Synthesis and characterization of MMP degradable and maleimide cross-linked PEG hydrogels for tissue engineering scaffolds

Polymer Degradation and Stability 133 (2016) 312e320

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Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab

Synthesis and characterization of MMP degradable and maleimide cross-linked PEG hydrogels for tissue engineering scaffolds Jingjing Yu a, Feng Chen a, Xichi Wang b, Nianguo Dong b, **, Cuifen Lu a, *, Guichun Yang a, Zuxing Chen a a

Hubei Collaborative Innovation Center for Advanced Organochemical Materials & Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei University, Wuhan 430062, China Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 June 2016 Received in revised form 22 August 2016 Accepted 6 September 2016 Available online 7 September 2016

A series of poly(ethylene glycol) (PEG) hydrogels were successfully prepared via Michael-type addition between 4-arm PEG-maleimide (PEG-4MAL) and MMP degradable peptide. The gelation time was significantly short (~10 min) in a low concentration of TEA (4 mM). Thermogravimetric analysis indicated the thermal stabilities of hydrogels. SEM images confirmed a porous structure and the pore size increased with the increase of the PEG chain length. Rheological measurements indicated that all the hydrogels exhibited the characteristics of elastomer and the cross-linking density had a correlation to the polymer weight percentage. After immersing in 0.9% sodium chloride injection, PEG hydrogels exhibited a good water absorption capacity, and their swelling ratio were directly related to the amount of crosslinking. Biological activities of the hydrogels were evaluated by in vitro enzymatic degradation and in vitro cell compatibility on mesenchymal stem cells (MSCs) and the results showed that the hydrogels were biocompatible and could be degraded by exogenously delivered MMPs or cell-secreted MMPs. Thus, PEG hydrogels exhibited the potential for tissue engineering scaffolds. © 2016 Elsevier Ltd. All rights reserved.

Keywords: PEG hydrogels MMP degradable Maleimide Michael-type addition

1. Introduction Poly(ethylene glycol) (PEG) hydrogels are attractive for use as tissue engineering scaffolds [1e4] because they are intrinsically biocompatible, resist non-specific protein adsorption and permit the rapid encapsulation of cells in cytocompatible conditions. The mechanical property of PEG hydrogels can be regulated by changing the molecular weight and concentration of PEG [5,6]. Moreover, the biodegradability of PEG hydrogels can be conferred by the incorporation of degradable components via enzymatic, hydrolytic or environmental pathways. A matrix metalloproteinase (MMP) degradable peptide is often chosen to crosslink PEG chains to create a hydrogel network that is degraded by cell-secreted MMPs [7e9]. The reported PEG hydrogels were formed by either Michaeltype addition polymerization [5,10] or free-radical initiated

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (N. Dong), [email protected] (C. Lu). http://dx.doi.org/10.1016/j.polymdegradstab.2016.09.008 0141-3910/© 2016 Elsevier Ltd. All rights reserved.

polymerization [3,8]. A major drawback of free-radical initiated polymerization is that it can significantly reduce encapsulated cell viability. In contrast, Michael-type addition polymerization avoids the use of cytotoxic free-radicals and UV light, but instead requires a nucleophilic buffering reagent [11] to facilitate the addition reaction. However, PEG hydrogels formed by Michael-type addition polymerization with the end functional group of acrylate or vinyl sulfone in the presence of high concentrations of nucleophilic buffering reagent have cytotoxic effects on several sensitive cells [12]. The maleimide group is extensively used in peptide bioconjugate chemistry because of its faster reaction kinetics than acrylate and vinyl sulfone, high specificity for thiols at physiological pH and requiring a low concentration of nucleophilic buffering reagent [13]. Although much research effort has been directed towards PEG hydrogels crosslinked by MMP degradable peptide as scaffolds, but a comprehensive investigation of mechanical property, degradation property and biocompatibility of PEG hydrogels crosslinked by MMP degradable peptide has not previously been reported. Therefore in this study, a series of PEG hydrogels were prepared from 4-arm PEG-maleimide (PEG-4MAL) macromer crosslinked

J. Yu et al. / Polymer Degradation and Stability 133 (2016) 312e320

with MMP degradable peptide under physiological conditions by Michael-type addition polymerization. Moreover, the FTIR spectra, morphologies, thermal properties, mechanical properties, swelling properties, degradation properties and in vitro cell compatibility of the hydrogels were characterized.

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containing peptide (molar ratio of MAL/SH was 1:1) into triethanolamine solution (TEA; pH 7.4, 4 mM) at a specific concentrations, e.g. 5%, 7.5% wt/v. The pre-polymer solutions were mixed by vortexing and then transferred to an injection syringe for gelation at 37
C. The gelation time was determined as the time when the hydrogel would no longer flow by the force of gravity.

2. Experimental 2.3. Chemical structure identification 2.1. Materials PEG-4MAL (5, 10 and 20 kDa, >95% end-group substitution) was purchased from the Jenkem Technology Co., Ltd. (Beijing, China). Recombinant Human MMP-2 (62 kDa, 98% purity by HPLC analyses) was purchased from the PeproTech (USA). MMP degradable peptide (Ac-GCRD-GPQGYIWGQ-DGCG-NH2, 1.7 kDa) was obtained from the GL Biochem Ltd. (Shanghai, China). Plastic cell culture dishes and plates were purchased from Wuxi NEST Biotechnology Co Ltd (Wuxi, China). Bone marrow-derived mesenchymal stem cells (MSCs) were obtained from Sprague-Dawley (SD) rats in Tongji Medical College (Wuhan, China). All other chemicals were purchased from Sinopharm Chemical Reagent (Wuhan, China) and were used without further purification. 2.2. Preparation of PEG hydrogels A schematic of the hydrogel formation is presented in Fig. 1. Hydrogels were prepared by Michael-type addition of thiolcontaining peptide (Ac-GCRD-GPQGYIWGQ-DGCG-NH2) onto PEG-4MAL, as described by García et al. [13]. First, the pre-polymer solutions were prepared by dissolving PEG-4MAL and thiol-

FTIR spectra were obtained on dried samples at room temperature using a Nicolet IS 50 spectrometer (ThermoFisher Scientific Co.), equipped with a diamond Attenuated Total Reflection (ATR). Spectra were obtained at a resolution of 2 cm1 in the range 4000600 cm1 for a total of 16 scans. The swollen hydrogels were frozen rapidly at 55
C and then dried in a freeze-dryer. 2.4. Thermal properties To examine thermal stability of hydrogels, the hydrogel samples were measured by thermal analysis system (TG/DTA), using a Mettler Toledo equipment (thermobalance sensitivity: 0.1 mg), which was previously calibrated in the temperature range of 30e800
C by running tin and lead as melting standards, at a heating rate (f) of 20
C/min, using open alumina crucibles and a dry nitrogen purge flow of 40 mL/min. Sample weights ranging from 8 to 10 mg were used. 2.5. Scanning electron microscopy To observe the interior morphologies of hydrogels, the swollen

Fig. 1. Schematic of PEG hydrogel formation.

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hydrogel samples were quickly frozen in liquid nitrogen and further freeze-dried in a Freeze Drier (Beijing boyikang Lab Instrument Co., Ltd.) under the vacuum at 55
C for at least 1 day until all the solvent was sublimed. The freeze-dried hydrogels were then fractured carefully, and the interior morphologies of hydrogels were visualized by using a scanning electron microscope (SEM) on a JEOL JSM-6510 at an acceleration voltage of 20 KV. Before SEM observation, the hydrogel samples were fixed on aluminum stubs and coated with gold. 2.6. Dynamic rheological measurements A Stress Tech rheometer (TA Instruments) with standard steel cone-plate geometry of 20 mm diameter was used for the rheological characterization of all hydrogel samples (after the hydrogels were immersed for 12 h in 0.9% sodium chloride injection, then removed from sodium chloride injection and carefully blotted with filter paper). The test methods employed were oscillatory stress sweep and frequency sweep. The stress sweep was performed on hydrogels to determine the linear viscoelastic region (LVR) profiles of them and compare the storage modulus (G0 ) under the same physical condition. The stress sweep was set up by holding the temperature (25
C) and frequency (1 Hz) constant while increasing the stress level from 0.1 to 100 Pa. We also subjected the hydrogels to a frequency sweep at a fixed shear stress (10 Pa) and temperature (25
C), the oscillatory frequency was increased from 0.01 to 20 Hz and the G0 was recorded. The plots of G0 versus shear stress or frequency from the two sweep tests were obtained directly from the software controlling the rheometer. 2.7. Measurement of equilibrium swelling degree To study the swelling properties of hydrogels, the dried hydrogels were immersed in 10 mL of 0.9% sodium chloride injection at 37
C. At predetermined times, the swollen samples were removed to record the weight until constant weight. The swelling ratio (SR) of hydrogels were determined using the following equation: SR ¼ [(WsWd)/ Wd]  100%

using LIVE/DEAD® assay and CCK-8 assay. For three-dimensional (3D) cell encapsulation, the mesenchymal stem cells (MSCs) in the third passage were embedded in the 64 mL pre-polymer solution, which was then crosslinked to form hydrogels in 96 well plates at the density of 1  106 cells/mL. MSCs seeded on 96 well plates at the same seeding density were performed in parallel as the control. After cultured for a certain period time ranged 1e10 days, the encapsulated MSCs were washed twice with PBS and then were stained by LIVE/DEAD® Viability/Cytotoxicity Assay Kit containing propidium iodide (PI) and Calcein AM (4 mmol/L and 2 mmol/L, Dojindo Molecular Technologies, Inc.) for 30 min in dark at room temperature to indicate the dead (with membrane rupture) and live cells, respectively. Then the populations of living (green cytoplasmic fluorescence) and death (red nucleus) of cells were evaluated by fluorescence microscopy (OLYMPUS BX71, Japan) with appropriate filter sets. In addition, CCK-8 assay was performed to further evaluated cell proliferation and viability. After each predetermined time interval, each sample was incubated with DEME containing 10% (v/v) CCK-8 (Dojindo Molecular Technologies, Inc.) in dark at 37
C for 2 h. After that, the CCK work solution was transferred to a new 96 well plates (100 mL per well) and absorbance was measured using a microplate reader (Biorad) at a wavelength of 450 nm. Each testpoint was repeated at least four times. 3. Results and discussion 3.1. Preparation of PEG hydrogels PEG-4MAL with different molecular weight (5, 10 and 20 kDa) were cross-linked into a hydrogel by addition of the dithiol MMPdegradable peptide cross-linker (Ac-GCRD-GPQGY IWGQ- DGCGNH2) at a 1:1 M ratio of remaining PEG reactive end groups to peptide thiols. The degree of gelation was estimated using the vial-tilting method, to confirm whether a macrogel had formed or not. Fig. 2 shows a phase diagram for the gelation between the polymer weight percentage and the molecular weight of PEG. PEG-20 kDa

(1)

Where Wd is the weight of the dry samples before immersion, and Ws is the weight of the swollen samples. Three measurements were done for each hydrogel. 2.8. In vitro enzymatic degradation In vitro enzymatic degradation tests were done in phosphate buffered saline (PBS; pH 7.4, 10 mM) at 37
C during 30 days. Hydrogels were immersed into PBS without and with Recombinant Human MMP-2. MMP-2 concentrations used in this study were 0, 100, 200 or 400 ng/mL. At predetermined periods of time, the samples were removed from PBS, washed with distilled water, and then dried under vacuum. The degree of degradation was estimated from the weight loss of the gels according to the following equation: Weight Loss ¼(WoWt)/ Wo  100%

(2)

Where Wo is the initial weight of the dry samples before immersion, and Wt is the dry samples weight after incubation for t days. Three measurements were done for each hydrogel. 2.9. In vitro cell viability In vitro viability of the 7.5% PEG-20 kDa hydrogels was studied

Fig. 2. Phase diagram of gelation. Influence of polymer weight percentage on gelation for networks made from PEG-4MAL with different molecular weight. Representative images are shown in the figure legend.

J. Yu et al. / Polymer Degradation and Stability 133 (2016) 312e320

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Fig. 3. FTIR spectra of (A) PEG hydrogels, (B) PEG-4MAL.

forms gels as low as 4%. We subsequently observed lower polymer weight percentage limits of 4% for PEG-10 kDa, 5% for PEG-5 kDa and PEG-20 kDa formed better gels at lower polymer concentration compared to PEG-5 kDa. These may due to the fact that the mechanical property of hydrogels was in general correlated to the crosslinking density and the inter-chain entanglements of component [14e16]. Although shorter PEG chain means higher

crosslinking density, the longer PEG chain might provide more entanglements in the hydrogels, which could result in better hydrogels. But these were so weak that it was difficult to handle then using tweezers. We improved the polymer weight percentage, mechanically hard gels were formed. Additionally, the time of gelation was significantly short in PEG-4MAL (~10 min) in 4 mM TEA, which is the reason that we chose maleimide group as the end

Fig. 4. Thermogravimetric analysis of hydrogels, (A) with different molecular weight, (B) with different weight percentage.

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Fig. 5. SEM images (cross-section view) of (A) PEG-20 kDa-10% hydrogel, (B) PEG-20 kDa 7.5% hydrogel, (C) PEG-20 kDa-5% hydrogel, (D) PEG-10 kDa-10% hydrogel, (E) PEG-10 kDa-7.5% hydrogel, (F) PEG-10 kDa-5% hydrogel, (G) PEG-5 kDa-10% hydrogel, (H) PEG-5 kDa-7.5% hydrogel.

3.2. FTIR spectra of PEG hydrogels Fig. 3 shows the FTIR spectra of the eight mechanically hard gels which are similar to some extent. The absorption peaks, observed

20kDa-10% 20kDa-7.5% 20kDa-5% 10kDa-10% 10kDa-7.5% 10kDa-15% 5kDa-10% 5kDa-7.5%

Storage Modulus G' (Pa)

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between 3500 cm1 and 3250 cm1, were assigned to stretching vibrations of N-H groups. The absorption peaks of double bonds near 3100 cm1 (Fig. 3B) were absence in Fig. 3A, so PEG-4MAL with different molecular weight have been completely reacted. The absorption peaks, observed between 3000 and 2840 cm1, were assigned to the asymmetric and symmetric stretching vibrations of CH2 groups in both cases of studied materials. In addition,

20kDa-10% 20kDa-7.5% 20kDa-5% 10kDa-10% 10kDa-7.5% 10kDa-5% 5kDa-10% 5kDa-7.5%

B Storage Modulus G' (Pa)

functional group. Fast gelation times are critically important for uniform distribution of encapsulated cells in 3D cultures.

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Fig. 6. Rheological behaviors of various hydrogel samples. (A) Oscillatory shear sweep, (B) Oscillatory frequency sweep.

J. Yu et al. / Polymer Degradation and Stability 133 (2016) 312e320 2500 20kDa-10% 20kDa-7.5% 20kDa-5% 10kDa-10% 10kDa-7.5% 10kDa-5% 5kDa-10% 5kDa-7.5%

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Time (h) Fig. 7. Swelling behavior of the hydrogel films in 0.9% sodium chloride injection at 37
C.

the very strong absorption peaks approach 1660 cm1 were attributed to carbonyl group of amide (-CONH), and the N-C of amide stretching vibration were observed as a peak at 1530 cm1, which were directly connected with N-H bonding plus C-N stretching in an amide (-CONH). The C-H bending, CH2 scissoring and CH2 wagging peaks were observed respectively at 1466, 1409 and 1342 cm1. The peaks observed at 1150 cm1 was related with extremely strong stretching in the soft segment of C-O-C groups from PEG. 3.3. Thermal properties of PEG hydrogels The thermal stability of PEG hydrogels with different molecular weight and different weight percentage were measured using TGA analysis. Fig. 4 shows the weight loss and weight loss rate curves recorded with a heating rate of 20
C/min in nitrogen atmosphere between 30 and 800
C range. Fig. 4A shows the thermogravimetric curves of PEG-20 kDa7.5%, PEG-10 kDa-7.5%, and PEG-5 kDa-7.5%. With the same weight percentage of the hydrogels, the numbers of cross-links in these samples are different from each other because of their different molecular weights. A higher molecular weight of PEG corresponds to the lower cross-linking density. Comparison among PEG hydrogels of the same polymer weight percentage, the hydrogel of PEG-20 kDa-7.5% with the highest molecular weight but lowest

A

60

cross-linking density has the highest thermal degradation temperature and the lowest weight loss. So among PEG hydrogels of the same polymer weight percentage, the thermal stability is mainly depended on the molecular weight of PEG. Meanwhile, the hydrogel of PEG-20 kDa-7.5% has the highest weight loss rate, which is consistent with the lowest cross-linking density. Then, the thermal stabilities for a variety of polymer weight percentages of PEG-20 kDa were measured. Fig. 4B shows the thermogravimetric curves of PEG-20 kDa-10%, PEG-20 kDa-7.5%, and PEG-20 kDa-5%. For the hydrogel films, the higher the weight percentage in the hydrogels were, the higher the thermal degradation temperatures were, and the less the weight loss of the hydrogel were. In particular, as the weight percentage of the hydrogels increasing from 5% to 7.5%, the thermal degradation temperature increased from 309.6 to 327.7
C, this is probably due to the high percentage of the hydrogels promoting the hydrated cross-linked polymer network of the hydrogel. Furthermore, the weight loss for PEG-20 kDa-10%, PEG-20 kDa-7.5%, and PEG-20 kDa-5% was 40%, 30% and 28%, respectively. The weight loss results indicated that the decomposition stage was depended on the weight percentage of the hydrogel. 3.4. Hydrogel morphologic analysis by SEM The cross-sectional morphology of the hydrogel networks in a

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Loss weight ratio (%)

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MMP-2 concentration (ng/mL) Fig. 8. Degradation studies in vitro for PEG hydrogels. (A) Weight loss of PEG-20kDa-7.5% hydrogel after degradation for 30 days in different concentrations (0, 50, 100, 200, 400 ng/ mL) of MMP-2 solution, (B) Weight loss after degradation for 30 days in MMP-2 solution (200 ng/mL) at 37
C.

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Fig. 9. Fluorescence micrographs of PI and Calcein AM stained MSCs at different periods of cell culture. Live cells are stained green, and dead cells in red. Scale bar represents 200 mm.

swollen state in ultrapure water was observed by SEM and is presented in Fig. 5. The SEM images of PEG-20 kDa hydrogels exhibited a highly macroporous spongelike structure and the average mesh size was about 30 mm (Fig. 5AeC). A lower molecular weight of PEG corresponds to a smaller pore size of the hydrogel, which is due to the higher cross-linking density. In the same weight percentage, the molecular weight of PEG is lower, the number of molecules and cross-links of PEG in the hydrogel are higher, so that the network structure is too tight. The porous structure of the hydrogel shows potential as scaffolds for cell infiltration and growth. 3.5. Dynamic-mechanical properties of PEG hydrogels The viscoelastic mechanical behavior of PEG hydrogels could be determined by rheological measurements. Oscillatory stress sweep allowed determination of the storage modulus (G0 ) of the hydrogels as a function of the weight percentage and molecular weight of PEG (Fig. 6). The loss modulus was lower than the storage modulus (G00 < G0 ) for all gel conditions, indicating that these gels primarily exhibit elastic (data not shown). It has been reported that the mechanical property of hydrogels was in general correlated to the crosslinking density and the inter-chain entanglements of component [14e16]. With the increasing cross-linking density of the hydrogel, the structure breakdown occurred at higher shear stress levels (Fig. 6A). On a basis of these data, a stress 10 Pa was chosen for comparison of various hydrogel samples. According to the previous literature, the G0 has a strong relationship to the number of effective intermolecular cross-links formed in the hydrogel network [17e19]. Hence, the evolution of G0 was monitored to

evaluate the extent of effective intermolecular cross-links formed in the hydrogel networks. As can be seen, the higher weight percentage in the hydrogels has a higher G0 , and when the weight percentage in the hydrogel is increased from 5% to 10%, it results a ten times storage modulus. PEG hydrogels with a longer PEG chain length had a lower crosslinking density which could not contribute to the higher mechanical property of PEG hydrogels. However, the longer PEG chain might provide more entanglements in the hydrogels, which could result in the higher mechanical property. The information about the stability of 3D cross-linked networks were obtain by Frequency sweep tests [10,20]. The data obtained for all the hydrogels is characterized by G0 exhibiting a plateau in the range of 0.01e2 Hz (Fig. 6B), indicating that hydrogels were well crosslinked. However, all the hydrogels show an increase in G0 at higher frequencies (2e20 Hz), the phenomenon mainly stems from the fact that long chains fail to rearrange themselves in the time scale of the imposed motion and therefore stiffen up at higher frequencies. Moreover, the higher cross-linking density makes higher rate of increase. 3.6. Swelling behavior of PEG hydrogels The swelling capacity of the hydrogels were evaluated in 0.9% sodium chloride injection, at 37
C. Fig. 7 shows the results of the swelling tests for the films of PEG hydrogels with different molecular weight and different weight percentage. As can be seen, all the hydrogels have a high rate of swelling ratio in the initial 10 h of swelling test and reach their equilibrium swelling ratio after 144 h (Fig. 7A). Moreover, the hydrogels prepared in this study have very

Fig. 10. SEM images of PEG hydrogels at different periods of cell culture.

J. Yu et al. / Polymer Degradation and Stability 133 (2016) 312e320

3.7. In vitro enzymatic degradation of hydrogels The biodegradation ability of biomaterials is one of the key parameter for biomedical applications. Hence, degradation studies were performed on MMP degradable peptide modified PEG hydrogels in activated MMP-2 solution (Fig. 8). In this case, hydrogels were degraded by exogenously delivered MMPs. PEG20kDa-7.5% hydrogels were treated with 0, 50, 100, 200 or 400 ng/mL MMP-2 and left to react 30 days. There was a negligible weight loss of <4% when the hydrogels were treated with 0 ng/mL MMP-2, which was probably because the residual monomers in the hydrogels were dissolved out. The percentage of weight loss increased with increasing the concentration of MMP-2. However, when the concentration of MMP-2 increased from 200 to 400 ng/ mL, the percentage of weight loss was equal (Fig. 8A). These results demonstrated that the degradation of gels was due to the presence of MMP-2 and the concentration of MMP-2 had an effect on the rate of hydrogel degradation. Hydrogel degradation is controlled by cross-linking density [25] and the degradation rate can be tuned by varying molecular weight of PEG-4MAL and concentration of prepolymer. Increasing the molecular weight of the PEG-4MAL or concentration of pre-polymer corresponds to a faster degradation rates (Fig. 8B), confirming the influence of crosslink density. 3.8. In vitro cell viability To examine the viability of these hydrogels to support cellular activities, the mesenchymal stem cells (MSCs) were encapsulated in 7.5% (wt/v) PEG hydrogels (64 mL) at 1  106 cells/mL and cultured for a certain period time ranged from 1 to 10 days. Fig. 9A depicts the morphology of MSCs after Calcein AM and PI staining. The stained living and dead cells are displayed in green and red color under a fluorescence microscope, respectively. As shown in Fig. 9, although lower than TCPS (control), a high percentage of viable cells could be clearly seen for MSCs in PEG

3.0

3D TCPS

2.5 2.0

OD

high equilibrium swelling ratios, ranging from 1100% to 2300% and increased with increasing concentration of pre-polymer or molecular weight of PEG-4MAL (Fig. 7B). In Flory-Rehner theory of infinite networks, the equilibrated swelling of a network depends on the effective molecular weight of chains between crosslinks [21], which is consistent with the phenomenon. For the hydrogel of PEG20 kDa at the same condition, the swelling ratio increased from ~1900% to ~2300% when increasing the concentration of prepolymer from 5% to 10%. Although high concentration of prepolymer leads to higher cross-linking density which leads to lower swelling ratio [22], the swelling ratio increased with increasing concentration of pre-polymer, this may be due to the increase of the number of hydrophilic groups in produced hydrogel [23] and the swelling behavior of this hydrogel mostly depended on the hydrogen bonds between the water molecule and the ether oxygen of PEG. Meanwhile, Fig. 7B shows the result of the swelling test for the hydrogels with the same weight percentage but different molecular weight of PEG-4MAL. It is obvious that, the higher the molecular weight of PEG is, the higher the swelling behavior of the hydrogel is. The increase in the water uptake is a consequence of the reduction in the cross-linking density and a greater hydrophilic constituent in the hydrogel network [24]. This is due to the fact, with the same weight percentage, when the molecular weight of PEG is lower, the number of molecules and cross-links of PEG in the hydrogel are higher, so that the network structure is too tight to hold water, thus, the hydrogel formed by high molecular weight PEG shows high water content. It is suggested that the length of PEG chain in the hydrogel also affects the swelling behavior.

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Time (day) Fig. 11. CCK-8 activities of MSCs cultured for up to 10 days.

hydrogels, and dead cells were seldom found after 7 days, indicating that PEG hydrogels had a very good biocompatibility. Moreover, an enzymatically-degradable peptide was incorporated within the network to allow cells to locally degrade the matrix to permit cell spreading [26]. Cells cultured on TCPS had elongated morphologies (Fig. 9A). In contrast, MSCs encapsulated within 3D hydrogels were smaller and had a more rounded morphology, however, cellular protrusions extending out into the matrix were observed after 10 days (Fig. 9B). MSCs were less elongated in 3D hydrogels because they must first degrade the local matrix before they can elongate. Loss of structural integrity was observed in PEG hydrogels undergoing 3D cell culture for 10 days, therefore, the samples were taken out and further viewed using SEM to observe the morphology changes due to degradation. As shown in Fig. 10, the porous structure of the hydrogel could be clearly seen in the first 7 days and then mostly loss of porosity after 10 days, suggesting that the hydrogels were being degraded by MSCs, as intended. It has been reported that MMP degradable peptide in the polymer network can be cleaved by MMPs to allow hydrogel degradation [27e29]. Encapsulated cells were also assayed for cell proliferation and viability by CCK-8 assay (Fig. 11). In the early days of cell culture, although MSCs seeded on TCPS or encapsulated in 3D hydrogels were at the same seeding density, the number of MSCs were less in 3D because most cells were encapsulated in the PEG hydrogels, insufficient contact agent. The OD of MSCs encapsulated in 3D hydrogels had been an upward trend and reached around 1.8 in day 10 while the OD of MSCs cultured on TCPS had experienced a relatively slow growth from approximate 1.3 in day 1e2.1 in day 10. Moreover, the OD under the 3D method had experience a stable growth during the first week. Afterwards, it shows a rapid rise and reaches around 1.8 in day 10, which is almost double than that in day 7. Obviously, MSCs maintained high cell viability on the duration of culture, which was in line with the result from the Live/Dead assay. 4. Conclusions We have successfully prepared PEG hydrogels with different PEG chain length and weight percentage via Michael-type addition between PEG-4MAL and MMP degradable peptide. All PEG hydrogels had a porous structure and the pore size increased with the increase of the PEG chain length. Their mechanical and swelling

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properties are readily controlled by PEG chain length and the crosslinking density of PEG hydrogels. Moreover, the hydrogels were degradable by exogenously delivered MMPs or cell-secreted MMPs and in vitro cell viability study on MSCs indicates PEG hydrogel has good compatibility.

[14]

[15]

Acknowledgments [16]

We gratefully acknowledge the National Natural Sciences Foundation of China (No. 31330029 and 51603064) for financial support.

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