Gelatin Methacryloyl based scaffolds by 3D Printing with potential application in tissue engineering [305021]

Gelatin Methacryloyl based scaffolds by 3D Printing with potential application in tissue engineering

Leu Rebeca1, Ghitman Jana1, Stavarache Cristina1,3, Raluca Ianchis4 , Horia Iovu 1,2*

1 [anonimizat], 1-7 Gh. Polizu Street, 011061, Bucharest, Romania

2 Academy of Romanian Scientists, 54 Splaiul Independentei, 050094, Bucharest, Romania
3"C.D. Nenitescu” Centre of Organic Chemistry, 202-B Spl. Independentei, RO-060023 Bucharest, Romania .

4National R-D Institute for Chemistry and Petrochemistry ICECHIM—Bucharest, Spl. Independentei 202, 6th district, P.O. Box 35/174, 060021 Bucharest, Romania

Abstract

Developing a material for 3D bioprinting with tailored of properties for tissue engineering represent one of the main concern in our days. [anonimizat], spread, proliferate and regenerate the tissue, a [anonimizat]. [anonimizat], [anonimizat]-[anonimizat]. In this study it was designed a 3D hydrogel scaffold based on gelatin methacrylate (GelMA) with potential application in tissue engineering. GelMa with three degree of methacrylation was synthesized by using methacrylic anhydride to covalently bind the methacrylate groups onto the gelatin surface.

The presence of methacriloyl groups bounded to the surface of GelMa was confirmed by FTIR and 1H-NMR. According to the mechanical test GelMA with 1% photoinitiator shows better mechanical properties than GelMA with 0.5%. [anonimizat]. The Isoelectric point (IP) [anonimizat]. In order to be used in biological applications it is important to see if the secondary structure of gelatin was affected during the methacrylation process. Using Circular Dichroism (CD), structures like random coil and β-sheets- specific to the secondary gelatin structure are observed to be present in GelMA spectra.

Keywords: Biomaterials, hydrogels, Gelatin, GelMA, 3D bio-printting, [anonimizat], [anonimizat], degradation and reasons for tissue damage should be well known.

[anonimizat] (ECM), because ECM modulates and directs cell behavior.

A powerful tool in the research field is represented by the modern technologies that are able to design scaffold based on biopolymers which can satisfy both the biological and mechanical (supportive) requirement [1].

3D-[anonimizat]. It’s a non-invasive method that allows the use of the material without affecting its vital properties that recommend it to be used in tissue engineering.

In order to produce 3D structures, a lot of bio-inks have been widely studied. To be employed in regenerative medicine, these materials should allow precise control of the tissue architecture for obtaining a personalized implant. Among materials that are studied, hydrogels are the most innovative biomaterials used to bioprint three-dimensional structures in tissue engineering. As bioprinting materials, hydrogels must present a shear thinning behavior, property that allows continuous flow and printing under a high shear rate [2, 3].

Also, in order to recreate the complexity of natural extracellular matrix (ECM), hydrogels must meet the following criteria: biocompatibility and biodegradability, tunable mechanical properties, precise control of the multi-scale internal architecture of scaffolds [4,5].

Gelatin is one of the most extensively used proteinaceous for bio-ink formulations used in 3D bioprinting. It’s a protein soluble in water and it is extracted from animal collagen through partial acid (type A) or alkaline (type B) hydrolysis [6]. Gelatin exhibits an amphoteric character having hydrophilic amino acid sequences like arginine-glycine-aspartic acid (Arg-Gly-Asp) in its structure, and hydrophobic groups in an estimate ratio of 1:1:1. The hydrophobic groups presented in gelatin structure are leucine, isoleucine, methionine, and valine [6, 7, 8]. Moreover gelatin is a biocompatible, non-toxic and nonimmunogenic polymer, a biomimetic peptide with the ability to prevent cell apoptosis [11, 13]. Additionally cell adhesion is improved by natural cell binding motifs, such as arginine-glycine-aspartic acid peptides that are retained in GelMA. Due to these properties, gelatin promotes cell proliferation and differentiation in a certain direction, an essential process for determining the behavior and functions of the tissue, also accelerating tissue regeneration [9-12]. Moreover, gelatin resembles the chemical structure and biological functions of collagen in the native ECM. Because of these characteristics, gelatin is considered to be an ideal material that could emulate the natural structure of the ECM [13-15].

Gelatin is a hydrophilic protein with low mechanical and thermal properties, which represents a drawback for a potential biomaterial with biomedical applications.

Both mechanical and thermal features of gelatin can be improved by crosslinking [16]. Gelatin exhibit a folding structure that can be easily modified by crosslinking its functional groups with Formaldehyde, Acetic acid, Potassium Iodide [37], or with different targeting ligands, -thiol groups, cationic agents, alendronate, PEG, and conjugating targeting EGFR peptide [9].

The main target of our study was to design a 3D hydrogel scaffold based on gelatin methacrylate (GelMA) with potential application in tissue engineering. Thus, gelatin with different degrees of methacrylation was synthesized in order to obtain a material with optimal properties to be used in bioprinting.

GelMA was synthesized by binding the methacrylic groups on the gelatin surface through a covalent reaction. Due to the grafted methacryloyl groups, the biopolymer can be crosslinked by a versatile photocuring process using biocompatible photoinitiators (TiO2, LAP, Irgacure 2959) [8]. Photopolymerization using UV light provides good temporal and spatial control over the crosslinking mechanism, in order to attain a 3D scaffold with unique properties. It’s a versatile method that can take place at room temperature, and studies showed that functions and viability of the cells are not compromised [8,35,36,38].

Irgacure 2959 (I-2959) was used because of its low cytotoxicity compared with others photoinitiators, and under UV-light dissociates into benzoyl and ketyl free radical [40].

In addition to the biological properties of gelatin, GelMA-based hydrogels exhibit high morphological and mechanical stability with tunable mechanical properties, inherent bioactivity and physicochemical tailorability and allow cell to attach, spread in GelMA scaffold, proliferate and regenerate tissue. Also these hydrogels based on GelMA confer a platform that alows the control of cell behavior [17,18,19]. All the materials obtained were characterized and tested in order to be used in applications in tissue engineering. The precursor solution and obtaind materials were characterized using different technics such as: 1H-NMR-spectrometry, for structural characterization, Fourier-transform infrared spectrometry (ATR-FTIR), Circular Dichroism (DC), Dinamic Light Scattering (DLS) to determine the isoelectric point, Contact Angle, Swelling Behavior to determine the hydrophilicity, Printability
for setting the printing parameters, Nanoindentation for mechanical properties and SEM for morphological characterization.

Materials and methods

2.1 Materials

Gelatin (from bovine skin, gel strength ~ 225g Bloom, type B), Methacrylic anhydride (MA) (MW=154.16g/mol) and 2-Hydroxy-4’-(2hydroxyethoxy)-2-methylpropiophenone(Irgacure 2959, (I-2959), (MW=224.25g/mol) were purchased from Sigma-Aldrich; ultrapure water and PBS solution pH=7.4 (8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4) were prepared in our laboratory.

2.2 Experimental

Functionalized gelatin (GelMA) was obtained according to the previously reported method [33-36]. The degree of methacrylation was controlled by the amount of methacrylic anhydride added (Table 1). Briefly, 5 g of gelatin was dissolved in 45 mL PBS (pH=7.4) at 50 0C for 1.5 h until total solubilization. Then, depending on the degree of metacrylation, different amounts of methacrylic anhydride was added to the gelatin solution. Afterwards, the reaction was maintained at 50 oC for 2 h under vigorously magnetic stirring, to allow methacrylic anhydride to react with amino functional groups from protein structure. Then the obtained solution was dialyzed against distilled water using dialysis cellulose bags (MWO =1200 Da) for 72 h. Functionalized gelatin was lyophilized at 0.28 bar for 72h (D-37520, Osterode am Harz, Germany) and was stored at room temperature.

Table1. GelMA composition and methacrylation degree

2.3 Preparation of the Hydrogel

To determine the optimal compositional mixture for the bioprinting, the precursor solutions were prepared by totally dissolving two concentration of GelMA with different methacrylation degree in PBS (pH=7.4) at 40 0C, under gentile stirring. Then, to the obtained solution, the photoinitiator (I-2959) was added in a concentration of 0.5% or 1% from the total amount of polymer (Table 2). The mixture was subjected to homogenization by magnetic stirring until the photoinitiator was completely dissolved, in order to obtain a perfectly homogeneous mixture.

Table2. The bioink preparation for 3D bioprinting

2.4 1H-NMR- spectrometry of gelatin and gelatin methacrylate (GelMA)

The 1H-NMR spectrometry was used in order to determine the methacrylation degree of GelMA. In order to make the analyses, 20mg GelMA were dissolved in 0,75mL D2O to clear solution. The spectrum was registered on a Bruker NMR 600MHz Advance spectrometer. The methacrylation degree was calculated using the equation (1):

(1)

2.5 Fourier-transform infrared spectrometry (ATR-FTIR)

The structural characterization of raw material and GelMA with different metacrylation degrees was performed using ATR-FTIR spectrometry.

In order to identify the methacrylate functional groups that were grafted onto the gelatin backbone, ATR- FTIR spectra were recorded on a Vertex 70 Bruker FTIR spectrometer equipped with an attenuated total reflectance (ATR) accessory. For all the formulation, the ATR- FTIR spectra were registered in the ATR-FTIR mode, at a resolution of 4 cm-1 in 600-4000 cm-1 wave-number region.

2.6 Determination of the isoelectric point (IP).

Isoelectric point (IP) of Gelatin and GelMA with different degrees of methacrylation was determined by DLS technique, monitoring the variations of Zeta Potential of samples in different pH media. Thus, different solutions consisting from 1mg/mL concentration of Gelatin, GelMA 1.5%, GelMA2.5% and GelMA 5% in deionized water with 1mM NaCl were prepared and were subjected to analysis. Samples were introduced in ZETASIZER instrument and were measured at different pH values, ranging from 2 to 8, at 250C in three cycles, each cycle with 50 measurements. All the measurements were performed in triplicate

2.7 Circular dichroism (CD)

Circular dichroism counts on the differential absorption of left-right polarized circular light of a chiral macromolecule. Spectroscopy acquisition was performed on a Jasco J-1500 Spectrophotometer, Japan (J-1500 Circular Dichroism Spectrophotometer) using a quartz cell with l = 1mm. For each reading, 500 μL of functionalized / non-functional gelatin aqueous solution was placed at a concentration of 0.005% and the spectra were registered by scans in triplicate, using a spectral range of 176-250 nm, at a scan rate of 100 nm / min.

2.8 GelMA-based Hydrogels Morphology and Printability

In order to characterize the material from point of view of the printability, a series of tests were performed using the direct dispensing print head of the bioprinter 3DDiscovery™ from RegenHU Ltd, Switzerland. Tests were repeated five times in order to verify the reproducibility of selected method and the stability of the 3D printed hydrogel. A syringe of 3 mL having attached cylindrical nozzle of 25 G (needle gauge ø0.25 mm, needle length 6.35 mm), or 23 G (ø 0.33mm, needle length 6.35 mm) was used. Different printing speeds ranging from 5mm/s to 10mm/s, and pressures in the range of 150-300 kPa were tested. All the samples were printed at room temperature. For scaffold architecture the special BIOCAD software was selected. This software allows drawing in 2D of objects that can be viewed in 3D after generating the G code. Scaffolds were printed layer by layer in 20 layers.

2.9 Scanning Electron Microscopy (SEM) Analysis

In order to perform SEM analysis, scaffolds obtained were frozen and lyophilized. After that, for a better observation of the sample morphology, the scaffold was broken manually. The broken samples were analyzed using Environmental Scanning Electron Microscopy (ESEM-FEI Quanta 200, Eindhoven, The Netherlands). Also, in order to obtain the secondary electrons images, a GSED detector was used in the following conditions: 25–30 KV accelerating voltage and a pressure of 2 torr.

2.10 Swelling and dissolvability of the 3D printed hydrogel based on GelMA

An important feature of hydrogels for bioprinting is their hydrophilic/hydrophobic character and their susceptibility to degrade by hydrolysis. These characteristics are measured through swelling ratio. The degree of swelling is influenced by the pore size of a polymer network, concentration of GelMA solution, the methacrylation degree of GelMA and the amount of photoinitiator used in the crosslinking step. In order to study the swelling behavior of obtained samples, hydrogel based on GelMA with 58% methacrylation degree was chosen. Samples were prepared from different concentration of GelMA (10%, 20%, 30%) using 0.5% and 1% I-2959 against the amount of polymer. The swelling ratios of hydrogels were measured at predetermined periods of time by determining the weight of each sample after removing the residual liquid [20].

Degree of swelling was calculated using the equation (2):

Degree of swelling (%) = (2)

Where: Ww = wet weight, Wd = dry weight

In order to study the dissolvability of the 3D printed biomaterials, lyophilized discs based on hydrogels were immersed in PBS for 24 h and then were subject to weighing. Before each weighing, the residual liquid from the discs was removed [20, 21].

Dissolvability was calculated using the equation (3):

Dissolvability (%) = (3)

Where: Wo=initial weight, Wd= dry weight

2.11 Contact Angle Measurements

The hydrophilicity of the crosslinked 3D printed scaffolds based on GelMA was determined by measuring their water contact angle using the Drop shape analyzer DSA100 equipped with a CCD video camera. A specific amount of each tested sample was attached on a glass slide and then was placed in the sample stage. The behavior of the droplet was measured, and the water contact angles were automatically calculated by the software with which the instrument is equipped.

2.12 Mechanical properties

The effect of methacrylation degree of GelMA on the mechanical properties of the crosslinked 3D bioprinted hydrogels was studied by nanoindentation technique. All analyses were performed on Nano Indenter® G200, and an Espress Test to a Displacement was taken. This method allows to “achieve rapid evaluation of Young’s modulus and hardness by performing an array of indents to a user specified depth.”[41] The principle of the indentation test is described as follows: The sample is moved by the system from the microscope to XP head when the test is initiated. After that, all indents are performed in a rapid succession; for each indentation, the sample is moved automatically under the Indenter tip. When all the analyses are complete, the head disengages and the sample is moved by the system under the microscope [41].

Results and discussion

GelMA was synthesized by binding the methacrylate groups on the gelatin surface through a covalent reaction, as it is shown in Figure 1a. Then the obtained GelMA was subjected to photocuring in order to obtain the 3D scaffold (Figure 1, b).

Figure1. a) Gelatin methacrylation reaction; b) Mechanism of GelMA crosslinking with photoinitiator

3.1 1H-NMR

The methacrylation degree of GelMA was determined by 1H-NMR spectrometry. 1H-NMR spectra registered for all analyzed samples are presented in Figure 2. Methacrylate functional groups were grafted onto the gelatin backbone through reactions between methacrylic anhydride and lysine residues. In this context, the modification of lysine residues with methacrylate groups was confirmed by a decrease in the lysine signal at 2.9 ppm, and the appearance of the methyl group signal at 1.8 ppm (in all spectra recorded for GelMA) [20].

Figure2. 1H-NMR spectra of Gelatin, GelMA 1.5%, 2.5%, 5%

Supplementary, in the GelMA registered spectra, it can be seen the presence of two new signals at δ=5.4ppm and δ=5.6ppm corresponding to the acrylic protons of methacrylic functions characteristic to MA structure which was grafted onto the gelatin backbone and led to the synthesis of GelMA. It is also important to mention that the area of these signals increases with the increase of MA concentration suggesting a high degree of methacrylation (methacrylation degree of GelMA with 1.5%, 2.5%, 5% MA was 37%, 59% and respectively 61%).

In order to normalize the amine signals (2.9ppm) of methacrylated lysine, the phenylalanine signals (7.0-7.5ppm) for 5 protons were set as internal reference.

3.2 ATR-FTIR

The ATR-FTIR spectra recorded for all the samples are presented in Figure 3.

Figure3. ATR-FTIR spectra of Gelatin, GelMA1.5%; GelMA2.5%; GelMA5%

As it can be observed the characteristic absorption bands of functional groups from gelatin structure are also found in the ATR-FTIR spectra recorded for GelMA with different metachrylation degrees, being positioned at the same wavelength. This indicates that the peptide bonds (-NH-CO-) of the amino acids from the primary structure of the protein were not affected in the methacrylate step. The spectra registered for all the hydrogels based on Gelatin, and GelMA with different degree of methacrylation show a peak at 3290 cm-1 related to the stretching vibration of H-O groups from the hydroxyproline amino acid, structure that is founded in the gelatin composition. The band from 3200cm-1-3400cm-1 represents the presence of peptide bond (N-H stretching) from amide A, as well as the signal at 3069 cm-1 attributed to the stretching vibration of the C-H bond from amide B which was also registered in all the spectra.

GelMA infrared spectrum showed peaks at 1640 cm-1, 1541 cm-1 and 1240 cm-1 related to the C=O stretching (amide I), N-H bending (amide II), and C-N stretching plus N-H bending (amide III). Moreover, a N-H stretching (amide A) could be observed at 3303 cm-1, and a stretching vibration of CH at 2934 cm-1 corresponding to the symmetric and antisymmetric stretching in the CH2 groups of alkyl chains (Figure 4) [20,22,23].

3.3 Isoelectric point

The IP of gelatin and GelMA with different methacrylation degrees was determined by measuring the zeta potential of each sample at various pH value. The obtained results are presented in Table 3 and Figure 4.

Table 3. Determination of the IP of Gelatin, GelMA1.5% , GelMA 2.5%, GelMA 5%

Figure 5. The IP of all analysed samples

From the results showed in Table 3 and Figure 4 it can be seen that as the degree of methacrylation reaction increases, the value of the isoelectric point decreases.

The functionalization reaction of the gelatin with MA takes place between amino (NH2) groups from lysine structure which is present in the chemical composition of gelatin. Therefore, an increase in the MA concentration determines a high number of amino and hydroxyl groups involved in the methacrylation reaction, respectively their consumption. The excess of the unreacted carboxyl groups from the GelMA structure give it an acidic character respectively a low value of IP. From Figure 5, it can be observed that the IP value is shifted to lower pH value with increasing the degree of methacrylation.

3.4 Circular dichroism

Circular dichroism (CD) is a method that provides the possibility to study the conformations adopted by proteins and nucleic acids in solution. This technique allows to notice any structural alterations that might result from changes in environmental conditions, such as pH, temperature, and ionic strength [24, 25]. CD is usually employed to analyze the secondary structure of the protein [26]. In Figure 6 the CD registered spectra of the all analyzed samples are shown.

Figure 6. Rough overlapping CD spectra (190-250) of gelatin and GelMA

solutions

After the methacrylation reaction, GelMA structure was studied in order to observe if the secondary structure of gelatin was retaining, or the methacryloyl functionalization of GelMA interfered with helix formation. As it can be seen in Figure 6, CD spectra of GelMA with different degree of methacrylation compared with gelatin spectra, displayed pronounced negative band around 199nm -201 nm, characteristic of a structure with random coil and β-sheets (structures formed by binding of two or more B-chains adjacent to each other via hydrogen bonds) specific to the secondary gelatin structure [27]. Functionalized samples showed negative peak amplitude at 198 nm (the higher the degree of methacrylation, the lower the ellipticity), this decrease may be caused by methacrylated pendant groups [28].

The intensity of negative band of the spectrum registered for GelMA with high methacrylation degree ascribing to a portion of random coil formation was slightly higher than that of GelMA with a low methacrylation degree, therefore the growth of methacryloyl functionalization of GelMA bring out random coil formation. Also, along with the increase in the degree of metacrylation of gelatin, the triple-helix contents of GelMA at 220 nm decreased.

As it can be seen, the intrachain or interchain hydrogen binding in the triple helix could be reduced by the methacrylation of hydroxyl or amino groups from gelatin chain. Thus, with the increase of the degree of methacrylation, the random coil increases [29].

3.5 Printability and morphology of GelMA hydrogels

The main concern of this work was to develop a printable bio-ink based GelMA hydrogel that will mimic the biological and functional complexity of native tissues. In order to achieve these objectives, materials with different concentration of GelMA and photoinitiator (I-2959) were developed. Also, the direct dispensing printhead that use extrusion technology was used with different needle shapes (conic and metallic) and dimensions (0.25mm, 0.33mm). The bioprinting process was performed at room temperature, and the photocrosslinking step was achieved under the UV light (Figure7a).

Figure7. a) Bioprinting process b) Printing tests

In order to study the effect of the precursor concentration on the printability properties and on the final characteristics of the 3D printed hydrogels, three concentration of GelMA solution were prepared and subjected to printing test. Initially, hydrogels with 10% GelMa and 0.5% or 1% I-2959 were prepared and subjected to 3D bioprinting process. The low concentration of GelMA leads to low viscosity of the bio-inks that induce instability during extrusion process, therefore irregular filament shapes. Regardless of the concentration of photocrosslinker agent used, the structural integrity of the filaments was not maintained because of the slow gelation time of the bio-ink, and the poor mechanical properties do not allow the scaffolds to maintain their initial structural integrity.

Then, the GelMA concentration in the precursor solution was increased to 20% keeping the same amount of the photoinitiator (0.5% and 1% I-2959). The viscosity of the bio-inks allows printing and obtaining stable filaments shapes. After that, the material was exposed to UV light and hydrogel scaffolds based on GelMA retained their initial shape integrity and geometry. Furthermore, in order to select the optimal polymer concentration for bio-ink printing, the GelMA concentration from the initial solution was increased to 30%.

The increased viscosity of the hydrogel leads to the impediment of printing, and wrinkled filaments resulted after printing process (Figure7b).

According to the obtained results, the 3D hydrogels consisting from GelMA 20% with 0.5% I-2959, 1% I-2959 presented the optimal characteristics in terms of filaments integrity and geometry shape stability and they were selected for further investigation in order to obtain a material to be used in tissue regeneration [31, 32].

Finally, a scaffold with a square shape and grids with 20 layers was designed in BIOCAD and printed. The resulted scaffold present homogeneity and stability, after 5 minutes UV light photo-curing.

3.6 Scanning Electron Microscopy (SEM) Analysis

SEM analyses were performed in order to observe the morphology of non-mineralized GelMA hydrogels and the morphology of GelMA hydrogels after mineralization. Both samples have a concentration of 20% GelMA and 1% photoinitiator.

As it can be seen in Figure 8a.b. before mineralization, the sample showed high porosities and interconnected macropores. These macropores are separated by smooth thin surface walls. Additionally the walls of scaffolds present a high porous structure. The pores show different size and were obtained during the photopolimerization process. The syneresis phenomenon prevents the system from developing a homogenous material.

One of the most important parameter for biopolymers use in tissue engineering is the roughness surface, because it affects the cell behavior- allows the cells to attach, spread and proliferate in GelMA scaffold.

In order to study the in vitro behavior of 3D materials, the mineralization of the hydrogel was performed. Lyophilized hydrogel disks were immersed in PBS at 370C, for 5 days. After that, the discs were washed, lyophilized, and stored at room temperature until analyses were performed.

After mineralization, we can observe that the hydrogel retained the 3D microstructure and it was covered with spherical agglomerates of minerals. So a good adherence of the material can be observed.

Figure 8. SEM Images showing the microstructure aspect of a) non-mineralized GelMA scaffold at 500μm and b) 50 μm as well as c) mineralized GelMA scaffold at a scale bar of 20 μm.

3.7 Swelling Behavior Studies

The swelling behavior and the degree of dissolvability of all tested hydrogels after the crosslinking process are presented in Figure 9. a) b)

Figure 9.a) Swelling ratio &b) Dissolvability of GelMA-based hydrogel

with different concentration of GelMA and I-2959

As it can be seen in Figure 9a), the capability of hydrogels to absorb water decreases with the increase of both the concentration of the crosslinking agent and the concentration of GelMA in the solution. The chains of the hydrogel become closer due to the new bond formation, making the mesh denser, with higher retraction forces [30]. So, hydrophilicity of the materials increases with decrease of GelMA concentration, also with the decrease of photo initiator. Then, in Figure 9b), the dissolvability tests of the hydrogels performed in PBS at 37 0C are presented. The stability of the material increases with addition of photoinitiator due to the solid bond resulted after photocrosslinking process, under UV light.

Thus, with the increase of GelMA and I-2959 concentration, hydrophilicity of the 3D printed hydrogel decreases, respectively swelling degree and dissolvability properties also decrease.

3.8 Contact Angle Measurements

The hydrophilicity of 3D printed scaffolds based on GelMA was studied by the means of contact angle. To evaluate the effect of the crosslinking agent upon the hidrophilicity of scaffolds, two samples with different amount of I-2959 (0.5, 1%) were subjected to analysis. The measurements were done in triplicate.

Figure10. Water contact angle on GelMA20%+ 0.5% I-2959

GelMA 20%+1% I-2959

As it can be seen from Figure 10, both GelMA-based scaffolds presented a hydrophilic character, regardless the amount of photoinitiator (55.45 for GelMA20%+ 0.5% I-2959 and 78.84 for GelMA 20%+1% I-2959). However the hydrophilicity of the biomaterial decreases as the concentration of the photoinitiator increases. This effect can be explained by the presence in the system of a high number of centers that initiate the photocuring process, determining the formation of a denser crosslinking network, respectively a less hydrophilic system.

3.9 Mechanical properties

In order to characterize mechanical properties of GelMA, it is important to mention that there are a few factors that influence them such as: degree of methacrylation, the precursor concentration, concentration of the photocrosslinker used, and UV exposure time [39].

In this research study, the mechanical properties were studied on two hydrogels based on 20% GelMA and 0.5% or 1% the concentration of I-2959.

Method used to evaluate the materials was “Express Test to a Displacement”. In order to performe the analyses an array of 100 indents was performed, to a specified depth of 200 nm and poisson ration 0.4, using XP head and Berkovich indenter.The results are presented as the average of Young’s modulus for all one hundred indents performed. In order to calculate the Young’s modulus from force –displacement curves, Oliver Pharr method was used. In Figure 11 are presented the results obtained for Young modulus (E) of the tested hydrogels.

Figure11. Modulus of GelMA with diffent concentration of photoinitiator

Results obtained from the analyses show that with increasing concentration of photo initiator, mechanical properties of the materials increases. A value of E = 0.393 GPa was obtained for GelMA with 1% I-2959, while the value of Young modulus determined for GelMA hydrogels with 0.5% I-2959 was with approximately 45 % lower (E = 0.22 GPa).

So, a high concentration of photoinitiator used leads to better mechanical properties because of the chains of the hydrogel that become closer due to the new bond formation.

Conclusion

The focus of this research was to develop a printable bio-ink that could be used in regenerative medicine. Because of its properties like: biocompatibility, biodegradability, good mechanical properties, gelatin was chosen like raw material. Due to its folding structure, in order to improve the stability and mechanical properties, gelatin was modified with methacrylic anhydride. In order to be used as bio-ink, GelMA with different concentrations and different degree of methacrylation was studied. The 1H-NMR investigation highlighted the functionalization of gelatin with methacrylate functional groups that were grafted onto its backbone.

Printability studies showed that the concentration of GelMA from the precursor is a key-parameter in obtaining a printable scaffold with optimal integrity and structural geometry.

SEM analysis highlighted the morphology of GelMA scaffolds that present high porous structure, property that allows the cell to attach, spread and proliferates in order to achieve the tissue. Mechanical properties are influenced by the concentration of the photocuring agent.

According to the obtained results, the 3D printed GelMA-based hydrogel can be a potential biomaterial for biomedical application in the field of tissue engineering.

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[41]Nano Indenter® G200 user manual