Innovative bioinks for 3D bioprinting: Exploring technological potential and regulatory challenges

Abstract

The field of three dimensional (3D) bioprinting has witnessed significant advancements, with bioinks playing a crucial
role in enabling the fabrication of complex tissue constructs. This review explores the innovative bioinks that are
currently shaping the future of 3D bioprinting, focusing on their composition, functionality, and potential for tissue
engineering, drug delivery, and regenerative medicine. The development of bioinks, incorporating natural and synthetic
materials, offers unprecedented opportunities for personalized medicine. However, the rapid technological progress
raises regulatory challenges regarding safety, standardization, and long-term biocompatibility. This paper addresses
these challenges, examining the current regulatory frameworks and the need for updated guidelines to ensure patient
safety and product efficacy. By highlighting both the technological potential and regulatory hurdles, this review offers a
comprehensive overview of the future landscape of bioinks in bioprinting, emphasizing the necessity for cross-disciplinary collaboration between scientists, clinicians, and regulatory bodies to achieve successful clinical applications.

Introduction

The techniques that have been widely used to fabricate scaffolds, with the aim of developing 3D structures for tissue engineering, include freeze-drying, leaching, and electrospinning. However, a number of major challenges existed in these methods, which had to do with reproducibility, structural versatility, and the capability to incorporate biological components. Scaffold fabrication is an important step in tissue engineering as it offers an essential 3D structure that promotes cell proliferation and facilitates tissue regeneration. Although conventional methods like gas foaming and solvent casting are relatively simple, they often need hazardous solvents and fail to provide precise control over pore topologies. On the other hand, methodologies such as rapid prototyping, freeze-drying, electrospinning, and thermally induced phase separation offer enhanced precision with interconnected porosity and controlled pore dimensions. These modifications are essential for optimizing tissue growth and enhancing cell infiltration.

A study by Taherkhani et al. demonstrates that techniques such as solvent casting and particle extraction are
useful in producing porous scaffolds for bone tissue engineering. Advancement in this field in terms of research and
developing new products illustrates the significant potential in the healthcare sector as this technique enables the
modifications required as per the conditions of the patient and the replication of authentic bone architecture. Vigani
et al. investigated the freeze-drying process, which creates sponge-like scaffolds by eliminating water from polymer
solutions. The amount of CaCl2 used to make these scaffolds can change the size of their pores. As a result, structures that are similar to skin gradients are created, which improves the mechanical strength and bioactivity of wound care applications. Another technique that may be used to make nanofiber mats that are comparable to the extracellular matrix is called electrospinning. In their study, Azarsa et al. emphasized the utilization of electrospinning with a mixture of polyvinyl alcohol, polyvinyl chloride, and gelatin. This method enhances the biocompatibility and degradation of the scaffold by adding alginate and decellularized extracellular matrix. Analysis using scanning electron
microscopy and histology demonstrated that these scaffolds are capable of facilitating cell penetration and proliferation, making them ideal for use in tissue engineering.

Conventional approaches are capable of producing functioning scaffolds however, they frequently struggle to
achieve complicated structures that may be customized with a considerable degree of accuracy. For example, electrospinning has the capability of producing nanofibers; yet, it is still difficult to regulate the alignment of the fibers,
the size of the pores, and the dispersion of the material over larger constructions. Although salt leaching can produce
porous structures, it does not provide exact control over the design and regularity of the pores.

In the fabrication of intricate scaffolds customized to the specific requirements of each patient, 3D printing is
significantly more efficient. It produces structures that resemble the native properties of the tissue by providing
meticulous control over structural complexities, porosity, and material layers. These processes permit the incorporation
of a wide range of materials into the structure. 3D printing is the preferred technology for regenerative medicine
and TE due to its advantages, which include scalability, personalization, and reduced material waste. Even though it must overcome challenges such as the construction of sophisticated tissue architectures, the realization of vascularization, and the augmentation of printing speed, 3D printing provides significant benefits over conventional
methods. These advantages are provided even though it must overcome these challenges. Innovative
solutions, such as the utilization of temporary materials for vascularization and sophisticated extrusion techniques,
can increase both the structural stability and the survivability of the cells. In addition, the combination of flexible hydrogels with stiff scaffolds increases the mechanical robustness of the material. These advancements in 3D bioprinting tend to increase the survival of cells as well as the functional mimicry of cells, which ultimately leads to the industry moving forward in the direction of individualized tissue engineering solutions.

The rapid development of additive manufacturing technologies has significantly affected this field, especially in
the development of 3D bioprinting capable of handling simultaneously and precisely with living cells, biomaterials, and bioactive molecules to create biologically functional and active constructs. 3D bioprinting solves the disadvantages of conventional processes; for instance, enabling complex scaffold architectures to be precisely designed and reproducibly fabricated.

3D bioprinting involves different techniques based on ASTM classification which provides a framework to
organize 3D bioprinting techniques based on their bioink requirements, principles, construct quality and effect on
cell viability and overview of 3D bioprinting technique is shown in Figure 1. ASTM classifies the techniques as
extrusion- based, jetting- based and vat polymerizationbased technique. Extrusion based bioprinting extrudes
bioinks through nozzle. It is the most commonly and widely technique due to its versatility and ability to handle
various bioinks having viscosities from 100 to 30,000 mPa.s. Printability of bioinks using extrusionbased
technique depends on the pressure and viscosity. The effectiveness and the quality of the fabricated construct
is dependent on two factors: shear stress and crosslinking. Nozzle diameter, extrusion pressure, speed,
and bioink’s viscosity decides the magnitude of the shear stress and excessive shear stress can lead to cell damage
which in turn will compromise the cell viability. Membrane rupture and apoptosis are the common cause of mechanical
damage that excess shear stress causes. Crosslinking is essential to maintain stability and mechanical strength of
the construct once printed and it is important to balance the shear stress and crosslinking to ensure maximum cell viability.

By selecting the correct parameters, bioink formulation, cell survival and functionality in the constructs can
increase and enhance. In one of the studies, nanoengineered granular hydrogel bioink was developed to enhance
the mechanical properties of the structure and preserve the interconnected microporosity that helps in the cell attachment and tissue regeneration. The granular bioink also reduced the shear stress while printing using extrusionbased technique. In another study, a biphasic system consisting of solid and liquid phases were used to achieve the optimal properties while using extrusion based bioprinting technique. The bioink had solid colloidal particles suspended in liquid matrix creating a biphasic structure that provides structural support and flexibility by tailoring the viscoelastic properties of the ink and giving a control over the rheological behavior of the bioink during the extrusion. Jetting based technique comprises of inkjet and laser assisted bioprinting where inkjet based bioprinting
utilizes droplet-based technique to deposit cell laden droplets that creates patterns. The requirement of this technique
is using bioink having viscosity around 3–50 mPa.s, for smooth ejection of the droplets. This type of printing
offers high resolution and can be used for fabricating small controlled structures.12 Laser assisted bioprinting transfer
bioink droplet using laser pulses onto a substrate and requires bioink with moderate viscosity that can be vaporized
using laser pulse.

The printability using this technique is excellent with high resolution and spatial control. Thevelocity of the droplets that hit the substrate determines the shear force or the impact related stress. Cells may facedamage such as cell death or altered cell behavior if the velocity is too high. By adjusting the jetting pressure andpulse frequency, the velocity of the droplet can be controlled. The size of droplet also plays an important role asthe cell loading is higher in larger droplets but they can showcase higher impact force when they hit the surface.

In one study, the researchers used polyvinylpyrrolidone as bioink additive that helped in enhancing the printability,
making it more stable and viscous. It is important while using jetting-based technique for bioprinting to take care
of the substrate surface as use hydrophilic surface encourages the droplet to spread out, leading to uneven deposition
of cells and hydrophobic surface leads to droplet bead up which affects the cell adhesion to the structure. Another factor that effects jetting based bioprinting is the droplet evaporation, as it leads to bioink hardening which
leads to cell death. To prevent droplet evaporation, bioprinting systems have controlled humidity chambers that
reduce the rate of evaporation of solvent. Vat polymerization uses photopolymerizable resin or bioink that solidifies
when come in contact to light. This technique includes stereolithography (SLA) and digital light processing

(DLP). SLA uses laser beam that cures layer by layer and solidifies the resin selectively while offering high resolution
and precision whereas DLP uses micromirror device that can project entire layer of light to the resin. It speeds
up the printing process and can fabricate large complex organ scaffolds. It is important to balance the concentration
of photoinitiator being used while printing as less concentration can lead to insufficient crosslinking and higher
concentration can lead to cell toxicity. Instead of UV light in spectrum range 350–400 nm, it is better to use visible
light (405–450 nm) that is biocompatible. The intensity of the light source use for curing is also important as
higher intensity led to localized heat generation and lower intensities may cause prolong printing times and reduce
resolution. All of these techniques are capable of delivering high precision during the construction of complicated
biological structures. In extrusion- based technique, bioinks are It begins with the design using computer-aided
design, whereby one develops a three-dimensional model of the intended structure of the tissue. Furthermore, a decision is made on a suitable bioink and its preparation for printing desired constructs through the collection of cellular and material components. The most significant factor in 3D bioprinting involves bioinks, living cells on a biomaterial base that offer an appropriate environment for cellular replication, adhesion, and differentiation; functional
tissue constructs can eventually be developed.

Such bioinks can be made from natural or synthetic material, their choice depending, of course, on the compatibility
with the cells and/or special requirements of use. The selection of a bioink is mainly influenced by the type of
bioprinter, printing technique, and required mechanical/ biological properties. It is critical that bioinks, for optimal
performance, have appropriate mechanical strengths, tunable gelation rates, biocompatibility, and be scalable
for large-scale production. A number of characteristics need to be considered during formulation, including biocompatibility and biodegradability. The proposed biomaterial should have low immunogenicity and controllable
biodegradation rate, matched by a tissue repair process. It is important that the bioink degrades itself, so it forms its
own ECM. Besides, the viscosity of the bioink is important for an accurate deposition process: too high viscosity
can block nozzles, and too low viscosity results in cell damage. Low viscosities can be further modified by chemical,
physical, or enzymatic crosslinking of the bioinks.

Water absorption and porosity are just a few of the factors involved in the mechanical integrity of the scaffold.
Mainly, hydrophilic polymers are used because they can swell and still be able to maintain the shape of the scaffold.
Moreover, the biomaterial to be used for such purposes should present biological interaction sites to integrate the
cells to proliferate or adhere. In the absence of such sites, synthetic scaffolds can be functionalized with bioactive
sequences, including RGD (arginine-glycine-aspartate) or MMP binding sites, to further enhance cellular interactions.

Figure 2 summarizes the major considerations for the choice of bioinks, and a number of bioink types commonly
applied in the context of tissue engineering applications are the subject of further discussion in the present
review and the challenges faced while working with bioinks along with few innovative bioinks is shown in
Table 1. The bioprinting techniques present significant challenges due to cell sensitivity which requires environmental
factors such as nutritional supply, temperature, and pH. Additionally, there are risk factors that affect cell survival
during the printing process, for instance, mechanical stress, viscosity, and shear-thinning, which are governed
by bioink parameters. Currently, available bioinks have low printability which translates to fewer options in
designing tissue constructs and, in a way, reduces cell functions because of post-printing operations like curing
and incubation.2

These issues are overcome by creating bioinks with good printability for next-generation bioprinting applications that do not sacrifice the proximity of the cell to the bioink. These advanced formulations aim to create ideal microenvironment for cells thus, speeding up the timeline for attachment, proliferation, and differentiation. Bioinks
derived from natural polymers such as alginate, collagen, silk, and gelatin demonstrate promising results in terms of
cellular activity and tissue functionality. Furthermore, enhanced mechanical properties of scaffolds improve the
transport of nutrients and elimination of waste which also promotes growth or improves health of the cell populations.
The additional supplementation of bioactive substances and growth factors further enhances cell activity
and survival rate promoting increased regeneration processes.

Studies conducted in vivo validate the effectiveness of bioprinted tissues in supporting regeneration when procedures are fine-tuned. These results highlight the importance of advancing bioink formulation and printing techniques to address current challenges and advance the clinical use of 3D constructs.

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Vidhi Mathur, Prachi Agarwal, Meghana Kasturi, Varadharajan Srinivasan, Raviraja N Seetharam and Kirthanashri S Vasanthan, Innovative bioinks for 3D bioprinting: Exploring technological potential and regulatory challenges, Journal of Tissue Engineering, Volume 16: 1–31, © The Author(s) 2025, Article reuse guidelines: sagepub.com/journals-permissions, DOI: 10.1177/20417314241308022 journals.sagepub.com/home/tej, Date received: 24 September 2024; accepted: 4 December 2024

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