glycerophosphate hydrogels and their biodegradation

Accepted Manuscript Injectable chitosan/dextran-polylactide/glycerophosphate hydrogels and their biodegradation Jingjing Wu, Ting Zhou, Jiaoyan Liu, Dr. Ying Wan PII:

S0141-3910(15)30049-5

DOI:

10.1016/j.polymdegradstab.2015.07.018

Reference:

PDST 7706

To appear in:

Polymer Degradation and Stability

Received Date: 21 May 2015 Revised Date:

13 July 2015

Accepted Date: 19 July 2015

Please cite this article as: Wu J, Zhou T, Liu J, Wan Y, Injectable chitosan/dextran-polylactide/ glycerophosphate hydrogels and their biodegradation, Polymer Degradation and Stability (2015), doi: 10.1016/j.polymdegradstab.2015.07.018. 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.

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Injectable chitosan/dextran-polylactide/glycerophosphate hydrogels and their biodegradation Jingjing Wu, Ting Zhou, Jiaoyan Liu, Ying Wan*

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College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China

*

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Corresponding author

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Dr. Ying Wan College of Life Science and Technology Huazhong University of Science and Technology Wuhan 430074, P. R. China Tel: +86 27 87792147 fax: +86 27 87792234 ying_x [email protected] (Y. Wan).

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Abstract: Dextran-polylactide (Dex-PLA) copolymers were synthesized and the selected Dex-PLA with water-soluble characteristics was used together with chitosan and glycerophosphate (GP) to produce injectable chitosan/Dex-PLA/GP hydrogels. Some chitosan/Dex-PLA/GP solutions with designated compositions were able to form into

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hydrogel in a temperature range between around 32 and 35 °C, and their pH values were found to alter between 7.0 and 7.2. Elastic modulus of the optimal chitosan/Dex-PLA/GP gel could reach about 1.0kPa or higher, and meanwhile, it was much higher than their viscous

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modulus, revealing that these chitosan/Dex-PLA/GP gels behave like mechanically strong ones. Compression measurements indicated that the certain chitosan/Dex-PLA/GP gels had

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around 8-fold modulus and strength higher than the chitosan/GP gel, confirming that greatly enhanced compressive properties for chitosan/Dex-PLA/GP gels have been achieved. After 8-week subcutaneous degradation in rats, some chitosan/Dex-PLA/GP gels showed significantly extended degradation endurance compared to the chitosan/GP gel, and the PLA

in

a

controllable

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content in the chitosan/Dex-PLA/GP gels was able to regulate the degradation rate of the gels manner.

These

results

suggest

that

the presently developed

chitosan/Dex-PLA/GP gels have promising potential for injectable gelling applications where

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the gel with mechanically strong features and degradation tolerance is needed. Keywords: chitosan; dextran copolymer; injectable hydrogel; mechanical property;

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degradation 1. Introduction

Stimuli-responsive hydrogels, which usually undergo a phase transition in response to external stimuli: changes in temperature, pH, solvent, ionic strength, electric or magnetic fields and light, have become a topic of extensive research [1-3]. Of them, thermosensitive hydrogels having in situ gelling properties near physiological pH and temperature have received growing attention as temperature is the sole stimulus for their gelation without other requirements for chemical or environmental treatment [3,4]. In general, the hydrogels with 2

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thermosensitive features can be injected into tissue or organ cavity in a minimally invasive manner, and form into solid-like fillers with specific shapes exactly matching with the cavity, which makes them attractive for various biomedical applications [5]. To date, several hydrogels prepared with synthetically sourced polymers, such as N-isopropylacrylamide poly(ethylene

oxide)-b-poly(propylene

oxide)-b-poly(ethylene

oxide)

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(NiPAAM),

(PEO-PPO-PEO) and poly(ethylene glycol)-biodegradable polyester copolymers, have been largely investigated [6-8]. Despite injectability, their gel formation usually requires higher

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polymer concentration, and the resulting hydrogels have slow gel-conversion rate, which leads to limitations of their applications [9]. In particular, NiPAAM and PEO-PPO-PEO are

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known to be non-biodegradable, which could result in resistance to metabolism for their in vivo applications [9,10].

As an alternative, much attention for injectable hydrogels has been given to certain natural polymers [11,12]. Among them, polysaccharides have been largely investigated for in situ forming hydrogels since they have good biocompatibility and biodegradable features that

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are not possessed by many synthetic polymers [12,13]. Of optional polysaccharides, chitosan has been used as hydrogels for varied applications due to its well-demonstrated advantages

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[12,14]. By adding certain polyol salts such as sodium glycerophosphate (GP) into chitosan solution, a type of chitosan/GP gel has been developed, which appears to be a viscous liquid

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at room temperature or below, and is able to convert into a solid-like gel near 37°C at physiological pH, meaning that they have well-defined injectability due to suitable pH and sol-gel transition temperature [15]. Nowadays, chitosan/GP hydrogels have been used as in situ gelling scaffolds for the repair or reconstruction of cartilage, bone, nerve and skin defects [11,16-18], and also, as vehicles for delivering drugs or bioactive molecules [19]. Despite mentioned advantages, the usage of chitosan/GP hydrogels is frequently limited because of their low mechanical strength and high in vivo degradation rate [14,20]. There have been a few efforts towards enhancing chitosan/GP gels so far. An effort was related to 3

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incorporation of collagen into the chitosan/GP gel considering that collagen is a natural polymer with doable tensile strength, and hence, it could be able to enhance the chitosan/GP gel while retaining the injectable nature of the resulting gel [21]. Another effort involved the addition of starch to the chitosan/GP gel, and the resulting chitosan/starch/GP gel had a

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lowered sol-gel transition temperature in comparison to chitosan/GP gel while showing certain enhanced strength [11,22]. In addition, some chopped silk fibers have also been incorporated into the chitosan/GP gel, and the resulting gels show enhanced strength while

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retaining thermosensitive characteristics [23]. However, addtion of silk fibers into the chitosan/GP gel seems to be unfavorable for the injection of gels.

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Although some other biomaterials can be used to mechanically enhance chitosan/GP gels, the resulting gels may lose their gelling properties or injectable characteristics, or otherwise, have an unsuitable sol-gel transition temperature at physiological pH if an inappropriate material is applied to the chitosan/GP gel. In the present study, an attempt was made to enhance the strength of chitosan/GP gel while slowing its degradation by using

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dextran-polylactide (Dex-PLA) as a complementary component. Polylactide (PLA) is known to be linear and biodegradable polyester, and has mechanically strong characteristics and slow

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degradation rate [24-26]. Nevertheless, it will be very difficult to directly incorporate PLA component into the chitosan/GP gel due to the water-insulable features of PLA. Accordingly,

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some Dex-PLAs with water-soluble properties were first synthesized, and they were then added into the chitosan/GP gel as an accessorial component. It is expected that Dex-PLAs will be able to enhance the strength of chitosan/GP gel while extending the degradation duration of the resulting gels. It was found that some chitosan/Dex-PLA/GP gels indeed showed well-defined phase-transition characteristics near physiological temperature and pH whilst having significantly enhanced mechanical strength and prolonged in vivo degradation in comparison to the chitosan/GP gel. Hence, some results for these gels were reported. 2. Experimental 4

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2.1. Materials

Chitosan was purchased from Aofulong Bio-Technology, China. To increase the degree of deacetylation (DDA) of chitosan, the received sample was treated in a 50 % NaOH aqueous solution for 2 h at 95 °C and the alkali treatment was repeated once. Viscosity-average

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molecular weight and DDA of processed chitosan were measured as 7.15(±0.16)×105 and 92.4(±1.8)%, respectively, following reported methods [24]. Dextran (Dex, Mw:23.8k), L-lactic acid (LA), N,N′-carbonyldiimidazole (CDI), 4-(N,N-dimethylamino) pyridine

grade and purchased from SinoPharm, China.

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(DMAP), β-glycerophosphate (GP) disodium salt, and other chemicals were of analytical

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Dex-PLA copolymers were synthesized according to some methods described elsewhere [27-29]. Lactic acid oligomers with terminated mono-hydroxyl groups were first synthesized by ring-opening polymerization of LA with ethanol as an initiator and stannous octoate as a catalyst, and the average degree of polymerization (DP) was tailored by the monomer

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/initiator ratio [27]. The resulting lactic acid oligomers were processed with preparative HPLC (column: ACE C18, 10µ, 21.2×250 mm) to remove short oligomers by using acetonitrile as the solvent, and a gradient was run from 100% A (water/actonitrile 95:5) to 100%

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B (acetonitrile/water 95:5) in 50 min at a flow rate of 5 mL/min [28,29]. The oligomers with DP changing between 20 and 22 were collected for the follow-up coupling reaction. The

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hydroxyl group of the oligomers was activated using CDI and Dex-PLAs were synthesized via a coupling reaction between dextran and activated oligomers [27]. In a typical procedure, 240 mg of Dex and 220 mg of DMAP were dissolved in dry DMSO (8 mL). To this mixture, hydroxyl-activated oligomers (DP: 20-22, 180 mg) dissolved in dry DMSO (5 mL) was added, and the reaction was allowed to perform with stirring at room temperature for 4 days in a nitrogen atmosphere. After that, the reaction was stopped by addition of HCl to neutralize DMAP and imidazole. The obtained precipitate was collected by centrifugation and was washed with acetone. The traces of uncoupled PLAs were removed by Soxhlet extraction using 5

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acetone and the product was dried under vacuum to yield Dex-PLA powder sample. Dex-PLAs with various PLA weight percentages were synthesized by mainly changing the feed ratio of Dex to oligomer, and in some cases, regulating the reaction time (4-6 days) and/or temperature (25-60 °C) [27,30]. The amount of lactic acid oligomers grafted to dextran

anomeric proton of dextran at 4.65 ppm in 1H NMR spectra [28]. 2.2. Preparation of chitosan/Dex-PLA/GP solution

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was calculated using the integral area ratio of CH3 of lactate oligomer at 1.41 ppm to the

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A serious of Dex-PLA solutions was prepared by dissolving from 50 to 200 mg of selected Dex-PLA in 9 ml of 0.1 M HCl at a step-size of 50mg. To each Dex-PLA solution,

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200 mg of chitosan was introduced with stirring, and accordingly, four types of chitosan/Dex-PLA solutions with varied weight ratios of chitosan to Dex-PLA at 4:1, 4:2, 4:3 and 4:4 were produced. These solutions were cooled down to ca. 4 °C, and to each of them, 1 mL of 50 (w/v)% GP solution in distilled water was added dropwise. The resultant

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chitosan/Dex-PLA/GP solutions were additionally stirred to obtain homogenous ones and each solution had a final volume of 10 mL. 2.3. Characterization

H NMR measurements were recorded on a Bruker AV 500 spectrometer using dimethyl

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1

sulfoxide-d6 (DMSO-d6) as solvent.

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Gelation performance was assessed using the inverted tube test. Typically, 2 mL of chitosan/Dex-PLA/GP solution were stirred for around 5 min in an ice/water bath and the solution was then introduced into a glass vial. Gelation time was measured as a function of incubation time that was recorded starting from the incubation of the glass vial in a water bath at 37 °C. The flowability of the solution was examined every 20 sec by inverting the vials. The time at which the gel stopped flowing was designated as the gelation time. 2.4. Rheological measurement Rheological measurements were carried out using a Kinexus Pro KNX2100 Rheometer 6

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(Malvern). The values of the strain amplitude were optimized to ensure that measurements were performed in a linear viscoelastic region in which the elastic modulus (G′) and viscous modulus

(G″)

were

independent

of

the

strain

amplitude.

Aliquots

of

the

chitosan/Dex-PLA/GP solution (2 ml) were introduced onto parallel plate and the temperature

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dependency of G′ and G″ was measured from 25 to 45 °C at a temperature-elevated rate of 1°C/min. To determine incipient gelation temperature (Ti), oscillatory measurements were performed. In regard to the isothermal frequency dependence, G′ and G″ were measured in the

2.5. Compression measurement

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1% strain. The chitosan/GP solution was used as control.

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frequency range changing from 0.1 to 100 Hz, at a fixed temperature of 37 °C and a constant

The above-prepared chitosan/Dex-PLA/GP solutions were injected into a 24-well culture plate and incubated at 37 °C for 20 min to form into gels. Cylinder-shaped gel samples with a diameter of 10 mm and a height of around 7mm were obtained using a hollow sample punch.

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Compressive modulus of gels was assessed in unconfined compression mode at room temperature using a MACH-1TM micromechanical testing system with a 15 N load cell. Samples were compressed at a constant deformation rate of 1.0 mm/min up to a strain of 20%.

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Data for load and displacement were converted into stress-strain values based on initial dimensions of samples. Compressive modulus (E) was determined using the slope of the

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initial linear region of stress-strain curves and the stress at 10% strain (σ10), which is another common indicator for the compressive mechanical properties of porous materials [24], was recorded for estimating compressive strength. 2.6. In vivo gel formation Animal experiments were performed following NIH standards as set forth in “the Guide for the Care and Use of Laboratory Animals”. In vivo gel formation of chitosan/Dex-PLA/GP solutions was examined by dorsal subcutaneous injection in mice (Balb/c, average body 7

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weight: ca. 28.3g). Sterile solution formulations were obtained by autoclaving chitosan/Dex-PLA solutions and filtrating GP solutions with 0.22 µm filtration. After being anesthetized with anhydrous ether, the mouse was injected with chitosan/Dex-PLA/GP solutions at the selected sites using a gauge 22 G needle and each injection was 0.3 mL in

chitosan/Dex-PLA/GP gels at injection area were removed. 2.7. In vivo degradation

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volume. 12 hours after injection, the mice were sacrificed and the solid-like

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Wistar rats (290-320 g, male) were used to evaluate the in vivo degradation of hydrogels. 0.8 ml of chitosan/GP or chitosan/Dex-PLA/GP solutions was placed in a cylindrical mold at

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37 °C for 20 min to form into gels and the resulting hydrogels were measured for their volume (V0) and wet weight (W0). 0.8 mL of sterilized chitosan/GP or chitosan/Dex-PLA/GP solutions was subcutaneously injected into the back subcuits of rats at both right and left sides in a zygomorphous manner. The resulting gel implants were allowed to develop in vivo. At

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the end of prescheduled degradation periods, rats were sacrificed and the hydrogels under skin were separated for volume and weight measurements. The volume (Vt) of degraded specimens was measured using vernier caliper, and was calculated via following estimation formula

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[14,31]:

Vt (mm3) = [length×width2]/2

(1)

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and the volume remaining ratio (VR) of specimens was determined using following equation: VR (%) = [Vt/V0] × %

(2)

The wet weight (Wt) of degraded specimens was also measured and the weight remaining ratio (WR) of specimens was calculated as follows: WR (%) = [Wt/W0] × %

(3)

2.8. Statistical analysis Data were presented as mean±standard deviation. In the case of independent groups, Student's t-test was used for the comparison between the means of two groups, and otherwise, 8

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analysis of variance was performed for multiple comparisons with p<0.05 as the criteria for statistical significance. 3. Results and discussion 3.1. Basic parameters of Dex-PLAs

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PLA side chains can be grafted onto Dex via ring-opening polymerization of LA monomer or conjugation of lactic acid oligomers using different techniques [27-29,32,33]. It was found that

the direct ring-opening polymerization would provide Dex-PLAs with polydisperse PLA side chains, and the short PLA side chains would not be able to significantly enhance Dex-PLAs

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while extending their degradation endurance. On the other hand, it was also observed that

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Dex-PLAs would have reducing solubility or even become insoluable in aqueous media if the PLA side chains were too long. To avoid the side-chain interferance and ensure the necessitated gelation performance of the resulting hydrogels, the lactic acid oligomers were

thus first synthesized and they were then defined following suggested methods [27,34]. Only some selected oligomers with DP of around 20 were employed for the synthesis of Dex-PLAs,

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based on many trials. By mainly changing the feed ratio of Dex to PLA oligomers while regulating the reaction time and temperature, a series of Dex-PLA samples was synthesized

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and relevant parameters are listed in Table 1.

Fig. 1 shows a representative 1H NMR spectrum of Dex-PLA samples. The chemical

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shifts at δ: ca.1.4 can be attributed to methyl protons located in the PLA backbone, and the shift at δ: ca.5.2 is ascribed to the methine protons in PLA units [27,34], respectively. The signals at 3.05-3.8 ppm and around 4.6 ppm are belonged to the protons in methine groups of Dex units, respectively [27,35,36]. These signals are basically consistent with the reported results for Dex-PLA copolymers [27], suggesting that PLA side chains have been successfully grafted on Dex main chains. PLA is a hydrophobic polymer, as a result, the obtained Dex-PLAs showed various soluble properties in aqueous media and DMSO, depending on the PLA content (see Table 1). 9

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In the present study, although the PLA content in the Dex-PLAs could reach around 63 wt% or even higher, the PLA content should be cautiously balanced because the resultant Dex-PLAs would show some characteristics more similar to pure PLA and become insoluble in aqueous media if the PLA content is too high. Considering mechanically strong

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characteristics and slow degradation rate of PLA component as well as the processing feasibility of Dex-PLAs, only some water-soluble Dex-PLAs with around 50 wt% PLA content were selected for further use in preparation of hydrogels unless otherwise stated.

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3.2. Chitosan/Dex-PLA/GP hydrogels and their rheological properties

To examine whether the ternary composites consisted of chitosan, Dex-PLA and GP

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could form into hydrogels near physiological pH and the temperature, various compositions for chitosan/Dex-PLA/GP solutions were formulated, and the thermosensitive behavior of resulting chitosan/Dex-PLA/GP solutions was examined. It was found that some chitosan/Dex-PLA/GP solutions were able to form into well-defined monolithic gels near physiological pH and temperature on condition that the GP content in chitosan/Dex-PLA/GP

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solutions is controlled in a region changing between about 4.6 and 7.0 (w/v)% while regulating the Dex-PLA percentages in chitosan/Dex-PLA/GP solutions from 0.5 to around 2

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(w/v)% or slightly higher.

In the present instance, to achieve improved mechanical properties and ensure the

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suitable gelling temperature and pH of chitosan/Dex-PLA/GP gels, the Dex-PLA content in chitosan/Dex-PLA/GP solutions was selected as 2 (w/v)% or lower while setting the GP content in chitosan/Dex-PLA/GP solutions at 5 (w/v)% that is close to the GP amount suggested for the chitosan/GP gel [15,37]. Some parameters for several types of chitosan/Dex-PLA/GP gels are summarized in Table 2. Fig. 2 shows several typical photos obtained from inverted tube testing for the chitosan/Dex-PLA/GP(I) gel formation (see Table 2). These photos show four different states : (1) fully flowable liquid (A); (2) partially formed gel with certain wall-mounted ability (B); (3) 10

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deformable gel with gravity-induced deformation and increasing wall-mounted ability (C); and (4) solid-like gel with a integrally wall-mounted feature (D). These photos confirm that the chitosan/Dex-PLA/GP(I) solution underwent clear phase transitions as incubation time was extended, revealing that the chitosan/Dex-PLA/GP(I) solution has thermosensitive

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features near physiological temperature. Temperature-dependent phase transition of the chitosan/GP system was reported to be associated with several types of interactions, including ionic bridging somewhat similar to the

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crosslinking linkage, hydrophobic effect and hydrogen bonding [12,15,37-40]. Since these interactions coexist with different intensities in the chitosan/GP system during phase

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transformation, finding the exact mechanism of gelation for the chitosan/GP system still remains an open-ended scientific challenge [12,42]. Despite complexity in phase transition mechanism, several attempts have been made to figure out the details for the thermosensitive behavior of the chitosan/GP gel [15,37,39,41,42]. A comprehensive explanation for the gelation of the chitosan/GP system can be summarized as follows. At relatively low

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temperatures, the addition of GP to the chitosan solution would cause the neutralization of positively charged ammonium groups (NH3+) by the negatively charged phosphate groups

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presenting in GP, resulting in decreasing electrostatic repulsions among chitosan chains, and on the other hand, the interaction between the glycerol moieties of GP and water would also

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promote the protective hydration of chitosan chains and facilitate them to stretch freely in solution even at a neutral pH [12,15,37,38]. As the temperature raises, the increasing internal energy in the chitosan/GP system would break abundant hydrogen bonds between chitosan and water while releasing the bound water, and meanwhile, a large decrease in the number of ionic bridging would also occur due to heat-induced transfer of protons from chitosan to GP [39-42], and consequently, predominant hydrophobic interactions among chitosan molecules allow the chitosan chains to become closer, leading to the gelation of chitosan/GP solutions [39,41-43]. With respective to chitosan/Dex-PLA/GP system, the Dex-PLA component would 11

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participate in the hydrogen bonding and hydrophobic interactions that exist in the original chitosan/GP system since the Dex main chains have a large number of hydroxyl groups, and the PLA side chains in the Dex-PLAs are hydrophobic. As a result, the chitosan/Dex-PLA/GP solution could still form into a gel following a similar mechanism mentioned above for the

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chitosan/GP system besides some changes in the gelation time and temperature. To make a comparison, temperature-dependence functions of G′ and G″ for three types of gels were measured and relevant results are illustrated in Fig. 3. It can be observed that Ti

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of the chitosan/GP gel was around 37°C; and in the cases of chitosan/Dex-PLA/GP(II) and chitosan/Dex-PLA/GP(IV) gels, Ti for each became relatively low, suggesting that the amount

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of Dex-PLA in the chitosan/Dex-PLA/GP gels can impose a significant impact on their Ti. Fig. 3 also shows that all these gels had very low G″ in the experimental temperature range whilst their G′ significantly increased starting from their respective Ti, signifying that the viscous liquid samples gradually became solid-like gels at the temperature starting from Ti. In

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addition, it can also be seen from Fig.3 that at 37°C, G′ of chitosan/Dex-PLA/GP(II) and chitosan/Dex-PLA/GP(IV) gels was much higher (p<0.01) than that of the chitosan/GP gel, indirectly revealing that the chitosan/GP gel can be mechanically enhanced by adding

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Dex-PLA as a complementary component because the magnitude of G′ of gels is closely correlated to the strength of hydrogels [44,45].

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Several sets of samples were measured for their pH, gelation time and Ti, and relevant data are listed in Table 2. Results in Table 2 indicate that pH of chitosan/Dex-PLA/GP(i) (i=I, II, III and IV) gels was slightly higher than that of the chitosan/GP gel, and it changed from ca. 7.0 to about 7.2 as the Dex-PLA content in chitosan/Dex-PLA/GP solutions was increased from 0.5 to 2.0 (w/v)%. Data in Table 2 confirm that these chitosan/Dex-PLA/GP solutions are able to form into gels near physiological pH. In addition, it can also be seen that Dex-PLA component imposed a strong impact on the gelation time and Ti of chitosan/Dex-PLA/GP gels. In comparison to the chitosan/GP gel, the gelation time of chitosan/Dex-PLA/GP(i) (i=I, II, 12

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III and IV) samples became significantly shortened (p<0.01) and the corresponding Ti showed a downtrend along with increasing Dex-PLA content in these samples. These results suggest that the Dex-PLA component in the chitosan/Dex-PLA/GP gels plays an important role in regulating their several key parameters such as pH, gelation time and Ti. In considering the

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possible in vivo applications of chitosan/Dex-PLA/GP gels, the Dex-PLA content in chitosan/Dex-PLA/GP gels was selected as around 2 (w/v)% or lower in order to ensure gel formation near physiological pH.

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Frequency-dependence of G′ and G″ is commonly used to assess rheological properties of hydrogels [37,44-46]. Approximate independence of G′ and G″ against frequency within a

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relatively low frequency range is considered to be important for evaluating the strength of hydrogels [37,47,48]. Fig. 4 presents several representative frequency-dependence curves of G′ and G″ at 37 °C for different samples. Fig. 4(A) exhibits that (1) G′ of these gels was nearly linear within the frequency sweep interval, (2) G′ of chitosan/Dex-PLA/GP(i) (i=I, II,

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III and IV) gels was much higher than that of the chitosan/GP gel, and (3) G′ increased along with increasing Dex-PLA content in these gels. In general, the magnitude of G′ together with the ratio of G′ to G″ can be used to

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estimate the strength of hydrogels [48,49]. In principle, mechanically strong hydrogels are associated with high G′ values, and meanwhile, G′ of strong hydrogels is usually one order or

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even two orders of magnitude greater than their G″ [37,44,50]. Fig. 4(A) and (B) explicate that both chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) gels had high G′, and the ratio G′/G″ for them was more than 30. In addition, G′ of chitosan/Dex-PLA/GP(III) or chitosan/Dex-PLA/GP(IV) gels was much higher than that of the chitosan/GP gel, suggesting that chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) behave like mechanically strong gels as compared to the chitosan/GP gel. To see the quantitative effect of the Dex-PLA content in chitosan/Dex-PLA/GP gels on the 13

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magnitude of G′, several sets of samples were measured for their G′ at 6.26 Hz according to the suggested measurement frequency [11,22], and relevant data are graphed in Fig. 4(C). It can be observed that the chitosan/Dex-PLA/GP(IV) showed its G′ much higher compared to the chitosan/GP. Some reported results for the chitosan/GP gel or other chitosan-based gels

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point out that the higher density in these gels can result in significantly larger G′ due to increasing intermolecular interactions during the gel formation [11,44,48]. In the present instance, addition of Dex-PLA into the chitosan/GP system with various Dex-PLA amounts

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would increase the density of chitosan/Dex-PLA/GP gels because the full volume for all gels was a constant value, as shown in Table 2. Consenquently, the G′ of chitosan/Dex-PLA/GP

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gels might change in a manner similar to that occurred in the chitosan/GP system, progressively increasing as the Dex-PLA amount in chitosan/Dex-PLA/GP gels was gradually raised. 3.3. Compressive mechanical property

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The presently developed chitosan/Dex-PLA/GP gels are intended to be used for in situ articular cartilage repair, and thus, their compressive property is of importance because in the case of articular cartilage repair, the suitable gels must be able to endure a certain pressure

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during implantation to help the seeded cells grow. Representative stress-strain profiles of several gels are shown in Fig. 5(A). It can be noted that the chitosan/GP gel exhibited a

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uniformly linear strain-stress relationship from 0 to around 10% strain, and its stress slightly deviated from linear tendency when the deformation percentage was further increased; and on the other hand, strain-stress curves for chitosan/Dex-PLA/GP(i) (i=I, II, III and IV) gels became steeper in comparison to the chitosan/GP sample and they showed nonlinear characteristics, depending on the Dex-PLA content in these gels. Several sets of gel samples were measured and collected data for their E and σ10 are depicted in Fig. 5(B). Clearly, the chitosan/GP gel had low E and σ10, indicative of a soft and weak gel. In contrast to this

14

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observation, chitosan/Dex-PLA/GP(i) (i=I, II, III and IV) gels showed much higher E and σ10, and their E and σ10 significantly increased as the Dex-PLA content in the chitosan/Dex-PLA/GP

gels

increased.

In

particular,

both

E

and

σ10

of

chitosan/Dex-PLA/GP(IV) gels were around 8 times higher than that of the chitosan/GP gel,

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and they reached more than 13 kPa and 1.2 kPa, respectively, confirming that chitosan/Dex-PLA/GP(IV) gels behave like relatively stiff and strong gels. Based on the results presented in Fig.5, it can be drawn that the compressive modulus and strength of

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chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) gels are comparative to that of some cartilage [51,52].

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Several studies have reported that certain amphiphilic Dex-PLA copolymers can be used to assemble hydrogels based on hydrophobic interactions, and the hydrophobic domains built by the PLA side chains actually function as physically cross-linked sites [27,28,30,34]. In principle, amphiphilicity of Dex-PLAs also provides a possibility for them to self-assemble

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micelles when dispersed in the aqueous media. Therefore, it can be inferred that the PLA side chains in the Dex-PLAs would be able to build some hydrophobic domains, which could act like physically cross-linked sites, if water-soluble Dex-PLAs are properly blended with some

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other aqueous polymer solutions.

In the present study, during the formation of chitosan/Dex-PLA/GP gels, the Dex-PLA

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component inside the gels will interact with chitosan, GP and water molecules, and on the other hand, the PLA side chains could form PLA-rich sites in a self-aggregated manner, functioning like physical crosslinking to mechanically enhance the chitosan/Dex-PLA/GP gels. On the basis of the early mentioned explanation for the gelation of the chitosan/GP system [15,37,41-43], a possible mechanism for the formation of chitosan/Dex-PLA/GP gels as well as their mechanical enhancement could be proposed as blow. At a relatively low temperature, numerous water-molecule-mediated hydrogen bonds connected to hydrophilic components, namely, chitosan, Dex backbones and the glycerol 15

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moieties of GP, would form inside the chitosan/Dex-PLA/GP solution, and at the same time, many ionic bridges spanned over the protonated amino groups in chitosan and the phosphate groups in GP would also coexist. Both hydrophilic and ionic interactions are able to keep chitosan and Dex-PLA chains stretched while allowing them to deform freely in the solution.

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As the temperature of the chitosan/Dex-PLA/GP solution is elevated to a critical value, the heat-induced phase transformation will occur. During the formation of gels, most hydrogen bonds are broken, and water molecules are thus released and move with freedom; increasing

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hydrophobic interactions will on the one hand allow chitosan to aggregate to form chitosan-rich portions in which the GP-involved ionic bridges can act like physical

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crosslinking while facilitating PLA side chains in Dex-PLAs to form hydrophobic aggregates that actually function as additional physical crosslinking. In addition, the physical entanglement between chitosan and Dex-PLAs will also contribute the mechanical stabilization of three-dimensional network of gels. As a result, chitosan/Dex-PLA/GP gels would be significantly enhanced in their mechanical strength due to presence of PLA-built

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hydrophobic domains and additional physical entanglement in comparison to chitosan/GP gels. To make this possible mechanism visible, a schematic representation for the formation

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of chitosan/Dex-PLA/GP gels is illustrated in Fig.6 with reference to some proposed schemes for the chitosan/GP gels [43,53]

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3.4. In vivo gelation

Some chitosan/Dex-PLA/GP solutions were tested in vivo via subcutaneous injection to see if they are able to effectively gel at the injection sites. Fig. 7 shows several representative photos for the chitosan/Dex-PLA/GP(IV) gel formation after subcutaneous injection on the back of mouse. It is well known that many types of thermosensitive hydrogels can fill the tissue cavity in way of site-specific in situ gel-formation, and thus, they are able to deliver therapeutic agents, drugs or living biologicals via injection administration in a minimally invasive manner [5,18]. The ability to rapidly form into gels after injection through a needle is 16

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therefore important for evaluation of an injectable hydrogel since slow gelation can cause delocalized gel formation due to the gel precursor diffusion [11,54]. In our cases, chitosan/Dex-PLA/GP(IV) solution was able to rapidly form into gel and localize to the injection

site

(Fig.7(B)),

and

the

average

in

vivo

gelling

time

for

the

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chitosan/Dex-PLA/GP(IV) gel was measured as around 8.2 min. After 12 h of administration, the chitosan/Dex-PLA/GP(IV) gel was found to slightly contract (Fig. 7 (B) and (C)), which can be attributed to the water loss from the gel due to in vivo metabolism and rebalance of

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body fluid. In addition, the sample shown in Fig. 7(D) confirms that after subcutaneous administration for 12 h, chitosan/Dex-PLA/GP(IV) gel already became a solid-like material

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for easy explantation. 3.5. In vivo degradation

The in vivo degradation performance of the hydrogel is crucial for its practical applications in vivo, specially in the situation where the hydrogel needs to function as a carrier for the sustained delivery of drugs and/or active molecules over a longer period of time, or to

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provide necessitated mechanical support for an expected duration during tissue repair. The changes in volume and weight of several types of hydrogels after in vivo degradation for a

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period up to 8 weeks were thus examined, and relevant data are presented in Fig.8 as functions of degradation time. Fig.8(A) shows that the volume the chitosan/GP gel altered

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roughly in a three-phase manner: it was slightly reduced during the initial stage of degradation, rapidly decreased since day 5, and changed in a slow downtrend starting from around day 21 along with the prolongation of degradation time. At the end of 5-week degradation, the chitosan/GP gel remained less than 10 % of its original volume, meaning that it was almost completely degraded. In contrast to this observation, chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) gels also showed similar three-phase degradation behavior but they had considerably slower decreases in their volume within the same degradation period. After being respectively in vivo degraded for 7 and 8 weeks, VR of chitosan/Dex-PLA/GP(III) 17

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and chitosan/Dex-PLA/GP(IV) gels still had their VR at around 19 and 24%. As mentioned in the experimental section, the remaining volume of degraded gels was determined using Equation (1), a suggested empirical formula for estimating the volume of the out-of-shape subcutaneous implants or solid tumor [14,31,55], and VR was calculated with Equation (2).

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The rough estimate for volume changes of degraded gels was reflected by irregular curves together with large standard deviations shown in Fig.8(A). Although the large standard deviations

might

obscure the differences and

VR

to

a

chitosan/Dex-PLA/GP(IV)

certain

extent

gels

or

between among

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chitosan/Dex-PLA/GP(III)

in

chitosan/Dex-PLA/GP(I), chitosan/Dex-PLA/GP(II) and chitosan/GP gels, Fig.8(A) confirms

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that chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) have definitely enhanced ability against in vivo degradation.

Fig.8(B) illustrates the time-dependent weight decrease of different gels. In comparison to the curves shown in Fig.8(A), similar trends for the weight decrease of the matched gels were recorded, demonstrating that there is a well correlated consistency between two types of In

these

cases,

it

can

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measurements.

be

noted

that

chitosan/Dex-PLA/GP(II),

chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) gels had significantly slower

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(p<0.01) decreases in weight than chitosan/GP gel starting from day 7, and of them, WR of the chitosan/Dex-PLA/GP(IV) gel was more than double as compared to chitosan/GP gel during

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the degradation period changing from day 14 to day 35. On the other hand, chitosan/Dex-PLA/GP(I) gel behaved in a way similar to chitosan/GP gel without significant differences during the same degradation period. In addition, it is also observed that starting from

day

14,

significant

differences

(p<0.05)

appeared

in

WR

among

chitosan/Dex-PLA/GP(II), chitosan/Dex-PLA/GP(III) and chitosan/Dex-PLA/GP(IV) gels, and the increasing PLA content in the gels would result in incremental WR. In vivo degradation of polysaccharides and polyesters usually involves various enzymes, such as lysozyme, amylase, parenzyme and lipase [56]. Although these enzymes can catalyze 18

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the degradation of chitosan, Dex and PLA components in chitosan/Dex-PLA/GP gels, of three components, PLA has much slower in vivo degradation rate, closely depending on its molecular weight [25,26,49]. In considering the hydrophobic features of PLA, the linear PLA side chains on different Dex-PLA molecules could be entangled together to build hydrophobic

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domains inside gels, and the resulting PLA domains would act as physically crosslinked sites inside gels to enhance the strength of gels. On the other hand, PLA-rich domains could also bring about a hindrance to the enzyme for its infiltrating into gels, resulting in slow

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degradation of chitosan/Dex-PLA/GP (i) (i=II, III and IV) gels as compared to the chitosan/GP gel. Accordingly, the increasing amount of PLA in chitosan/Dex-PLA/GP gels

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will lead to concomitantly decreasing degradation rates when PLA content in chitosan/Dex-PLA/GP gels was higher than a threshold (see Fig.8 (B)), suggesting that the degradation rate of chitosan/Dex-PLA/GP gels can be regulated by PLA content in a controllable manner.

The three-phase degradation behavior of gels may be attributed to several facets involved

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in infiltrated enzymes and invaded cells. In the first a few days, different enzymes would diffuse into gels with various speeds due to the enzyme concentration difference between the

EP

interior of gels and the surrounding degradation environment, and thus, the surface layer of gels could only be degraded without significant changes in their volume and weight, which is

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signified by a plateau region in each curve with a time span less than 5 days for all gels (see Fig.8). As the enzyme concentrations inside different gels increased to a certain level and continued to be elevated to their respective dynamic equilibrium values, degradation of gels would be thus speeded, and in turn, the rapid decreases in volume or weight of gels would take place, as indicated by the steep descent regions with various widths depending on the Dex-PLA content (see Fig.8). During the fast degradation period, more porous spaces inside gels were yielded because the degradation products would be removed by macrophages and phagocytes [57,58], and the resulting porous spaces would allow different cells to migrate into 19

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gels and to proliferate there, or otherwise, the residual porous spaces without cell residence became collapse. The gradual invasion of cells into hydrogels along with degradation prolongation of gels has already been commonly observed [14,57,58]. Accordingly, after being fast degraded for a certain period of time, the degraded gels could be filled to a certain

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extent by different cells coming from the degradation environment, resulting in slow changes in their volume or weight even though the gels were still being degraded, as manifested by the relatively flat regions in curves shown in Fig.8.

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On the basis of above-presented results, it can be reached that presently developed chitosan/Dex-PLA/GP composites are able to form into gels near physiological pH and

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temperature, and the optimal gels can function as injectable in situ gelling materials with enhanced strength and extended degradation endurance. Some chitosan/Dex-PLA/GP gels carried with chondrocytes and certain growth factors are being investigated for in situ articular cartilage repair where mechanically strong and slowly degradable gels are preferably needed.

Relevant results will be presented in separate reports.

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4. Conclusions

A new type of injectable hydrogel with mechanically strong features was successfully

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produced using chitosan, selected dextran-polylactide copolymer and glycerophosphate. The ternary composite solutions with suitable proportions showed well-defined ability to form into

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gels near physiological pH and temperature. It was found that the dextran-polylactide component

exerted

significant

impacts

on

some

properties

of

chitosan/dextran-polylactide/glycerophosphate gels, and the increasing dextran-polylactide content in the gels could result in lowered incipient gelation temperature, shortened gelation time and significantly increased viscoelastic modulus of gels. Compression measurements confirmed that some optimal chitosan/dextran-polylactide/glycerophosphate gels had greatly enhanced compressive modulus and strength that are around 8 times higher than that of chitosan/glycerophosphate gel. In addition, in vivo degradation confirmed that the optimal 20

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chitosan/dextran-polylactide/glycerophosphate gels had significantly prolonged degradation endurance in comparison to the chitosan/glycerophosphate gel, and the dextran-polylactide content in the gels was able to regulate the degradation rate of the gels in a controllable manner. These results suggest that besides the regular usage, the newly developed gels have

characteristics and degradation tolerance is required. Acknowledgement

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promising potential for applications where the gel concurrently having mechanically strong

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This work was funded by the National Natural Science Foundation of China (Grant no. 81371705).

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References

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Hennink WE. Novel self-assembled hydrogels by stereocomplex formation in aqueous solution of enantiomeric lactic acid oligomers grafted to dextran. Macromolecules 2000; 33:3680-6. [31] Unezaki S, Maruyama K, Hosoda JI, Nagae I, Koyanagi Y, Nakata M, Ishida O, Iwatsuru M, Tsuchiya S. Direct measurement of the extravasation of polyethyleneglycol-coated liposomes into solid tumor tissue by in vivo fluorescence microscopy. Int J Pharm 1996; 144:11-7. [32] Nouvel C, Dubois P, Dellacherie E, Six JL. Silylation reaction of dextran: effect of experimental conditions on silylation yield, regioselectivity, and chemical stability of silylated dextrans. Biomacromolecules 2003; 4:1443-50. [33] Ouchi T, Kontani T, Ohya Y. Modification of polylactide upon physical properties by solution-cast blends from polylactide and polylactide-grafted dextran. Polymer 2003; 44: 3927-33. [34] Thanki PN, Dellacherie E, Six JL. Prevailing mechanisms of the hydrolytic degradation of oligo(D,L-lactide)-grafted dextrans. Euro Polym J 2005; 41:1546-53. [35] Wang H, Han S, Sun J, Fan T, Tian C, Wu Y. Preparation of dextran-poly(lactide)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine copolymer and its micellar characteristics. Carbohydr Polym 2011; 83:1408-13. [36] Shi HY, Zhang LM. Phase-transition and aggregation characteristics of a thermoresponsive dextran derivative in aqueous solutions. Carbohydr Res 2006; 341:2414-9. [37] Chenite A, Buschmann M, Wang D, Chaput C, Kandani N. Rheological characterization of thermogelling chitosan/glycerol-phosphate solutions. Carbohydr Polym 2001; 46:39-46. [38] Ruel-Gariepy E, Chenite A, Chaput C, Guirguis S, Leroux JC. Characterization of thermosensitive chitosan gels for the sustained delivery of drugs. Int J Pharm 2000; 203:89-98. [39] Cho J, Heuzey MC, Begin A, Carreau PJ. Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature. Biomacromolecules 2005; 6:3267-75. [40] Cho J, Heuzey MC, Begin A, Carreau PJ. Chitosan and glycerophosphate concentration dependence of solution behaviour and gel point using small amplitude oscillatory rheometry. Food Hydrocolloids 2006; 20:936-45. [41] Lavertu M, Filion D, Buschmann MD. Heat-induced transfer of protons from chitosan to glycerol phosphate produces chitosan precipitation and gelation. Biomacromolecules 2008; 9:640-50. [42] Aliaghaie M, Mirzadeh H, Dashtimoghadam E, Taranejoo S. Investigation of gelation mechanism of an injectable hydrogel based on chitosan by rheological measurements for a drug delivery application. Soft Matter, 2012; 8:7128-37. [43] Moura MJ, Faneca H, Lima MP, Gil MH, Figueiredo MM. In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking. Biomacromolecules 2011; 12:3275-84. [44] Martınez-Ruvalcaba A, Chornet E, Rodrigue D. Viscoelastic properties of dispersed chitosan/xanthan hydrogels. Carbohydr Polym 2007; 67:586-95. [45] Banerjee A, Arha M, Choudhary S, Ashton RS, Bhatia SR, Schaffer DV, Kane RS. The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells. Biomaterials 2009; 30:4695-9. [46] Qiu X, Yang Y, Wang L, Lu S, Shao Z, Chen X. Synergistic interactions during 23

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thermosensitive chitosan-β-glycerophosphate hydrogel formation. RSC Adv 2011; 1:282-9. [47] Hernandez R, Lopez D, Mijangos C, Guenet JM. A reappraisal of the ‘thermoreversible’ gelation of aqueous poly(vinyl alcohol) solutions through freezing-thawing cycles. Polymer 2002; 43:5661-3. [48] Lejardi A, Hernandez R, Criado M, Santos JI, Etxeberria A, Sarasua JR, Mijangos C. Novel hydrogels of chitosan and poly(vinyl alcohol)-g-glycolic acid copolymer with enhanced rheological properties. Carbohydr Polym 2014; 103:267-73. [49] Clark AH, Ross-Murphy SB. Structural and mechanical properties of biopolymer gel. Biopolym. Adv Polym Sci 1987; 83:57-192. [50] Kavanagh GM, Ross-Murphy SB. Rheological characterisation of polymer gels. Prog Polym Sci 1998; 23:533-62. [51] LaPorta TF, Richter A, Sgaglione NA, Grande DA. Clinical relevance of scaffolds for cartilage engineering. Orthop Clinics North Am 2012; 43:245-54. [52] Shepherd DET, Seedhom BB. The ‘instantaneous’ compressive modulus of human articular cartilage in joints of the lower limb. Rheumatology 1999; 38:124-32. [53] Kim GO, Kim N, Kim DY, Kwon JS, Min BH. An electrostatically crosslinked chitosan hydrogel as a drug carrier. Molecules 2012; 17:13704-11. [54] Gupta D, Tator, CH, Shoichet MS. Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the injured spinal cord. Biomaterials 2006; 27:2370-9. [55] Naito S, von Eschenbach AC, Giavazzi R, Fidler IJ. Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice. Cancer Res 1986; 46:4109-15. [56] Dee KC, Puleo DA, Bizios R. An introduction to tissue-biomaterial interactions. New York: John Wiley & Sons; 2002. [57] Tan R, Feng Q, She Z, Wang M, Jin H, Li J, Yu X. In vitro and in vivo degradation of an injectable bone repair composite. Polym Degrad Stab 2010; 95:1736-42. [58] Anderson JM. Biological responses to materials. Annu Rev Mater Res 2001; 31:81-110.

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Figure captions Fig. 1

1

Fig. 2

Several typical photos for the gel formation of the chitosan/Dex-PLA/GP(II) solution at 37 °C (see Table 1 for its composition).

Fig. 3

Typical temperature-dependence functions of G′ and G″ for chitosan/GP, chitosan/Dex-PLA/GP(II) and chitosan/Dex-PLA/GP(IV) gels.

Fig. 4

Representative frequency-dependence of G′ (A) and G″ (B) as well as average value of G′ (C) at 6.25 Hz for different gels (37 °C, ∗∗, p<0.01; ∗, p<0.05).

Fig. 5

Representative strain-stress curves (A) and compressive modulus (B) for different gels (∗∗, p<0.01; ∗, p<0.05).

Fig. 6

Schematic representation of chitosan/Dex-PLA/GP gel formation.

Fig. 7

In vivo gel formation after subcutaneous injection of chitosan/Dex-PLA/GP(IV) solution on the back of mouse: before injection (A), after injection for 10 min (B), after 12 h of administration (C) and the explanted gel after injection for 12 h (D).

Fig. 8

Time-dependent in vivo volume remaining ratio (A) and weight remaining ratio (B) of different hydrogels.

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H NMR spectrum of a Dex-PLA copolymer in DMSO-d6.

25

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Table 1. Parameters of Dex-PLA compolymers DS(c)

1.0 : 0.75

35.3±1.17

6.23

+

Dex-PLA(B)

1.0 : 1.0

46.7±1.31

9.85

+

Dex-PLA(C)

1.0 : 1.25

53.2±1.53

13.05

+

Dex-PLA(D)

1.0 : 1.5

59.5±1.49

17.36

±±

Dex-PLA(E)

1.0 : 1.75

63.1±1.65

19.71

±

Dex-PLA(A)

(a)

Solubility(d) Water 0.1 M HCl

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PLA content in Dex-PLA (wt%)(b)

DMSO

+

+

+

+

+

+

±±

+

±

+

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Feed ratio of Dex to oligomer (wt/wt)

Sample name

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(a) The PLA content in Dex-PLAs was regulated by the reaction time and temperature. (b) Estimated from 1H NMR. (c) Degree of substitution (the number of oligo-PLAs per 100 glucopyranose units of dextran) (d) “±”, “±±” and “+” indicate that Dex-PLAs are swelled, partially soluble or highly swelled, and soluble, respectively.

Table 2. Parameters of chitosan/Dex-PLA/GP hydrogels.(a) Concentration of chitosan (w/v %)

Concentration of Dex-PLA (w/v %)

pH

Gelation time at 37 °C (sec)

Ti (°C)(d)

chitosan/Dex-PLA/GP(I)(b)

2.0

0.5

6.91±0.05

528±16

35.84±1.14

chitosan/Dex-PLA/GP(II)

2.0

1.0

7.02±0.08

437±13

34.61±1.27

EP

Sample name

chitosan/Dex-PLA/GP(III)

2.0

1.5

7.11±0.07

379±8

33.54±1.06

chitosan/Dex-PLA/GP(IV)

2.0

2.0

7.18±0.06

352±5

32.97±1.28

(c)

2.0

−

6.79±0.06

762±19

37.39±1.33

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chitosan/GP

(a) The full volume of chitosan/Dex-PLA/GP solutions for gel preparation was 10 ml, and the concentration of GP for all gel samples was 5.0 (w/v %); (b) Dex-PLA with PLA content of around 50 wt% was used for producing chitosan/Dex-PLA/GP(i)(i=I, II, III and IV) gels (see Table 1); (c) This gel was used as control; (d) Ti indicated the incipient gelation temperature.

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DOH

DMSO

2,3,4,5,6

6

5

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1

a

4

3

2

b

c

1

0

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EP

ppm

Figure 1

1

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Figure 2

2

ACCEPTED MANUSCRIPT Figure 3 (A) 3500

chitosan/GP G' G"

3000

2000

1500

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G' and G" (Pa)

2500

1000

500

0 25

30

35

40

45

o

SC

Temperature ( C)

Figure 3 (B) 3500

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chitosan/Dex-PLA/GP(II) G' G"

3000

G' and G" (Pa)

2500

2000

1500

1000

0 25

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500

30

35

40

45

o

Temperature ( C)

EP

Figure 3 (C) 3500

chitosan/Dex-PLA/GP(IV) G' G"

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3000

G' and G" (Pa)

2500

2000

1500

1000

500

0 20

25

30

35

40

45

50

0

Temperature ( C)

3

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Figure 4(A)

chitosan/GP chitosan/Dex-PLA/GP(I) chitosan/Dex-PLA/GP(II) chitosan/Dex-PLA/GP(III) chitosan/Dex-PLA/GP(IV)

1600 1400 1200

800 600 400 200 0 1

10

100

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0.1

Frequency (Hz)

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Figure 4(B) 100

chitosan/GP chitosan/Dex-PLA/GP(I) chitosan/Dex-PLA/GP(II) chitosan/Dex-PLA/GP(III) chitosan/Dex-PLA/GP(IV)

80

60

40

20

0 0.1

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G" (Pa)

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G' (Pa)

1000

1

10

EP

Frequency (Hz)

Figure 4(C)

AC C

1400

*

1200

**

G'(Pa)

1000

800

*

600

** 400

200

0 P(I) n/GP P(III) P(IV) GP(II) chitosa an/Dex-PLA/Gn/Dex-PLA// /Dex-PLA//G /Dex-PLA//G n chitos chitosa chitosa chitosan

Name of sample

4

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Figure 5 (A) 3500

chitosan/Dex-PLA/GP(IV) chitosan/Dex-PLA/GP(III) chitosan/Dex-PLA/GP(II) chitosan/Dex-PLA/GP(I) chitosan/GP

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2500

2000

1500

1000

SC

Compressive stress (Pa)

3000

0 0

5

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500

10

15

20

Strain (%)

14 12

8 6

2500

** 2000

** **

**

EP

E (kPa)

10

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16

* **

1500

1000

σ10(Pa)

Figure 5(B)

AC C

**

4

**

500

2 0

0

(IV) P(I) /GP P(II) P(III) chitosasnan/Dex-PLA/Gan/Dex-PLA/Gan/Dex-PLA/G n/Dex-PLA/GP chito chitos chitos chitosa

5

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 6

6

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 7

7

ACCEPTED MANUSCRIPT

Figure 8(A) 100

RI PT

chitosan/Dex-PLA/GP(IV) chitosan/Dex-PLA/GP(III) chitosan/Dex-PLA/GP(II) chitosan/Dex-PLA/GP(I) chitosan/GP

80

SC

VR (%)

60

40

0 0

10

20

M AN U

20

30

40

50

60

70

Time (day)

Figure 8(B)

TE D

100

80

chitosan/Dex-PLA/GP(IV) chitosan/Dex-PLA/GP(III) chitosan/Dex-PLA/GP(II) chitosan/Dex-PLA/GP(I chitosan/GP

EP

40

AC C

WR (%)

60

20

0

0

10

20

30

40

50

60

70

Time (day)

8