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3D bioprinting of low concentration cell-laden gelatin methacrylate (GelMA) bioinks with two-step crosslinking strategy Jun Yin, Mengling Yan, Yancheng Wang, Jianzhong Fu, and Hairui Suo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16059 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Applied Materials & Interfaces

3D bioprinting of low concentration cell-laden gelatin methacrylate (GelMA) bioinks with two-step crosslinking strategy Jun Yin1,2,*, Mengling Yan1,2, Yancheng Wang1, Jianzhong Fu1,2, Hairui Suo1,2,*

1

The State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou 310028, China 2

Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering, Zhejiang University, Hangzhou 310028, China

*Corresponding authors. E-mail: [email protected] (J.Y.), [email protected] (H. S.)

1

ACS Paragon Plus Environment

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Abstract: Methacrylated gelatin (GelMA) has been widely used as the tissue engineered scaffold material, but only low concentration GelMA hydrogels were found to be promising cell-laden bioinks with excellent cell viability. In this work, we reported a strategy for precisely deposition of 5% (w/v) cell-laden GelMA bioinks into controlled micro-architectures with high cell viability using extrusion-based three-dimensional (3D) bioprinting. By adding gelatin into GelMA bioinks, a two-step crosslinking combining the rapid and reversible thermo-crosslinking of gelatin with irreversible photo-crosslinking of GelMA was achieved. The GelMA/gelatin bioinks showed significant advantages in processability, since the tunable rheology and the rapid thermo-crosslinking of bioinks improved the shape fidelity after bioprinting. Here, the rheology, mechanical properties, and swelling of GelMA/gelatin bioinks with different concentration ratios were carefully characterized to obtain the optimized bioprinting setup. We successfully printed the 5% (w/v) GelMA with 8% (w/v) gelatin into 3D structures, which had the similar geometrical resolution as that of the structures printed by 30% (w/v) GelMA bioinks. Moreover, the cell viability of 5/8% (w/v) GelMA/gelatin bioinks was demonstrated by in vitro culture and cell printing of bone marrow stem cells (BMSCs). It was found the larger BMSCs spreading area on 5/8% (w/v) GelMA/gelatin scaffolds, and the BMSCs viability after printing of 5/8% (w/v) GelMA/gelatin cell-laden bioinks was more than 90%, which was very close to the viability of printing pure 5% (w/v) GelMA cell-laden bioinks. Therefore, this printing strategy of GelMA/gelatin bioinks may extensively extend the applications of GelMA hydrogels for tissue engineering, organ printing or drug delivery.

Keywords: bioink, gelatin, 3D bioprinting, processability, two-step crosslinking

2


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1. Introduction The capability of patterning soft biopolymer materials to three-dimensional (3D) structures as the scaffolds is critical for emerging tissue engineering technology.1 On one hand, in the conventional process cells donated by patients are expanded in culture and then transferred to the scaffolds,2 where porous engineered scaffolds provide the physical and chemical cues to guide cell differentiation and assembly into 3D tissues.3 On the other hand, the recent cell printing technology provides the potential to incorporate cells into the scaffold forming process simultaneously, termed as a "bottom-up" approach of tissue/organ fabrication.4,5 The basic requirements for 3D engineered scaffolds to achieve the functionality of tissue-engineered construct are concluded as: a) biocompatibility and biodegradability;6 b) tunable mechanical properties;7 c) precise control of the multi-scale internal architecture of scaffolds to represent the complexity of natural extracellular matrix (ECM):8 macro-scale pores to accommodate cell attachment and growth, and micro-scale pores to accommodate growth factors release and nutrient transport to surrounding cells.9 The biocompatible hydrogels are considered as appealing scaffold materials because they show physical or structural similarity with natural tissues,9,10 and have great potential to satisfy the basic requirements of tissue engineered scaffolds listed above. However, current studies reflected one of the major obstacles that these hydrogels generally have poor processability to form complex 3D structures.11 The conventional scaffold fabrication techniques, i.e. fiber meshes and bonding, gas foaming, phase separation, freeze drying and particulate leaching,6 can only regulate the overall scaffold morphological features,12 but have limited capability of control over scaffold architecture, composition, pore shape, size and distribution.13 Therefore, how to precisely control the specific distribution of appropriate biomaterials, mimicking the biological and functional complexity of native tissues is regarded as the major challenge in the full regeneration of tissues.11 Currently, additive manufacturing (or termed as 3D printing) technologies are recognized as promising innovative technologies in the emerging field of regenerative medicine,14 since they allow for accurate deposition of bioinks and cells to build tissue-like constructs at high resolution.15 Fiber extrusion-based 3D printing has been widely used to deposit biomaterials into porous 3D scaffolds by multiple filaments,16 since the wide viscosity range of the bioinks can be used and high cell densities 3



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can be achieved in this method.17 Gelatin with methacryloyl side groups (GelMA), which forms covalently crosslinked hydrogels under UV light exposure with the presence of a photoinitiator, has recently gained increasing attention, especially in the field of biomedical applications.18,19 Recent investigations have shown GelMA hydrogels are promising bioinks to build constructs with various 3D architectures using additive manufacturing strategies and photopolymerization under UV light.14,20-23 Also, it has been demonstrated that GelMA hydrogels with low concentration (i.e. ≤ 5% w/v) are more suitable cell-laden bioinks due to their high cell stability and viability.7,24 However, the low concentration GelMA bioinks suffer the poor processability during fiber extrusion-based 3D printing process due to three major reasons. First, the low concentration leads to low viscosity of GelMA bioinks, so that the process-induced instability occurs during extrusion, leading to irregular filament shapes. Second, the gelation rate of low concentration GelMA hydrogels from liquid to (semi-)solid is too slow to maintain the structural integrity and precision after printing. Third, the gel phase of low concentration GelMA hydrogels does not have adequate mechanical strength to allow the scaffold to retain its initial geometry. Although, GelMA bioinks with high concentrations (i.e. > 15% w/v) have excellent printability,25 high concentration of GelMA can decrease the cell viability of bioinks. The central issue of GelMA hydrogels application as the tissue engineered scaffolds is how to balance the physical printability and biological functionality, which remains a challenge in 3D printing of GelMA bioinks.15 The strategy used in this study to enhance the processability of low concentration GelMA bioinks was blending GelMA with gelatin. Similar strategies also have been applied in pervious

investigations

of

printing

GelMA/methacrylated

hyaluronic

acid,26

alginate/gelatin,27 alginate/gelatin/hydroxyapatite,28 or GelMA/alginate blends.7 As the hydrolysis product of collagen, gelatin has excellent ability to support cell adhesion and spreading with the presence of cell adhesion domains.29 Moreover, gelatin has low antigenicity and better solubility compared to collagen.18 Here, blending of gelatin not only can regulate the viscosity of GelMA bioinks, it also provided an additional reversible thermo-crosslinking mechanism upon irreversible photo-crosslinking of GelMA.14 Thus, the GelMA/gelatin bioinks were rapidly held by thermo-crosslinking after printing, and further 4


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stabilized by photo-crosslinking; where the two-step crosslinking processing significantly improved the processability of low concentration GelMA/gelatin bioinks. Then, gelatin gradually dissolved away when temperature rose above the gel point of gelatin, leaving the low concentration GelMA scaffold for cell culture, and the loss of gelatin has no effect on scaffold geometry.28 In addition, residual gelatin also improved the cell viability of GelMA bioinks. In this study, the rheology of GelMA/gelatin bioinks was measured and the optimized printing conditions were obtained. Finally, by testing the cell morphology and viability after 3D printing of cell-laden GelMA/gelatin bioinks, the biocompatibility of GelMA/gelatin bioinks was demonstrated.

2. Materials and methods 2.1 GelMA/gelatin bioink preparation Gelatin from porcine skin (Bloom 250, type A) and methacrylic anhydride were obtained from

Aladdin

Industrial

Inc.

The

photoinitiator,

lithium

phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), was synthesized as described before.30 Phosphate buffered saline (PBS) was purchased from HyClone Laboratories Inc. Methacrylated gelatin (GelMA) was synthesized as described in a previous literature.31 Briefly, methacrylic anhydride was added at a rate of 0.5 mL/min to a 10% (w/v) solution of gelatin in PBS under constant stirring. After reaction at 50 °C for 3 hours, the reacted polymers solution was dialyzed against distilled water for 7 days at 40 °C to remove methacrylic acid and anhydride, filtered through a 0.22 µm membrane, freeze-dried and finally stored at -80 °C before use. The degree of methacrylic anhydride substitution of GelMA was measured as described before,32 and the substitution degree of GelMA used in this work was about 96%. GelMA/gelatin hydrogel solutions were prepared by dissolving freeze-dried GelMA and gelatin polymers in deionized water at 40 °C, and kept for 2 hours to form homogeneous solutions. The photoinitiator (LAP) was then added to the prepolymer solutions at a concentration of 0.5% (w/v). In this paper, the m/n% GelMA/gelatin bioink indicates that GelMA and gelatin concentrations are m% (w/v) and n% (w/v), respectively.

2.2 3D bioprinting 5



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A custom-made 3D pneumatic dispensing system (Fig. 1) was used to deposit GelMA/gelatin bioinks. The system consisted of a computer controlled three-axis positioning stage, which had a working space of 150×150×150 mm3 and a controllable pneumatic extruder (0~1 MPa). The temperature controller was equipped around the syringe to ensure a homogeneous plotting temperature; while a cooling system was also implemented under the receiving platform to maintain the stable temperature after the deposition of GelMA/gelatin bioinks. An UV light source (365 nm, OmniCure S2000) was equipped upon the receiving platform to photo-crosslink bioinks for long-term stability. The whole printing system was covered within a plexiglass box to prevent temperature disturbance.

Syringe temperature controller

Cooling receiving platform

Pneumatic extruder

UV light source 3D working platform

Fig. 1. Schematic of 3D bioprinting system: the temperature controller on the syringe regulated the viscosity of GelMA/gelatin bioinks; the cooling system under the receiving platform temporarily thermo-crosslinked gelatin in the bioinks; and the UV light permanently photo-crosslinked GelMA in the bioinks.

The bioinks with different GelMA/gelatin concentration ratios were prepared. In order to investigate the printability of bioinks, the GelMA concentrations varied from 5~30%, while the gelatin concentrations varied from 0~10%. The GelMA/gelatin bioinks were printed using a 5-mL syringe equipped with a 260 µm inner diameter needle. A two-step crosslinking strategy was implemented during 3D bioprinting of each layer (Fig. 2). In the first step, GelMA/gelatin bioinks underwent a rapid thermo-crosslinking by adjusting the syringe temperature controller between 15 to 25 °C as the appropriate temperature for stable filament 6


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deposition, and the temperature of receiving platform was kept 2~5 °C lower than the syringe temperature to maintain the printed hydrogel. In the following step, printing structures on the receiving platform were under UV light source (365 nm, 0.5 W/cm2) for a long-term stability by photo-crosslinking of GelMA. After the entire printing process, the whole scaffold was exposed to UV light (1 W/cm2) for 2 minutes for further photo-crosslinking. All the 3D bioprinting processes were performed under room temperature. The setup of 3D bioprinting process was listed in Table 1.

Step 1: Reversible thermo-crosslinking of gelatin Temperature

Temperature Temperature

Step 2: Irreversible photo-crosslinking of GelMA

GelMA/gelatin in gel state (below the gel point )

UV

GelMA/gelatin in gel state (up the gel point)

Fig. 2. Two-step crosslinking of GelMA/gelatin bioinks.

Table 1. Printing parameters. Parameter

Value

GelMA concentration (w/v)

5~30%

Gelatin concentration (w/v)

0~10%

Extrusion pressure (MPa)

0~0.3

Syringe temperature (℃)

15~25

Cooling receiving platform temperature (℃)

10~20

XY plotting speed (mm/min)

100~500

UV light intensity (W/cm2)

0.5~2

UV exposure time after printing of scaffolds (min) 2 Nozzle inner dimeter (mm)

0.26

2.3 Material characterization 7



2.3.1 Rheology Rheological properties of different bioinks were evaluated by a rheometer (MCR302, Anton Parr, Austria) equipped with a Peltier element for temperature control. A plate-plate geometry with a diameter of 50 mm was used in all measurements. At first, all hydrogel samples were placed on the plate at 40 °C to completely fill the gap (size of 1 mm) between two plates. The measurements of viscosity and shear stress were performed by varying the shear rate from 1 to 500 s-1 with a rotational test at 15 and 25 °C, respectively. Then, the viscosity also was measured with a temperature ramping from 37 to 15 °C at the rate of 1 °C/min and the shear rate was kept at 1 s-1. Storage modulus (G’) and loss modulus (G’’) were measured as a function of temperature at a constant frequency of 1 Hz and a constant strain of 0.1%, while the hydrogel samples were equilibrated at 40 °C and then cooled at a rate of 1 °C/min from 40 to 10 °C.

2.3.2 Mechanical properties The mechanical properties of photo-crosslinked GelMA/gelatin hydrogels were characterized by measuring the compression and uniaxial tensile tests using a dynamic mechanical analysis instrument (ElectroForce, TA Instruments, America) at 25 and 37 °C, respectively. For unconfined compression tests, the GelMA/gelatin bioinks were cured in the disks (2 mm thick, diameter = 12 mm) by being exposed to 365 nm UV light (1 W/cm2) for 2 minutes. Each sample was placed between two compression plates and compressed at a displacement rate of 1 mm/min. While the samples were cut into 4×28×4 mm3 strips after photo-crosslinking and stretched at a rate of 1 mm/min to explore the tensile properties. The compression modulus and Young’s modulus were calculated as the slope of the linear region in the 0-10% strain range of the stress-strain curves.

2.2.3 Swelling In order to measure the equilibrium swelling of hydrogels, GelMA/gelatin bioinks were photo-crosslinked into the disk shape (thickness = 1 mm, diameter = 12 mm). Then, the gel disks with different compositions were immersed into 2 mL PBS at 37 °C for 24 hours to reach equilibrium swelling. After being removed from PBS, the gel disks were gently blotted 8


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with a KimWipe to remove the residual liquid, and the swollen weight of each disk was recorded (Ws). The samples were subsequently lyophilized and weighed again to determine the dry weight of each disk (Wd). The swelling ratio was then calculated as Q = (Ws - Wd)/Wd.

2.4 Cell culture and printing Bone marrow stem cells (BMSCs) were isolated from femora of 4-6-week old S-D rats as described before.33 Briefly, both ends of the femora were cut away from the epiphysis, and the bone marrow was flushed out of the diaphysis using a syringe with the media of DMEM supplemented with 10% FBS (v/v). The marrow was collected and cultured for 3 days in a humidified atmosphere of 95% air and 5% CO2. The first medium change was done after 4 days and twice a week thereafter. BMSCs were passaged once they reach 80-90% confluence. BMSCs between the passages 4 and 9 were used for all experiments. BMSCs with a density of 5.0 × 106 cells/mL were cultured on the printed scaffolds and co-printed with the bioinks into scaffolds, respectively. Three types of bioinks (5% GelMA, 5/8% GelMA/gelatin, 30% GelMA) were prepared with 0.5% (w/v) photoinitiator (LAP) for printing in sterilized environment. 3D scaffolds with inner grid structure were printed in petri dish, lyophilized and stored at -20 °C for further use. The scaffolds were further sterilized by UV irradiation (254 nm) and finally supplemented with cell suspension and cultured in a CO2 incubator. For 3D cell printing, BMSCs were trypsinized, centrifuged and then suspended with bioinks prepared as above. 3D grid scaffolds with cells were printed in petri dish and immersed in culture medium after UV exposure (365 nm) for 30 seconds. During the 7-day culture, the culture medium was pipetted out and the samples were stained with 1 µg/mL Calcein-AM and 5 µg/mL PI for 30 minutes. The samples were then observed and imaged using a florescence microscope (Ti-S, Nikon, Japan).

2.5 Statistical analysis Unless otherwise stated, all characterizations were performed using data analysis software OriginPro 2016 and all data were expressed as mean ± standard deviation. Differences between groups were explored by one-way analysis of variance (ANOVA) followed by Student’s t-tests and significance was determined at p < 0.05. 9



3. Results 3.1 Printability of GelMA/gelatin hydrogel solutions

Unextrudable

Printable

Irregular

Gelatin concentration (%, w/v)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

GelMA concentration (%, w/v)

Fig. 3. Phase diagram of GelMA/gelatin bioinks printability.

To precisely print 3D scaffolds with various GelMA concentrations, the printing parameters were optimized by assessing the printability of GelMA/gelatin bioinks as a function of GelMA and gelatin concentrations, while the other printing conditions were listed in Table 1. Here, the printability was directly verified by the shapes of gel fibers at the nozzle outlet, where only bioinks forming regular and smooth fibers (Fig. 3) were considered as printable bioinks. As shown in Fig. 3, in the cases of lower GelMA and gelatin concentrations, i.e. 0~10% GelMA, or 0~2% gelatin, the longitudinal instability occurred at the nozzle outlet leading to spindle shaped filaments, even breaking to form droplets; while in the cases of extremely high concentration GelMA/gelatin solutions, i.e. 20% GelMA with 6~10% gelatin or 30% GelMA with 4~10% gelatin, the bioinks were too viscous and wrinkled filaments formed at the nozzle outlet. Therefore, these two regions of GelMA and gelatin concentration combinations were considered as unprintable regions (grey regions in Fig. 3). While by 10


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adjusting suitable concentration ratios of gelatin and GelMA, GelMA/gelatin bioinks could form smooth and uniform filaments steadily even with low GelMA concentrations (≤ 5%), thus the concentration ratios of GelMA and gelatin in this region (green region in Fig. 3) were considered as printable.

Fig. 4. Microscopy images of printed 3D grid structures (9×9×5 mm3). Overall structure (a), top view (b) and side view (c) of 5/8% GelMA/gelatin scaffold; overall structure (d), top view (e) and side view (f) of 30% GelMA scaffold. The scalar bar is 500 µm.

To further demonstrate the feasibility of 3D printing technology with two-step crosslinking process on GelMA/gelatin bioinks, the 3D grid structures were printed with different GelMA/gelatin concentration ratios. Fig. 4 presents the printing structures (designed structure: 9×9×5 mm3, fiber spacing: 0.75 mm) with 0-90° square grid using 5/8% GelMA/gelatin and 30% GelMA bioinks, respectively. Both two bioinks were printed into fully interconnected grid structures without internal pore collapse. Comparing the top view and side view of two structures, 5/8% GelMA/gelatin and 30% GelMA bioinks had very similar printing resolution that the printed fiber diameter was about 260 µm and the fiber spacing was about 750 µm, which were close to the design dimension.

3.2 Rheology The rheological properties of bioinks were measured in terms of different GelMA/gelatin 11



concentration ratios and temperatures. Fig. 5(a) presents the shear rate dependent behavior of bioinks with different concentrations and temperatures. The shear-thinning behavior was observed for all bioinks with different GelMA/gelatin concentration ratios; while it was also found that higher concentration bioinks (5/8% GelMA/gelatin, 30% GelMA) showed more obvious shear-thinning behavior than 5% GelMA bioink. In addition, the viscosity of all bioinks had an obvious decrease when temperature increased from 15 to 25 °C. The temperature dependence of the bioinks viscosity was shown in Fig. 5(b), where the viscosity decreased with increasing temperature. The viscosity of 5% GelMA bioink significantly increased when 8% (w/v) gelatin was added. Additionally, both 5/8% GelMA/gelatin and 30% GelMA bioinks had a more obvious thermo-responsive behavior than 5% GelMA bioink around 20 to 25 °C. Figure 5(c) shows the variation of storage modulus (G’) and loss modulus (G’’) of bioinks with the change of temperature. Two types of bioinks exhibited very close gelation temperatures that the gel points (G’ = G’’) of 30% GelMA and 5/8% GelMA/gelatin bioinks were approximately 22.5 and 23.5 °C, respectively. Below the gel point (G’’ > G’), both 30% GelMA and 5/8% GelMA/gelatin bioinks showed a typical liquid-like behavior with G’ close to zero when the temperature > 25 °C. Upon cooling, G’ of both bioinks increased rapidly and finally crossed over G”, indicating characteristics of a gel-like structure. It has to be noticed that both G’ and G’’ of 5% GelMA bioink were much lower than those of 30% GelMA and 5/8% GelMA/gelatin bioinks; while the gel point of 5% GelMA bioink was about 12 °C.

12


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104

Viscosity (Pa·s)

10

5% GelMA at 25 °C 5% GelMA at 15 °C 5/8% GelMA/gelatin at 25 °C 5/8% GelMA/gelatin at 15 °C 30% GelMA at 25 °C 30% GelMA at 15 °C

3

102 101 100 10-1 10-2

(a)

10-3

1

10

100

1000

Shear rate (1/s) 104 5% GelMA 5/8% GelMA/gelatin 30% GelMA

Viscosity (Pa.s)

103 102 101 100 10-1 10-2

(b)

10-3

15

20

25

30

35

40

Temperature (°C) 105 5% GelMA (G') 5% GelMA (G'') 5/8% GelMA/gelatin (G') 5/8% GelMA/gelatin (G'') 30% GelMA (G') 30% GelMA (G'')

4

10 Storage and Loss Modulus (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


103

Gel point

102 101 100

Gel point 10-1

Gel point

10-2 10-3

(c) 10

15

20

25

30

35

40

Temperature (℃ )

Fig. 5. Rheological properties of the three hydrogel composites: 5% GelMA, 5/8% GelMA/gelatin, 30%GelMA. (a) Viscosity as a function of shear rate at 15 and 25 °C. (b) Viscosity as a function of temperatures. (c) Effect of temperature on storage modulus (G’) and loss modulus (G’’). 13



3.3 Mechanical properties

160

160

T = 25 ℃

140

5% GelMA 5/8% GelMA/gelatin 30% GelMA

120

100 80 60

100 80 60

40

40

20

20

(a)

0 0

10

20

30

40

50

60


T = 37 ℃

140

Stress (KPa)

Stress (KPa)

120

(b)

0

70

0

10

20

Strain (%)

30

40

50

60

70

Strain (%)

1000

1000

T = 25 ℃


T = 37 ℃


100

Stress (KPa)

100

Stress (KPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10

1

10

1

(c)

(d)

0.1

0.1

0

50

100

150

200

250

0

50

Strain (%)

100

150

200

250

Strain (%)

Fig. 6. Mechanical tests of GelMA/gelatin hydrogels with different concentrations. Compressive stress-strain curves at 25 (a) and 37 °C (b); tensile stress-strain curves at 25 (c) and 37 °C (d).

The unconfined compression and uniaxial tensile tests were performed to characterize the mechanical properties of GelMA/gelatin hydrogels at 25 and 37 °C. Compressive and tensile stress-strain curves of GelMA/gelatin hydrogels with three different concentrations were shown in Fig. 6. Mechanical properties were characterized in terms of compressive and Young’s moduli as displayed in Table 2. It was found that GelMA/gelatin hydrogels became stiffer when GelMA concentration increased, i.e. 30% GelMA hydrogels had a much higher compressive modulus (288.24±62.34 KPa) and Young’s modulus (264.74±11.08 KPa), which were almost 100 times greater than those of 5% GelMA hydrogels (compressive modulus：2.86±0.10 KPa and Young’s modulus：2.08±0.43 KPa) at 25 °C. While by adding gelatin into GelMA, 5/8% GelMA/gelatin hydrogels had a significant increase of 14


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compressive and Young’s moduli compared to 5% GelMA hydrogels at 25 °C. The same tendency was also observed for hydrogels at 37 °C (Table 2). Furthermore, it was found that all GelMA/gelatin hydrogels became softer when temperature increased from 25 to 37 °C. It needs to be noticed that compared to other two hydrogels, both compressive and Young’s moduli of 5/8% GelMA/gelatin hydrogels had the largest reduction (24.5% and 19.8% for compressive and Young’s moduli, respectively) when temperature increased, since physically embedded gelatin diffused out of the gel at 37 °C.

Table 2. Mechanical properties of GelMA/gelatin hydrogels at 25 and 37 ℃. Compressive modulus (KPa)

Young’s modulus (KPa)

25 ℃

37 ℃

25 ℃

37 ℃

5% GelMA

2.86±0.10

2.41±0.38

2.08±0.43

1.67±0.56

5/8% GelMA/gelatin

4.53±1.87

3.42±0.85

4.85±0.41

3.89±0.85

30% GelMA

288.24±62.34

216.81±10.28

264.74±11.08

226.80±39.97

3.4 Swelling behavior

60

GelMA with gelatin GelMA

* 50

Swelling ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40

* *

*

30 *

* 20 * * 10 0 n n n MA MA MA MA lati lati lati /ge /g e /ge Gel Gel Gel Gel A A A % % % % M M M 5 10 20 30 el Gel Gel %G 6% 2% 5/8 10/ 20/

Fig. 7. Equilibrium swelling properties of GelMA/gelatin hydrogels. Asterisk indicates significant differences (P < 0.05).

15



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The swelling behavior of various combinations of GelMA/gelatin hydrogels was presented in Fig. 7. The swelling ratio decreased significantly from 44.57±3.05 to 4.05±1.19 with GelMA concentration increasing from 5% to 30%. Also, adding gelatin evidently reduced the swelling ratio of all GelMA hydrogels. The possible reason is that a part of gelatin is undissolved into the PBS and entangled in the GelMA network, leading to a lower swelling ratio.

3.5 Cell culture and cell printing Representative fluorescence images of 3-day BMSCs culture on the surface of three types of 3D printed GelMA/gelatin scaffolds were shown in Fig. 8. The similar cell viability of BMSCs was observed attaching on the surface of scaffolds printed using 5% GelMA and 5/8% GelMA/gelatin bioinks; while the living BMSCs attached to 30% GelMA scaffols were much less.

(a)

(c)

(b)

Fig. 8. BMSCs on the surface of 3D printed scaffolds: (a) 5% GelMA; (b) 5/8% GelMA/gelatin; (c) 30% GelMA.

The representative fluorescence images of 7-day culture of BMSCs cell-laden printed two-layer constructs were presented in Fig. 9; while the cell viability of 3D printed cell-laden constructs within 7 days was listed in Table 3. The viability of BMSCs in both 5% GelMA and 5/8% GelMA/gelatin constructs was always higher than 90% within 7 days after printing, which was much higher than BMSCs viability in 30% GelMA constructs. Also, more dead cells (red points) were observed in the constructs of 30% GelMA (Fig. 9). Another interesting point is that the BMSCs viability in 30% GelMA constructs was 62.7±2.8% right after 16


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printing, and gradually increased to 70.4±0.44% after 7 days culture; which indicates BMSCs were recovering after a few days of culture.

5% GelMA

5/8% GelMA/gelatin

30% GelMA

Right after printing

3-day of culture

7-day of culture

Fig. 9. 3D printing of BMSCs cell-laden constructs using 5% GelMA, 5/8% GelMA/gelatin, and 30% GelMA bioinks (dead cells were highlighted with arrows). Living cells are depicted in green and dead cells in red. The scale bar is 500 µm.

Table 3. Cell viability of 3D printed constructs within 7 days of cell culture. 5% GelMA 5/8% GelMA/gelatin 30% GelMA Right after printing (%)

92.5±2.1

93.1±3.5

62.7±2.8

3-day of culture (%)

94.7±2.4

94.7±1.8

67.9±2.9

7-day of culture (%)

94.4±2.7

92.9±2.6

70.4±0.44

17



Page 18 of 25

4. Discussion Biofabrication of hydrogels encountered two basic requirements on the biomaterial properties: processability

for

building

high

resolution

tissue-like

structures

and

cytocompatibility to facilitate migration, proliferation and differentiation of the embedded and endogenous cells.11 The traditional approach to improve the hydrogel processability was increasing the polymer concentration and crosslinking density.34 For higher GelMA concentrations, the shear-thinning behavior was more pronounced (Fig. 5a), leading to stable filament formation at the nozzle outlet during printing; and highly crosslinked hydrogels served as stiff construction materials (Fig. 6), providing better shape fidelity after printing. While high concentrations or high levels of crosslinking resulted in lower swelling ratios (Fig. 7) and smaller pore size, reducing the diffusion of oxygen and nutrients required for the cells to survive.35 Thus, the requirements towards the hydrogels properties regarding processability and cytocompatibility were often opposing, resulting in the lack of such versatile hydrogels. As a widely used biomaterials, GelMA has been printed alone, but normally with concentrations higher than 10%.14,21,25 Although, Bertassoni et al. were able to print GelMA hydrogels with 7% concentration using the noncontinuous approaches,20 researchers were still seeking the simple and effective method to transform GelMA based-inks into biologically functional 3D structures. Here, we provided a promising solution by blending GelMA with gelatin. The strategy of adding gelatin to improve the printability of low concentration GelMA bioiks had the advantages in three aspects. First, the rheology of GelMA/gelatin bioinks can be easily regulated by adjusting the temperature of syringe and nozzle in the printer head. As shown in Fig. 5(b), comparing to the viscosity of 5% GelMA bioinks, adding 8% gelatin significantly enhanced the thermo-responsive behavior that the viscosity of 5/8% GelMA/gelatin bioinks had a sudden decrease when temperature increased from 20 to 25 °C, which was very similar to 30% GelMA bioinks. Thus, a tunable viscosity of GelMA/gelatin bioinks is favorable to form the stable filaments by changing the syringe and nozzle temperature within a friendly temperature region for living cells. Second, Fig. 5(a) indicates that adding gelatin enhanced the shear-thinning behavior of 5% GelMA bioinks. Hence, the low viscosity within the nozzle ensured good printability for the GelMA/gelatin 18


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bioinks to be extruded into smooth filaments (Fig. 3); while the high viscosity after printing gave shape fidelity to maintain the structures with high resolution. Third, as a thermo-reversible crosslinking material, gelatin induced a rapid thermo-crosslinking of GelMA/gelatin bioinks, immediately maintaining the shape of printed filaments and leading to a more accurate structure. As shown in Fig. 2, the photo-crosslinking of GelMA occurred after the thermo-crosslinking of gelatin. Although, the rate of photo-crosslinking was much slower than thermo-crosslinking; the photo-crosslinking took place below the gel temperature (Fig. 2), leading to the permanent stable structures. In the previous study, it was noticed that the photo-crosslinking of whole structure after printing process may limit the dimension of the whole structure, since UV may not penetrate deeply into thick structures.25 In our study, this problem was solved by inducing photo-crosslinking during printing of each layer. In a word, gelatin provided both the tunable rheology and instantaneous crosslinking to the GelMA/gelatin bioinks, significantly improving the processability of GelMA bioinks, leading to higher printing resolution. As a typical cell-laden bioink, GelMA hydrogels with low concentrations were particularly attractive due to their ability to recapitulate the cellular microenviroment.13 In our study, the cell printing of GelMA/gelatin bioinks was performed (Fig. 9) and the printed GelMA/gelatin scaffolds were also applied for cell culture (Fig. 8). Since GelMA/gelatin scaffolds were placed in the incubator at 37 °C, which was above the gel temperature of all GelMA/gelatin bioinks (Fig. 5c); the gelatin gradually dissolved out from scaffolds (Fig. 2). By measuring the weight of gelatin dissolution (Supplemental Information), it was found the released amount of gelatin from 5/8% GelMA/gelatin hydrogels increased within first 24 hours, and the released amount was larger than the amount released from 5% GelMA hydrogels (Fig. S1); which means physically embedded gelatin dissolved out from GelMA/gelatin hydrogels. During cell culture, the scaffolds lost the temporary stabilizer gelatin, but the long-term stability provided by photo-crosslinking of GelMA still kept the initial geometry of scaffolds (Fig. 8). In this study, BMSCs showed both better cell spreading on the surface (Fig. 8a, b) and higher viability in the printed scaffolds (Fig. 9) when GelMA concentrations were lower. Since higher GelMA concentrations induced the abundant covalent bonds, leading to high rigidity and low porosity of scaffolds, the cell viability of 19



GelMA scaffolds was significantly reduced (Table 3). On the other hand, even though some residual gelatin may be embedded in the 5/8% GelMA/gelatin scaffolds during cell culture, high swelling ratio (Fig. 7) still resulted in enough pores (Fig. S2) for the transportation of nutrition and oxygen. Moreover, BMSCs exhibited obvious spreading morphology on the edge of 5/8% GelMA/gelatin constructs after 7 days of culture (Fig. 9). Thus, cells encapsulated in 5/8% GelMA/gelatin scaffolds stayed in the similar micro-environment as in pure 5% GelMA scaffolds. It can be conclude that the scaffolds fabricated with 5/8% GelMA/gelatin bioinks not only showed similar cell viability as 5% GelMA scaffolds (Table 3), but also had the similar structural resolution as that of 30% GelMA scaffolds (Fig. 4).

5. Conclusion In summary, this work developed the strategy for 3D bioprinting of low centration cell-laden GelMA bioinks by adding gelatin, and the 5% GelMA bioinks with gelatin were successfully extruded into stable 3D constructs using a two-step thermo-photo-crosslinking strategy. Adding gelatin in GelMA bioinks simultaneously achieved optimizing bioink rheology for extrusion and the rapid crosslinking for holding initial structures. This strategy was demonstrated by obtaining similar printing resolution of 5/8% GelMA/gelatin as that of 30% GelMA bioinks. Moreover, 5/8% GelMA/gelatin scaffolds showed excellent cell viability as that of 5% GelMA scaffolds. This work provided a simple yet effective approach to fabricate biomimic scaffolds with low concentration of GelMA bioinks, and would further contribute to develop novel heterogeneous structures for tissue engineering and drug delivery.

Acknowledgement This study was supported by the National Natural Science Foundation of China (NSFC, Grant Nos. 11402056, 51605426), the Key Research and Development Program of Zhejiang Province (Grant No. 2017C01063), and the Fundamental Research Funds for the Central Universities.

Supporting Information Figure S1: Gelatin dissolution behavior of 5% GelMA and 5/8% GelMA/gelatin hydrogels. 20


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Figure S2: SEM images of freeze-dried 5/8% GelMA/gelatin hydrogels before and after swelling at 37 ℃.

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Table of Contents (TOC) Graphic Syringe temperature controller

Step 1: Reversible thermo-crosslinking of gelatin Temperature

Temperature Temperature

Cooling receiving platform

UV light

Step 2: Irreversible photo-crosslinking of GelMA GelMA/gelatin in gel state (below the gel point )

UV

5% GelMA

5/8% GelMA/gelatin

25


30% GelMA

GelMA/gelatin in gel state (up the gel point)

3D Bioprinting of Low-Concentration Cell-Laden ... - ACS Publications

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