Highly methacrylated gelatin bio-ink for bone tissue engineering - ACS

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg

Tissue Engineering and Regenerative Medicine

Highly methacrylated gelatin bio-ink for bone tissue engineering Gulseren Irmak, Tu#rul Tolga Demirta#, and Menemse Gumusderelioglu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00778 • Publication Date (Web): 02 Dec 2018 Downloaded from http://pubs.acs.org on December 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41 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

ACS Biomaterials Science & Engineering

Highly methacrylated gelatin bio-ink for bone tissue engineering (Highly methacrylated gelatin bio-ink)

Gülseren Irmak1‡, Tuğrul Tolga Demirtaş1‡, Menemşe Gümüşderelioğlu1,2* 1

Hacettepe University, Bioengineering Department 06800, Beytepe, Ankara, TURKEY

2

Hacettepe University, Chemical Engineering Department 06800, Beytepe, Ankara, TURKEY

Submitted to: ACS Biomaterials Science & Engineering *To whom correspondence should be addressed:

Prof. Dr. Menemşe Gümüşderelioğlu Hacettepe University, Faculty of Engineering, Chemical Engineering Dept 06800, Ankara, TURKEY. Tel: +90 312 297 74 47 Authors e-mail :

[email protected] [email protected] [email protected]

‡

These authors contributed equally.

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

Abstract Methacrylated gelatin (Gel-MA) is a commonly used biomaterial in bioprinting applications. Gel-MA synthesize procedure is inadequate and needs to be improved, particularly from the point of optimization and efficacy. We report a significantly faster (by 5 min) and effective method to controllably synthesize Gel-MA using microwave energy (Mw at 1,000 W power) with  90% degree of methacrylation (DM) even with the use of a very low concentration of methacrylic anhydride (MA). Rheological and mechanical analyses indicated that Gel-MA synthesized by Mw-assisted methacrylation enabled the formation of hydrogels that are more elastic, stronger and have a lower degradation rate (27% at 35 days) than Gel-MA synthesized by the conventional method. The viscosity values of the Gel-MA bio-ink were in the range applicable for use in 3D bioprinters. Additionally, Mw-assisted methacrylated GelMA hydrogels that have mechanically superior properties significantly enhanced the viability, attachment, proliferation, alkaline phosphatase (ALP) activity, mineral deposition, and mRNA expression levels of osteogenic genes of MC3T3-E1 pre-osteoblastic cells. Key words: Microwave energy; methacrylated gelatin (Gel-MA); bioprinting; bone tissue engineering.

1. Introduction Gelatin obtained by the denaturation of collagen which is the main component of the extracellular matrix

1

is biocompatible, biodegradable, and has great capacity for the

modification of amino-acid groups 2. Gelatin in the triple helix structure becomes gel form physically as a result of the stabilization of the tertiary structure of the molecule at temperatures below 37C . However, the non-covalent links are easily broken at temperatures above 37C; thus, the physical network disappears3. To obtain enhanced mechanical properties for gelatin hydrogels, various crosslinking methods including the use of


Page 2 of 41

Page 3 of 41 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


crosslinking chemicals (glutaraldehyde 4, genipin 5, and glyoxal 6) and chemical modifications have been applied. The gelatin can be photocrosslinked with UV light in the presence of a photo-initiator after the chemical modification of gelatin with methacrylic anhydride to enable more stable hydrogel formation at body temperature (MA) 7. Methacrylated gelatin (Gel-MA) was first proposed by Van Den Bulcke and co-workers 7. In the conventional method, the methacrylate chain reacts with primary amine (lysine and hydroxyl lysine) groups of the gelatin macromolecule at 50°C for 1-3 h

7-12.

However, there

are some deficiencies and problems in the conventional Gel-MA production method: i) In theory, one MA molecule can react with one lysine unit. Nevertheless, in studies, excess methacrylic anhydride (10-30%, v/v) (which has a toxic effect on cells) is used to achieve high macromere conversion expressed as a degree of methacrylation (DM) (40-85%) 7, 8, 10, 13. For example, Hoch and co-workers (2012) reported nearly complete conversion of type B gelatin into Gel-MA using a high molar excess (20–30-fold excess) of MA over the free amino groups of type B gelatin. ii) As a result of using excess MA and the low reaction efficiency

14.

Gel-MA is exposed to long purification periods (1-2 weeks) to remove

unreacted methacrylic anhydrate. iii) Gel-MA hydrogels exhibit insufficient mechanical and rheological properties for use in tissue engineering. iv) Gel-MA hydrogels show different characteristics (e.g. different gelling times under UV light, mechanical properties, and DM) depending on the person being used to synthesize the sample. Shirahama, Lee, Tan, & Cho (2016) developed highly methacrylated Gel-MA (DM: 95%) by adjusting the pH to 9 and then adding MA during the reaction to keep the free amino groups of lysine neutral in order to allow them to react with MA. Then, Gel-MA in the presence of a high concentration of photo-initiator (1%, w/v) was exposed to UV (2-8 min) to obtain photocrosslinked hydrogels. However, these conditions may adversely affect cell life for tissue engineering applications 15.



Gel-MA has been used in several micro-scale techniques including photo-patterning, micromolding, self-assembly, microfluidics, and bioprinting. Bioprinting technologies have been used to produce Gel-MA-based 3D structures with inspected architectures for several tissue engineering applications 16. Microwave heating originates from the ability of a particular substance to absorb microwave energy and transform electromagnetic energy to heat. Under microwave energy, molecules with a dipole moment readily absorb this energy and a large number of molecules are excited rotationally. As they strike other molecules, the rotational energy is transformed to kinetic energy resulting in heating

17,18.

Benefits of microwave heating over conventional heating

include selectiveness, effectiveness, and speed, which allow energy and time to be saved. In addition, the use of microwave energy can enhance the efficiency of the chemical crosslinking reaction. Microwave irradiation has been used for polymer synthesis and functionalization 19-23.

We demonstrate the first reported microwave-assisted (Mw) method to obtain functionalized gelatin using methacrylic anhydride and highlight common advances over conventional methacrylated gelatin (C-Gel-MA) synthesis. We investigated the effects of varying the Mw power and concentrations of MA (8% and 4%, v/v) on the degree of methacrylation (DM) using 1H-NMR analysis. The highest power of microwave energy (1,000 W) accelerated the reaction rate and reduced the reaction time to the minute range (5 min) while the degree of methacrylation was significantly increased to 98%. An extruder-based bioprinter (Fab@HomeTM Model 3) was used to print hydrogels in dimensions of 6 mm × 1 mm. GelMA hydrogels were crosslinked by photo-crosslinking in the presence of Irgacure 2959, which is a photo-initiator, using UV (200 mW/cm2, 40 s) irradiation. We characterized the microwave-assisted methacrylated gelatin (Mw-Gel-MA) hydrogels by their rheological, mechanical, morphological, and degradation properties. In addition, morphological


Page 4 of 41

Page 5 of 41 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


characterization, proliferation, and osteogenic differentiation of encapsulated MC3T3-E1 cells in the Mw-Gel-MA hydrogels were compared with the cells in C-Gel-MA hydrogels. 2. Materials and Methods 2.1. Materials Gelatin (Type A, 300 bloom from porcine skin), methacrylic anhydride (MA), Irgacure 2959 (2-hydroxy-4′-(2-hydroxyethoxy)-2 methylpropiophenone) and phosphate-buffered saline (PBS, pH: 7.4) tablets were purchased from Sigma-Aldrich (USA). Minimum essential medium-α (α-MEM) was obtained from Biowest (France). Fetal bovine serum (FBS), Lglutamine, penicillin–streptomycin solution, trypsin, ascorbic acid, β-glycerol phosphate, paraformaldehyde, and propidium iodide were supplied by Sigma-Aldrich (USA). Glutaraldehyde (25 wt% in H2O) was purchased from Merck (Germany). Presto Blue, Alexa Fluor 488 Phalloidin, and Trizol were obtained from Invitrogen (USA). A Live/Dead assay kit was purchased from Life Technologies (USA). Alkaline phosphatase (ALP) assay and Ca assay kits were obtained from Biovision (USA). 2.2. Microwave-assisted synthesis of methacrylated gelatin Conventional methacrylated gelatin (C-Gel-MA) was synthesized as described previously 7. Briefly, a solution of gelatin in phosphate buffer was reacted with methacrylic anhydride (MA) at 50 °C, for 3 h. Then, the Gel-MA solution was dialyzed against deionized water for 14 days at 40 °C to remove unreacted MA7. The dialyzed Gel-MA solutions were freezedried. In this study, Gel-MA was also synthesized by the microwave-assisted reaction of gelatin with methacrylic anhydride. Type A gelatin was dissolved in PBS (pH: 7.4) at 50 ºC and stirred for 40 min to obtain 10% (w/v) gelatin solution. This solution was transferred to a reaction vessel (250 mL borosilicate beaker) and the reaction vessel was placed on the turntable of a microwave-assisted synthesis system (Milestone, Italy) (Supplementary Figure



Page 6 of 41

S1A). Different concentrations of methacrylic anhydride (4 and 8%, v/v) were added to the solution at 0.4 mL/min using a syringe pump under stirring in the microwave oven. Microwave irradiation was applied for 5 min with different periods and different powers (100 W, 600 W, 800 W, and 1,000 W) (Table 1). The reaction was carried out at 50-60 ºC. The microwave irradiation was paused periodically and the system was cooled between cycles. After reaction completed, the mixture was dialyzed against distilled water using 12-14 kDa dialysis tubing for different lengths of time (1 day and 14 days) at 40 ºC to remove salts and unreacted methacrylic anhydride (Figure1A, Supplementary Figure S1A). The reaction product was dried in a freeze-dryer (Christ, Germany) and stored at -80 ºC until use.

Table 1: Synthesizing conditions of Gel-MA. Synthesis method

MA concentration

Reaction Time

Dialysing Time

of Gel-MA

(%)

(min)

(day)

Conventional

8

-

150

14

Conventional

4

-

150

14

Microwave

8

100

5 (30s x10)

2

Microwave

8

600

5 (30s x 10)

1

Microwave

8

800

5 (20s x 15)

1

Microwave

8

1,000

5 (20s x 15)

1

Microwave

4

100

5 (30s x10)

2

Microwave

4

600

5 (30s x10)

1

Microwave

4

800

5 (20s x15)

1

Microwave

4

1,000

5 (20s x15)

1

Power (W)


Page 7 of 41 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


Figure 1: Schematic diagrams illustrate the manufacturing processes of microwave-assisted methacrylated Gel-MA hydrogels. A) Microwave-assisted methacrylated gelatin synthesis and purification steps. Gelatin macromers that contain primary amine groups are reacted with methacrylic anhydride under microwave energy to form Gel-MA that has a high degree of methacrylation. The purification period significantly decreased as a result of highly efficient reaction. B) MC3T3-E1 pre-osteoblast cells are mixed with printable Gel-MA solution in the presence of a photo-initiator (Irgacure 2959) and the mixture is placed in a 3D printer syringe. A 3D bioprinter is used to print the mixed hydrogel solution to disk shape (6 mm diameter × 1 mm thickness). The printed hydrogel was crosslinked using UV (300-500 nm, 200 mW/cm2) irradiation to create a hydrogel network. 2.3. 1H-NMR spectroscopy The methacrylation degree of gelatin was determined using 1H-NMR spectroscopy (400 MHz). Gel-MA (30 mg) was dissolved in 600 L deuterium oxide (D2O) and analyzed at 40ºC. Three 1H-NMR spectra were collected from each sample. The degree of methacrylation (DM) was identified as the percentage of conversion of the lysine and hydroxyl lysine methylene groups of gelatin. The peak area of aromatic acids in the Gel-MA sample was



Page 8 of 41

employed as a reference in each spectrum. The peak area of acrylic protons of the methacrylated compound appeared at around 5.5-6.3 ppm in the methacrylated gelatin spectrum. The peak area of methyl proton signals appeared at around 1.99-2.2 ppm. The peak area of lysine methylene signals appeared at around 2.83-3.0 ppm in the gelatin and methacrylated gelatin spectrum was used for calculation of the DM. Detailed information of 1H-NMR

spectra of all groups are given in Supplementary Figure S3 and S4. The DM of the

different Gel-MA batches were calculated using Equation. 1 14. DM%= [1-(peak area of lysine methylene groups of Gel-MA)/ (peak area of lysine methylene groups of gelatin)] × 100

(Equation 1)

2.4. Preparation and printing of bio-ink solutions without cells Hydrogels including 15% (w/v) Gel-MA with different DMs were prepared by photocrosslinking with different concentrations of Irgacure (0.1, 0.3 and 0.5 w/v). Irgacure was completely dissolved in PBS (pH: 7.4) at 50 ºC. Gel-MA was dissolved in this solution at 37C (pH: 5.5-6.0) and the pH was adjusted to 7.0 with 1 M NaOH. Each Gel-MA solution was loaded into a bioprinting syringe and the syringe temperature was kept at 25-30 C. After printing, hydrogels were immediately crosslinked using a UV-A (300-500 nm) light source (Tanses Technologies, Canada) at an intensity of 200 mW/cm2 for 40 s. UV was applied 5 cm from the outer shield of the light source (Figure1B and Supplementary Figure S1B). 2.5. 3D bioprinting set up The Fab@Home™ (The Seraph Robotics, USA) open source RP platform Model 3 with a position accuracy of 100 microns was used for printing the hydrogels. The hydrogel precursor solution was loaded into the deposition syringes (tip diameter: 1.2 mm) and extruded along the X–Y-Z target paths to build cylinder-shaped disks (6 mm diameter and 1 mm thickness) (Supplementary Figure S1B). Printing process of hydrogel was shown in Supplementary Figure S2a and Supplementary Video S1.


Page 9 of 41 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


Six groups of Gel-MA (15%, w/v) hydrogels without cells were prepared as bio-ink (Table 2). Table 2. Groups of Gel-MA (15%, w/v) hydrogels without cells. Group No I II III IV V VI

Production MA content Method % (v/v) Conventional/8 Microwave (600W)/8 Microwave (1,000W)/8 Conventional/4 Microwave (600W)/4 Microwave (1,000W)/4

Abbreviation C/8%MA 600W/8%MA 1,000W/8%MA C/4%MA 600W/4%MA 1,000W/4%MA

2.6. Hydrogel characterization 2.6.1. Biodegradation study Printed hydrogel discs (6 mm × 1 mm) were placed into 24-well tissue plates with 1 mL 2 U/mL collagenase type I solution

24

for 30 days and then, the degradation medium was

replaced with 0.2 U/mL collagenase type I 11. In vitro degradation studies were carried out at 37°C. The collagenase solution was refreshed every 2-3 days to preserve constant enzyme activity. The collagenase solution was removed at specific time points and the samples were freeze-dried and weighed. Photographs of hydrogels were taken to observe their morphologies. The percentage mass loss of each gel was determined using Equation 2 11. Mass loss % = (W0-Wt)/W0 x 100

(Equation 2)

where W0 is the initial sample dry weight and Wt is the dry weight after time t. 2.6.2. SEM analysis The surface and cross-section morphologies of the printed Gel-MA hydrogels were examined using scanning electron microscopy (SEM). The hydrogel discs were lyophilized and placed on aluminum slabs. Then, they were coated with gold–palladium. The coated samples were evaluated using SEM (Zeiss, Germany) at 5-20 kV.



2.6.3. Mechanical properties The compressive moduli of printed hydrogels (7 mm thickness and 12 mm in diameter, n = 3) were determined by applying uniaxial compression force using a universal testing machine (Zwick, Germany) at room temperature. Compression force (load of 0.05 N) was exerted at a displacement rate of 1 mm/min. Hydrogels were stored in PBS (pH: 7.4) before measurements. The compressive modulus was assessed from the slope of the linear elastic region of the stress-strain curve, which was between 20% and 30% strain for all samples. 2.6.4. Rheological properties The rheological characteristics of Gel-MA hydrogels were evaluated on a rheometer (Ares Instruments, USA). A photo-crosslinked Gel-MA hydrogel (12 mm diameter and 2 mm thickness) was placed between parallel circular plates that had a 400 m gap size. The rheological properties were examined using oscillatory shear measurements. Dynamic frequency sweeps were performed and the storage (G) and complex (G*) moduli were detected in the range of 0.5-200 rad/s at 0.2% strain (at 37 °C). For strain sweep analysis of Gel-MA hydrogel, G’ was determined by increasing the strain from 0 to 2 (%) at a constant frequency (1 Hz) at 37 °C. The viscosities of gelatin (10%, w/v), 1,000 W/4%MA, and 1,000 W/8% MA solutions before crosslinking were determined by dynamic measurements with a rotatory test setup by varying the temperature from 15 to 45 °C. In addition, the shear stress was determined at different temperatures from 15 to 45 °C for the 1,000 W/4%MA and 1,000 W/8%MA solutions. 2.7. Cell culture Cell culture studies were conducted with the MC3T3-E1 mouse pre-osteoblast cell line (No: RCB1126, Riken cell bank, Tsukuba, Japan). The cells were cultured in a base medium of αMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin in a CO2 incubator (Heraus Instruments, Germany). The cultured cells were detached with 0.25%


Page 10 of 41

Page 11 of 41 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


trypsin–EDTA and centrifuged before mixing with bio-ink. The cell culture studies were performed using three experimental groups of hydrogel (C/8%MA, 1,000 W/8%MA, and 1,000 W/4%MA). Gel-MA solution was prepared under sterile conditions as described in the “Preparation and printing of bio-ink solutions without cells” section. Irgacure and Gel-MA solutions were filtered through a 0.22 μM filter. MC3T3E1 cells were gently dispersed and mixed into sterile hydrogel solutions at a density of 2 × 107 cells/mL. A cell-bio-ink mixture was loaded into the deposition syringes and printed along the X–Y–Z target paths. After printing, cell-laden hydrogel disks (6 mm × 1 mm) were immediately crosslinked with UV (300-500 nm) light at an intensity of 200 mW/cm2 for 40 s (Figure 1B). 2.7.1. Cell viability analysis The cell viability of the hydrogel was examined using a Live/Dead viability kit and Presto Blue analysis. The Live/Dead assay was evaluated on the 1st, 3rd and 10th days after printing. The cell-laden hydrogels were washed in PBS and incubated in 2 µM Calcein AM and 4 µM ethidium homodimer (Ethd-1) solution for 30 min. The cell-loaded Gel-MA hydrogels were observed under a confocal microscope (Zeiss, LSM 510, Germany). Three images were taken from the top, middle, and bottom piles of the bioprinted hydrogels. To evaluate cell viability, the numbers of green (live) and red (dead) cells were counted using ImageJ software. 2.7.2. Cell proliferation assay A Presto Blue assay was performed on the 1st, 3rd, 7th, 14th, and 21st days after printing. The culture medium was replaced with Presto Blue Cell Viability Reagent diluted with culture medium (10%, v/v). After 2 h incubation, the UV absorbance (570 nm) of the medium was determined using a microplate reader (UVM 340; Asys Hitech, Austria). 2.7.3. Cell morphology The surface and cross section morphologies and attachments of printed MC3T3-E1 cells in



hydrogels were observed by SEM (Zeiss, Germany). The culture medium was removed and the hydrogel was washed three times with PBS (pH: 7.4) on the 7th, 14th, and 21st days of culture. The cells were fixed in glutaraldehyde (2.5%, v/v) for 30 min and then the hydrogel was washed thrice in PBS. The hydrogel was lyophilized with a freeze-dryer and coated with gold-palladium layer before SEM analysis. Energy-dispersive X-ray spectroscopy (EDX) was used to record the element distribution maps and calculate the Ca/P ratio. To observe the cytoskeletal organization of MC3T3-E1 cells in the Gel-MA hydrogels, the culture medium was removed and the hydrogels were washed three times with PBS (pH: 7.4) on the 4th, 7th, and 14th days of culture. The cells were fixed with 10% (w/v) paraformaldehyde for 10 min at 4 C. The fixed hydrogels were permeabilized with 0.1% Triton-X 100 in PBS for 15 min. Cell cytoskeletal filamentous actin (F-Actin) was imagined by treating the cells with 2.5% (v/v) Alexa Fluor 488 Phalloidin for 20 min. The samples were washed with bovine serum albumin (BSA) solution (1%; w/v). The hydrogels were incubated with 10 μg/mL propidium iodide for nucleus staining over 5 min. The samples were then thoroughly rinsed with 1% (w/v) BSA solution. The samples were observed under a laser scanning confocal microscope (LSM 510 Carl Zeiss Micro imaging) at 20× and 40×. Images were taken from the middle piles of the bioprinted hydrogels. 2.7.4. Cell differentiation analysis 2.7.4.1. Gene expression analysis for cells printed with hydrogels On days 7, 14, and 21, the ribonucleic acid (RNA) was extracted from MC3T3-E1encapsulated Gel-MA hydrogels using Trizol® (UK) reagent. RNA was purified at each time point using an RNeasy® Mini Kit (Qiagen, UK). The concentration and purity of the isolated RNA were detected by Nanodrop (Thermo scientific 2000c, USA). RNA was reverse transcribed into cDNA and amplified by a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). The real-time reverse transcription-polymerase chain reaction


Page 12 of 41

Page 13 of 41 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


(RT-PCR) was performed using Hot FirePol Eva Green qPCR Mix Plus (Solis Biodyne, Estonia) with cDNA and forward-reverse primers. Following an RT-PCR initial activation step at 95 C for 15 min, amplification was performed for 40 cycles of denaturation at 95 C for 15 s, annealing at 60 C for 20 s, and extension at 72 C for 20 s using Light Cycler Nano (Roche, Switzerland). Gene expression levels were standardized to those of β-actin, which is commonly used as a housekeeping gene. The target genes and primer sequences used in this study are listed below: β-actin forward: 5'-GTGCTATGTTGCCCTAGACTTCG-3'; reverse: 5'-GATGCCACAG GATTCCATACCC-3'; Collagen type 1 (col 1) forward: 5'-CAAGATGTGCCACTCTGACT3';

reverse:

5'-TCTGACCTGTCTCCATGTTG-3';

Osteocalcin

(Ocn)

forward:

5'-

CTTTCTGCTCACTCTGCTG -3'; reverse: 5'-TATTGCCCTCCTGCTTGG-3'; Run X2 forward:

5’-GCATGGCCAAGAAGACATCC-3';

CCTCGGGTTTCCACGTCTC-

3');

and

reverse:

Osteopontin

CACTTTCACTCCAATCGTCCCTAC-3’;

(Opn)

reverse:

forward:

5’5’5’-

ACTCCTTAGACTCACCGCTCTTC-3’. To submit relative gene expression, the comparative CT method (also referred to as the 2−ΔΔCT method) was used. 2.7.4.2. Biochemical analysis The ALP enzyme activity and Ca content were analyzed to determine the osteogenic differentiation of the cells. The ALP activity of the cells was determined with an ALP assay kit on the 7th, 14th, and 21st days of culture. Cytoplasmic ALP was extracted using Triton X-100 (0.01%, v/v in PBS) at 4 °C. The extracts were centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatants were collected and analyzed using an ALP assay kit (Biovision, USA) according to the manufacturer's instructions.



The Ca deposition produced by cells was analyzed with a calorimetric Ca assay kit on days 14 and 21. Hydrogels were incubated in tricholoroacetic acid (5%, v/v) overnight at 4 C to extract Ca. The extracts were centrifuged at 4 C and 12,000 rpm for 10 min. The supernatants were collected and analyzed using the Ca assay kit (Biovision, USA) according to the manufacturer's instructions. 2.8. Statistical analysis All data are expressed as mean ± standard deviation of representative experiments performed in triplicate. Statistical analysis was carried out using GraphPad InStat software. A Student’s t-test was used to determine the differences among groups, and p-values less than 0.05 were indicated as significant. 3. Results 3.1. Synthesis of Gel-MA and determination of DM Gelatin and MA were successfully integrated via microwave-assisted methacrylation—in comparison with the conventional method—using two different concentrations of MA (4%, v/v and 8%, v/v) to create polymers with different degrees of methacrylation (DM) (Figure 1A). Quantitative proton nuclear magnetic resonance (1H-NMR) analysis was used to determine the extent of conversion of the free amine groups in Gel-MA samples. The peaks of lysine methylene protons (2H) around 2.9-3.0 ppm (Figure 2b, e) decreased significantly in the spectra of Mw-Gel-MA, exhibiting the complete linkage of lysine groups with MA by Mw energy. 1H-NMR

peak of methylene protons on MA appears on δ ≈ 5.4 and 6.4 ppm 25,26. The acrylic

protons (2H) from -CH2 functional groups of the methacrylate around 5.5-6.3 ppm (Figure 2a, d) and those of the methyl protons (3H) of methacrylate around 1.8-2.1 ppm (Figure 2c, f) were higher in the Mw-Gel-MA. These values confirm that MA was successfully grafted onto


Page 14 of 41

Page 15 of 41 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


the gelatin molecule. The appearance of additional acrylic protons (2H) at 5.5- 6.27 ppm, when MW is applied, indicates that the all of lysine and hydroxyl lysine groups were methacrylated. Free amino groups to be completely consumed and additionally some hydroxyl groups on amino acids reacted with MA, as interpreted from the 1H-NMR data showing peaks at 5.03-5.64 (1H) ppm (Supplementary Figure S3e and S4f).



Figure 2. 1H-NMR spectra of gelatin and Gel-MA produced by the conventional method and with microwave irradiation using 8% (v/v) MA and 4% (v/v) MA. The signals of the acrylic protons of methacrylate grafts of lysine groups and those of hydroxyl lysine groups for GelMA 8%MA (a) and 4%MA (d); methylene protons (2H) of unreacted lysine groups for GelMA 8%MA (b) and 4%MA (e); methyl protons (3H) of methacrylate grafts for Gel-MA 8%MA (c) and 4%MA (f).

The degree of methacrylation of Gel-MA synthesized by two different methods was calculated using equation (1), as mentioned in Section 2, and is given in Table 3. The 1HNMR results show that DM of Mw-Gel-MA was higher than that of C-Gel-MA (Figure 2). Nearly complete substitution (DM: approximately 98%) was achieved at 600 W, 800 W, and 1,000 W with 8% MA. We also investigated the effect of different concentrations of MA on the DM. 1H-NMR results showed that Mw-Gel-MA with 4% MA had a much higher DM (DM: 89.0 % ± 1.4) than CGel-MA (DM: 23.1±5.6). According to the 1H-NMR results, DM of Mw-Gel-MA increased with increasing power of Mw when using 4% MA (Table 3). As a result of increasing reaction efficiency, the purification step was reduced to 1 day owing to the Mw energy (Table 1). 1H-NMR results showed that there is no difference in DM between the Mw-Gel-MA dialyzed for 1 day and the Mw-Gel-MA for 14 days (data not shown).


Page 16 of 41

Page 17 of 41 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


Table 3. Characteristics of printed Gel-MA hydrogels. Hydrogel

Degree of Methacrylation (%)

Storage Modul (kPa)

Loss Modul (kPa)

Complex Modul (kPa)

Compressive Modul (kPa)

C/8%MA

76.0±2.5

30.6

0.6

30.7

37.310.5

600W/8%MA

98.2±0.5

39.0

0.1

39.1

49.25.2

1,000W/8%MA

98.6±1.4

39.1

0.1

39.2

55.7 5.8

C/4%MA

23.1±5.6

23.0

0.5

23.5

25.67.2

600W/4%MA

62.0±2.8

27.5

0.1

27.6

37.15.0

1,000W/4%MA

89.0±1.4

41.1

0.1

41.2

60.39.1

*Storage modulus (G'), Loss modulus (G'') and Complex modulus (G*) were obtained at frequency of 100 rad/s in rheological frequency sweep analysis. 3.2. Hydrogel formation Gel-MA hydrogel precursor solution was printed with a Fab@Home bioprinter as a hydrogel disk (6 mm × 1 mm) and polymerization of Gel-MA was realized via photo-crosslinking in the presence of Irgacure 2959 and UV-A. Via radical polymerization, gelatin chains are connected through short poly (methacryloyl) chains (Figure 1). Gel-MA hydrogels were successfully crosslinked with 0.3% (v/v) Irgacure via UV-A (200 mW/cm2) for 40 s (Supplementary Figure S1B and S2b) while they were not crosslinked at concentrations of 0.05% and 0.1% (v/v) Irgacure. 3.3. Hydrogel characterization 3.3.1. Biodegradation assay To evaluate biodegradation, Gel-MA hydrogels were incubated in type I collagenase solution (2 U/mL)

11, 24

for 35 days (n=3 for each group). The degradation rate was determined by

measuring weight loss (Figure 3g and h) over time. Biodegradation results showed that the degradation rate decreased with microwave induced methacrylation of Gel-MA.



Statistical calculations showed that there are no significant differences of biodegradation rates between 600 W/4%MA and 1,000 W/4%MA hydrogels and between 600 W/8%MA and 1,000 W/8%MA hydrogels. However, the rates of degradation of microwave-assisted methacrylated Gel-MA hydrogels are significantly lower (p

Highly methacrylated gelatin bio-ink for bone tissue engineering - ACS

Recommend Documents