Synthesis of in-situ formable hydrogels with collagen and hyaluronan through facile Michael addition

Accepted Manuscript Synthesis of in-situ formable hydrogels with collagen and hyaluronan through facile Michael addition

Riwang Li, Zhengwei Cai, Zhiwen Li, Qian Zhang, Shuyun Zhang, Li Deng, Lu Lu, Lihua Li, Changren Zhou PII: DOI: Reference:

S0928-4931(17)30279-5 doi: 10.1016/j.msec.2017.04.046 MSC 7869

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

23 January 2017 3 April 2017 6 April 2017

Please cite this article as: Riwang Li, Zhengwei Cai, Zhiwen Li, Qian Zhang, Shuyun Zhang, Li Deng, Lu Lu, Lihua Li, Changren Zhou , Synthesis of in-situ formable hydrogels with collagen and hyaluronan through facile Michael addition. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.04.046

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.

ACCEPTED MANUSCRIPT

Synthesis of in-situ formable hydrogels with collagen and hyaluronan through facile Michael addition Riwang Li*, Zhengwei Cai*, Zhiwen Li, Qian Zhang, Shuyun Zhang, Li Deng, Lu Lu, Lihua Li**, Changren Zhou Department of Material Science and Engineering, Engineering Research Center

PT

of Artificial Organs and Materials, Jinan University, Guangzhou, 510632,

RI

China

SC

* Riwang Li and Zhengwei Cai contributed equally to this work

NU

** Corresponding authors: Professor Lihua Li

MA

Tel.:0086-20-85226663, Fax: 0086-20-85223271

AC

CE

PT E

D

Email address: [email protected]

ACCEPTED MANUSCRIPT Abstract： To mimic the natural extracellular matrix (ECM) and to facilitate tissue regeneration, in-situ formable collagen/hyaluronan composite hydrogels were prepared by a facile approach via Michael addition reaction, with maleilated collagen (Col-MA) and thiol derivatized hyaluronan (HA-SH). The hydrogels

PT

were denoted as CHG-1, CHG-2, CHG-3 and CHG-4 by vinyl/free thiol molar ratio (f) of 1:1, 1:2, 1:3 and 1:4, respectively. Results showed that with

RI

decrease of the f values, the gelation time decreased from 43 s to 15 s, Young’s

SC

modulus increased from 1671.65 Pa to 9105.86 Pa, and the swelling ratio increased from 1.5 to 12.7. SEM images of the air-dried samples revealed that

NU

the chemical modification process did not denature the collagen, and the interwoven collagen fibrils were indeed observed. Cell culture confirmed that

MA

the CHG facilitated the growth and proliferation of MC3T3-E1. Cells displayed spreading morphology and formed nearly aligned cell layers with the extension

D

of culture time. Therefore, it is suggested that CHG can be used as injectable

PT E

materials for tissue regeneration. Keywords

AC

CE

Collagen; Hyaluronan; In-situ formable hydrogels; Michael addition.

ACCEPTED MANUSCRIPT 1. Introduction In tissue engineering (TE), three dimensional scaffolds act as artificial extracellular matrixes (ECM), allowing cells to adhere, migrate, proliferate and maintain their specific functions in the provided niche, and serve as a template for new tissue formation [1, 2]. Among different biomaterials being developed

PT

and utilized for TE, hydrogels have attracted considerable attention owing to their capability to mimic the physiochemical and biological properties of the

RI

natural ECM. Furthermore, in-situ formable or injectable hydrogels have

SC

shown significant advantages, which allow easy and homogenous distribution of bioactive molecules or cells within any defect size or shape and minimize

NU

the invasiveness of the surgical techniques, and have been prepared by various chemical and physical crosslinking methods [3]. It is vital for an in-situ

MA

forming hydrogel system to cross-link under mild conditions or similar physiological conditions. Physical cross-linking hydrogels can be quickly

D

formed by electrostatic, hydrophilic and hydrophobic interactions etc, but the

PT E

stability is poor. Thus, chemical cross-linking methods such as Schiff base reaction and photo cross-linking have been often utilized [4, 5]. The main challenges of chemical cross-linking are the biocompatibility of the 3D

CE

microenvironment of the cell, the biodegradability of the materials, and the transport constraints of nutrients and metabolites [6]. Michael addition reaction

AC

is an increasing popular method to prepare hydrogel by addition reactions between nucleophiles (e.g. thiol groups) and electrophiles (e.g. vinyl/acrylate groups) [7, 8], and can be carried out rapidly in the human physiological conditions with a high degree of chemical selectivity. Collagen (Col) is the main component of ECM, in which type I collagen is the most abundant that filled in the skin, bone and connective tissue, and has been widely used in TE [9]. The collagen matrix material interacts well with the surrounding cell environment, either before it is absorbed as a skeleton to form a new tissue, or absorbed and assimilated as a part of the host tissue [10].

ACCEPTED MANUSCRIPT Collagen modification can give it many new properties, particularly collagen free amino group is prone to acylate [11-13]. Hyaluronan (HA) is a naturally non-sulfated glycosaminoglycan composed of β-1,3-N-acetylglucosamine and β-1,4-D-glucuronic acid, which is also widely distributed in ECM of many tissues [14, 15] with good biocompatibility and viscoelasticity [16]. Despite

PT

their excellent performance, they are limited by poor mechanical properties when used alone [17].

RI

Therefore, in this paper, maleilated collagen (Col-MA) was prepared by treating collagen with maleic anhydride, to introduce double bonds and

SC

carboxyl groups. And the thiol derivatized hyaluronan (HA-SH) was synthesized by the reaction of hyaluronan with cysteine. The double bonds on

NU

Col-MA and the thiol groups on HA-SH will form a macromolecular structure through a rapid Michael addition reaction under the physiological condition at

MA

37°C, to obtain an injectable macromolecular network hydrogel. Furthermore, HA-SH will form intermolecular and intramolecular disulfide bonds under

D

neutral condition, to increase the crosslinking degree, and enhance the

PT E

viscoelasticity and the mechanical strength of hydrogels [18].

CE

2. Materials and methods 2.1. Materials

AC

Chitosan (CS, degree of deacetylation=75-85%, viscosity=200-800cps), maleic anhydride (MA) and L-cysteine hydrochloride monohydrate (CYS) were purchased from Sigma-Aldrich. Beta-glycerophosphate disodium salt (β-GP),

N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide

hydrochloride

(EDAC) and N-Hydroxysuccinimide (NHS) were of analytical grade and purchased from Qi Yun Biotechnology Co., Ltd. (Guangzhou, China). Hyaluronan (HA, Mw=20-40kDa) was obtained from Bloomage Freda Biopharm Co., Ltd. (China). Collagen (Col, Mw=100kDa) were purchased from Huang Yao Biotechnology Co., Ltd. (Guangzhou, China). Dimethyl

ACCEPTED MANUSCRIPT sulfoxide (DMSO), 2.5% glutaraldehyde solution and 4% paraformaldehyde solution were of analytical grade and purchased from Sijia Biotechnology Co., Ltd. (Guangzhou, China). MTT cell proliferation and cytotoxicity assay kit were

of

analytical

grade

and

purchased

from

Nanjing

Jiancheng

Bioengineering institute. Fluorescein isothiocyanate isomer I (FITC) was

PT

purchased from Nanjing Keygen Biotech Co., Ltd. and 4’,6-diamidino-2phenylindole (DAPI) was purchased from Guangzhou Jetway Biotech Co., Ltd.

RI

All other reagents were commercially available, of analytical grade, and used

SC

without further purification.

NU

2.2. Synthesis of thiol derivatized hyaluronan (HA-SH) HA-SH was synthesized with HA and L-cysteine hydrochloride

MA

monohydrate (CYS), according to the literature with slight modification [8, 19]. 0.4 g HA was dissolved in 100 mL deionized water, EDAC and NHS were then added in at final concentrations of 50 mmol/L. The pH was adjusted to 5.50

D

with 1 mol/L HCl solution, and the reaction was carried out at room

PT E

temperature for 15 min. Afterwards 0.8 g CYS was added to the above solution and the pH was readjusted to 4.75 with 1 mol/L HCl solution. After incubation for 5 h at room temperature under stirring in the dark, the resulting conjugate

CE

was dialyzed (MWCO 8~14 kDa) at 4°C for three days in the dark with pH=5.0

AC

HCl solution, pH=5.0 HCl solution containing 1% NaCl, pH=5.0 HCl solution, respectively, and was finally lyophilized. Samples were stored at 4°C for further use.

2.3 Characterization of HA-SH 2.3.1 Nuclear magnetic resonance (NMR) spectroscopic analysis 5 mg HA and HA-SH placed in a centrifuge tube, respectively, completely dissolved in deuterated water (D2O) and then added to the nuclear magnetic

ACCEPTED MANUSCRIPT tube. 1H NMR (500 MHz) spectra were recorded on an AVANCE III 500 NMR spectrometer (Bruker, Germany). 2.3.2 Determination of the free thiol group of HA-SH The degree of sulfhydryl modification of HA-SH was determined by

PT

Ellman's method according to Gucci et al. [20]. 1 mg HA-SH was dissolved in 250 μL deionized water, and then was added in 250 μL of 0.5 mol/L pH=8.0

RI

phosphate buffer and 500 μL DTNB. After incubation in the dark for 2 h at

SC

room temperature, absorbance at 450 nm was measured with the UV-Vis absorption spectra with a UV-2550 spectrophotometer (Shimadzu, Japan). The

NU

amount of free thiol groups was calculated using a standard curve obtained by the sulfhydryl group determination of a series of solutions containing CYS.

MA

2.4. Synthesis and characterization of maleilated collagen (Col-MA)

PT E

formaldehyde titration

D

2.4.1 Determination of free amino group content in collagen by

0.2 g sample (accurate to 1 mg) and 5 mL 10% acetic acid solution were put into the mortar, and grinded. The mixture was transferred to 50 mL

CE

volumetric flask, diluted with water to the mark, shaken well, and filtered

AC

finally. Afterwards, 2 mL filtrate, 4 mL water and 3 drops of phenolphthalein indicator were added to the flask, with following shaking. The solution was then titrated to a reddish color with 0.01 mol/L NaOH standard solution. After 2 mL neutral formaldehyde solution was following added to the flask, NaOH standard solution was continued to be dropped, until the solution was changed to red. The consumption volume of NaOH standard solution after addition of formaldehyde was marked as V. 6 mL distilled water was tested as blank control. Free amino content of collagen was calculated by the following formula:

ACCEPTED MANUSCRIPT Amino nitrogen content (%)  (V  V 0)  C  0.014  (1 / 2)  50  (1 / m) 100 Where, V = Consumption volume (mL) of NaOH standard solution after the adding formaldehyde; V0 = Consumption volume (mL) of NaOH standard solution after adding formaldehyde in the blank control; C = Concentration of NaOH

PT

standard solution (mol/L); 0.014 = Consumption of 1ml 1 mol/L NaOH standard solution equivalent to the quality of nitrogen (g); 2 = Volume of

RI

sample filtrate (mL); 50 = Total volume of diluent sample (mL); m = Mass of

SC

the samples (g).

NU

2.4.2 Synthesis of Col-MA

1 g collagen was dissolved in 100 mL 0.5 mol/L acetic acid solution.

MA

Maleic anhydride was then added in three ratios of 1:1, 1:2 and 1:3 (n-NH2/nMA), respectively [21]. The solution were kept stirring in N2 atmosphere for 30 min at room temperature, and were then closed for 8 h, 16 h and 24 h, respectively.

D

The resulting solution was lyophilized and washed with acetone. The resulting

PT E

product was denoted as Col-MA.

CE

2.4.3 Nuclear magnetic resonance (NMR) spectroscopic analysis To confirm the immobilization of MA onto the pure collagen, 5 mg

AC

collagen and maleilated collagen placed in a centrifuge tube, respectively, completely dissolved in 1 mL of HCl (37%) in deuterated water (D2O) [22] and then added to the nuclear magnetic tube. 1H NMR (500 MHz) spectra were recorded on an AVANCE III 500 NMR spectrometer (Bruker, Germany). 2.4.4 Determination of the degree of substitution (DS) of Col-MA under different reaction conditions Maleic anhydride reacts with the free amino groups of the lysine of

ACCEPTED MANUSCRIPT collagen. 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to determine the degree of reaction [23]. TNBS reacts with the unreacted free amino groups on lysine in maleilated collagen to form soluble complexes. The degree of substitution can be calculated by measuring the absorbance (ABS) of the collagen and maleilated collagen. The DS was calculated via the following equation: 𝐴𝐵𝑆𝐶𝑜𝑙 × 100% 𝐴𝐵𝑆𝐶𝑜𝑙−𝑀𝐴

PT

DS(%) =

RI

Where ABSCol was the absorbance of the collagen and ABSCol-MA was the

SC

absorbance of maleilated collagen.

1 mL 0.5%wt of freshly prepared TNBS solution and 1 mL 4%wt

NU

NaHCO3 solution were firstly mixed, and then different Col-MA (Table 1) samples were added and reacted at 40°C for 2 h. Afterwards, 2 mL 6 mol/L

MA

HCl solution was added to the reaction solution, and the reaction was carried out at 60°C for 90 min. The resulting solution was diluted with 5 mL deionized water and the ABS was measured at 345 nm. Pure collagen solution sample as

PT E

D

a control.

Table 1. The degree of substitution of Col-MA under different reaction conditions. Collagen (mg)

MA (mg)

n-NH2:nMA

Reaction time(h)

1

1

0.1

0.01

1:1

8, 16, 24

2

1

1

0.1

0.02

1:2

8, 16, 24

3

1

1

0.1

0.03

1:3

8, 16, 24

Group

AC

1

CE

TNBS (mL)

NaHCO3 (mL)

2.4.5 TG and DSC analysis 5-10 mg of collagen and Col-MA were measured respectively with a TGA209F thermogravimetric analyzer and the temperature raised from 25°C to 900°C under a nitrogen atmosphere at a rate of 10 °C/min. Differential scanning calorimetry (DSC) was measured with a differential

ACCEPTED MANUSCRIPT scanning calorimeter (DSC-2000PC). Samples were dissolved in 0.1 mol/L acetic acid solution, and 800 μL 15 g/L samples were placed in sealed aluminum pans, 800 μL 0.1

mol/L acetic acid in aluminum pans as a

control. The following protocol was used for all experiments: the first cooling run from room temperature to 20°C at a rate of 1 °C/min followed by 10 min

PT

isothermal step, then heating from 20°C to 70°C and cooling to 20°C at 1 °C/min.

2.5.1 CHG formation and gelation time

SC

RI

2.5 Preparation and characterization of collagen/hyaluronan gel (CHG)

NU

2%wt Col-MA and 4%wt HA-SH aqueous solutions were mixed by vortexing with vinyl/free thiol molar ratio of 1:1, 1:2, 1:3 and 1:4, respectively.

MA

Afterwards, 58%wt β-GP aqueous solution was added to adjust the pH to 7 and the mixtures were incubated at 37°C to form hydrogel. The hydrogel were

D

denoted as CHG-1, CHG-2, CHG-3 and CHG-4, respectively. The gelation

PT E

time of 2%wt Col-MA and 4%wt HA-SH solution was determined by inverting the glass bottle to observe whether the liquid flowed[8]. The preparation scheme of the CHG as Fig.1. The thiol adds to the α-C to -COOH or α-C to

AC

CE

-CONH- to form the hydrogels network.

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig.1. Preparation scheme of the CHG

MA

2.5.2 Rheology analysis

Rheological experiments were performed with a Kinexus Pro rheometer

D

(Malvern, UK) using parallel plates (Ø 20 mm). The time-sweep of precursor

PT E

solution was carried out at 37°C, a frequency of 1 Hz, and a strain of 1%.

CE

2.5.3 Compressive strength test Cylindrical hydrogel samples (Ø14mm×6mm) were placed on a PHILIPS

AC

TECNAI 10 testing machine for compression testing at a rate of 1mm/s (n=3). 2.5.4 Swelling ratio The lyophilized samples with accurate weigh (Wd) were immersed in 6.0 mL pH=7.4 PBS buffer solution and taken out at predetermined intervals. After the water on the surface was gently removed with filter papers, the weight of the swollen hydrogel (WS) was accurately weighed. The swelling ration (Q) was calculated by the following formulate (n=3): Q = (Ws/Wd) ×100%

ACCEPTED MANUSCRIPT 2.5.5 Microstructure analysis Scanning electron microscopy (SEM, PHILIPS Hitachi S-4700) was employed to determine the morphology of freeze-dried and air-dried hydrogels. Briefly, the freeze-dried samples were frozen rapidly in liquid nitrogen and wetting-off. Then the cross-sections of the freeze-dried samples and the surface

PT

of the air-dried samples were followed by gold coating using a sputter coater

RI

for 30 s prior to SEM imaging and observed at an accelerating voltage of 5 kV.

SC

2.6 Cells experiment

The biocompatibility of the CHG-4 was investigated with MC3T3-E1

NU

pre-osteoblastic cells according to the protocol reported previously [24]. The cell culture medium used was modified Eagle’s minimum essential medium

MA

with 4.5 g/L glucose, 10% fetal calf serum, 10 μg/ mL ascorbic acid and 30 μg/ mL gentamicin. About 5×105 cells were suspended in culture medium, with the

PT E

2.6.1 MTT assay

D

medium changed twice per week.

MTT assay was carried out to study the proliferation of the cells. Briefly,

CE

at predetermined time intervals (1, 3, 5 and 7days), 0.4 mL MTT solution (5 mg/mL) was injected into the hydrogel and incubated for 4 h. Thereafter, 4 mL

AC

of DMSO was added into each well and allowed to react until dissolution of the formazan pigment. Afterwards, 200 μL of pigment solution of each sample was transferred to a new 96-well plate and the absorbance was measured. The MTT absorption was measured at 570 nm with a background subtraction at 630 nm. A higher absorbance indicates that there are either more cells or that they have an increased metabolic activity.

ACCEPTED MANUSCRIPT 2.6.2 Cell morphology and cell proliferation The morphology of the cells seeded in the gels was investigated by SEM after 3, 5 and 7 days. The samples were fixed with 2.5% glutaraldehyde solution for 3 h. Then, the samples were sequentially dehydrated in 75% and 95% aqueous ethanol solution, each for 15 min. They were further dehydrated twice

PT

in absolute ethanol, each for 10 min. After that, the samples were freeze-dried

RI

and coated with Au by a sputter coater and examined by SEM.

SC

2.6.3 Confocal laser scanning microscopy (CLSM)

The cell morphology and cytoskeletal arrangement of the MC3T3-E1s

NU

seeded onto the hydrogels surfaces was measured by confocal laser scanning microscopy (CLSM; 510 Meta Duo Scan; Carl Zeiss, Germany). After being

MA

cultured for 6 h and 12 h, the cells were fixed in 4% paraformaldehyde solution for 10 min at room temperature, washed twice with PBS and then 0.1% Triton

D

X-100 was used to permeate the cells for 5 min at room temperature. The cells

PT E

were stained with 50 mg/ml FITC (actin filaments, red fluorescence; Invitrogen) for 20 min at room temperature in the dark. Then, DAPI (red fluorescence;

CE

Invitrogen) was used to stain the nuclei for 5 min. 3. Results and discussion

AC

3.1 Characterization of HA-SH HA-SH was synthesized via the carboxylic acid groups of HA and amine groups of L-cysteine. 1H NMR spectra in Fig.2. (B) revealed that the new peak of methylene protons on –CH2SH at δ=2.81 indicated the successful modification [25]. The substitution degree of HA-SH determined from 1H NMR was 45%, by comparing the peak area of methylene protons on –CH2SH and N-acetyl methyl protons (δ=2.03) [26]. According to the standard Ellman’s assay, the free thiol groups standard

ACCEPTED MANUSCRIPT curve was A = 3.157C-0.012, R = 0.9996, and the free thiol of HA-SH was

SC

RI

PT

calculated as 225.7±4.4 μmol/g.

3.2 Characterization of Col-MA

NU

Fig.2. Synthesis scheme (A) and 1H NMR spectra of HA-SH (B).

MA

3.2.1 Free amino group content in collagen

D

Formaldehyde titration method was used to determine the amino nitrogen, the total amino acid content of the samples can be measured. The principle is

PT E

the α-amino of amino acid react with aldehydes to form Schiff base in neutral or weak alkaline aqueous solution: α amino acids and formaldehyde react to

CE

produce a methyleneimino derivative. The methyleneimino derivative is titrated with the base to determine the total amino acid content of the sample. The free

AC

amino content of collagen calculated was 1.78% ± 0.04%, which means the mass percentage of free amino groups contains in per gram of collagen, and this can be used as the important reference for further collagen modification with maleic anhydride. 3.2.2 NMR spectroscopic analysis By comparing the 1H NMR spectra of collagen and Col-MA in Fig.3 (B), the new chemical shift at δ=6.24 [22] were found in Col-MA, corresponding to the -HC=CH- proton chemical shift, revealing that collagen was successfully

ACCEPTED MANUSCRIPT

MA

NU

SC

RI

PT

modified by maleic anhydride.

Fig.3. Schematic representation (A) and 1H-NMR (B) of the Col-MA (n-NH2/nMA

D

=1:2 and reaction time of 24 h).

PT E

3.2.3 DS of Col-MA under different reaction conditions The feed ratios and the reaction time affect the DS of Col-MA. Table 2

CE

shows that the DS values increased with prolonging of reaction time. When the reaction time was prolonged from 8 h to 16 h, the DS of Col-MA (n-NH2/nMA =1:

AC

1) increased dramatically from 5.43% up to 10.13%, but the other two products increased slightly. When the reaction time was 24 h, the DS of the three Col-MA products increased rapidly, especially the DS of n-NH2/nMA =1: 2 sample reaching about 35%. As the reaction time was short (8 h and 16 h), the feed ratio had little effects on the DS. However, when the reaction time was prolonged to 24 h, the DS increased first and then decreases with the increasing content of maleic anhydride. The main reason may be that with the increase content of maleic

ACCEPTED MANUSCRIPT anhydride, the contact chance between amino group and acid anhydride is increased and the reaction efficiency is therefore improved. While collagen fibers are macromolecules with triple helixes, so as the reaction time goes by, the steric hindrance increases and reaction efficiency will decrease with the increasing maleic anhydride.

PT

Therefore, n-NH2/nMA = 1:2 and reaction time of 24 h will be used for the following experiment.

RI

Table 2. Effects of the n-NH2/nMA ratio and reaction time on the substituting degree

DS of Col-MA samples

n-NH2/nMA =1:1 5.43%+ 0.12%

16 h

10.13%+0.29%

24 h

16.667%+0.34%

2.30%+0.15%

2.496%+0.11%

2.43%+0.19%

34.875%+0.17%

27.174%+0.26%

D

3.2.4 TG and DSC analysis

n-NH2/nMA =1:3

1.47%+0.34%

MA

8h

n-NH2/nMA =1:2

NU

Time

SC

of maleilated collagen

PT E

The first peak appears in the TG curve at about 100°C, which was the heat-absorption peak of free (before 105°C )and binding water (above 120°C)

CE

[6]. The main mass loss at 250°C to 380°C was due to the breakage of the covalent bond between the amino acid chains. The second peak of the TG

AC

curve of collagen and Col-MA appeared at 320°C, and the obtained values are similar to the data reported in literature [27]. The mass loss of collagen was about 52.81%, which was slightly higher than 47.24% of Col-MA. The residual mass of Col-MA and collagen were 15.91% and 8.73%, respectively, indicating that collagen decomposed more thoroughly when the temperature was higher. This may be the presence of amide bonds, more cross-linking points in the molecular chain of Col-MA to form more intermolecular crosslinking structure and improved the thermostability.

RI

PT

ACCEPTED MANUSCRIPT

Fig.4. TG characterization of the collagen (A) and Col-MA (B)

SC

In addition, DSC results (Fig.5) verified the improvement of the thermostability of Col-MA. There is a significant change in the modified

NU

collagen solution, indicating that the collagen intermolecular structure does modified. The peak of Col-MA at 40.5°C and became more extensive, while

MA

the unmodified collagen was 34.5°C. Based on the experiments of NMR and

AC

CE

PT E

D

DSC, it was found that the modified collagen had a stable covalent bond.

Fig.5. DSC characterization of the collagen and Col-MA

ACCEPTED MANUSCRIPT 3.3 Characterization of collagen/hyaluronan hydrogel 3.3.1 Influences of n-C=C- /n-SH values on the gelation time The gelation time of 2%wt Col-MA and 4%wt HA-SH solution was determined by inverting the glass bottle to observe whether the liquid flowed as

PT

the Fig.6. The results are shown in Table 3. In the 37°C water bath, gelation occurs very quickly, resulting in a white translucent hydrogel. By adjusting the

RI

n-C=C-/n-SH values, the gelation time changed, which was shortened with

SC

decrease of the n-C=C-/n-SH values, possibly due to the increase in the number of reactive sulfhydryl groups per unit volume. While with the ratio of HA-SH

NU

increased higher than 4, the hydrogel formed too fast to be manipulated,

PT E

D

MA

therefore we did not try the other higher ratios.

Fig.6. Images of CHG before (A) and after (B) gelation.

f (n-C=C- /n-SH)

Gelation time (s)

AC

CE

Table 3. Influences of f (n-C=C- /n-SH) values on the gelation time

Group 1

1:1

43

2

1:2

37

3

1:3

24

4

1:4

15

3.3.2 Rheological and mechanical properties Rheological properties (viscosity and fluidity) are important properties of

ACCEPTED MANUSCRIPT injectable materials in the process of injection gelation and have important practical significance in clinical application. Fig.7 (A) shows the storage modulus (G') and loss modulus (G'') of CHG-4 over time, and the lines intersect at about 40 s, which reveals the fast formation of hydrogel. Thereafter G' is maintained above G''. The results show that Col-MA/HA-SH has a good

PT

gelation performance. Fig.7 (B) shows the compressive elastic modulus of the hydrogels. With

RI

increases ratio of the HA-SH, hydrogel got stiffer, the Young’s modulus increased from 1671.65±546.23 Pa to 9105.86±2064.15 Pa, and the CHG-4

SC

possessed the highest modulus in all the four samples. This may be because the increase of HA-SH increased the numbers of the active groups, the

NU

cross-linking degree of the hydrogel increased and therefore the interaction

PT E

D

MA

between molecular chains was strengthened.

CE

Fig.7. Mechanical behavior of the hydrogels. (A) Storage modulus G’ and loss modulus G’’ analyses during gelation process (37°C; frequency: 1.0 Hz; strain: 1.0%;

AC

CHG-4). (B) The compression strength of hydrogels with different n-C=C- /n-SH values (37°C).

3.3.3 Swelling ratio The equilibrium swelling ratios of the dry hydrogels were measured using a gravimetric method. Fig.8 shows the swelling curves of different hydrogel samples. All the samples absorbed water rapidly within 40 min, then slow down and reached the balance gradually. Hydrogel has many hydrophilic

ACCEPTED MANUSCRIPT groups which can form hydrogen bonds with water molecules. On the other hand, the negative charge between the network molecules increases and electrostatic repulsion increases, which promotes the extension of the chains in the network. Gel network expansion is conducive to water molecules into the gel and the swelling rate is further increased, can reach the balance quickly.

PT

CHG-1 has the worst water-absorption capacity, with Qmax of 1.5, which may be the insufficient Michael reaction, the gel network can hold less water. With

RI

the increase of the proportion of HA-SH in the hydrogel, the water absorption capacity of the hydrogel gradually increased, and the CHG-4 water absorption

SC

capacity was the strongest, the Qmax was as high as 12.7. Hyaluronan is a highly water-absorbent and highly water-retaining natural polymer material, and with

NU

the increase of the HA-SH ratio in the network, the association between the gel molecular chains occurs and cross-linked to form a stable water-retaining

MA

network. According to the Fickian diffusion theory, the first step is to diffuse the solvent molecule into the polymer network. The second step is to make the

D

polymer chain to relax from the glassy state to the rubbery state by the

PT E

solvation. The third step is the polymer network diffuse into the solvent. When the first-step process determines the reaction rate, the amount of solvent absorbed by the gel is very small, the polymer chain is hardly relaxed, or the

CE

polymer chain relaxed very fast and the swelling of the gel is controlled by the diffusion of solvent molecules into the network. From Fig.8 (B), we can also

AC

find that CHG-4 hydrogels absorb water at 15 min with a fast rate and then enter a relatively slow water absorption process, which may be the diffusion of water and gel network diffusion.

PT

ACCEPTED MANUSCRIPT

RI

Fig.8. Equilibrium swelling ratio of CHG composite hydrogels incubated in PBS at

SC

37°C.

NU

3.3.4 Microstructure analysis

The morphological characteristics of hydrogels are an important factor in

MA

tissue engineering applications, and specifically porous structures are critical for the growth of tissues, the diffusion of nutrients and metabolite. In addition, the percentage of porosity, pore size and interconnection are critical parameters

D

in determining the performance of the hydrogel. Fig.9 (A) is the SEM image of

PT E

the freeze-dried samples, wherein the porous structure can be clearly seen, which confirms the open and interconnecting porosity of the CHG hydrogel.

CE

Although the morphology of the freeze-dried hydrogel cannot reflect the real porous structure of the wet hydrogel, it is a very important indicator for water

AC

volume, and it is valuable for cell inoculation, nutrient and metabolite diffusion. It is notable that the interwoven and coalescing collagen fibrils can be obviously observed on the surface of the air-dried samples, which reveals that the modification and following Michael addition did not denature the collagen.

ACCEPTED MANUSCRIPT Fig.9. SEM images of the freeze-dried (A) and air-dried (B and C) CHG-4 samples.

3.4 Cell culture 3.4.1 MTT assay An MTT assay was performed to determine cell growth in the CHG-4.

PT

Fig.10 shows that the MTT absorption of all the samples increased with time,

RI

indicating cell proliferation in the hydrogels. The results also confirmed that the CHG-4 facilitated the growth and proliferation of MC3T3-E1. This

SC

indicates that the collagen/hyaluronan composite network, which acted as a

CE

PT E

D

MA

NU

native ECM, promoted the proliferation of the cells.

Fig.10. MTT absorbance at 570 nm of MC3T3-E1 after 1, 3, 5 and 7 days culture on

AC

control and CHG-4. Results present means ± SD of three independent experiments.

3.4.2 Cell morphology SEM and CLSM images after cells cultured for 3, 5 and 7 days are shown in Fig.11. Cells displayed flatten shapes and a spreading morphology on the CHG-4, and formed confluent and nearly aligned cell layers with the extension of culture time, which might be created by the contact guidance of the collagen fibrils. Numerous well defined actin filaments were observed, and the actin

ACCEPTED MANUSCRIPT microfilament system ran parallel to the long axis of the cells. As well known, the natural structure of collagen fibers have been proved to enhance cell attachment and it has been reported that cells align along oriented collagen

MA

NU

SC

RI

PT

fibril matrices via contact guidance[28].

Fig.11. SEM images (A-C) and CLSM images (D-F, with original magnification of

PT E

4. Conclusions

D

400) of MC3T3-E1 cells on the surface of CHG-4 cultured for 3, 5 and 7d.

In this study, the collagen/hyaluronan in situ composite hydrogel was

CE

prepared through mild and rapid Michael addition reaction between the -C=Cand -SH. The results showed that the gelation time of CHG could be controlled,

AC

and the gel time was 45 s at f =1:4 by rheological experiments. The water absorption was rapid within 15 min and reached equilibrium within 40 min, the Qmax was 12.7. Although the mechanical properties of gel need to be improved, the hydrogel is expected to be used in the field of minimally invasive treatment. Cell culture revealed that CHG facilitated the growth and proliferation of MC3T3-E1. Therefore, it is suggested that CHG can be used as injectable materials for tissue regeneration.

ACCEPTED MANUSCRIPT Acknowledgments：The work is supported by the National Natural Science Foundation of China (31270021), Science and Technology Project of Guangdong Province (2014A010105031), the Natural Science Foundation of Guangdong Province (2014A030313379) and Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation

AC

CE

PT E

D

MA

NU

SC

RI

PT

(pdjh2016b0065).

ACCEPTED MANUSCRIPT References [1] A. Sionkowska, A. Kamińska, Thermal helix-coil transition in UV irradiated collagen from rat tail tendon, International Journal of Biological Macromolecules, 24 (1999) 337-340. [2] J. Radhakrishnan, U.M. Krishnan, S. Sethuraman, Hydrogel based injectable

PT

scaffolds for cardiac tissue regeneration, Biotechnology Advances, 32 (2014) 449-461.

RI

[3] A. Sivashanmugam, R.A. Kumar, M.V. Priya, S.V. Nair, R. Jayakumar, An overview of

SC

injectable polymeric hydrogels for tissue engineering, European Polymer Journal, 28 (2015) 543-565.

NU

[4] J.A. Yang, J. Yeom, B.W. Hwang, A.S. Hoffman, S.K. Hahn, In situ-forming injectable hydrogels for regenerative medicine, Progress in Polymer Science, 39 (2014)

MA

1973-1986.

[5] C.H. Lee, A. Singla, Y. Lee, Biomedical applications of collagen, International Journal of Pharmaceutics, 221 (2001) 1-22.

D

[6] E.A.A. Neel, L. Bozec, J.C. Knowles, O. Syed, V. Mudera, R. Day, J.K. Hyun,

PT E

Collagen-Emerging collagen based therapies hit the patient, Advanced Drug Delivery Reviews, 65 (2013) 429-456.

CE

[7] L. Yu, J. Ding, ChemInform Abstract: Injectable Hydrogels as Unique Biomedical Materials, ChemInform, 37 (2008) 1473-1481.

AC

[8] B. Ye, L. Meng, Z. Li, R. Li, L. Li, L. Lu, S. Ding, J. Tian, C. Zhou, A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability, Materials Letters, 183 (2016) 81–84. [9] H.Ç. Arca, S. Şenel, Chitosan Based Systems for Tissue Engineering Part 1: Hard Tissues, Fabad Journal of Pharmaceutical Sciences, 33 (2008) 35-49. [10] C.D. Moreira, S.M. Carvalho, H.S. Mansur, M.M. Pereira, Thermogelling chitosan-collagen-bioactive glass nanoparticle hybrids as potential injectable systems for tissue engineering, Materials Science & Engineering C Materials for Biological Applications, 58 (2016) 1207-1216.

ACCEPTED MANUSCRIPT [11] Y.B. Truong, V. Glattauer, K.L. Briggs, S. Zappe, J.A. Ramshaw, Collagen-based layer-by-layer coating on electrospun polymer scaffolds, Biomaterials, 33 (2012) 9198-9204. [12] S.H. Kim, J.H. Lee, S.Y. Yun, J.S. Yoo, C.H. Jun, K.Y. Chung, H. Suh, Reaction monitoring

of

succinylation

desorption/ionization

mass

of

collagen

spectrometry,

Rapid

with

matrix-assisted

Communications

Mass

PT

Spectrometry, 14 (2000) 2125-2128.

in

laser

RI

[13] R. Kumar, R. Sripriya, S. Balaji, M.S. Kumar, P.K. Sehgal, Physical characterization of succinylated type I collagen by Raman spectra and MALDI-TOF/MS and in vitro

SC

evaluation for biomedical applications, Journal of Molecular Structure Theochem, 994 (2011) 117-124.

NU

[14] Y. Luo, K.R. Kirker, G.D. Prestwich, Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery, Journal of Controlled Release Official Journal of

MA

the Controlled Release Society, 69 (2000) 169-184.

[15] J.A. Burdick, G.D. Prestwich, Hyaluronic acid hydrogels for biomedical

D

applications, Advanced Materials, 23 (2011) 41-56.

PT E

[16] G. Kogan, L. Soltes, R. Stern, P. Gemeiner, Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications, Biotechnology Letters, 29 (2007) 17-25.

CE

[17] J. Zhang, X. Ma, D. Fan, C. Zhu, J. Deng, J. Hui, P. Ma, Synthesis and characterization of hyaluronic acid/human-like collagen hydrogels, Materials Science

AC

& Engineering C, 43 (2014) 547-554. [18] A. Polák, Collagen functionalized with unsaturated cyclic anhydrides-interactions in solution and solid state, Biopolymers, 101 (2014) 228–236. [19] J. Borek, W. Osoba, Influence of the plasticization on free volume in polyvinyl chloride, Journal of Radioanalytical and Nuclear Chemistry, 211 (1996) 61-67. [20] D. Guggi, M.K. Marschütz, A. Bernkop-Schnürch, Matrix tablets based on thiolated poly(acrylic acid): pH-dependent variation in disintegration and mucoadhesion, International Journal of Pharmaceutics, 274 (2004) 97-105. [21] Y. Dong, A.O. Saeed, W. Hassan, C. Keigher, Y. Zheng, H. Tai, A. Pandit, W. Wang,

ACCEPTED MANUSCRIPT “One-step” Preparation of Thiol-Ene Clickable PEG-Based Thermoresponsive Hyperbranched Copolymer for In Situ Crosslinking Hybrid Hydrogel, Macromolecular Rapid Communications, 33 (2012) 120-126. [22] S. Potorac, M. Popa, V. Maier, G. Lisa, L. Verestiuc, New hydrogels based on maleilated collagen with potential applications in tissue engineering, Materials Science & Engineering C, 32 (2012) 236–243.

PT

[23] W.A. Bubnis, C.M. Ofner, The determination of ϵ-amino groups in soluble and

RI

poorly soluble proteinaceous materials by a spectrophotometrie method using trinitrobenzenesulfonic acid, Analytical Biochemistry, 207 (1992) 129-133.

SC

[24] M. Zhao, L. Li, X. Li, C. Zhou, B. Li, Three-dimensional honeycomb-patterned chitosan/poly(L-lactic acid) scaffolds with improved mechanical and cell compatibility,

NU

Journal of Biomedical Materials Research Part A, 98 (2011) 434-441. [25] J. Ding, R. He, G. Zhou, C. Tang, C. Yin, Multilayered mucoadhesive hydrogel films

MA

based on thiolated hyaluronic acid and polyvinylalcohol for insulin delivery, Acta Biomaterialia, 8 (2012) 3643-3651.

D

[26] Y. He, G. Cheng, L. Xie, Y. Nie, B. He, Z. Gu, Polyethyleneimine/DNA polyplexes

PT E

with reduction-sensitive hyaluronic acid derivatives shielding for targeted gene delivery, Biomaterials, 34 (2013) 1235-1245. [27] M.M. Horn, V.C.A. Martins, A.M.D.G. Plepis, Interaction of anionic collagen with

CE

chitosan: Effect on thermal and morphological characteristics, Carbohydrate Polymers, 77 (2009) 239-243.

AC

[28] S. Guido, R.T. Tranquillo, A methodology for the systematic and quantitative study of cell contact guidance in oriented collagen gels. Correlation of fibroblast orientation and gel birefringence, Journal of Cell Science, 105 (Pt 2) (1993) 317-331.

ACCEPTED MANUSCRIPT Highlights  We have synthesized in-situ formable collagen/hyaluronan composite hydrogel to mimic the natural extracellular matrix (ECM) with great application prospect.  The in-situ hydrogels were prepared by a facile approach via Michael addition reaction, without any extraneous chemical crosslinking agents.

PT

 Physical and chemical properties of the hydrogels can be tuned by the

AC

CE

PT E

D

MA

NU

SC

RI

molar ratio of vinyl/free thiol groups.