A BIOINK FOR FABRICATING SKELETAL MUSCLE TISSUE CONSTRUCTS AND METHOD OF PREPARATION THEREOF

Abstract

A bioink, for fabrication of skeletal muscle tissue constructs, and method of preparation thereof, are disclosed. Said bioink broadly comprises: methyl cellulose; gelatin; and alginate, with: ratio of the gelatin to the methylcellulose being 1:0.8; ratio of the gelatin to the alginate being 1:0.4; and said bioink being dually crosslinked through 1% microbial transglutaminase and 0.1 M calcium chloride. The disclosed bioink (and/or method of preparation) offers at least the following synergistic advantages and effects: improved mechanical properties; improved stability; is suitable, for bioprinting of complex shapes and organ models; allows cell proliferation, indicating suitability, for bioprinting of thick cellular constructs; requires a low pressure, for dispensing; and/or promote skeletal muscle regeneration.

Specification

Description:TITLE OF THE INVENTION: A BIOINK FOR FABRICATING SKELETAL MUSCLE TISSUE CONSTRUCTS AND METHOD OF PREPARATION THEREOF
FIELD OF THE INVENTION
The present disclosure is generally related to skeletal muscle tissues. Particularly, the present disclosure is related to fabrication of skeletal muscle tissue constructs. More particularly, the present disclosure is related to: a bioink, for fabrication of skeletal muscle tissue constructs; and method of preparation thereof.
BACKGROUND OF THE INVENTION
Extrusion bioprinting is a commonly used printing method, due to its ability to print complex geometries. Though bioinks are known in the art, limitations restrict their applicability, in the fields of tissue engineering and regenerative medicine. These limitations include, but are not limited to: high costs; poor availability; poor immunogenicity; inferior mechanical properties; and/or the like.
There is, therefore, a need in the art, for: a bioink, for fabrication of skeletal muscle tissue constructs; and method of preparation thereof, which overcome the aforementioned drawbacks and shortcomings.
SUMMARY OF THE INVENTION
A bioink, for fabrication of skeletal muscle tissue constructs, and method of preparation thereof, are disclosed.
Said bioink, for fabrication of skeletal muscle tissue constructs, comprises: methyl cellulose; gelatin; and alginate, with: ratio of the gelatin to the methylcellulose being about 1:0.8; ratio of the gelatin to the alginate being about 1:0.4; and said bioink being dually crosslinked through 1% microbial transglutaminase and about 0.1 M calcium chloride.
The disclosed bioink (and/or method of preparation) offers at least the following synergistic advantages and effects: improved mechanical properties; improved stability; is suitable, for bioprinting of complex shapes and organ models; allows cell proliferation, indicating suitability, for bioprinting of thick cellular constructs; requires a low pressure, for dispensing; and/or promote skeletal muscle regeneration.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A, Figure 1B, Figure 1C, Figure 1D, Figure 1E, and Figure 1F illustrate results of temperature sweep, viscosity, amplitude sweep, frequency sweep, shear thixotropy, and oscillation thixotropy analyses, respectively, assessed for 4MC-5Gel and 4MC-5Gel-2Alg bioinks, in accordance with an embodiment of the present disclosure;
Figure 2A and Figure 2B illustrate constructs printed using 4MC-5Gel and 4MC-5Gel-2Alg bioinks, respectively, at different pressures and speeds, in accordance with an embodiment of the present disclosure;
Figure 2C (i) and Figure 2C (ii) illustrate results of pore factor analyses, of 4MC-5Gel and 4MC-5Gel-2Alg bioinks, respectively, in accordance with an embodiment of the present disclosure;
Figure 3A, and Figure 3B illustrate results of strand width printability analyses, of 4MC-5Gel and 4MC-5Gel-2Alg bioinks, respectively, in accordance with an embodiment of the present disclosure;
Figure 3C, and Figure 3D illustrate results of spreading ratio (*p < 0.05) and pore factor analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 4A illustrates results of stability of printed constructs after about 30 minutes, of 4MC-5Gel bioink, in accordance with an embodiment of the present disclosure;
Figure 4B and Figure 4C illustrate stability of printed constructs at about 0 minutes and after about 30 minutes, respectively, of 4MC-5Gel-2Alg bioink, in accordance with an embodiment of the present disclosure;
Figure 5 illustrates results of handleability of a 4MC-5Gel-2Alg bioink printed construct, after ionic and dual crosslinking at different time points, in accordance with an embodiment of the present disclosure;
Figure 6A illustrates results of rheological property analyses, of uncrosslinked and crosslinked 4MC-5Gel bioink, in accordance with an embodiment of the present disclosure;
Figure 6B illustrates results of rheological property analyses, assessed for uncrosslinked, crosslinked, and dual crosslinked 4MC-5Gel-2Alg bioink, in accordance with an embodiment of the present disclosure;
Figure 6C and Figure 6D illustrate results of Fourier-Transform Infrared Spectroscopy (FTIR) analyses, in accordance with an embodiment of the present disclosure;
Figure 7A, Figure 7B, Figure 7C, Figure 7D, Figure 7E, and Figure 7F illustrate photographs of 3D printed constructs (nose, kidney, hand, team, jaw, and tubular constructs), respectively, in accordance with an embodiment of the present disclosure;
Figure 8A, Figure 8B, Figure 8C, and Figure 8D illustrate results of swelling ratio, degradation, porosity, and scanning electron microscopy analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 9A and Figure 9B illustrate results of swelling of crosslinked constructs and changes in strand width of 3D printed constructs analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 10A and Figure 10B illustrate results of in vitro cytotoxicity analyses, in accordance with an embodiment of the present disclosure;
Figure 11A illustrates live-dead images of C2C12 cells in printed constructs, observed using laser scanning confocal microscopy, in accordance with an embodiment of the present disclosure;
Figure 11B and Figure 11C illustrate results of myoblasts viability and cell proliferation (*p < 0.05) analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 12A (i) and Figure 12A (ii) illustrate results of cytoskeletal staining analyses, of ionically crosslinked constructs, at about day 3 and at about day 7, respectively, in accordance with an embodiment of the present disclosure;
Figure 12A (iii), Figure 12A (iv), and Figure 12A (v) illustrate results of cytoskeletal staining analyses, of dual crosslinked constructs, at about day 3, at about day 7, and at about day 14, respectively, in accordance with an embodiment of the present disclosure;
Figure 12B (i) and Figure 12B (ii), illustrate results of scanning electron microscopy (SEM) analyses, of ionically crosslinked constructs, at about day 3 and at about day 7, respectively, in accordance with an embodiment of the present disclosure;
Figure 12B (iii), Figure 12B (iv), and Figure 12B (v) illustrate results of scanning electron microscopy (SEM) analyses, of dual crosslinked constructs, at about day 3, at about day 7, and at about day 14, respectively, in accordance with an embodiment of the present disclosure;
Figure 12C (i) and Figure 12C (ii) illustrate results of aspect ratio and myoblasts circularity analyses, in accordance with an embodiment of the present disclosure;
Figure 13A and Figure 13B illustrate results of expression of MHC analyses, in dual crosslinked constructs at about day 0 and about day 7, respectively, in accordance with an embodiment of the present disclosure;
Figure 13 C and Figure 13D illustrates results of the expression of myosin heavy chain (MHC) and sarcomeric α-actinin, alongside the formation of elongated multinucleated myotubes after about 7 days in bioprinted constructs, in accordance with an embodiment of the present disclosure;
Figure 13E, Figure 13F, Figure 13G, Figure 13H illustrate results of percentage of MHC positive cells, myotube length, myotube width, and relative fold change analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 14 illustrates results of myotube formation analyses in two dimensional controls, in accordance with an embodiment of the present disclosure;
Figure 15A and Figure 15B illustrate images of volumetric muscle loss developed in C57BL/6 mice and results of defect size analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 15C illustrates images of the operated and contralateral of tibialis anterior (TA) muscles after about 4 weeks post-surgery, in accordance with an embodiment of the present disclosure;
Figure 15D and Figure 15E illustrate results of muscle weight recovery and cross-sectional area of the TA muscle analyses (*p < 0.05), respectively, in accordance with an embodiment of the present disclosure;
Figure 16 illustrates results of changes in body weight of animals in different groups, in accordance with an embodiment of the present disclosure;
Figure 17A (i) and Figure 17 B illustrate a wall hanging test and results of fall latency assessed using the wire hanging test (*p < 0.05) analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 17A (ii) and Figure 17C illustrate treadmill test and results of recovery of maximum distance covered analyses (*p < 0.05), respectively, in accordance with an embodiment of the present disclosure;
Figure 17A (iii) and Figure 17D illustrate grip strength test and results of grip strength measurement analyses (*p < 0.05), respectively, in accordance with an embodiment of the present disclosure;
Figure 18A and Figure 18B illustrate results of hematoxylin and eosin staining analyses, at lower and higher magnification, respectively, in accordance with an embodiment of the present disclosure;
Figure 18C and Figure 18D illustrate results of Masson's trichrome staining analyses, at lower and higher magnification, respectively, in accordance with an embodiment of the present disclosure;
Figure 18E, Figure 18F, Figure 18G, and Figure 18H illustrate results of myofiber diameter, myofiber cross sectional area, percentage of newly regenerated myofibers, and percentage of collagen deposition area (*p < 0.05) quantitative analyses, respectively, in accordance with an embodiment of the present disclosure;
Figure 19A illustrates results of immunohistochemical staining analyses, observed using confocal microscope, in accordance with an embodiment of the present disclosure; and
Figure 19B illustrates results of quantitative analyses, of MHC, CD31, and β III - tubulin staining (*p < 0.05), in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this specification, the use of the words "comprise" and "include", and variations, such as "comprises", "comprising", "includes", and "including", may imply the inclusion of an element (or elements) not specifically recited. Further, the disclosed embodiments may be embodied, in various other forms, as well.
Throughout this specification, the use of the word "fabricate", and its variations, is to be construed as: "produce; manufacture; and/or the like".
Throughout this specification, the words "hydrogel" and "bioink" may be used interchangeably.
Throughout this specification, the use of the acronym "MC", is to be construed as: "methylcellulose".
Throughout this specification, the use of the acronym "Gel", is to be construed as: "Gelatin".
Throughout this specification, the use of the acronym "Alg", is to be construed as: "Alginate".
Throughout this specification, the use of the acronym "BMTC", is to be construed as: "Bioprinted Muscle Tissue Constructs (for example, 4MC-5Gel-2Alg bioink)".
Throughout this specification, the use of the acronym "VML", is to be construed as: "Volumetric Muscle Loss".
Throughout this specification, the use of the word "Sham", is to be construed as: "uninjured mice".
Throughout this specification, the use of the acronym "MHC", is to be construed as: "Myosin Heavy Chain".
Throughout this specification, the use of the acronym "DMEM", is to be construed as: "Dulbecco's Modified Eagle Medium".
Throughout this specification, the abbreviation "DPBS" is to be construed as: "Dulbecco's Phosphate Buffered Saline".
Throughout this specification, the abbreviation "TCPS" is to be construed as: "Tissue Culture Polystyrene".
Throughout this specification, the use of the acronym "4MC-5Gel" is to be construed as: "methylcellulose-gelatin bioink".
Throughout this specification, the use of the acronym "4MC-5Gel-2Alg" is to be construed as: "methylcellulose-gelatin-alginate bioink".
Throughout this specification, the use of the acronym "4MC-5Gel constructs" is to be construed as: "constructs that were printed using the fabricated 4MC-5Gel bioink".
Throughout this specification, the use of the acronym "4MC-5Gel-2Alg constructs" is to be construed as: "constructs that were printed using the fabricated 4MC-5Gel-2Alg bioink".
Throughout this specification, the use of the word "constructs" is to be construed as: "bioprinted constructs that are made with "4MC-5Gel" or "4MC-5Gel-2Alg", as the case may be".
Throughout this specification, the use of the acronym "mTG" is to be construed as: "Microbial Transglutaminase".
Throughout this specification, the use of the acronym "CaCl2" is to be construed as: "Calcium Chloride".
Throughout this specification, the use of the acronym "w/o" is to be construed as: "without".
Throughout this specification, the disclosure of a range is to be construed as being inclusive of: the lower limit of the range; and the upper limit of the range.
Throughout this specification, the words "the" and "said" are used interchangeably.
Also, it is to be noted that embodiments may be described as a method. Although the operations, in a method, are described as a sequential process, many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of the operations may be re-arranged. A method may be terminated, when its operations are completed, but may also have additional steps.
A bioink, for fabrication of skeletal muscle tissue constructs (also referred to as "bioink"), and method of preparation thereof, are disclosed. The disclosed bioink was prepared, and tested, as follows:
Bioink Preparations
Preparation of 4MC-5Gel Bioink
About 0.5 g of gelatin was dissolved in about 10 mL of DPBS to obtain a mixture, and stirred in a magnetic stirrer at about 60°C for about 1 hour to obtain about 5% (w/v) gelatin solution.
About 0.4 g of methylcellulose was added to the obtained gelatin solution, and stirred in a magnetic stirred overnight at about 40°C to obtain 4MC-5Gel bioink.
Preparation of 4MC-5Gel-2Alg Bioink
About 0.5 g of gelatin was dissolved in about 10 mL of DPBS to obtain a mixture, and stirred in a magnetic stirrer at about 60°C for about 1 hour to obtain about 5% (w/v) gelatin solution.
About 0.4 g of methylcellulose and about 0.2 g of alginate were added to the obtained gelatin solution, and stirred in a magnetic stirrer for overnight at about 40°C to obtain 4MC-5Gel-2Alg bioink. Ratio of the gelatin to the methylcellulose was about 1:0.8. Ratio of the gelatin to the alginate was about 1:0.4.
The obtained bioinks were stored at about 4°C for about 2 hours, allowing swelling of MC and incubated at about 37°C for about 30 minutes before printing.
Bioink Characterisations
Viscoelastic Property Analyses
Temperature sweep analysis was performed from about 5°C - 40°C to identify the temperature-dependent gelling behaviour of the 4MC-5Gel bioinks and 4MC-5Gel-2Alg bioinks.
As illustrated, in Figure 1A, addition of alginate in the 4MC-5Gel-2Alg bioink, showed an about two-fold increase in the storage modulus (G'). The 4MC-5Gel bioink's storage modulus and loss modulus indicated gel-like behaviour.
Both the bioinks showed gel-like behaviour (G′ > G′′) throughout the temperature ranges, and the storage modulus was reduced with an increase in temperature.
As illustrated, in Figure 1B, the 4MC-5Gel bioink showed very less shear thinning behaviour than the 4MC-5Gel-2Alg bioink, due to the addition of alginate. The addition of alginate increased the shear thinning behaviour of the 4MC-5Gel-2Alg bioink due to the presence of non-thermoresponsive components (alginate & methylcellulose) in higher proportion.
As illustrated, in Figure 1C and Figure 1D, during amplitude and frequency sweep measurements, the 4MC-5Gel and 4MC-5Gel-2Alg bioinks exhibited gel-like behavior, with their strength increasing as the frequency increased, at different strains (about 1% to about 1,000%) and at different frequencies (about 0.1 rad/s to about 100 rad/s) respectively.
As illustrated, in Figure 1E, it was observed that the 4MC-5Gel and the 4MC-5Gel-2Alg bioinks maintained a consistent viscosity when subjected to a low shear rate of about 1 s⁻¹ for approximately 60 seconds. A rapid decrease in viscosity was observed when the shear rate was increased to about 100 s⁻¹ for about 5 seconds indicating shear thinning property of the bioinks.
When the shear rate was further reduced, the viscosity of the 4MC-5Gel and the 4MC-5Gel-2Alg bioinks were recovered up to about 76.43% and about 66.80% when compared to initial viscosity.
As illustrated, in Figure 1F, it was observed that the 4MC-5Gel bioink achieved about 90% of its initial storage modulus after about 10 seconds, while the 4MC-5Gel-2Alg bioink reached the same recovery level in about 4 seconds indicating improved viscoelastic properties due to alginate addition.
Printability Analyses
A printing temperature was maintained at about 24 degrees Centigrade and a nozzle size of about 0.4 mm (inner diameter) were constant throughout the studies/analyses.
As illustrated, in Figure 2A and Figure 2B, it was observed that the minimum pressure required for extruding the 4MC-5Gel bioink and the 4MC-5Gel-2Alg bioink was about 0.8 bars, and about 1 bar, respectively. This pressure difference between both the 4MC-5Gel bioink and the 4MC-5Gel-2Alg bioink was due to the addition of alginate, which increased the total polymer concentration to about 11% in the 4MC-5Gel-2Alg bioink when compared to about 9% of total polymer concentration in the 4MC-5Gel bioink.
Figure 2C (i) and Figure 2C (ii) illustrate results of pore factor analyses, of the 4MC-5Gel and the 4MC-5Gel-2Alg bioinks, respectively, printed at different pressures and different speeds. The conditions for printing were determined by calculating the pore factor values, which were close to about 1. It was found that all the conditions used for printing showed values between 0.9 and 1. Further tests were conducted to identify and analyse suitable conditions.
Figure 3A and Figure 3B illustrates variations in strand width of the 4MC-5Gel and 4MC-5Gel-2Alg bioinks, respectively when printing speed was varied from about 3 mm/s to about 12 mm/s, and pressure from about 0.8 to about 1.2 bars.
After analysing the strand width of printed constructs, spreading ratio was determined by comparing the actual strand width to the nozzle diameter used for printing. The 4MC-5Gel-2Alg bioink exhibited significantly (*p < 0.05) lower spreading ratios (about 1) when compared to the 4MC-5Gel bioink as illustrated in Figure 3C.
As illustrated, in Figure 3D, during pore factor analyses, the 4MC-5Gel-2Alg bioink demonstrated higher printability of about 1.02 ± 0.03 when compared to the 4MC-5Gel of about 0.98 ± 0.03, when extruded at about 1.2 bars and about 8 mm/s speed (strand diameter of about 645.04 ± about 77.88 µm), indicating better performance in maintaining shape fidelity during printing.
Stability Analyses
The fabricated bioinks were crosslinked improve their stability, mechanical properties and cell supportiveness. Crosslinking conditions for the constructs are given in the below table.

Constructs Enzyme/Compound Temperature Time
4MC-5Gel 1% mTG 25oC 30 minutes
4MC-5Gel-2Alg 0.1 M CaCl2 - ionic crosslinking 25oC 30 minutes
1% mTG + 0.1 M CaCl2 - dual crosslinking
Figure 4A illustrates stability of the 4MC-5Gel constructs after about 30 minutes in a medium with about 1% mTG. After incubation of the 4MC-5Gel printed constructs at about 25 oC for about 30 min with about 1% mTG slight shape deformation was observed. The 4MC-5Gel constructs lost their structural integrity due to the rapid release of gelatin and MC when transferred to culture media at about 37 oC
Stability of the 4MC-5Gel-2Alg constructs at about 0 min and about 30 min in the medium with about 1% mTG is illustrated in Figure 4 B and 4C respectively (scale bar - about 1 cm; arrows indicate deformed printed constructs).
Constructs printed using the 4MC-5Gel-2Alg bioink were crosslinked using about 0.1 M calcium chloride (CaCl2) referred as ionic crosslinking and about 1% mTG + about 0.1 M CaCl2 referred as dual crosslinking, to assess the effect of crosslinking on the stability.
As illustrated, in Figure 5, it was observed that the dual crosslinked constructs demonstrated improved stability and better handleability, for up to about 21 days, while the constructs that were ionic crosslinked lost handleability after about 7 days.
Mechanical Property Analyses
Mechanical properties of the bioinks before and after crosslinking were analysed using a non-destructive mechanical analyser.
As illustrated, in Figure 6A, the storage modulus of the 4MC-5Gel bioink was about 4,837.89 ± 366.22 Pa at about 25°C and further increased when incubated with about 1% mTG for about 30 minutes. However, when the same hydrogel was placed at about 37oC for about 24 h, there was a significant (*p < 0.05) decrease in the storage modulus (about 712. 16 ± 175.05 Pa).
This significant difference might be due to the release of both MC and gelatin at about 37oC, which decreased the final polymer concentration of the bioink, which were directly related to the gelation behaviour of polymers.
Figure 6B illustrates that after incubating the dual crosslinked hydrogels in culture medium supplemented with about 1% mTG (at about 37°C), there was a significant (*p < 0.05) increase in the storage modulus (about 5,771.90 ± 660.08 Pa) when compared to the ionically crosslinked hydrogels (about 4,533.65 ± 519.20 Pa) incubated at about 24°C. as illustrated in Figure 6B. This may be due to the crosslinked gelatin in the dual crosslinked hydrogels which was entrapped by quick gelation of alginate, further stabilised through the electrostatic and hydrogen bonding interactions between the components of the bioinks.
Figure 6C and Figure 6D illustrate results of FTIR spectra of individual components used for bioprinting (Alginate, Gelatin, MC, mTG), and the 4MC-5Gel-2Alg bioink that was uncrosslinked, ionically crosslinked, and dual crosslinked hydrogels, respectively.
Complex Shape Printability Analyses
The printability of the 4MC-5Gel-2Alg bioink was tested by extruding at the printing pressure of about 1.2 bars and speed of about 8 mm/s.
As illustrated, in Figure 7A, Figure 7B, Figure 7C, Figure 7D, Figure 7E, and Figure 7F, complex structures of about 2 cm in height and about 1 cm in diameter were printed, confirming structural similarity with the digital models and demonstrating the feasibility in printing complex structures.
 
Swelling, Degradation, and Porosity Analyses
As illustrated, in Figure 8A, the ionically crosslinked constructs had higher swelling ratio of about 7.86 ± 1.48 when compared to the dual crosslinked constructs (5.07 ± 1.11).
As illustrated, in Figure 8B, the dual crosslinked constructs exhibited significantly (*p < 0.05) lower mass loss at about day 1, whereas the ionically crosslinked constructs had more mass loss than he dual crosslinked constructs after about 7 days and about 14 days.
As illustrated, in Figure 8C, the ionically crosslinked constructs had higher porosity, when compared to the dual crosslinked constructs.
Figure 8D illustrates degradation of crosslinked constructs at about day 0, about day 1, about day 3, about day 7, and about day 14.
As illustrated, in Figure 9A, the dual crosslinked constructs showed less degradation compared to the ionically crosslinked constructs, when incubated in DPBS up to about 14 days.
As illustrated, in Figure 9B, it was observed that there were no significant changes in the strand width of the constructs, after immersing the printed constructs before and after crosslinking and after immersing in culture media for about 21 days.
In vitro Cytotoxicity Analyses
Cytotoxicity of the ionically and dual crosslinked 4MC-5Gel-2Alg were analysed using in vitro cytotoxicity analysis according to: ISO standards 10993-5 (ISO 10993-5:2009).
To study the cytotoxic effects, hydrogels crosslinked with the about 0.1 M CaCl2 were removed from the crosslinker solution, washed with DPBS, and incubated in a DMEM medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (Penicillin-Streptomycin) for about 24 h to collect the extracts. Dual crosslinked hydrogels were removed and maintained in DMEM medium supplemented with about 1% mTG, about 10% FBS and about 1% P/S for about 24 h to collect the extracts. Further, the extracts were diluted into four different concentrations: about 12.5%, about 25%, about 50% and about 100% in culture medium as illustrated in Figure 10A.
Figure 10B illustrates the viability of C2C12 cells after treating with the different extract concentrations for about 24 h (evaluated by MTS assay). Cell viability in all the extract treated wells were comparable with the wells cultured with only DMEM medium (negative control). However, cells cultured in about 1% Triton X-100 (positive control) had low viability due to the lysis of the cell membrane.
Bioprinting of C2C12 cells
Viability and Proliferation of Myoblasts:
Initially, C2C12 cells were trypsinised and mixed with bioink solution at about 37 oC with a seeding density of about 5 x 106 cells/mL and maintained at about 24 oC to initiate mild gelation of bioink due to thermoresponsive nature of gelatin. Further, the bioink was extruded into grid patterns of dimension (1 cm x 1 cm x 1 mm) using a pressure of about 1.2 bars and bioprinting speed of about 8 mm/s to evaluate the effect of printing parameters & bioink on maintaining the myoblast's viability and proliferation. Finally, the constructs were crosslinked through ionic crosslinking and dual crosslinking methods to stabilise the printed constructs during the culture period.
Crosslinked constructs were stained with calcein & ethidium bromide and observed using laser scanning confocal microscope to determine the cell viability in the printed constructs. After about 1 h, both crosslinking conditions did not affect the viability of the cells in the printed constructs. Similarly, the viability of myoblasts was maintained in both of the constructs on days 1, 3, and 7 without any significant differences, confirming the cytocompatibility of the bioink and crosslinking strategies. However, gelatin in the printed constructs enhanced cell adhesion and proliferation owing to the presence of RGD motifs.
The morphology of myoblasts was observed through fluorescent cytoskeletal staining, and with scanning electron micrographs.
Figure 11A illustrates live-dead images of C2C12 cells in printed constructs obtained using Laser Scanning Confocal Microscope (LSCM) from about 1 h to about 7 days.
Figure 11B illustrates the quantification of myoblasts viability (% of live cells) at various time intervals. Morphology of the cells in the printed constructs revealed that cells in the dual crosslinked constructs exhibited characteristics resembling the native myoblasts.
As illustrated, in Figure 11C, from about day 7 to about day 14, the dual crosslinked constructs showed a significant (*p < 0.05) increase in cell proliferation compared to the ionically crosslinked constructs. No notable differences in cell proliferation were observed between the constructs crosslinked using ionic and dual crosslinking strategies from about day 1 to about day 3.
Figure 12A (i) and Figure 12A (ii), illustrate results of cytoskeletal staining analyses, of the ionically crosslinked constructs, at about day 3 and at about day 7.
Figure 12A (iii), Figure 12A (iv), and Figure 12A (v) illustrate results of cytoskeletal staining analyses, of the dual crosslinked constructs, at about day 3, at about day 7, and at about day 14, respectively.
Figure 12B(i) and Figure 12B(ii) illustrate SEM images of the ionically crosslinked bioprinted constructs at about day 3 and about day 7. respectively.
Figure 12B(iii), Figure 12B(iv) and Figure 12B(v) illustrate SEM images of the dual crosslinked constructs at about day 3, about day 7, and about day 14, respectively.
Figure 12C (i) illustrates aspect ratio of the ionically crosslinked bioprinted constructs and dual crosslinked constructs at about day 3 and about day 7.
Figure 12C (ii) illustrates circularity of myoblasts in the ionically crosslinked constructs and the dual crosslinked constructs at about day 3 and about day 7 (*p < 0.05).
 
Myotube Formation Analyses
As illustrated, in Figure 13A, prior to differentiation, the cells did not express the myosin heavy chain marker, and no formation of multinucleated myotubes were observed, indicating that the cells remained undifferentiated at about day 0.
Figure 13B(i) and Figure 13B(ii) illustrate differentiation observed at 10x and 40x magnification (where MHC in green colour; nuclei in blue colour), respectively.
Figure 13C(i) and Figure 13C(ii) illustrate expression of sarcomeric α-actinin (green colour) and nuclei (blue colour) in the dual crosslinked constructs observed at about day 7 in 10x and 40x magnification, respectively.
Figure 13D illustrates 3D reconstructed images of myotubes stained with MHC (green colour) and nuclei (blue colour) at about day 7 (40x magnification).
Figure 13E, Figure 13F, and Figure 13G, illustrate the percentage of myosin heavy chain-positive cells, myotube length, myotube width, respectively, when compared to the control (tissue culture polystyrene).
Gene expression analyses were performed in the dual crosslinked 3D bioprinted muscle tissue constructs (BMTCs) to assess changes expression pattern of key genes involved in the differentiation of myoblasts into myotubes (MyoD, MyoG, and MHC). The results showed upregulation of all the three genes on day 7, compared to day 0, with MHC exhibiting high level of expression after 7 days of culture among all the genes.
Figure 13H illustrates relative fold change in expression of genes: MyoD; MyoG; and MHC with respect to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in dual crosslinked BMTC constructs (*p < 0.05).
Figure 14 illustrates myotube formation in TCPS controls where green colour represents Myosin Heavy Chain (MHC) and blue colour represents nuclei.
 
In vivo studies
For in vivo studies, four groups of C57BL/6 mice: uninjured (sham); untreated (VML injury) controls; autologous minced muscle (as a clinical equivalent control); and bioprinted muscle tissue construct (BMTC) were utilised as experimental groups to evaluate the efficacy of BMTC. Volumetric muscle loss (VML) model was developed in each of the experimental groups.
Figure 15A(i), Figure 15A(ii), Figure 15A(iii), Figure 15A(iv) illustrate VML surgery procedure comprising steps of: hair removal and disinfection; creation of a defect in tibialis anterior (TA) muscle following a precise incision through the skin and fascia; suturing of the fascia; and suturing of the skin; in different experimental groups used in the study.
Figure 15B illustrates defect size (%) of surgically created VML injury in C57BL/6 mice by removing about 20-25% of the TA muscle.
Figure 15C illustrates gross images of the operated and contralateral of TA muscles after about 4 weeks post-surgery (scale bar - 1 cm).
Figure 15D illustrates percentage recovery in muscle wet weight (*p < 0.05).
Figure 15E illustrates cross-sectional area of the TA muscle (*p < 0.05).
Figure 16 illustrates changes in body weight of C57BL/6 mice in different experimental groups during the study period (about 4 weeks). Post-surgery, body weight remained stable across all the groups during about 4-weeks study period.
Figure 17A(i), (Figure 17A(ii) and (Figure 17A(iii) illustrate results of various tests utilised to assess functional recovery: wire hanging test; treadmill test; and grip strength test respectively.
The wire hanging test results at week 1 showed an initial decrease in hanging time across all injured groups due to the impact of VML injury. At week 4, a significant (*p < 0.05) improvement in the latency to fall was observed in both the minced muscle and BMTC-treated groups, which were comparable to the sham control. In contrast, the untreated group exhibited no significant improvement in fall latency due to poor muscle regeneration as illustrated in Figure 17B.
Figure 17C illustrates recovery of maximum distance covered by mice at weeks 1 and 4 compared to baseline (before surgery) (*p < 0.05). On a relative scale, there was a decrease in total running distance of mice in untreated group, however there was no statistical significance among the groups.
Figure 17D illustrates the functional recovery analysis of animals treated with BMTCs showed muscle tissue regeneration similar to the sham control and minced muscle-treated animals after 4 weeks. At week 4, the BMTC group showed a significant (*p < 0.05) improvement in grip strength, reaching levels comparable to both the sham control and minced muscle groups, showing that the BMTCs are equivalent to minced muscle in providing grip strength recovery.
Hematoxylin and Eosin (H&E) staining was performed to the excised TA muscles to analyse the muscle regeneration by measuring the total cross-sectional area of TA muscle, myofiber diameter & cross-sectional area (CSA) and the percentage of newly regenerated myofibers.
Figure 18A and Figure 18B illustrate hematoxylin and eosin staining of the experimental groups at lower magnification (about 500 μm) and higher magnification (about 100 μm) respectively.
Figure 18C, Figure 18D and Figure 18H illustrates masson's trichrome staining at lower magnification (about 500 μm) indicates higher collagen deposition in the untreated group relative to the sham control, minced muscle, and BMTC groups respectively due to excessive fibrosis.
Figure 18E & Figure 18F illustrate results of myofiber diameter and myofiber cross sectional area (CSA) showed no significant difference between the sham control, minced muscle-treated, and BMTC-treated groups, suggesting that both treatments are effective in maintaining muscle fiber size and help in preventing atrophy following VML injury.
Figure 18G illustrates the percentage of newly regenerated myofibers in the minced muscle and BMTC groups were higher compared to the untreated and sham controls. This result reveals the efficacy of the minced muscle and BMTC groups in promoting muscle regeneration, restoring muscle mass and functions.
Immunohistochemical Analyses
Figure 19A illustrates immunohistochemical staining of various markers observed using a confocal microscope: Myosin heavy chain (MHC) (Figure 19A (i)); CD31 (Figure 19A (ii)); and β III - tubulin (Figure 19A (iii)) with a scale bar of about 50 µm.
Figure 19B(i) illustrates quantification of MHC, where higher MHC expression in minced muscle and BMTC groups suggested that these treatments promoted the formation and maturation of new muscle fibers, leading to improved muscle mass and function.
Figure 19B(ii) illustrates quantification of CD31, where higher expression of CD31-positive area in the BMTC and minced muscle groups compared to untreated and sham controls demonstrate the ability of BMTCs in enhancing vascularization which is imperative to support angiogenesis and reduce fibrosis by preventing scar tissue formation.
Figure 19B(iii) illustrates quantification of β III - tubulin the higher expression of β III - tubulin is observed in the BMTC, minced muscle, and sham control groups compared to the untreated VML injury group confirmed the successful nerve integration with the scaffolds (or minced muscle).The results indicates that BMTCs (4MC-5Gel-2Alg) has the potential to replace the gold-standard procedure done in clinics which has disadvantages such as limited availability, size mismatch, expensive procedure, and creation of additional surgical damage to healthy muscle tissues.
The disclosed bioink (and/or method of preparation) offers at least the following synergistic advantages and effects: improved mechanical properties; improved stability; is suitable, for bioprinting of complex shapes and organ models; allows cell proliferation, indicating suitability, for bioprinting of thick cellular constructs; requires a low pressure, for dispensing; and/or promote skeletal muscle regeneration.
It will be apparent to a person skilled in the art that the above description is for illustrative purposes only and should not be considered as limiting. Various modifications, additions, alterations, and improvements, without deviating from the spirit and the scope of the disclosure, may be made, by a person skilled in the art. Such modifications, additions, alterations, and improvements, should be construed as being within the scope of this disclosure. , Claims:1. A synergistic bioink, for fabrication of skeletal muscle tissue constructs, comprising methyl cellulose, gelatin, and alginate, with: ratio of the gelatin to the methylcellulose being 1:0.8; ratio of the gelatin to the alginate being 1:0.4; and said bioink being dually crosslinked through 1% microbial transglutaminase and 0.1 M calcium chloride.

2. The synergistic bioink, for fabrication of skeletal muscle tissue constructs, as claimed in claim 1, wherein: weight of said gelatin is 0.5 g; weight of said methylcellulose is 0.4 g; and weight of said alginate is 0.2 g.

3. A method of preparing a synergistic bioink for fabricating skeletal muscle constructs, comprising steps of:

dissolving gelatin in 10 mL of Dulbecco's Phosphate Buffered Saline, followed by stirring at 60°C for 1 hour to obtain a 5% (w/v) gelatin solution;
adding methylcellulose and alginate to the gelatin solution, and stirring overnight at 40°C to obtain 4MC-5Gel-2Alg bioink, with: ratio of the gelatin to the methylcellulose being 1:0.8; and ratio of the gelatin to the alginate being 1:0.4;
dually crosslinking the bioink through 1% microbial transglutaminase and 0.1 M calcium chloride.
4. The method of preparing a synergistic bioink for fabricating skeletal muscle constructs, as claimed in claim 3, wherein: weight of said gelatin is 0.5 g; weight of said methylcellulose is 0.4 g; and weight of said alginate is 0.2 g.

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