Report on various kind of bio-inks development process for bioprinting Applications

Abstract

Bio-ink is a relatively recent field of study. As the area of 3D printing matures, it throws up plenty of new opportunities for artificial tissue development. However, because we're working with a substance whose end component will be implanted within the human body, finding a decent ink is usually tough.  In this review, we provide an in-depth discussion on various types of bio-ink available, compare their properties, check their use cases, and their development procedure for bioprinting applications.

Key Words:-  bio-ink, bioprinting, tissue-development, polymer, 3D printer

1. Introduction

The increase in demand for organ and tissue regeneration has been always challenging. It is always a matter of concern whether the donated organ or tissue will be biocompatible with the patient or not. 3D bioprinting has emerged to be a good solution to tackle this issue. Like a normal 3D printer it also requires ink, the composition of the ink should be suitable for the environment on which we are attaching. It should not or minimal exert external forces at the encapsulated cell during the printing procedure. Its physical property must allow it to form a well-defined structure. Most of the Bio-inks are highly viscous and are supposed to be that, but it is a matter of concern to many researchers to keep control of viscosity so that it can adopt the biological environment of the host body. Although an ideal Bio-ink is yet to be achieved, we can compare Bio-ink on the basis of its biocompatibility, mechanical, rheological, and biological properties on which organ or tissue it will be targeting [1]. In this study we will discuss various types of Bio-ink available, comparing biomaterial, its target site, and its printing procedure. We will also look at some of the development procedures of Bio-inks.(i.e. Clay-based, Gelatin-based, Polyvinylpyrrolidone-Based, etc.) 

2. Types of Bio-Ink

Based on the material used to develop bio-ink, it is broadly classified as Natural polymer-based and Synthetic polymer-based, but recent development shows that hybrid bio-ink is very good at certain points of scaffold development. The study by Lee et al. [2] shows that Hyaluronic Acid-based hydrogel bioink has a gelatin time of 30 min, while Hyaluronic Acid-based  hybrid bio-ink has gelatin time of 200 s. Table 1 shows the various biomaterials used in bio-ink, their advantages, and disadvantages.

2.1. Natural Polymer Based Bio-ink

Natural polymer has very good potential and it offers a suitable scaffolding system for the structure and function organisation of the cell. Natural polymers like agarose, gelatin, collagen, etc. have shown a good result and play a key role in the 3D bioprinting of tissues and organs. They all have distinct qualities including nontoxicity, biocompatibility, and biodegradability, making them ideal for a variety of tissue engineering applications. 

2.1.1. Collagen

In mammalian cells, collagen type I is one of the most essential organic elements of the extracellular matrix (ECM). Hydrogels made of collagen type I have high biocompatibility and are frequently used in biomedical applications. Under the neutral pH and at 37°C, Collagen molecules self-organise into fibrils and form a hydrogel. Osidak et al. [3] study show that existing collagen type I has low mechanical properties and is considered to be the big issue in bioprinting. A suggested approach acquired by Osidak et al. was using supporting subordinate hydrogel to solve the mechanical properties. Since we are hybridising the Bioink, It will be discussed further in more detail in the Hybrid Bioink section. 

2.1.2. Gelatin

t's a collagen-based denatured protein that's been dissolved via acids and alkalis. By changing collagen's electrical charge distribution, a new isoelectric point of gelatin is created. Gelatin is utilised in a variety of industrial, medicinal, and pharmaceutical applications. Having ecological, and non-toxic properties gives it an upper edge for its use in clinical application. The capacity to successfully manufacture cartilage and liver tissue constructions was proven in early research using methacrylate gelatin.

2.1.3. Fibrin

It is the major component of the coagulation cascade and is produced from soluble circulating fibrinogen in the blood. Shpichka et al. [4] studies show that, With increasing concentration, pure fibrinogen solutions show a nonlinear rise in viscosity. Furthermore, the fibrinogen content in blood plasma is related to plasma viscosity. We can control the gelatin time by the concentration of thrombin and temperature, this will make the bio-ink more approachable for bioprinting. 

2.1.4. Silk

It is a protein thread or filament produced by silkworms. Natural silk is highly biocompatible, hypoallergenic, and is highly used in medicine for wound dressing.  Chawla et al. [5] studied silk-based bio-ink was extensively on worm silk and spider silk. The studies show that most of the natural polymeric bio-inks are fragile, can easily deform, in vivo degradation is higher, and thus can’t maintain good structure fidelity of the construct printed. On the other hand, Silk is authorised by  the food and drug administration(FDA). Silk-based bio-inks are amphiphilic in nature, meaning they can be used to produce protein drops in the pico-litre to nano-litre range (for droplet printing) or extruded filaments (for extrusion-based printing) by adjusting ink rheologic with variable pH and ionic strength. 

2.1.5. Alginate

Bacteria and sea algae are the two primary sources of biopolymer alginate. This natural polymer is found in the cell walls of marine brown algae (Phaeophyceae) and in the capsular polysaccharides of certain bacteria. Alginate hydrogels are commonly utilised as biomaterials in tissue engineering, drug delivery, and ECM models in fundamental biological research. Its application requires significant attention and control of certain properties like degradation, swelling, mechanical properties, and interaction with bioactive compounds. Alginate can be used as pure or hybrid for various material properties requirements. 

2.2. Synthetic Polymer-based Bio-ink

Synthetic polymer has certain advantages over natural polymer, in terms of various material properties and synthesis cost. It is widely used in 3D bioprinting, including Polyethylene glycol(PEG), polyvinylpyrrolidone (PVP), polylactic acid(PLA), etc. They may be fine-tuned to meet the target tissues and organs tissue-specific degradation and mechanical property requirements. 

2.2.1. Polyethylene Glycol

PEG is a popular hydrogel for cell research and medication delivery in tissue engineering scaffolds. PEG is a synthetic compound produced by copolymerization ethylene oxide that is highly valued for its hydrophilicity, which allows for easier exchange of nutrients and waste in cells due to its molecular structure. For the on-site manufacture of scaffolds, photopolymerization is the most prevalent approach for developing PEG hydrogels, which can enable better spatial and temporal control. Chemical modification methods can quickly change the properties of PEG-based derivatives. It has good mechanical stability. Although it has many advantages some of the notable disadvantages of PEG such as they do not provide any biological cues for cell proliferation, and also Photocrosslinking duration, light intensity, and photoinitiator all affect cell survival. 

2.2.2. Polyvinylpyrrolidone

It is a non-toxic, nonpolar, water-soluble amorphous polymer with strong polar solvent solubility. polyvinylpyrrolidone is widely employed in a variety of industries, including medicines, tissue engineering, and cosmetics. Ng et al. [6] studies where they evaluated the cell output for polyvinylpyrrolidone-based bio-ink for the period of 30 minutes. They found that precoating of polyvinylpyrrolidone solution in the printing magazine increases cell production. The use of polyvinylpyrrolidone-based bio-inks reduced cell bond and alluviation during the printing method, resulting in more consistent cell production over time.

2.2.3. Poly-L-Lactic Acid

PLA is a biodegradable acyclic polyester made from lactic acid extracted from reusable sugarcane or maize starch sources. PLA is a lactic acid cyclic dimer that emerges from a cyclization and oligomerization phase. PLA is a biocompatible polymer that is frequently utilised in biomedical applications since it has no carcinogenic or toxic effects on local tissue.

2.3. Hybrid Bioink

Conventional approach of making bio-ink suitable for various bioprinters faces severe issues, to tackle this problem many researchers applied and combined two different types of material either natural-natural, synthetic-synthetic, or Natural-synthetic. This approach of mixing two materials leads to the development of Hybrid Bioink, Hyaluronic Acid-based hybrid bio-ink is one such example where Hyaluronic Acid is immobilised with bio-active peptides for fast gelation. This hybridisation is mostly based on the requirement like the previous case was of gelation time[11]. Same as in the case of hybrid chitosan/acrylamide bio-ink is for DLP-based 3D bioprinting where printed assemblies are conferred with high-strength and acceptable biocompatibility.

Table 1

Materials	Crosslinking Method	Advantages	Disadvantages
Collagen	pH Temperature Vitamin Riboflavin Tannic acid	Easy degeneration promotes cell adhesive and cell attachment Easy to alter with other polymers With the addition of different polymers, it will be possible to increase its mechanical and biological characteristics.	Time-consuming for gelation refinement is a difficult procedure. Low mechanical properties biohazard
Silkworm Silk	Enzymatically Temperature pH value changes Sonication Salt filter Photo-crosslink	Ease of structure modification disciplined degradation Higher cellular survival variety of methods for crosslink or sol-to-gel Magnificant potency and strength Enclosed aquation qualities ample sources	Rheology needs to be optimised as bio-ink Low viscosity Hard to print individually
Fibrin	Enzymatic treatment	splendid biocompatibility and biodegradation quick gelation	frail mechanical stuff Severe immunogenic reciprocation So briskly for its degradation
Alginate	Ionic(Ca2+)	Ease of cross-linking solidity of construct Biocompatible ease cell entanglement Ease of manufacturability	Fast deterioration in vitro, needs supplementary dopants Low cell bond and protein surface assimilation absence of satisfactory mechanical properties
Agarose	Low temperature	innocuous Biological properties can be enhanced with another hydrogel easily appropriate mechanical properties for cartilage tissue restoration	Non-degradable Not appropriate for injecting printing with high viscosity Low cell bond and scattering

3. Development procedure

3.1. Development of Polyvinylpyrrolidone-Based Bio-Ink

Ng et al. study on the advancement of Polyvinylpyrrolidone-Base Bio-ink is one of its kind attempts where they were able to refine cell viability. The development of bio-ink consists of five processes including cell culture, Bio-ink synthesis, Bio-ink characterization, Drop-On-Demand Printing of Cell Droplets, expanding with statistical analysis.

3.1.1. Cell Culture

In the study conducted by Ng et al., human foreskin fibroblasts were used. The cells were grown in a full growth medium containing 15 percent FBS(fetal calf serum) and high glucose DMEM with L-glutamine. The cells were consistently passed in tissue culture flasks  and the culture media was replaced every three days. At 90 percent confluency, adherent HFF-1 cells were collected using one-fourth of trypsin/ethylene diamine tetraacetic acid (EDTA).

3.1.2. Bio-Ink Synthesis

Ng et al. prepared distinct types of  bio-ink based on PVP, varying the weight to volume percentage. The authors were trying to assess the persuade of cell denseness on the Z values. The Z value is the inverse of the Ohnesorge number(Oh),  as shown in equation 2, which is derived from equation 1. 

$Oh = {\mu \over \sqrt{\rho \sigma L}}={\sqrt{We}\over{Re}} \sim {Viscous \ force \over{inertia . surface \ tension}}$

$\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ...eq(1)$

$Z = {1\over Oh}={\sqrt{\rho \sigma L}\over \mu}={Re \over{\sqrt{We}}} \ \ \ ...eq(2)$

Where Re is referring to Reynolds number and We is the Weber number.

To evaluate the relation of polymer denseness to the Z value, they differentiate the concentration of PVP.

3.1.3.  Bio-Ink Characterisation 

As previously stated, the authors were evaluating the Z value with respect to the concentration of PVP polymer, for that mostly they were varying the concentration of PVP polymer keeping the persistent cell denseness of one million cells/mL in a complete growth medium. The effect of altering polymer concentration on viscosity, interfacial tension, and compactness of the different polyvinylpyrrolidone-based bio-inks and their associated Z values was then evaluated using measurements on the different polyvinylpyrrolidone-based bio-inks. The Discovery hybrid rheometer was used to test the rheological characteristics of the polyvinylpyrrolidone-based bio-inks. To guarantee that all computations were done inside the linear viscoelastic zone, the strain amplitude values were first validated. Following that, the viscosities of several polyvinylpyrrolidone-based bio-inks were tested at constant temperatures of 27°C(the temperature is the same as that of the printer) with shear rates spanning from 0.1 to 1000 $s^{-1}$. The interfacial tension of the bio-inks was evaluated using the capillary rise procedure, and the density of the bio-inks was assessed using a weighing scale. All measurements were carried out three times. 

3.2. Development of clay-based hybrid bio-ink

Natural hydrogel is biocompatible, but the mechanical integrity of such scaffolds is still challenging.  Habib and Khoda [7] in 2019 came up with an approach of hybrid bio-ink consisting of alginate, carboxymethyl cellulose, and montmorillonite clay. They conducted many quantified tests to examine and evaluate the bio-ink compatibility with bioprinting shape constancy and cell viability. The bio-ink generated by them achieves 84% living cells after seven days of bioprinting, this shows its efficient cell survival. The material and the composition used by them are mentioned in Table 2. Where A, C, M represents Sodium alginate, carboxymethylcellulose, and sodium montmorillonite respectively; and the appendix represents the weight percentage of the corresponding material. 

Table 2

Label	Aliginate(A) %(w/v)	Carboxymethyl cellulose(C) %(w/v)	Sodium montmorillonite(M) %(w/v)
A4C0 M0	4	0	0
A4C0 M4	4	0	4
A4C1 M4	4	1	4
A4C2 M4	4	2	4
A4C3 M4	4	3	4

 As illustrated in Figure 1(a) , alginate is a biopolymer made up of two organic acid monomers: (1 – 4) - linked  β- D mannuronic (M) and α - L guluronic acids (G). This substance is a water-soluble, negatively charged linear copolymer (G and M blocks). This material's G-block produces a gel, while the GM and M blocks promote flexibility. As demonstrated in Figure 1(b), carboxymethyl cellulose (CMC) is an anionic hydro soluble biopolymer generated naturally or via a chemical process from cellulose. It's a β-1,4-glycosidic compound of β-D-glucose and β-D-glucopyranose- 2-O-(carboxymethyl)-monosodium salt that's joined by glycosidic links [8]. As seen in Fig. 1(c), Na-MMT has a 2:1 layered structure, with an octahedral alumina layer sandwiched between two silicon tetrahedral layers [9]. MMT with a layered structure has a negative surface charge that attracts $Na^+$ or $Ca^{2+}$ to neutralise it. To make the bio-ink gel, the percentages of material listed in Table 1 are dissolved in 0.2 m purified deionized (DI) water and mixed overnight. To ensure fast gelation of the hoarded bio-ink, 4 percent (w/v) $CaCl_2$ (Sigma-Aldrich) is produced with 0.2 μm  purified deionized (DI) water and utilised as a chemical cross-linker. 

Due to its polar nature alginate, MMT and CMC are water-soluble. It is also found that MMT and alginate have chemical interaction, this is due to intermolecular hydrogen bond and electrostatic forces. Furthermore, alginate is said to draw CMC into the solution through a hydrogen bond [10]. As a result, it's possible that these materials interact in the hydrous solution through intermolecular hydrogen bonds and static electricity attraction at several locations to create a gel, as illustrated in Figure 2, which makes this blend bio-ink printable.

Figure 1(a) Chemical formula of Alginate

Figure 1(b) Chemical formula of Carboxymethyl Cellulose

Figure 1(c) Chemical Structure of Na-MMT

Figure 2 Chemical interaction Among alginate, carboxymethyl cellulose, and Na-montmorillonite

4. Conclusion and Future direction

Bioprinting technologies are still in their early phases of development, and there is still a lot of studies to be done on many unresolved problems. Bio ink design should continue to be a top concern, as it is one of the most important aspects of bioprinting. In the extrusion-based approach, the adoption of gel-based formulations appears to be the proper path to go. Since numerous advancements geared to the most diverse human tissues and organs by using bio-inks have been noticed in the literature, the results provided in this paper constitute a chance for additional investigations so that improvements may be achieved in any of the various elements of bioprinting. Implementation of  the hybrid bio-ink can be a good approach to solve various mechanical and rheostatic problems in the traditional bio-ink. This study has provided in-depth insights regarding existing bio-inks in light of the aforementioned concerns, and it is expected that this will assist a large audience in the field of bioprinting and tissue engineering.

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