Integration of 3D printed GelMA in rat liver: A pilot study

Abstract

Liver transplantation is the only treatment for patients with end-stage liver diseases yet the limited availability of donor organs greatly restricts this treatment option. One innovative approach to mitigate this challenge involves biofabrication of tissues through 3D Bioprinting technology. Gelatin Methacrylamide (GelMA), a modified form of gelatin is one of the extensively studied hydrogel for bioprinting of soft tissues. This study reports a non-cytotoxic, printable bioink formulation composed of 10% GelMA, 3% Gelatin and 0.25% LAP (GelMA-G-L). We have studied the in vivo integration of the 3D printed construct within rat liver tissue following upto a 30-days implantation period. Histological examination revealed that the 3D printed GelMA-G-L construct had successfully integrated with the surrounding liver tissue, displaying adequate vascularization with no indications of adverse immune responses or rejection signs.

Keywords

3D bioprinting bioink GelMA implantation liver

Introduction

The liver is the largest gland in the human body that carries out multiple physiological functions in which few to mention are protein synthesis, metabolism, detoxification, bile production, synthesis of blood coagulation factors, glycogen storage and biotransformation of drugs.¹ It comprises of hepatocytes as parenchymal cells supported with liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells and biliary epithelial cells. The cells in the liver are organized within a hexagonal structure known as the hepatic lobule, which is the fundamental functional unit of the liver. Apart from its normal physiological activities, liver possesses immense potential of regeneration even after losing more than 70%–80% of its mass due to injury or toxicity. In patients with end-stage liver disease and early hepatic cancers in cirrhotic livers, orthotopic liver transplantation is currently regarded as the gold standard when traditional medical treatments are ineffective.² The shortage of sufficient donor organs for meeting the escalating number of waiting list of patients impose requirement for finding alternative strategies for liver transplantation. Numerous biotechnological and biomedical strategies have been explored to generate alternative donor tissues, including the use of genetically modified pigs and bioengineered liver.³ The significant growth of tissue engineering over the past three decades is attributable to its potential to create artificial organs and tissue constructs, thereby addressing the shortage of organs available for transplantation.⁴ Tissue engineering (TE) strategies attempt to organize cells within temporary biomaterial scaffolds by providing appropriate mechanical support and nutrients to the cells enabling them to mature into functional tissue which is achieved through the formation of scaffolds, it becomes possible to assess cell behaviour and explore interactions between cells as well as between cells and the matrix.^5,6 However, lack of tissue specific biomaterial, cell distribution within the scaffold, vascularization, tissue formation proportionate to biodegradation of scaffold and functional cells representing organ function have limited the translation of TE to clinics.^7
–9 Recently, advancements in TE have led to the development of a promising and innovative approach known as three-dimensional (3D) bioprinting. Compared to conventional tissue engineering where cells are seeded on prefabricated scaffolds, 3D bioprinting allows, precise cell distribution with high cell density, mimicking microarchitecture of native tissues.¹⁰ This technology facilitates the fabrication of highly precise and complex living functional tissue constructs, aimed at regenerating lost organ function.¹¹ The three main categories of 3D bioprinting technologies are jetting-based, extrusion-based, and vat photopolymerization-based (VP based) bioprinting. The selection of an appropriate bioprinting method is largely influenced by the specific application, the characteristics of the bioink, as well as the distinct benefits and constraints associated with each bioprinting technique.¹² Extrusion bioprinting has been extensively used to deposit continuous filament of bioink, which consists of combination of biomaterials with differing viscosities at an elevated fabrication speed and living cells, in a layer-by-layer fashion to create three-dimensional structures.^13,14 Whereas in VP-based bioprinting technique distinguished by its enhanced resolution and precision relative to other bioprinting method.¹⁵ The bioprinting technology that allow precise spatial arrangement of live cells and biomaterials is jetting-based method where there is significant level of control over number of cells dispensed as droplets at designated locations.¹⁶ Among the technologies mentioned above, extrusion based bioprinting is the most widely used method for bioprinting liver models.

One of the important components in 3D bioprinting is the bioink which is a mixture of cells, a hydrogel and the growth supplements. The selection of tissue compatible hydrogel for the bioink is a critical factor that determines the success of tissue bioprinting.¹⁷ Many synthetic and natural hydrogels have garnered widespread interest in bioinks due to their resemblance to extracellular matrix, high water content and, capacity to facilitate cell signalling.¹⁸ Among the bioinks reported in the literature gelatin methacrylamide (GelMA) has emerged as a promising material for tissue bioprinting, and offers a versatile platform for creating functional liver tissues.¹⁹ Investigators have looked into various advancements in bioprinting technologies and bioink formulations to overcome current limitations, paving the way for successful clinical applications in liver regeneration and disease modelling. For instance, Pai et al.,²⁰ formulated a radical scavenging formulation of GelMA bioink (ultra-violet safe) which could protect the embedded cells from the harmful effects of UV during the crosslinking. Anupama et al.,²¹ loaded GelMA with a cocktail of antioxidant and evaluated the biocompatibility of the novel GelMA bioink formulation.

In this study we propose a formulation for hydrogel comprising of GelMA, Gelatin and Photoinitiator. The hydrogel was evaluated for its physicochemical characteristics such as rheology and printability and in vitro cytotoxicity. The formulation was 3D printed, crosslinked to get hydrogel and implanted in rat liver to ensure the tissue integration with native tissue with sufficient vascularization.

Materials and methods

Gelatin (Porcine skin, Type A, 175 Bloom), Methacrylic anhydride (MAA), Dialysis tubing (12–14 kDa), Minimum Essential Medium (MEM), Harris’ Haematoxylin and Eosin were obtained from Sigma-Aldrich Chemicals Pvt Ltd, India; Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) procured from Tokyo Chemical Industry (India) Pvt. Ltd.; Foetal Bovine Serum (FBS), Trypsin-EDTA, Penicillin-Streptomycin antibiotic and xylene were purchased from Thermofisher Scientific; Phosphate buffered saline (PBS) was procured from TaKaRa (Shiga, Japan); The mouse subcutaneous fibroblast cell line (L-929) was procured from the American Type Culture Collection (USA); All the cell culture plasticware such as Petri dishes, T25 flasks and multi-well plates were purchased from CytoOne^® Starlab International GmbH; The consumables for 3D printing were procured from regenHU, Switzerland; Ketamine was from Miracalus Pharma Pvt. Ltd., Xylazine from Indian Immunologicals Limited, Ceftriaxon from Li Med Labs Pvt. Ltd, and meloxicam was from Intas Pharmaceuticals Ltd.; Tisseel (Fibrin Sealant) was purchased from Baxter India Pvt Ltd; Reagents like Isopropyl alcohol was from Spectrochem Pvt. Ltd., mountant from Dako North America, Inc.; Triton-X-100 were from Sisco Research Laboratories Pvt. Ltd.; Bovine Serum Albumin was purchased from Central Drug House, India, Rabbit anti-CD31 antibody was from NOVUS, NB100-2284 and Anti-Rabbit secondary antibody was from Elab Sciences-E-AB-1053.

Cell culture

Fibroblast L929 cells in T25 flask was maintained in MEM supplemented with 5% FBS and 1% Penicillin-Streptomycin antibiotics inside a carbon dioxide (CO₂) incubator set at 37°C, 5% CO₂ and 95% relative humidity.

Synthesis of GelMA and preparation of GelMA hydrogel

Gelatin Methacrylamide (GelMA) was synthesized according to previously described method.²⁰ GelMA was dialyzed, lyophilized and subsequently sterilized by ethylene oxide gas at 37°C. GelMA and Gelatin was taken at 3:1 weight ratio in a glass vial and sufficient volume of serum free MEM was added to prepare polymer solution with final concentration of 10% GelMA and 3% Gelatin. The vial was incubated at 37°C until the contents were completely dissolved. The photoinitiator was prepared at a stock concentration of 2% by dissolving LAP in PBS and was filter sterilized using 0.22 µm syringe filter. Then LAP was added to GelMA-Gelatin polymer solution get a final concentration of 0.25%. The samples for rheological analysis were prepared in PBS instead of serum free MEM. The final formulation of 10% GelMA, 3% Gelatin and 0.25% LAP hereafter referred as GelMA-G-L.

Biological evaluation – in vitro

An in vitro cytotoxicity test of GelMA-G-L was performed using Direct Contact and Test on Extraction method based on ISO standard 10993 – Part 5. GelMA-G-L hydrogel was prepared by exposing GelMA-G-L solution to 365 nm UV light for 3 min at an intensity of 8–12 mW/cm². The GelMA-G-L hydrogel samples of dimensions (φ = 4 mm, height = 2 mm) were prepared for in vitro direct contact cytotoxicity test. The hydrogel samples for the extraction method having dimensions 10 mm diameter and 2 mm thickness was prepared using a Teflon mould.

Direct contact

The L-929 cell monolayer was trypsinized and seeded in a 24 well plate at a cell density of 3 × 10⁴ cells/well. After the cells attained 70% confluency, GelMA-G-L hydrogel disc was placed over the cells at the centre of the well and sufficient medium was added. Ultra-high molecular weight polyethylene (UHMWPE) and Copper discs of same dimensions were used as negative and positive controls, respectively. The cells were incubated for 24 h at 37°C in a CO₂ incubator. The morphology of cells in the vicinity of test and control samples was observed under a phase contrast microscope (Nikon TS 100, Japan). Based on the cell morphology, cell detachment and vacuolization, cytotoxic activity was graded as 0, 1, 2, 3, 4 representing none, slight, mild, moderate and severe, respectively. The viability of the cells was determined by incubating with neutral red stain (1 mg/mL in saline) for 10 min at 37°C. After removing the stain, cells were rinsed gently with PBS and observed under microscope.

Test on extract

L929 cells were seeded in 96 well plate at a density of 1 × 10⁴ cells/well and allowed to attain sub-confluency. The extract of GelMA-G-L was prepared by incubating hydrogel discs in a serum-containing culture medium at an extraction ratio of 1.25 cm²/mL, using a shaker incubator at 37°C for 24 h. Cells in normal culture medium served as the control group, while cells treated with 1.3 mg/mL phenol in culture medium were used as the positive cytotoxic control. The 100% extract of GelMA-G-L was diluted with culture medium to obtain 50%, 25% and 12.5% concentrations. The cells were then exposed to GelMA-G-L and control extracts (n = 8) and incubated for 24 h 37°C in CO₂ incubator. Viability of the cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Following 24 h incubation the extract was removed and the cells were incubated with 50 µl MTT reagent (1 mg/ml in Serum free Medium) for 2 h at 37°C in a CO₂ incubator. Then the MTT reagent was removed and the violet crystals formed was dissolved by adding 100 µl isopropanol. The absorbance of colour developed was read spectrophotometrically at 570 nm. Then cytotoxicity was determined by calculating the percentage cell viability by normalizing the optical density values of GelMA-G-L and positive control with that of the cell control.

Rheology

Flow properties of GelMA-G-L was analysed using a rheometer (AntonPaar, Germany). Temperature sweep, viscosity curve and time oscillatory sweep tests of the GelMA-G-L solution were performed using parallel plate geometry (PP25) with 0.5 mm measuring distance. Temperature sweep test was conducted at varying temperatures ranging from 25°C to 10°C at a constant strain of 1% to determine the physical gelation temperature. The shear thinning behaviour GelMA-G-L was studied from the viscosity curve below the gelation temperature (18°C) at a variable shear rate (0.01–1000 per second). To investigate the photo-polymerization of GelMA-G-L the sample was exposed to 365 nm UV light source (Omnicure 1000, Lumen Dynamix LDGI) at 37°C under a constant shear strain of 1% and 10 Hz frequency. The variation in the storage modulus was recorded up to 360 s at an interval of 10 s.

3D printing

The printability of the GelMA-G-L bioink was evaluated using 3D bioprinter (RegenHU 3D Discovery, Switzerland). Single layer of different geometric patterns such as zigzag, circle, triangle and square were designed using the 3D BioCAD software (regenHU, Switzerland) and G-codes were generated. GelMA-G-L bioink was taken in a 3 ml cartridge and kept at 4°C for 2–3 min for physical gelation. The cartridge was then loaded in the extrusion head of Bioprinter and the air pressure was adjusted from 80 to 90 kPa until a smooth continuous filament of GelMA-G-L was extruded. The pressure was noted and used for further printing at a speed of 10 mm/s at temperature of 20°C. The single layer patterns were printed on a glass microslide. Multilayer square grid patterns of dimensions of 10×10×2 mm with internal perpendicular filament orientation was printed in a plastic petri dish. The printed multi-layer constructs were crosslinked using UV light of 365 nm for a duration of 3 min.

Circular construct of 10 mm diameter × 5 mm height was 3D printed using GelMA-G-L and UV crosslinked for pilot rat liver implantation study. 3D printed construct was transferred to sterile PBS in a 5 mL glass vial and kept at 37°C in CO₂ incubator. The implant was used for implantation on the third day.

Animal model

This study aimed to assess how a normal liver will interact with the 3D printed GelMA-G-L when implanted following partial hepatectomy. There are no previous reports on implanting 3D printed GelMA hydrogel in liver and hence we consider this as the pilot study in a rat liver resection model (n = 1). The implantation was performed after prior approval from the Institutional Animal Ethics Committee (SCT/ABS/IAEC-109/02). After anaesthetizing the animal by intramuscular administration of ketamine (70 mg/kg body weight) and xylazine (7 mg/kg BW). The hair at surgical site was clipped and skin was wiped with povidone-iodine solution. Laparotomy was performed by creating an incision of approximately 2 cm in length on the skin and muscle to expose the liver. Liver tissue of 10 mm × 5 mm was resected from the left lateral lobe using a 10 mm diameter trephine. The edges of the biopsy site was cleared using fine tipped forceps and gentle pressure was applied at the resected site using a sterile cotton pad to enhance clotting and reduce bleeding. The 3D printed GelMA-G-L construct scaled to fit into the resection site was placed in position by applying TISSEEL fibrin sealant. The incision on muscle and skin were sutured using non-absorbable nylon suture thread. As post implantation care, the animal was given meloxicam (0.5 mg/kg BW) as analgesic and antibiotic injection (25 mg/kg BW) for next 3 days starting from date of implantation. The animals were observed for 30 days in individual isolated cage and aseptic condition with separate controlled humidity, temperature and HEPA filtered air. After 30 days, the animal was euthanized by CO₂ asphyxiation and implant was excised with surrounding tissues and collected in 10% neutral buffered formalin. The fixed tissue was processed for standard paraffin embedding and 5 µm thick sections were taken. The sections were mounted on slides for Haematoxylin and Eosin (H&E) staining and immuno-fluorescence staining.

H&E staining

The paraffin sections were de-paraffinized at 60°C and treated with xylene for 10 min. The sections rinsed with 90% and 75% alcohol and exposed to Harris’ Haematoxylin for 30 min. After rising with acid alcohol, tap water and Scott’s tap water, the sections were treated with 70% alcohol. The sections were stained with 0.5% eosin for 10 min and finally rinsed with 100% alcohol. Stained sections were then treated with xylene and mounted on glass slide using DPX.

Immunostaining

The paraffin sections were de-paraffinized by heating to 60°C for 1 h. After cooling, the sections were treated with xylene for 10 min and sequentially treated 5 min each with 90% and 75% ethanol. The sections were washed with water and heated in sodium citrate buffer at 100°C for 20 min for antigen retrieval. The sections were washed with Tris Buffered Saline (TBS) containing 0.025% Triton X-100 and non-specific binding sites were blocked with 1% BSA in TBS for 2 h at room temperature. The sections were exposed overnight to rabbit anti-rat CD31 antibody at 4°C. Sections were rinsed with TBS and were treated with secondary anti-rabbit IgG antibody conjugated with rhodamine for 1 h at room temperature in the dark. The vasculature around the implant site was observed under a fluorescence microscope (Leica DMI6000, Switzerland). The implant site was identified in the section under bright field mode and the vascularization was observed under fluorescence mode.

Statistical analysis

All the experiments except implantation were performed in triplicate (n = 3). The results are presented as mean ± standard deviation. The significance was calculated from Student’s t-test and was considered significant when the p value is less than 0.05.

Results

Synthesis of GelMA and preparation of GelMA hydrogel

Gelatin was solubilized in CB buffer at 50°C to obtain a 10% solution (Figure 1(a)). MAA (1 ml) was added intermittently at an interval of 30 min to obtain methacrylated gelatin (Figure 1(b)), the methacrylated gelatine was dialysis membranes and dilaysed against of distilled water to remove all the unreacted MAA (Figure 1(c)). The purified GelMA was lyophilized, packed at sterilized by ethylene oxide gas (Figure 1(d) and (e)).

In vitro cytotoxicity

Direct contact

In vitro cytotoxic effect of 10% GelMA hydrogel was analysed by direct contact test with L-929 fibroblast cells based on ISO 10993 part 5. No cytotoxic reaction was observed around or below the GelMA-G-L hydrogel after 24 h with a cytotoxic reactivity grading of 0. The cells retain normal fibroblast morphology without detachment or lysis of the cells, colour change of the media similar to observation in cell control and negative control (UHMWPE) (Figure 2). Whereas the positive control (Copper) showed severe cytotoxic reactivity with a grade 4.

Test on extract

The cytotoxicity was also assessed by extraction method. The L-929 cells exposed to different dilutions of GelMA-G-L extract showed normal spindle like morphology similar to cell control (Figure 3(a)). The cells that were exposed to dilute phenol (positive control) showed severe cell destruction and toxicity with a grade of 4. The cell control and the GelMA-G-L samples obtained a cytotoxicity grading of 0 representing non-cytotoxic nature. Cytotoxicity was quantified by MTT assay. The absorbance of cells exposed to GelMA-G-L and dilute phenol was normalized to that of cell control and presented as percentage metabolic activity (Figure 3(b)). The average metabolic activity of different dilutions of GelMA-G-L showed a non-significant difference among themselves and with cell control. This clearly demonstrate that the GelMA-G-L is non-cytotoxic to fibroblast cells.

Rheology

The effect of temperature, shear rate and time on the flow properties of GelMA was evaluated by analysing the rheological properties. The gelation temperature of the 10% GelMA solution was analysed by temperature sweep (25°C–10°C) is shown in Figure 4(a). Viscosity is inversely proportional to temperature, as the temperature decreases the viscosity of the solution increases at constant strain of 1%. At gelation point a sudden increase in the viscosity can be observed which was in the range of 21°C–20°C. Shear thinning is an important property of a bioink to use it for 3D bioprinting as it imparts the viability of the cell. As the shear rate increases, the viscosity of the bioink decreases and exhibits shear thinning property. Hence 10% GelMA biopolymer has good shear thinning property, hence it is suitable for use in 3D printing Figure 4(b). Photocrosslinking property of the bioink was analysed from Time oscillatory sweep test. When the hydrogel was expose to UV irradiation at 365 nm for 360 s the storage modulus was increased. As the time of exposure increases the storage modulus also increases linearly up to 3 min. Then the curve plateaued indicating the formation of a stable crosslinked hydrogel Figure 4(c). In the absence of UV irradiation even under applied pressure the storage modulus was constant without any change. Hence from Figure 4(c), it is evident that the hydrogel is photocrosslinkable under UV exposure and within 3 min will form stable hydrogel. Since Time oscillatory sweep test was conducted at 37◦C, it is also clear that crosslinking not because of the influence of gelatin present in it.

3D printing

Printability is an ideal characteristic of a bioink, in which extrudability is used to assess printing ability of a bioink. To analyse the extrudability, filament extrusion test of the formulated 10% GelMA with LAP bioink was done and was extruded as fine filament under pressure which is an ideal characteristic for extrusion bioprinting (Figure 5(a)). Various geometrical patterns like square, triangle, circle and zig zag were printed using 410 µm nozzle based on CAD drawings to analyse the printability (Figure 5(b)). The thickness of the filaments was same as the nozzle diameter and the filaments was flexible to print all the designed patterns and throughout the printing the filament was stable without any disturbance in the filament. Then shape fidelity, and structural integrity was evaluated by printing multilayer grid constructs. The 3D printed 2, 4 and 6 layer constructs were maintained their shape as the CAD design (Figure 5(c)). And in two layers construct each filament in the two layers can be separately visible. In 4 and 6 multilayer constructs the different layers can be observed and maintained its square structure without collapse. As the number of layer increases height of the construct also increases that proven the shape fidelity after 3D printing. After expose to UV at 365 nm it took 3 mins to crosslink and the crosslinked constructs were strong enough to handle.

Animal model and implantation

The 3D printed construct was implanted in a partial liver resection rat model (Figure 6(a) and (b)). Since the size of the trephine is already known, the 3D construct was designed to match the resected tissue’s dimensions in the liver (Figure 6(c) and (d)). In order to avoid tearing of tissue due to suturing, fibrin tissue sealant was used to secure in place (Figure 6(e) and (f)). It was observed that the tissue glue has effectively maintained the 3D printed GelMA-G-L construct in its position over a 30-day period without any issues of movement-induced displacement, loosening, or dislodgement in the animal. It was observed that the surgical wound on the skin was completely healed on day 5 indicating good health and proper wound healing in the 30-day study (Figure 6(g), (h) and (i)). Throughout the observation period, the rat remained active and consumed food and water as normal. The weight of the animal increased from 190 to 280 g by the end of the observation period.

Histology

In gross observation, remnants of the GelMA-G-L construct was observed which was in integration with native liver tissue at the end of 30 days after implantation. The H&E staining of implant site showed fragments of GelMA in the liver Figure 7(a). A connective tissue capsule was visible showing multinucleated giant cells and macrophages around it. GelMA-G-L construct was slowly absorbed without any profound inflammatory response. There were no adverse responses in and around the implanted GelMA-G-L indicating the integration of material with host tissue Figure 7(b). The presence of monocyte/macrophage was noted which is a normal foreign body reaction. There was healing response around the implant evidenced by the presence of macrophages and presence of vascular structures in the GelMA-G-L construct, but no signs of rejection were observed at the implant site. Here fibrin glue acts as channel for entry of blood vessels into the implanted construct. The blood vessels from the native tissue were branching into the implant through fibrin glue Figure 7(c).

Immunostaining

Staining of endothelial cell marker, CD 31, in the implanted site of rat liver after 30 days of post-implantation showed characteristic vascular patterns around the GelMA-G-L implant (Figure 8). The bright field image shows the implant site and presence of GelMA-G-L. The same field under fluorescence mode showed the CD31 positive endothelial cells around the sample and characteristic staining pattern in liver. The presence of blood vessels observed around GelMA-G-L construct confirms the seamless integration with the host tissue.

Discussion

Bioartificial organs is a promising alternative to donor organs in transplantation medicine. Attaining cellular level architecture in the artificial tissue is challenging with the present knowledge and technology. Large organs like liver require both extracellular matrix and various types of cells in high density with very complex arrangement of cells at micron level. Engineering high cell density tissues is possible by the additive manufacturing technology, which is popularly known as 3D bioprinting. The main requirement to make 3D Bioprinting to action is the bioink that contains cells, biomaterial and nutrients for the tissue growth. GelMA is an extensively studied hydrogel that exhibits great potential as a component of bioink for biofabrication of tissues.²² GelMA possess various properties that provides advantages over other hydrogel such as presence of Arg-Gly-Asp (RGD) domains that helps in cell adherence, biocompatibility, biodegradability, fine tuning of mechanical properties, stability at physiological temperature.²³ We have previously reported the use of 10% and 5% GelMA based bioink formulations with Irgacure as photoinitiator for 3D bioprinting of liver tissues. Similarly, there have been reports on GelMA based bioinks crosslinked with LAP as photoiniator.²⁴ The choice of a suitable photoinitiator in a bioink is of extreme significance. Some of the required characteristics of photoinitiators are solubility in aqueous medium, non-cytotoxicity and crosslinking of bioink material without compromising the cell viability. This is because photoinitiators can have a significant impact on the cytotoxicity of the bioink. The inherent toxic effects differ among PIs and the chemical structure of the PI molecule is closely related to its toxicity, specifically its hydrophobic characteristics, which enhance its capacity to penetrate the cellular membrane which can be reduced by choosing the lowest feasible concentration.²⁵ Nguyen et al.²⁶ studied the viability of cells in the extract of GelMA prepared with varying concentrations of LAP and found that < 0.5% is non-cytotoxic. In this study, bioink with 10% GelMA and 0.25% LAP was selected as the final formulation.

The bioink should possess certain biological and physical properties that are contributed by the bulk component of the formulation. The bulk component of the bioink formulation designed for the extrusion printing is a hydrogel, which is generally stabilized by crosslinking during or after printing. Most of the biological and physicochemical evaluation of the bioink is conducted without cells to study the suitability of the selected formulation for 3D printing and biocompatibility. In the biological evaluation of bioink, the formulations without cells has to be initially tested at target site for complete understanding of biocompatibility and material-tissue integration. The GelMA-G-L formulation, thus has to be evaluated without cells for the in vivo application, especially to know how the host immune system will respond to it. First, the crosslinked formulation without cells was evaluated in vitro using L-929 fibroblast cells for the cytotoxicity test by direct contact as well as with extract of hydrogel. The results of GelMA-G-L was comparable to that of previous report on GelMA-Gelatin formulation with Irgacure and LAP as photoinitiator.^20,26

Understanding the rheological properties of bioink formulation is essential for the precise fabrication of tissues through the adjustment of their physical attributes.²⁷ Temperature sweep analysis of GelMA-G-L showed that physical gelation occurs between 20°C and 21°C. This observation is within the same range of physical gelation temperature reported previously.²⁸ In another study, at constant shear rate of 50−1 it was observed that the GelMA at a concentration 10% displayed a physical gelation temperature similar to our results.²⁹ Shear-thinning (pseudoplastic) properties is an essential property required in a bioink as it facilitates smoother deposition of filaments. However, shear thinning at very high pressure could adversely affect viability of cells in the bioink.³⁰ Examining the change in viscosity of bioink in response to different shear rates could give better understanding the shear thinning at suitable pressure and temperature. Our formulation of GelMA-G-L with 10% GelMA and 3% gelatin demonstrates excellent shear-thinning characteristics, making it a suitable bioink for 3D bioprinting. The results of the time oscillatory sweep analysis clearly showed that 10% GelMA-G-L undergoes photocrosslinking under UV irradiation in the presence of the photoinitiator LAP within 360 s. With longer exposure to UV light, there is a noticeable rise in the storage modulus. Conversely, in the absence of UV irradiation, the storage modulus stays around 0 and remains constant. The findings of O’Connel et al.³¹ validate our results, as they investigated the variation in storage modulus over time. The findings of Lee et al.,²⁹ also supporting our result that GelMA synthesized from both types A and B gelatin underwent rapid photocrosslinking within 20–30 s after being exposed to UV irradiation.

Printability is an important parameter of a hydrogel to be qualified as bioink for 3D bioprinting. In extrusion based bioprinting the bioink is extruded as a uniform smooth filament. The tests like filament extrusion, filament strength, physical gelation during printing and shape fidelity are essential for 3D printing.³² Printing at its physical gelation temperature and 85 MPa air pressure, the GelMA-G-L bioink was extruded as fine smooth continuous filaments. Single layer and multilayer with internal grid patterns didn’t show filament fusion indicating good printability.³² The results showed that multilayer grid constructs printed with GelMA-G-L exhibits good printing accuracy, shape fidelity and stability before and after crosslinking.

Studies on implantation of scaffold in liver tissue engineering is very limited. Most of the study is based on implantation of decellularized liver scaffold. There are reports of subcutaneous implantation of GelMA for the biocompatible evaluation the material.²¹ Shimoda et al.³³ investigated the liver regeneration in porcine partial hepatectomy model using liver scaffolds. They found that in 28 days post implantation, there were vascular structures with blood supply. In order to confirm the tissue compatibility of GelMA-G-L hydrogel for organ or tissue printing, it is necessary to evaluate how the native liver tissue respond to the 3D printed construct. In previous studies biocompatibility of 10% GelMA was analysed by implanting subcutaneously in white male rats and GelMA scaffold of size 8 mm in diameter and 2.1 mm high was moulded in a PLA mould and implanted for 33 days.³⁴ Another study by Kurian et al.³⁵ studied nanomaterial imparted GelMA for its tissue compatibility and immune responses by subcutaneous implantation in SD male rat for 4 weeks. The bone regeneration capacity of these hydrogels were also analysed by implanting in critical-sized cranial bone defects male SD rat for 12 weeks. All the above studies used hydrogels casted using a mould were used, where as in this study we used 3D printed GelMA-G-L. The printed construct have very precise, controlled and specific configurable features compared to casting method.³⁶

Biological response towards a bioink is the first step in risk analysis, which is usually done as subcutaneous implantation as per international guidelines. The evaluation of compatibility of 3D printed bioink without cells in the target organ is also essential. Engineered hepatic construct can be studied in vivo at hepatic or extra hepatic site.³⁷ Liu et al.³⁸ reported different methods of implantation of tissue engineered liver in rat model, namely “ectopic vascular anastomosis, liver cross-section suture transplantation, intrahepatic insertion and mesenteric transplantation”. Since the GelMA-G-L is intended to use as bioink for 3D bioprinting of liver construct, we opted for implanting the 3D printed GelMA-G-L construct in rat liver. Organ specific implantation helps to analyse the host response of a particular organ towards the 3D printed construct. In this study we used the intrahepatic insertion method due to its high rate of survival compared to other methods. After partial resection of liver tissue with a trephine, a construct of dimension similar to the size of resected tissue was placed and held in position using fibrin tissue glue. Fibrin glue supported to hold the material in place initially that helped the implant to remain in place throughout the period of 30 days without displacement, loosening or dislodgement due to movement of animal. The histopathology analysis show that GelMA-G-L is tolerated by host liver tissue and supports vascularization. Immunostaining with endothelial cell marker CD31 supports the finding of vascularization around implant site and intact material integration with host tissue. Hence in this study 3D printed GelMA-G-L was implanted in normal rat liver to understand the tissue integration and vascularization. The presence of inflammatory cells surrounding the implant site over a longer duration is a normal response especially when a biodegradable scaffolds are implanted. A proper tissue integration was observed in the H&E staining with surrounding blood vessels. Vascularization is essential when a degradable scaffold is implanted as the material has to be removed from the site. The CD31 staining confirmed the presence of endothelial cells around the implant as shown in Figure 8. Despite using normal Wistar rats instead of immune-deficient animals for the experiment, no negative immune responses from the host were detected. In summary, the present study show that 3D printed GelMA-G-L integrated with liver tissue over 30 days’ duration with distinct degradation of material and vascularization.

Conclusion

This study reports the tissue integration of GelMA-G-L with liver tissue in normal Wistar rat model. The 3D printed GelMA showed tissue integration and vascularization with the host tissue without signs of rejection or adverse immune reactions. This is a pilot study performed in one animal that give promising insights into the potential of further implantation studies with 3D bioprinted cell laden constructs. The future prospects will be to conduct experiments with more number of animals.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors acknowledge the funding received from the Science and Engineering and Research Board (Grant # CRG/2020/002669), Government of India. ASJ thanks the Council of Scientific and Industrial Research, Government of India for PhD fellowship (09/523(0096)/2019-EMR-1)

Ethical approval

The animal experiments were performed after prior approval from the Institutional Animal Ethics Committee of Sree Chitra Tirunal Institute for Medical Sciences and Technology with number SCT/ABS/IAEC-109/02.

ORCID iDs

Sangeetha Suresh

Anupama Sekar J

PR Anil Kumar

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