2D Gelatin Methacrylate Hydrogels with Tunable Stiffness for

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2D Gelatin Methacrylate Hydrogels with tunable stiffness for investigating cell behaviors Yupeng Sun, Ruijie Deng, Xiaojun Ren, Kaixiang Zhang, and Jinghong Li ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00712 • Publication Date (Web): 24 Dec 2018 Downloaded from http://pubs.acs.org on December 26, 2018

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ACS Applied Bio Materials

2D Gelatin Methacrylate Hydrogels with tunable stiffness for investigating cell behaviors Yupeng Sun, Ruijie Deng, Xiaojun Ren, Kaixiang Zhang, and Jinghong Li* Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China. Supporting Information Placeholder

ABSTRACT: Cellular microenvironment has played a critical role in cell behavior regulation, natural tissue forming and development. Specifically, the stiffness of extracellular matrix (ECM) not only can help cells to maintain their morphology and location, but also provides physical cues to regulate cellular functions. Nevertheless, it’s still hard for conventional matrix materials to explore cell behaviors and functions under their physical microenvironments due to potential long-term cytotoxicity or unphysiological stiffness. Herein, a biocompatible stiffness-tunable 2D gelatin methacryloyl (GelMA) hydrogel matrix is fabricated to explore the influence of ECM stiffness on cell morphology as well as cellular gene expression. GelMA, as a derivative of gelatin, can not only serve as cell culture matrixes due to the existence of bio-active peptide sequences and biocompatibility, but also mimic the stiffness of native ECMs. As a result, the stiffness of GelMA matrix can regulate cytoskeleton assembly and cell morphology via mechanotransduction-related genetic pathways (RhoA/ROCK and PI3K/Rac1 signaling pathway). Therefore, the 2D GelMA hydrogel matrix with tunable stiffness can be regard as an alternative cellular matrix, and has a potential to reveal the fundamental principle of ECMs defect-associated diseases. Keywords: gelatin methacrylate, hydrogel, tunable stiffness, cell behaviors, gene expression INTRODUCTION During the natural tissue forming and development, cellular microenvironment has emerged as an important determinant of cellular behaviors and tissue functions.1,2 Not only components, topographical, and geometrical features of extracellular matrix (ECM) have great impacts on cellular behaviors and functions, but also the stiffness of ECM plays a crucial part in cell functions and the formation of tissues.3-5 However, a challenge in tissues engineering is developing ECM-mimicking biomaterials with specific compositions and well-defined stiffness to stim-

ulate tissue regeneration, providing a more detailed description of how materials features regulate cellular behaviors and functions.6 Traditional cellular matrix, for instance, glass or polystyrene, belongs to extra-stiff materials (beyond physiological stiffness) from the perspective of cells and native tissues, and cell behaviors grown on these substrates tend to emerge abnormal state: flattened shape, loss of differentiated phenotype, and aberrant polarization.7 Recently, polyacrylamide (PAAm) hydrogels have been employed as an alternative platform to elucidate fundamental phenomena, direct stem cell differentiation and regulate cell behaviors, which beyond the capacity of traditional cellculture substrates.8,9 But, the potential cytotoxicity and non-biodegradable property would hinder their applications in tissue engineering.10 Collagen is the primary structural component of native tissues, which contains the target sequences of matrix metalloproteinase (MMP) which promote cell remodelling,11 as well as arginine-glycine-aspartic (RGD) sequences which benefit cell adhesion.12 This ubiquity makes collagen an attractive material for cell studies from carcinoma cell reprogramming to mesenchymal stem cell (MSC) differentiation.13 However, collagen suffers from drawbacks, including limited long-term stability, low stiffness and the variability of batch-to-batch synthesis.14 Carbon materials such as carbon nanotubes (CNTs) and graphene oxides (GOs)15-17 have already been incorporated to strengthen the stiffness of hydrogel materials. For example, GO-reinforced collagen/nano-hydroxyapatite composite hydrogels with high mechanical and bioactive properties are potential candidates for bone tissue engineering.18 Nevertheless, this composite hydrogels still suffered potential long-term cytotoxicity (hard to metabolize from the body).19 Gelatin methacryloyl (GelMA), a derivative of gelatin (the amorphous form of collagen with the same amino acid sequence),20 can serve as cell culture matrixes, due to the existence of bio-active sites and the excellent biocompatibility. Besides, gelatin can be derived easily and inexpensively from various sources.21 Furthermore,

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GelMA hydrogels hold capabilities to simulate the stiffness of native ECMs by virtue of tunable mechanical and photocrosslinkable properties.22 Herein, a practical and uncomplicated strategy was reported to investigate cell behaviors and gene expression programs using a stiffness-tunable 2D GelMA hydrogel matrix. To mimic the stiffness of natural human tissue, a series of GelMA hydrogel matrixes were constructed to explore the effects of ECM stiffness on cellular behaviors and gene expression. It was found that GelMA hydrogel matrix with tunable stiffness could influence the formation of cytoskeleton, cell morphology and cellular gene expression. Thus, the GelMA-based matrix with tunable physical properties provides a promising platform for investigating cellular behaviors and gene expression profiles under their physical microenvironments. RESULTS AND DISCUSSION Overview of 2D GelMA hydrogel matrix with tunable stiffness. The stiffness-tunable 2D GelMA hydrogel matrixes were fabricated by regulating the pre-polymer concentration and exposing under UV irradiation (Scheme 1A). 2D GelMA hydrogel matrixes could regulate cell behaviors: the cells grown on soft matrix demonstrate a small cell spreading area with a round morphology, whereas the cells on stiff matrix present a large spreading area with a spindle morphology, besides there are distinct stress fibers appeared in the cells on stiff matrix. Scheme 1B illustrates the hypothetic mechanism of cell F-actin forming and growth. Briefly, the force generated by the interaction between F-actin/integrin and ECM

cell growth associated with cellular signaling RhoA/ROCK pathway and PI3K/Rac1 pathway.23

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would push cell spreading and growth. F-actin forming includes the rapid degradation and formation of the actin filaments (F-actin), in this process, cofilin (CFL1) would depolymerize the old F-actins into actin monomers, which subsequently would be used to construct new Factins by profiling (PFN1). During the formation of Factins, cellular signaling pathways (RhoA/ROCK and PI3K/Rac1) play essential roles in regulating cell behaviors. Fabrication and characterization of GelMA hydrogels. Methacrylate groups were added to the amine-containing side chains of gelatin to synthesize GelMA,24 which could develop into hydrogels via a photo-crosslinking process (Figure 1A). As a result, GelMA can provide cell-responsive features such as proteolytic degradability and suitable cell adhesion sites.25 The pristine gelatin appeared layered stacked structure, meanwhile the synthesized GelMA was shown to be homogeneous fibers with diameters ranged from 50 nm to 100 nm after modification with methacrylate group. The GelMA hydrogel revealed porosity and pore size distributed uniformly, which could be attributed to the photo-crosslinking of GelMA fibers via the methacrylate pendant groups (Figure 1B).

Figure 1. Fabrication and characterization of GelMA hydrogels. (A) Synthetic procedure of GelMA hydrogel: methacrylic anhydride (MA) was added to the primary amine groups of gelatin to generate methacrylate pendant groups, and the methacrylated gelatin was photo-crosslinked to produce the hydrogel networks. (B) SEM images of pristine gelatin, synthesized GelMA and GelMA hydrogels. Scale bar, 50 μm. (C) The NMR spectra of pristine gelatin and synthesized GelMA. (D) FTIR spectra of the GelMA hydrogel compared to pristine gelatin and synthesized GelMA. Scheme 1. Schematic illustration of cell behaviors regulated by the stiffness of 2D GelMA hydrogel matrix. (A) The fabrication of stiffness-tunable 2D GelMA hydrogel matrix and the influence on cell behaviors: cell morphology, spreading area and the formation of F-actins. (B) Hypothetic mechanism of cell F-actin forming and

Furthermore, 1H NMR spectra were applied to confirm the synthesized GelMA pre-polymer (Figure 1C). Briefly, compared to the gelatin backbone spectra, the methyl


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acrylamide-modified gelatin appeared two new peaks at 5.3 ppm and 5.5 ppm, which belong to the methacrylamide carbon-carbon double bond, indicating that the methyl acrylamide (MA) groups have been successfully modified to gelatin molecules. As shown in Figure S1 (SI), mass spectrometry analysis revealed that gelatin and GelMA presented the characteristics of molecular weight distribution, and the molecular weight of GelMA increased compared to that of gelatin. Specifically, by further comparing the main molecular ion peaks of gelatin and GelMA, we calculated that the amount of methyl acrylamide group modified to gelatin was from 1 to 10 (Table S1 shows the calculation process in detail, SI). Besides, the surface charge of gelatin, which showed positive charges (11.2 mV), shifted to negative charges (-3.9 mV) after grafting of the methyl acrylamide group (Figure S2, SI). This result may be due to the methyl acrylamide group instead of original amino groups, so that a negatively charged shuttle formed on the surface of GelMA. Possible changes in composition were evaluated by comparing FTIR spectra obtained from gelatin, GelMA to GelMA hydrogel (Figure 1D). These materials exhibited amide vibrations at 1640 cm-1 for C=O and 1538 cm-1 for N-H characteristic of peptide groups, with no significant difference between GelMA and GelMA hydrogel.26 But, for GelMA hydrogel, the vibrations between 1251 cm-1 and 1164 cm-1 significantly enhanced than those of GelMA. It might result from the photocrosslinking of acryloyl group of GelMA. Together, we fabricated gelatin-based GelMA hydrogels by a simple photo-crosslinking method.

flexible, thin wall of hole to a tough, thick well. The increase of GelMA concentration enhanced the stiffness of hydrogel as illustrated in the representative stress–strain curve (Figure 2A). In-depth quantitative analysis showed that the increasing concentration leaded to a higher compressive modulus, ranging between 1.04 ± 0.09 kPa, 5.76 ± 0.25 kPa, 12.01 ± 0.34 kPa, 20.59 ± 0.51 kPa, and 34.03 ± 1.21 kPa for 4%, 6%, 8%, 10%, 12% of GelMA, respectively (Figure 2B). As expected, the stiffness of GelMA hydrogels with varying concentration were matched to the stiffness of human organs, such as brain, adipose, muscle and osteoid, respectively.28-30 Another important hydrogel property is swelling, which is crucial to surface properties, solute diffusion, surface mobility, and mechanical properties.22,31 Figure S4 (SI) shows a trend to decrease swelling ratio with the increasing concentration of GelMA. Besides, the swelling ratio revealed a significant change between 4% GelMA and 6% GelMA. There were distinct hysteresis loops, as an indicator of energy dissipation, in the loading–unloading cycle of GelMA hydrogels (Figure S5A, SI). In the process of force-loading, the stress–strain curves existed a linear region at initial strains of 0-20% and an increasing slope region at strains of 20-40%. After force-unloading, the GelMA hydrogels nearly recovered their initial shapes without seriously plastic deformations. Furthermore, the fine hysteresis loops and reversible deformations of the GelMA hydrogels at different concentration were also demonstrated in the loading–unloading cycles (Figure S5B-F, SI). Besides, the cycle stability of GelMA hydrogels can contribute to investigate cell behaviors on different stiff substrates in a long-term.

Figure 2. Mechanical features of GelMA hydrogels with different concentrations. (A) Representative stress and strain curves and (B) compressive modulus of GelMA hydrogels with varying concentrations. Error bar indicates the standard deviation of measurements carried on six samples. The compressive modulus demonstrated significant changes (*p < 0.05) among GelMA hydrogels.

Fabrication of GelMA hydrogel matrix with tunable stiffness. The biomechanical features of the ECMs play a significant role in cell viability, behavior and function.27 To simulate the stiffness of different human organs, five various concentrations of GelMA were photo-crosslinked to yield hydrogels with varied stiffness (Table S2, SI). As illustrated in Figure S3 (SI), the pore size of hydrogel decreased when the concentration of GelMA increased, and the porous network structure gradually changed, from a

Figure 3. Cell behaviors on stiffness-tunable GelMA hydrogel matrix. (A) Fluorescence images of cell morphology features on the matrixes of varied stiffness: 1 kPa, 6 kPa, 12 kPa, 21 kPa, 34 kPa. Scale bar, 20 μm. The cell nuclei were stained by DAPI (blue), Factins were stained by Alexa 488-phalloidin (green) after culturing on the substrates for 24 h. (B) The normal distribution of cell spreading area on the matrixes of varied stiffness after cell-seeding for 24 h. (C) The mean value of cell spreading area on varied matrixes (mean ±s.d.; * P < 0.05).

The influence of substrate stiffness on cell behaviors. The ability of cells to grow and survive is of fundamental significance for cell behavior and function. To evaluate



the suitability of the GelMA hydrogel for the fabrication of cell culture matrix with tunable stiffness, human breast cancer cell lines MCF-7 were seeded on different stiffness GelMA hydrogels. Cell viability was detected by CCK-8 assay compared to those on traditional plastic substrate. It`s found that the cell viability on the GelMA hydrogel substrate with varied stiffness did not differ significantly from that of cells on traditional cell culture matrix (Figure S6, SI). The results suggest that GelMA hydrogel with tunable stiffness can be regarded as an excellent substrate in terms of cell viability. Besides, GelMA hydrogel, as a cell-responsive and biodegradable hydrogel, was benefit to tissue engineering applications.32 Therefore, GelMA hydrogel is a desired cell-cultured matrix for investigating the effect of ECM stiffness on cell behaviors. To investigate the effect of matrix stiffness on cell spreading behavior and morphology, we cultured MCF-7 cells on the different stiffness of GelMA hydrogel matrix. After incubation for 24 h, we labeled the nuclei and actin filaments with DAPI (blue) and Alexa 488-modified phalloidin (green), respectively. As illustrated in Figure 3A, distinct stress fibers were observed in MCF-7 cells cultured on a 34 kPa matrix, while those were not found on a softer matrix (1 kPa, 6 kPa, 12 kPa, 21 kPa). Stress fiber plays an important role in cell spreading and growth. They not only sense and transmit signals of matrix stiffness, but also provide cells with the biological forces to maintain cell morphology.33 According to the statistical analysis of cell spreading area, we found that the cell spreading area showed a normal distribution (Figure 3B). As the substrate stiffness increasing, the normal distribution gradually became wider and shifted to the direction of increasing the spreading area. Figure 3C shows that cells grown on softer matrix (1 kPa) were spherical and with a smaller cell spreading area (300 μm2), while cells on stiffer matrix (34 kPa) were spindle and with a larger spreading area (1600 μm2). Therefore, the cell morphology analysis shows that the stiffness of the GelMA hydrogels significantly affected the cytoskeletal assembly, cell shape and spreading area. The influence of ECM stiffness on cellular gene expression. Gene expression programs would imply the roles of ECM stiffness in regulating cell function.34-36 In order to explore the influence of matrix stiffness on cellular gene expression, as shown in Figure S7 (SI), we employed RT-qPCR to analyze the specific gene expression levels related to cytoskeletal assembly and cell signaling on different stiffness matrix. It has been reported that the change of expression of these genes participated in the formation of F-actin, resulting in the changes in cell morphology, cell spreading or cell growth behaviors.37 The above results indicate that the stiffness of matrix can influence the formation of stress fibers, and cells could form stress fibers on stiff substrates but not on soft substrates. The genesis of stress fiber involves the rapid formation

and degradation of actin filaments (F-actin). In this process, actin (translated by ACTB gene) is regarded as a basic building block of F-actin, cofiline (translated by CFL1 gene) promotes depolymerization of old F-actins into actin monomers, while profilin (translated by PFN1 gene) can use the depolymerized monomers to prolong a new F-actins, which in turn promotes cell spread and grow.38,39 As shown in Figure 4, from the point of view of F-actin assembly, the expression of the gene ACTB was significantly reduced on the soft matrix, the expression of PFN1 was increased, and the expression of CFL1 was basically unchanged. Since the gene ACTB is directly involved in the formation of F-actin, the decrease in expression level leads to lower production efficiency of actin filaments. However, surprisingly, the expression of PFN1 was increased on soft substrates. This may be due to the fact that the formation of F-actin involves other cellular regulatory mechanisms: the RhoA/ROCK signaling pathway and the PI3K/Rac1 signaling pathway.40,41

Figure 4. The relationship between gene expression programs and the stiffness of GelMA hydrogel matrix (stiff matrix, 34 kPa; soft matrix, 1 kPa). The signaling pathway associated with cell adhesion and cell spread: F-actin assembly (ACTB, PFN1, CFL1); RhoA/ROCK signaling pathway (RhoA, ROCK, MYLK) and PI3K/Rac1 signaling pathway (PI3K, FAK, Rac1). The gene expression is normalized by GAPDH on the stiff matrix. Student's unpaired t-test was used to evaluate the significance (*p < 0.05).

For the RhoA/ROCK signaling pathway, the expression level of the gene MYLK was significantly decreased on the soft substrate. Because the gene encodes troponin light-chain kinase, it is capable of phosphorylating the


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myosin-modulating light chain and stabilizing the unphosphorylated phosphate.42 Due to the action of myosin filaments, a decrease in the expression level on soft substrates results in instability of F-actin and failure to form stress fibers. It has been reported that phosphoinositide 3kinase (PI3K) regulates cell metabolism, gene expression, and cytoskeleton rearrangement.43 In addition, Mooney demonstrated that even in soft rBM matrices, overexpression of Rac1 and PI3K in MCF10As cells led to a malignant phenotype.44 From the analysis of PI3K/Rac1 signaling pathway, the expression of gene PI3K on the soft substrate was significantly increased, which favored cell spreading; however, the decreased expression of Rac1 was not conducive to the formation of F-actins and cell spreading. This may be due to the formation of a complex signal network by intracellular signaling pathways and the interaction of different signaling pathways.45,46 For example, the influence of ECM stiffness on directional migration of cells relied on the balance of the two signaling pathways of PI3K and ROCK triggered by ECM.47 Together, the force stimulus (matrix stiffness) of the cell microenvironment induced cytoskeletal assembly by activating a specific gene expression program, promoting stress fiber formation and cell spreading. Therefore, the stiffness-adjustable GelMA hydrogel matrix provides a powerful platform for the study of the potential mechanism of how matrix stiffness regulates cell behavior in the physical microenvironment. CONCLUSIONS In summary, a 2D stiffness-tunable cellular culture matrix based on GelMA hydrogel was fabricated and enabled the investigation of cell behaviors at physiological stiffness. The designed GelMA hydrogel, as an alternative biomaterial, could not only provide appropriate cell adhesion sites, but also adjust the stiffness of substrate by regulating the concentration of pre-polymer. The specific gene expression programs related to the formation of Factin was investigated, and it was found that the matrix stiffness could efficiently regulate cell morphology and F-actins assembly. In addition, cytoskeleton-associated genes (ACTB, PFN1 and CFL1) involved in the formation of F-actins, and cellular signaling pathways (RhoA/ROCK and PI3K/Rac1) play a crucial part in regulating cell behaviors. This 2D GelMA hydrogel matrix with tunable stiffness would be regard as a promising platform for exploiting new therapies for diseases related to ECMs defect. MATERIALS AND METHODS Materials. Photoinitiator Irgacure 2959 was bought from CIBA Chemical. Methacrylic anhydride (MA) and gelatin (type A) were bought from Sigma Aldrich (Wisconsin, USA). Coverslips and glass slides were bought from Fisher Scientific (Philadelphia, USA). Dulbecco's

modified eagle medium (DMEM), Triton-X 100, fetal bovine serum (FBS), 4% paraformaldehyde in PBS buffer, DPBS, Trypsin and Penicillin-Streptomycin were bought from Solarbio (Beijing, China). DAPI and FITCphalloidin were bought from Sigma-Aldrich (St. Louis, USA). A Millipore System (Millipore Q, USA) generated ultra-pure water (18 MΩ cm). The oligonucleotides (Table S3, SI) were synthesized by Sangon (Shanghai, China). All other reagents (analytical grade) were purchased from Sinopharm Chemical Reagent (Beijing, China). Preparation of gelatin methacrylate (GelMA). Typically, Gelatin was first dissolved in DPBS buffer at 60 °C. Then, methacrylic anhydride (0.8 mL/g) was slowly added to the gelatin solution (10% w/v) at 60 °C. After stirring for 2 h, DPBS was used to abort the reaction. To eliminate excess salts and methacrylic anhydride, the diluted mixture was dialyzed in ultra-pure water bath at 45 °C for 4 days (molecular weight cut-off, 12-14 kDa). The dialyzed solution was filtered (0.2 μm) and freezedrying for 5 days, then a white foam of GelMA was obtained for future use. Preparation of 2D GelMA hydrogel matrix. In a typical synthesis (Figure S8, SI), 0.5% (w/v) photoinitiator Irgacure 2959 was added to a certain concentration of GelMA macromolecules solution at 80 °C, stirred continuously until complete dissolution. The polymerizing solution was placed between amino-silanated coverslip and chloro-silanated glass slide, like a sandwich. After 365 nm UV irradiation for 30 min, we peeled off the 2D GelMA hydrogel matrix from the chloro-silanated glass slide, then stored in PBS solution for future use. The stiffness of the hydrogel matrix was controlled by adjusting the concentration of GelMA. Characterization of GelMA hydrogel. To investigate the surface topography of GelMA hydrogels, the samples were immersed in PBS overnight, then lyophilized and carved to expose their surfaces and cross-sections. At last, the samples were coated with Pt/Pd via a plasma sputtering method. Detailed structural information was performed using a SEM (SU-8010 HITACHI, Japan). To investigate the mechanical features of GelMA hydrogels, the 500 μL pre-polymer solution was added to a centrifuge tube of 1.5 mL, and exposed to ultraviolet light (6.9 mW / cm2, 365 nm) at room temperature for a particular time, then the sample was immersed in DPBS for 24 h. For compression test, a cylindrical hydrogel (diameter 9 mm, thickness 5 mm) was performed on an Instron 3342 mechanical analyzer at 10% strain/minute. The linear region (5-15% strain) of stress–strain curves was used to calculate the compression modulus of GelMA hydrogels. To investigate the swelling character, the GelMA hydrogels with different stiffness were placed in DPBS overnight and wiped the remaining liquid with KimWipe, then recorded the swelling weight. The samples were lyophi-



lized, and then recorded the dry weight. The mass swelling ratio is defined by the ratio of swelling weight to dry weight of hydrogels. Biocompatibility Test. MCF-7 cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM) of 10% FBS, 1% penicillin/streptomycin at 95% air humidity, 5% CO2, and 37 °C. Prior to cell seeding, the GelMA hydrogel matrixes with different stiffness were placed under UV for 30 min to sterilize their surfaces. The biocompatibility of the different matrix materials was characterized by determining the cell viability by the Cell Counting Kit (CCK-8). Specifically, MCF-7 cells were cultured in 96-well plates containing the GelMA hydrogel matrix with different stiffness. After incubation for 24 h, cell culture solution (containing 10% CCK-8) was added to the 96-well plates, then continuously cultured for 150 min at 37 °C. The group without a matrix material was regarded as control group (Ac), the group containing GelMA hydrogel matrix with different stiffness was regarded as an experimental group (As) and the well without cells was regarded as a blank group (Ab), then recorded the absorbance at 450 nm. At last, (As-Ab)/(Ac-Ab) was determined as the cell viability. Cell Staining and Image Analysis. MCF-7 cells were incubated for 24 h, washed twice with PBS, then fixed with paraformaldehyde for 15 min at room temperature (25 °C). The cell membrane was permeabilized for 3 min with 0.5 % v / v Triton-X 100, then FITC conjugated to phalloidin was used for actin staining performance. For nuclear staining, cells were immersed in DAPI after washing with PBS. For the quantitative analysis of cell morphology, only those cells that did not contact each other were considered. Cell outlines were manually marked according to the cell image and Image J software was used to determine the cell spread area. Real-time quantitative PCR (RT-qPCR) analysis. Total RNA extraction was performed by TransZol. cDNA was synthesized by TransScript one-step gDNA removel and cDNA synthesis kit. Specifically, 20 μL of the mixture solution was maintained at 42 °C for 15 min, heated to 85 °C and maintained for 5 s to inactivate the enzyme. Reaction system contains 7 μL total RNA (50 ng-5 μg), 10 μL 2×TS reaction mixture, 1 μL oligo (dT)18 (0.5 μg/μL), 1 μL gDNA remover and 1 μL TransScript RT/RI enzyme mix. cDNA samples could store at -80 °C for later use. In this work, the expression level of intracellular mRNA was investigated by RT-qPCR. The reaction system (20 μL) contained 10 μL 2×SYBR Select master, 2 μL cDNA sample, 2 μL upstream primer (5 μM), 2 μL downstream primer (5 μM) and 4 μL DEPC-treated water. The reaction conditions for qPCR were: 50 °C kept for 2 min, annealed at 95 °C for 2 min, and performed 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The Ct value was obtained by measuring the fluorescence quantitation curve. Finally, the expression levels of target mRNAs ΔΔ were assessed by the 2- Ct method.48

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ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge via the Internet at http://pubs.acs.org. MALDI-TOF MS and zeta potential of GelMA; morphology, equilibrium swelling properties, mechanical properties and biocompatibility of GelMA hydrogel; RT-qPCR data; synthetic scheme of 2D GelMA hydrogel matrix.

AUTHOR INFORMATION Corresponding Author [email protected]. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (No. 21621003, No. 21235004, No. 21327806), and Tsinghua University Initiative Scientific Research Program.

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