N -acryloyl L-lysine and
ɛ
N,N'-bis(acryloyl)-(L)-cystine by photo-crosslinking. The resultant pLysAAm/HA hydrogels had a
good shape recovery property after loading and unloading for 1.5 cycles (up to 90%) and displayed a
highly porous microstructure. Their compressive moduli were at least 5 times higher than that of HA
hydrogels. The pLysAAm/HA hydrogels had an equilibrium swelling ratio of up to 37.9 and
displayed a glutathione-responsive degradation behaviour. The results from in vitro biocompatibility
evaluation with pre-osteoblasts MC3T3-E1 cells revealed that the pLysAAm/HA hydrogels could
support cell viability and proliferation. Hematoxylin and eosin staining indicated that the
pLysAAm/HA hydrogels allowed cell and tissue infiltration, confirming their good in vivo
biocompatibility. Therefore, the novel pLysAAm/HA IPN hydrogels have great potential for bone
tissue engineering applications.
Keywords
Hyaluronic acid; poly(Nɛ-acryloyl L-lysine); hydrogel; bone tissue engineering; biomaterials
Introduction
Researchers involved in tissue engineering are intensively investigating materials that have the
potential to repair and replace lost or damaged tissues. A desired material for bone repair not only
must be biocompatible, bioactive and osteoinductive, but also should have composition and structure
similar to bone (Chen et al., 2011; Jee, Thula, & Gower, 2010; Abdal-hay, Vanegas, Hamdy, Engel &
Lim, 2014). Hydrogels, which are hydrophilic polymers of three-dimensional networks that are able
to swell in water without changing their integral structure (W. Wei et al., 2015; Ryu et al., 2014; Heo
et al., 2014), have been attracting increasing attention in biomedical fields such as tissue engineering
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(Daniele, Adams, Naciri, North, & Ligler, 2014; Munoz-Pinto, Jimenez-Vergara, Gharat, & Hahn,
2015) and drug delivery system (Matricardi, Meo, Coviello, Hennink, & Alhaique, 2013; Wu, Zhao,
Wu, & Chu, 2014; Barbucci, Giani, Fedi, Bottari, & Casolaro, 2012). However, most hydrogels
suffer from insufficient strength or toughness and cannot meet the requirement of daily compressive
stress during the bone healing (Bose, Roy, & Bandyopadhyay, 2012; K. Yasuda et al., 2005; Sharifi,
Blanquer, van Kooten, & Grijpma, 2012). Therefore, preparing hydrogels with excellent mechanical
properties for bone tissue engineering is still a big challenge.
Recently, interpenetrating network (IPN) hydrogels have attracted a great deal of attention due to
their high fracture toughness and strength while providing a temporary mechanical support, and
various IPN hydrogels synthesized from different polymers have been reported (Fan, Liao, Zhang, &
Liu, 2013; Teng et al., 2014). In most cases of IPN hydrogels, the first network is stiff and brittle
whereas the second network is soft and ductile (Hattori, Ishii, Tominaga, Osada, & Tahara, 2013;
Wang, & Han, 2011; Haque, Kurokawa, & Gong, 2012). Hyaluronic acid (HA), one of natural
polysaccharides, exhibits more versatile biological functions than synthetic polymers (Zawko, &
Schmidt, 2010). HA hydrogels are widely used to mimick the structure and biological functions of
native extracellular matrix (Tokatlian, Cam, Siegman, Lei, & Segura, 2012; Tokatlian, Cam,
Siegman, Lei, & Segura, 2012). However, the mechanical weakness of HA hydrogels limits their
application in bone tissue engineering (Lam, Truong, & Segura, Acta Biomater.,2014). Recently, HA
has been used as one precursor to fabricate IPN hydrogels due to its good toughness and
cytocompatibility (Fan, Liao, Zhang, & Liu, 2013).
Amino-acid-based polymers can be divided into two groups, depending on whether the amino acid
moieties are located in the main chains or side chains (Dutta, Dey, Ghosh, & Nayak, 2009;
Hashidzume, Kawata, Tanaka, Takabayashi, & Sato, 2010). Many synthetic polypeptides that contain
amino acid moieties in their main chains are synthesized by the ring-opening polymerization of
N-carboxy-amino acid anhydrides (Xu et al., 2015; Qian et al., 2015; Zhang, Fu, & Li, 2014). In
recent years, much interest has also been devoted to preparing well-defined polymers called
poly(N-acryloyl amino acid)s with amino acid moieties in their side chains, which are usually
synthesized by free radical polymerization of N-acryloyl amino acids (Buruiana, Buruiana, & Hahui,
2007; Karbarz, Pyrzynska, Romanski, Jurczak, & Stojek, 2010; Liu, Jin, Zhang, Han, & Ji, 2015).
Compared with the ring-opening polymerization to synthesize polypeptides, the free radical
polymerization of N-acryloyl amino acids has some advantages, such as facile molecular design, easy
operation, mild reaction conditions and being able to react in aqueous solutions (Oi, Hashidzume, &
Harada, 2011). However, the heterogeneous distribution of crosslinking points in the
photo-crosslinked poly(N-acryloyl amino acid) hydrogels would lead to the appearance of stress
concentration around the positions with a high crosslinking density, causing the hydrogels to break at
a low stress (Liu, Jin, Zhang, Han, & Ji, 2015; Hashidzume, Tanaka, & Sato, 2010). As we know,
poly-L-lysine (PLL), as a cationic polymer, has an outstanding ability to promote cell attachment and
proliferation through electrostatic interactions (Amorim, Martins, Neves, Reis, & Pires, 2014; Qi,
Yuan, Yan, & Wang, 2014). Poly(N -acryloyl L-lysine) (pLysAAm) was expected to show the same
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properties since it possesses side chains containing free amino groups similar to PLL. In addition,
polyacrylic acid (PAA) has also been used to fabricate the hydrogel to facilitate cell infiltration due to
its electroresponsive nature (Rahimi, Molin, Cleij, van Zandvoort, & Post, 2012). Therefore, it is
postulated that PLysAAm shows good cell attachment behavior like PLL and PAA because its
chemical structure is similar to those of PLL and PAA. On the other side, HA hydrogels are often
prepared for bone tissue engineering applications, but their mechanical strength is not satisfied (Bae
et al., 2011; Bae et al., 2014). In the present study, plysAAm was first synthesized and used to modify
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HA hydrogels for bone tissue engineering. As far as we know, an IPN hydrogel composed of
pLysAAm and HA has not been reported.
In this work, we designed and fabricated novel pLysAAm/HA IPN hydrogels. The first network HA
hydrogel was synthesized according to our previously reported method (Cui, Qian, Liu, Zhao, &
Wang, 2015). The second network pLysAAm hydrogel was photosynthesized from monomer
N -acryloyl L-lysine using N,N'-bis(acryloyl)-(L)-cystine (BACystine) as the crosslinker. The effects
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of the crosslinking density of pLysAAm hydrogel on the morphology, viscoelasticity, swelling and
degradation properties of the pLysAAm/HA hydrogels were studied. The in vitro biocompatibility of
the pLysAAm/HA hydrogels was evaluated with MC3T3-E1 cells by the live/dead cell viability and
MTT assays. Hematoxylin and eosin (H&E) staining was employed to characterize their in vivo
biocompatibility. The results showed that the pLysAAm/HA hydrogels had desired mechanical
strength, glutathione (GSH)-responsive degradation behaviour and good biocompatibility.
Experimental
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Materials
L-Lysine hydrochloride, L-cystine dihydrochloride, acryloyl chloride, hyaluronic acid sodium (HA,
molecular weight: 300 kDa), 3,3'-dithiodipropionic acid, levulinic acid (LEV),
N-hydroxysuccinimide (NHS) and GSH were purchased from Aladdin Reagent Inc. (Shanghai,
China). Polyethylene glycol (PEG, 2000 Da), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC·HCl), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
and 2-hydroxy-4ʹ-(2-hydroxyetho
-xy)-2-methylpropiophenone (Irgacure 2959) were purchased from Sigma-Aldrich. The live/dead
viability/cytotoxicity kit for mammalian cells was purchased from Invitrogen (Carlsbad, CA, USA).
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from
Procell (Wuhan, China). H&E staining kit was purchased from BOSTER (Wuhan, China). Both
MC3T3-E1 cells and Sprague Dawley (SD) rats were supplied by Medical Center of Xi'an Jiaotong
University (Xi'an, China). All other chemicals were of analytical grade and used without further
purification. All aqueous solutions were prepared using ultrapure water with a resistance of 18.25
MΩ.
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1 Fabrication of pLysAAm/HA hydrogels
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1.1 Synthesis of HA hydrogels
To synthesize HA hydrogels, 3,3'-dithiodipropionate hydrazide (DTPH), 3,3'-dithiodipropionate
hydrazide-modified HA (DTPH-HA) and polyethylene glycol dilevulinate (LEV-PEG-LEV) were
first synthesized according to the reported procedures with some changes (Cui, Qian, Liu, Zhao, &
Wang, 2015; Xu et al., 2013). The procedure of synthesizing DTPH was as follows. In brief,
3,3'-dithiodipropionic acid (10 g, 47.6 mmol), ethanol (22.1 g, 480 mmol) and p-toluenesulfonic acid
(0.4 g, 2.1 mmol) were dissolved in toluene (30 ml). The reaction mixture was stirred for 24 h under
reflux using a Dean-Stark trap, and the product dithiodipropionate diethyl was obtained after the
reaction mixture was washed with NaHCO solution and water to pH 7. After this, dithiodipropionate
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diethyl (10 g, 37.5 mmol) and hydrazine (12 g, 240 mmol) were dissolved in ethanol (20 mL), and the
mixture was stirred for 5 h at 85░°C to produce DTPH. Pure DTPH was obtained after the crude
product was recrystallized from ethanol-water solution (5% v/v) three times. Yield: 70%, m.p.
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131-132░°C. H NMR (DMSO-d , δ ppm): 2.53 (t, -SCH CH -), 2.85 (t, -SCH CH -), 4.2 (s, -NH ),
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9.1 (s, -NH-). The procedure for synthesizing DTPH-HA was as follows. Briefly, HA (300 mg, 0.75
mmol repeating units) and DTPH (802.4 mg, 3.37 mmol) were first dissolved in deionized water (60
ml), and the pH of the solution was adjusted with 1 M HCl to 4.8-5.0. After EDC·HCl (72.8 mg, 0.38
mmol) and NHS (43.7 mg, 0.38 mmol) were added, the mixture solution was stirred for 24 h at room
temperature. The resulting solution was dialyzed (MWCO 3500) against distilled water for 3 days and
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lyophilized, obtaining the product of DTPH-HA. H NMR (D O, δ ppm): 1.9 (s, CH CO-), 2.53 (t,
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-SCH CH -), 2.85 (t, -SCH CH -), 3.2-3.8 (m, the proton chemical shifts of the skeleton of HA).
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Solid state C NMR (δ ppm): 23.5 (s, CH CO-), 32.6 (s, -SCH CH -), 35.6 (s, -SCH CH -), 74.6 (m,
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the carbon chemical shifts of the skeleton of HA), 102.7 (s, -O-CH-O-), 174.2 (s, -CONH-). The
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degree of substitution of DTPH-HA was determined to be about 40% by H NMR spectrum using the
ratio of the methylene protons of DTPH to the acetyl methyl protons of HA. To synthesize crosslinker
LEV-PEG-LEV, a mixture of PEG (5 g, 2.5 mmol), LEV (2.88 g, 12.5 mmol) and p-toluenesulfonic
acid (9.5 mg, 0.05 mmol) was dissolved in 50 ml of toluene in a 250-ml three-neck flask, and the
solution was heated under reflux for 12 h. After the reaction had been completed, the majority of the
solvent was removed under vacuum, and the residue was poured into an excess amount of cold
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diethyl ether. The product was obtained as a faint yellow solid. H NMR (CDCl , δ ppm): 2.22 (s,
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CH CO-), 2.63 (t, -CH CH CO-), 2.79 (t, -CH CH CO-), 3.7 (m, -OCH CH -), 4.27 (s, -COOCH -).
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C NMR (CDCl , δ ppm): 28.2 (s, CH CO-), 30.3 (s, -CH CH CO-), 38.3 (s, -CH CH CO-), 63.9 (s,
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-CH COOCH -), 70.7 (s, -OCH CH -), 172.4 (t, -CH CH CO-), 206.6 (s, CH CO-).
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HA hydrogel was prepared as follows: DTPH-HA (60 mg, 129 µmol) was dissolved in water (1.6 ml),
and then 0.4 ml of aqueous solution containing LEV-PEG-LEV (6.45 µmol) was added. The mixture
was vigorously vortexed, defoamed and left to stand. A light yellow gel was formed within 30 min.
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Solid state C NMR (δ ppm): 22.9 (s, CH CO- in the pendant groups of HA), 27.7 (s, CH COCH -),
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29.6 (m, -CH CH CO- and -SCH CH -), 37.7 (m, -CH CH CO- and -SCH CH -), 63.9 (s,
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-CH COOCH -), 70.7 (s, -OCH CH -), 71-74.6 (m, the carbon chemical shifts of the skeleton of
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HA), 102.8 (s, -O-CH-O-), 166.8-169.7 (s, CH C=NNHCO-), 174.2 (s, CH CO-, -CO-NHNH-,
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-CH CH COO-).
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Synthesis of pLysAAm/HA hydrogels. To obtain the second network pLysAAm hydrogel, a
monomer LysAAm and a crosslinker BACystine needed to be synthesized according to the reported
procedures with some minor changes (Emilitri, Ranucci, & Ferruti, 2005). The monomer LysAAm
was synthesized as follows. L-Lysine hydrochloride (5.3 g, 29 mmol) and basic cupric carbonate
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(4.78 g, 20 mmol) were dissolved in water (50-60 ml) and the system was stirred at 101-105░°C for 3
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h. After the insoluble residue was removed by filtration, the pH of the solution was adjusted to 9-9.5
with solid NaHCO . Then, acryloyl chloride (3.54 ml, 43.5 mmol) was added dropwise into the
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solution at 0-5░°C and the pH of the mixture was maintained at 8.5-9 by adding 2 M NaOH aqueous
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solution. After stirring at room temperature for another 12 h, the blue precipitates were filtrated,
washed successively with water, methanol and ether, and dried in vacuum. The obtained blue solid (2
g, 4.33 mmol) was dispersed in water (30 ml) in a separation funnel, and a solution of 8-quinolinol
(1.89 g, 13 mmol) in chloroform (30 ml) was added. After shaking for 12 h, a green precipitate in the
chloroform phase was removed by filtration. Three extractions with chloroform were performed to
remove traces of 8-hydroxy quinoline. The aqueous phase was lyophilized, obtaining a white
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powdered solid. Yield: 92%. The chemical structure of LysAAm was characterized by H NMR using
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D O as the solvent. H NMR (D O, δ ppm): 1.37 (m, CH ), 1.57 (m, CH ), 1.83 (m, CH ), 3.24 (t,
2 2 2 2 2
CH ), 3.69 (t, CH), 5.71 (d, CH =CH [trans]), 6.18 (d, CH =CH [cis]), 6.21 (dd, CH =CH). The
BACystine was synthesized as follows. L-cystine dihydrochloride (5 g, mol) and NaOH (1.7 mg)
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were dissolved in water (50 mL), and the solution was cooled to 0-5░°C in an ice bath. A solution of
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acryloyl chloride (10 M) in dichloromethane (10 mL) and a NaOH aqueous solution (1 M) were
added simultaneously dropwise under stirring over a total time span of 1.5 h. During the process, the
pH of the reaction mixture was maintained at 9-10. After the addition had been completed, the
reaction mixture was stirred at room temperature for an additional 2 h. Then the pH of the reaction
mixture was adjusted with 2 M HCl to 2 and the crude product was precipitated in the excess of
acetone. The solid product was further purified by repeating the precipitation process thrice. Yield:
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56%. The chemical structure of BACystine was characterized by H NMR using D O as the solvent.
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H NMR (D O, δ ppm): 2.91-3.23 (m, CH ), 4.55 (m, CH), 5.73 (d, CH =CH [trans]), 6.14 (d,
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CH =CH [cis]), 6.29 (dd, CH =CH).
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PLysAAm/HA hydrogels were fabricated according to the following procedure. The lyophilized HA
hydrogels were immersed with an aqueous solution of LysAAm (2 mmol), BACystine (0.2 mmol, 0.4
mmol or 0.6 mmol) and photoinitiator (Irgacure 2959, 0.01 mmol) in 2 ml water at room temperature
in dark. White pLysAAm/HA hydrogels were formed after exposure to UV light radiation (365 nm)
for 30 s. According to the weight percentages of BACystine relative to LysAAm (10%, 20% and
30%), the formed pLysAAm/HA hydrogels were designated as pLysAAm10/HA, pLysAAm20/HA
and pLysAAm30/HA, respectively.
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2 Structural characterization of pLysAAm/HA hydrogels
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H NMR spectra of LysAAm and BACystine were recorded on a Bruker 400 MHz spectrometer
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(Bruker, Germany). Solid state C NMR spectrum of HA hydrogel was recorded on a Bruker
AVANCE Ш HD 400 MHz spectrometer (Bruker, Germany). To observe the structure and
morphology of pLysAAm/HA hydrogels, pLysAAm/HA hydrogel samples were prefrozen for 2 h at
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-50░°C at a cooling rate of 2░°C/min and freeze-dried at -50░°C for 72 h. The lyophilized samples
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were sliced, sputter-coated with gold and observed by scanning electron microscopy (SEM, S-3400
N, Hitachi, Japan) at an accelerating voltage of 20 kV. The compressive behavior was measured using
an Instron 5943 universal testing instrument at a constant rate of 1 mm/min. The compressive moduli
were calculated according to the data of strain range of 10-20% in stress-strain curves. Rheological
characterization was performed on a TA instrument DHR-2 rheometer using a parallel plate with a
diameter of 20 mm and a sample gap of 1000 µm) in a frequency range of 0.1-10 Hz. The
measurements were performed using a dynamic frequency sweep test, which covered a frequency
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range from 0.1-100 rad/s at a shear amplitude of 1% at 37░°C. Degradation tests were carried out in
PBS (pH 7.4) containing GSH (5 mM). Swelling tests were carried out in PBS (pH=7.4) solution at
37░°C. The swelling ratio was calculated according to the following equation:
Swelling ratio (%)=[(W -W )/W ]×100
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Where, W and W were the mass of dried and swollen pLysAAm/HA hydrogels, respectively.
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In vitro cell culture
MC3T3-E1 cells were cultured in DMEM supplemented with 10% FBS and 1%
penicillin/streptomycin in a humidified atmosphere with 5% of CO at 37 °C for 1 day and 3 days.
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Lyophilized pLysAAm/HA hydrogel samples were sterilized with ethylene oxide and then placed in a
48-well culture plate. MC3T3-E1 cells were seeded onto the surface of dry pLysAAm/HA hydrogel
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samples at a concentration of 2×10 cells/well suspended in 0.2 ml media. After 4 h of culture, 1 ml of
culture medium was added. The culture medium was replaced every day.
Live/dead cell viability assay
The viability of MC3T3-E1 cells cultivated on pLysAAm/HA hydrogels was evaluated using the
live/dead viability/cytotoxicity assay. At days 1 and 3, the pLysAAm/HA hydrogels were clearly
rinsed with PBS and stained with calcein AM and ethD-1. The living cells were stained green and the
dead cells were stained red. The concentration of both calcein AM and ethidium homodimer (ethD-1)
was fixed at 10 µM. The hydrogel/cell constructs were visualized using a confocal laser scanning
microscope (Leica, TCS SP5 II, Germany).
MTT assay
MTT assay was employed to further evaluate the viability and proliferation of MC3T3-E1 cells on the
pLysAAm/HA hydrogels. After 1 day and 3 days of cultivation, the culture supernatants were
removed and the hydrogel/cell constructs were washed with PBS for three times. MTT (100 µl)
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solution was added to each well and the hydrogel/cell constructs were incubated at 37░°C for 4 h.
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Then the culture medium was removed and the blue formazan product was dissolved with
dimethylsulphoxide. The optical absorbance value of the solution in each well was measured at 490
nm by using a microplate reader (PerkinElmer, EnSpire, America). The control experiments were
carried out in 48-well plate without pLysAAm/HA hydrogels.
In vivo evaluation of biocompatibility
In vivo biocompatibility and vascularization of the hydrogels were evaluated using the dorsal skinfold
chamber model. To assess in vivo inflammatory and angiogenic host tissue response to implanted
pLysAAn/HA hydrogel constructs, SD rats with a body weight of 190-200 g were used. All rats were
purchased from Medical Center of Xi'an Jiaotong University (Xi'an, China). All experiments
involving animals and their care were approved by the local governmental animal care committee and
were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Before the surgeries, the grafts of four groups including pLysAAm10/HA, pLysAAm20/HA and
pLysAAm30/HA hydrogels were prepared and swollen in DMEM medium for 3 days. The
pLysAAm/HA hydrogels of three groups were surgically implanted into the subcutaneous pockets on
the backs of the SD rats after anesthetized by chloral hydrate solution (10% wt/wt, 4 µl/g body
weight). Three rats were used for each experiment. After the surgeries, cefazolin (25 µg/g body
weight) and buprenorphine (0.1 µg/g body weight) were administered and each rat was individually
housed. After 7 days of carefully nurtured, all SD rats were sacrificed for histological analysis. At day
7 after implantation, the rats were euthanized and the hydrogel implants were removed and fixed
overnight at room temperature in buffered formalin (37%, pH 7.2-7.4; Sigma-Aldrich), and
embedded in paraffin, then 7 µm sections were prepared. Hematoxylin and eosin (H&E) staining was
performed on 7 µm thick sections for the observation. The obtained H&E staining images were
analysed by Image-Pro Plus software.
Statistical analysis
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Experimental data were expressed as mean ± standard deviation. The statistical analysis for
significance between the groups was performed by means of Student's t-test using SPSS13.0
software. A confidence level of 95% (p<0.05) was considered to be statistically significant. Each
experiment was done in triplicate.
Results and discussion
Synthesis of pLysAAm/HA IPN hydrogels
The schematic chemical structure of the pLysAAm/HA IPN hydrogels is shown in Fig. 1. The first
network referred to the HA hydrogel that was formed through the hydrazone bond crosslinking
reaction between the ketone groups of LEV-PEG-LEV and the hydrazide groups of DTPH-HA.
DTPH-HA was first dissolved in ultrapure water under stirring to form a solution with a
concentration of 3% w/v. An aqueous solution of LEV-PEG- LEV was mixed vigorously with the
above DTPH-HA solution. HA hydrogel could be formed within 30 min due to the hydrazone bond
crosslinking. The second network involved in the pLysAAm hydrogel that was produced from
monomer LysAAm and crosslinker BACystine via photo-crosslinking reaction. To form the IPN
hydrogel, lyophilized HA hydrogel was infiltrated with the aqueous solution of LysAAm, BACystine
and photoinitiator in dark and then exposed to UV light for 30 s. The second network pLysAAm
hydrogel was formed by free radical polymerization. The final pLysAAm/HA IPN hydrogels were
white and freeze-dried for use.
Morphology of pLysAAm/HA IPN hydrogels
The morphology of lyophilized pLysAAm/HA hydrogels was observed by SEM, and the results are
shown in Fig. 2. All the pLysAAm/HA hydrogels with different degrees of crosslinking showed a
highly porous morphology and good interconnectivity, and their pore sizes varied in the range of 50
µm to 500 µm. Such porous characteristics could be in favour of the attachment and proliferation of
cells (Foss, Merzari, Migliaresi, & Motta, 2013; Qian, Yong, Xu, & Jin, 2013; Xiao et al., 2011).
Moreover, the composition of pLysAAm/HA hydrogels has obvious implications in relation to the
morphology of the pores in terms of size and shape. As shown in Fig. 2a, HA hydrogel presented a
relatively homogeneous porous morphology (50 µm to 200 µm). PLysAAm10/HA hydrogel (Fig. 2b)
showed a non-homogeneous porous morphology (50 µm to 400 µm), which was due to the low degree
of crosslinking of pLysAAm10 hydrogel. PLysAAm20/HA hydrogel (Fig. 2c) displayed a relatively
homogeneous, porous morphology with a pore size range of 50-400 µm. PLysAAm30/HA (Fig. 2d)
hydrogel possessed thick-wall pores with an average size of about 400 µm which were separated by
walls having a thickness of around 50 µm. Therefore, it can be concluded that the porous structure of
the pLysAAm/HA IPN hydrogels strongly depended on the degree of crosslinking of pLysAAm
network.
Rheological analysis
To reveal the effect of degree of crosslinking of the pLysAAm network on the viscoelasticity of the
pLysAAm/HA hydrogels, the rheological behavior of these pLysAAm/HA hydrogels was
characterized by a rheometry, and the corresponding rheological profiles are illustrated in Fig. 3. As
displayed in Fig. 3, the rheological data indicated the frequency dependence of the elastic storage and
viscous loss moduli (G', G''). All the pLysAAm10/HA, pLysAAm20/HA and pLysAAm30/HA
hydrogels exhibited a similar nonlinear rheological behavior, and both the G' and G'' values increased
with increasing shear rate. It was also noted that the G' value increased significantly with increasing
degree of crosslinking while the G'' value changed slightly. For each pLysAAm/HA hydrogel, the G'
value was much higher than the G'' value over the entire range of frequencies. The G' values and
G''/G' ratios of HA, pLysAAm10/HA, pLysAAm20/HA and pLysAAm30/HA hydrogels were 580
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Pa/0.045, 2686 Pa/0.028, 4640 Pa/0.019 and 5891 Pa/0.016, respectively. The higher the G' value
was, the better the elasticity of pLysAAm/HA hydrogel would be (Wang, Zhao, Zhang, Yang, & Jia,
2014; Huang, Ravindran, Yin, & George, 2014; Anderson et al., 2008). The G''/G' ratio significantly
decreased as the degree of crosslinking increased, suggesting that the elasticity increased with
increasing crosslinking density (Anderson et al., 2008; Doran, Ryan, & Nilsson, 2014; Seekell,
Dever, & Zhu, 2014).
Compressive test
In order to obtain the shape recovery property and compressive modulus of pLysAAm/HA hydrogels,
the hydrogels were characterized by loading and unloading for 1.5 cycles in the strain range of
0-45%. As shown in Fig. 4, the residual strain of HA, pLysAAm10/HA, pLysAAm20/HA and
pLysAAm30/HA hydrogels was 2.6%, 4.5%, 9.4% and 14.1%, respectively, when the compressive
stress decreased to zero. The corresponding percentages of shape recovery were 94.2%, 90%, 79.1%
and 68.7%, respectively. It was obvious that the pLysAAm10/HA hydrogel had better shape recovery
capacity and smaller hysteresis loop area in comparison with the other two pLysAAm/HA hydrogels,
The reason for this is that the pLysAAm10/HA hydrogel had lower degree of crosslinking, allowing
easier shape recovery (Zhou, Wu, Yue, & Zhang, 2011; Shin, Olsen, & Khademhosseini, 2012, 33,
3143-3152). By comparing all the curves in Fig. 4, it could be found that the pLysAAm30/HA
hydrogel showed the biggest hysteresis loop area and the highest ultimate stress, which meant the
most energy had been stored in the hydrogel after loading and unloading cycle (F. Yu et al., 2014;
Zhang et al., 2011). In addition, the compressive moduli of the four hydrogels were 8.1±0.2 kPa,
49.2±0.5 kPa, 51.7±0.8 kPa and 77.8±0.5 kPa, respectively. The increasing degree of crosslinking of
plysAAm network could improve the compressive moduli of the pLysAAm/HA hydrogels, which
was consistent with the rheological data.
Swelling analysis
The degree of crosslinking of the pLysAAm network had an obvious effect on the swelling
equilibrium of the pLysAAm/HA hydrogels (Fig. 5). As shown in Fig. 5, the swelling ratio of the
pLysAAm/HA hydrogels increased nearly linearly with swelling time within the first 10 h. The
swelling ratio increased slightly from 10 to 36 h and then almost kept constant from 36 to 48 h, which
meant that the swelling equilibrium was reached. The equilibrium swelling ratios of the
pLysAAm10/HA, pLysAAm20/HA, pLysAAm30/HA hydro-gels were 37.9, 30.6 and 23.3,
respectively, which were much lower than that of HA hydrogel (48.7). The results showed that the
equilibrium swelling ratio decreased with increasing degree of crosslinking of the pLysAAm
network, which is consistent with the phenomena reported in the literature (Buller, Laschewsky, &
Wischerhoff, 2013; Fan, Zhang, Jing, Liao, & Liu, 2013).
Degradation evaluation
The hydrogels containing disulfide bonds are expected to be degradable in response to some reducing
agents such as GSH, which can cleave the disulfide bonds. Here, in order to reveal the degradation
behavior of the pLysAAm/HA hydrogels, their degradation behaviors in response to GSH in PBS
were tested. At predetermined time intervals, the pLysAAm/HA hydrogels were taken out from the
degradation media, dried and weighed, and the results are shown in Fig. 6. It can be seen from Fig. 6
that the residual mass of the pLysAAm/HA hydrogels in the presence of 5 mM GSH decreased nearly
linearly with the degradation time. The reason for this is that the low molecular weight GSH (307 Da)
could rapidly infiltrate into the pLysAAm/HA hydrogels and cleave the disulfide bonds (Choh, Cross,
& Wang, 2011). Furthermore, the degradation rate of the pLysAAm/HA hydro-gels decreased with
increasing degree of crosslinking at the same degradation time, because the pLysAAm/HA hydrogel
with higher degree of crosslinking contained a larger number of disulfide bonds to be cleaved.
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Live/dead cell viability assay of MC3T3-E1 cells on the pLysAAm/HA hydrogels
The viability of MC3T3-E1 cells cultured on the pLysAAm20/HA hydrogels for 1 day and 3 days
was studied by the live/dead cell viability assay. Live/dead staining method is a popular method to
evaluate the cell viability and proliferation in vitro reported in the literature (Cao et al., 2015). In this
study, the viability and proliferation of MC3T3-E1 cells on the pLysAAm20/HA hydrogels were
investigated to confirm the feasibility of the pLysAAm/HA hydrogel to support cell proliferation and
differentiation. Live cells were stained green with calcein AM while dead cells were stained red with
ethD-1. As shown in Fig. 7, the viability of MC3T3-E1 cells on the pLysAAm20/HA hydrogels had
an obvious increase with the increasing culture time. Green fluorescent points representing viable
cells were uniformly distributed inside and on the surface of the pLysAAm20/HA hydrogels. After 1
day of incubation, a few red points existed in the pLysAAm20/HA hydrogel (Fig. 7a), which
indicated that some cells were dead. After 3 days of culture (Fig. 7b), more green fluorescent points
were observed on the pLysAAm20/HA hydrogel, and some green fluorescent points became larger.
Compared with the HA hydrogel (Fig. 7c, d), the cells on pLysAAm20/HA hydrogel displayed a
higher viability ratio, demonstrating the good cytocompatibility of the pLysAAm/HA hydogels. This
may be related to the reciprocal interaction between surface charge of hydrogels and cells (Mekhail,
Jahan, & Tabrizian, 2015; Chong, Chan, Choolani, Lee, & Teoh, 2009). In addition, the higher
mechanical strength and fine porous structure of pLysAAm/HA hydrogel are other two key factors to
accelerate cell proliferation (J. Zhang et al., 2015; Xie et al., 2015).
MTT assay of MC3T3-E1 cells on pLysAAm/HA hydrogels
Generally, a MTT method is used to evaluate the proliferation of cells within hydrogels (Koo et al.,
2015). To further confirm the biocompatibility of the pLysAAm/HA hydrogels, the mitochondrial
activity of MC3T3-E1 cells on the HA and pLysAAm20/HA hydrogels was measured using the MTT
assay, and the results are displayed in Fig. 8. Overall, the statistical analysis revealed that
mitochondrial activity of MC3T3-E1 cells on the HA and pLysAAm20/HA hydrogels obviously
increased with the increasing culture time, which is consistent with that reported in literature (Pan et
al., 2014). There was no statistically significant difference between the pLysAAm/HA hydrogel and
the control group (p>0.05), indicating that the pLysAAm/HA hydrogel has no obvious negative effect
on cell viability. However, there was statistically significant difference between the pLysAAm/HA
hydrogel and the HA hydrogel (p<0.05), which indicated that the pLysAAm/HA hydrogel were more
beneficial to support the viability and proliferation of MC3T3-E1 cells than HA hydrogels. It was
found that the absorbance at 490 nm for HA hydrogel increased from 0.14 to 0.35 when the culture
period was prolonged from 1 day to 3 days. At the same culture intervals, the absorbance at 490 nm
for pLysAAm20/HA hydrogel increased from 0.19 to 0.45. This result meant a significant increase in
the cell population, confirming the great proliferative potential of MC3T3-E1 cells on the
pLysAAm/HA hydrogels. This result was consistent with that of live/dead cell viability assay. After 3
days of culture, the optical absorbance at 490 nm and cell viability of the cells on the
pLysAAm20/HA hydrogel were 0.45 and 90%, respectively, which were higher than those (0.35 and
70%) of the cells on the HA hydrogels. The results suggested that the incorporation of pLysAAm
hydrogel into HA hydrogel could enhance the cell viability of MC3T3-E1 cells, which confirmed the
results of live/dead staining.
Histological analysis
After 7 days of implant placement, the pLysAAm/HA hydrogels were extracted, sliced and stained
with H&E. Representative H&E images of pLysAAm/HA hydrogels at low magnification (a, b and c,
40×) and high magnification (e, f and g, 400×) were shown in Fig. 9. The images showed full
cross-section and surrounding tissue. The penetrating tissue was evident from the periphery, although
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the central void indicated incomplete infiltration. Even though there was a substantial penetration of
tissue, the hydrogels were not completely infiltrated with tissue, as evident from the central voids in
the sections. The presence of neutrophils showed the foreign body reaction of rat model to the
pLysAAm/HA hydrogels. It was also seen from Fig. 9, the pLysAAm/HA hydrogel samples were
stained dark blue and their pores were filled by erythrocytes (red) and neutrophils (blue). All the
hydrogels showed a continuous porous structure after 7 days of implant placement. SD rat cells
invaded the edge zones in the pLysAAm10/HA hydrogel samples (Fig. 9a, e). An unexpected
discovery is that more cells invaded into the pLysAAm/HA hydrogels with the increasing degree of
crosslinking of pLysAAm hydrogel network (Fig. 9b, f and Fig. 9c, g). The results demonstrated that
the amount of infiltrated cells was related to the dense structure and mechanical property of
pLysAAm/HA hydrogels (Kang, Mochizuki, Khademhosseini, Fukuda, & Yang, 2015; Chuang et al.,
2015). Compared with the results of HA hydrogels (Fig. 9d, h), pLysAAm/HA hydrogels could better
support the penetration of tissue and cells. In addition, the ratios of erythrocytes (red) and neutrophils
(blue) in pLysAAm10/HA, pLysAAm20/HA, pLysAAm30/HA and HA hydrogels approximated
1.10:1, 1.05:1, 0.98:1 and 0.33:1, respectively. These results demonstrated that the addition of
pLysAAm to HA hydrogels could effectively improve the biocompatibility.
Conclusions
In this study, the novel pLysAAm/HA IPN hydrogel were synthesized by sequent hydrazone bond
crosslinking and photo-crosslinking reactions. The pLysAAm/HA hydrogels exhibited a highly
porous structure, a high equilibrium swelling ratio and a reduction-responsive degradation behavior.
The pLysAAm/HA hydrogels displayed a better elastic behaviour and a higher compressive modulus
than HA hydrogels. In addition, the pLysAAm/HA hydrogels were more beneficial to support the
viability and proliferation of MC3T3-E1 cells in vitro than HA hydrogels. Preliminary in vivo
evaluation indicated that the pLysAAm/HA hydrogels showed better biocompatibility than HA
hydrogels. Therefore, the novel pLysAAm/HA IPN hydrogels might be a promising candidate in
bone tissue engineering.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (50773062,
50603020) and the Natural Science Basic Research Plan in Shaanxi Province of China (2013K09-27)
and the Fundamental Research Funds for the Central Universities (XJJ2014124, XJJ2013130).
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Fig. 1 Schematic chemical structure of the pLysAAm/HA IPN hydrogel.
Fig. 2 SEM images of lyophilized (a) HA, (b) pLysAAm10/HA, (c) pLysAAm20/HA and (d)
pLysAAm30/HA hydrogels.
Fig. 3 (a) Gʹ and (b) Gʺ of HA, pLysAAm10/HA, pLysAAm20/HA and pLysAAm30/HA hydrogels
versus shear frequency.
Fig. 4 Shape recovery capacities of (a) HA, (b) pLysAAm10/HA, (c) pLysAAm20/HA and (d)
pLysAAm30/HA hydrogels after loading and unloading for 1.5 cycles.
Fig. 5 Swelling kinetics of HA, pLysAAm10/HA, pLysAAm20/HA and pLysAAm30/HA hydrogels
(n=3).
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Fig. 6 In vitro degradation kinetics of HA, pLysAAm10/HA, pLysAAm20/HA and pLysAAm30/HA
hydrogels (n=3).
Fig. 7 Confocal laser scanning micrographs of the MC3T3-E1 cells cultured on the pLysAAm20/HA
(a, b) and HA (c, d) hydrogels for 1 day and 3 days.
Fig. 8 MTT results from the MC3T3-E1 cells cultured on the pLysAAm20/HA and HA hydrogels for
1 day and 3 days (n=3). *p<0.05.
Fig. 9 H&E staining images of (a, e) pLysAAm10/HA, (b, f) pLysAAm20/HA, (c, g)
pLysAAm30/HA and (d, h) HA hydrogels after 7 days of implant placement.
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