3D Bioprinting is a revolutionary technology that has created an immense impact in the field of medicine and healthcare. It has enabled us to mimic the natural tissue microenvironment into an in-house printed three-dimensional tissue model. This groundbreaking technology has enabled researchers to expand the horizon of their multidisciplinary research areas. Currently, the focus is on the development of application-specific bioinks and new biomaterials, which play a crucial step for the advancements in bioprinting tissues and organs for clinical use.

3D bioprinting is a multidisciplinary field, drawing expertise from computation, instrumentation, material science, chemistry, cell biology, and medicine. Printer development aims to cater to specific needs, such as portability, versatility, and improved resolution.

Besides the printer itself, researchers are developing highly portable imaging modalities to generate on-the-fly 3D models for intra-operative bioprinting. The quest for modern bioinks too is on. Tunable mechanical properties, selective immobilization of small molecules and growth factors, cytocompatible crosslinking, and stimulus-responsive release/degradation are active areas of research, relying on modifying existing natural biopolymers as well as developing novel gelators through organic synthesis.

Innoskin®, developed by Next Big Innovation Labs®, is an alternative to animal testing for cosmetic, chemical and agro-chemical testing applications.

One exciting research area is that of 4D bioprinting, wherein printed structures evolve to assume predicted shapes and morphologies. 4D bioprinting also aims to harness the inherent ability of cells to self-assemble and organize into biologically relevant tissue architectures. In this way, researchers are pushing the envelope of tissue engineering through bioprinting.

There have also been advances in 3D photogrammetry. Technologies like artificial intelligence for image segmentation/path planning are also finding their way into bioprinting practices. Co-axial nozzle bioprinting is in use to print hollow vascular structures. Researchers have also developed handheld 3D bioprinters for intra-operative applications such as burns, trauma and repairing cartilage defects.

The series of urinary bladder transplants done at Wake Forest since 2004 perhaps marks the entry of bioprinting in Healthcare. Since then, many cutting edge devices and techniques have been developed. A portable 3D printer with extrusion and inkjet nozzle heads, coupled to a 3D wound scanner, has been used to repair full-thickness skin wounds in pigs.

However Challenges Remain;

The indispensability of vascularization is still a challenge. Numerous efforts are underway to generate perfusable constructs with interconnected blood vessels. Intraoperative bioprinting almost always requires printing on an uneven surface, which is markedly different from conventional flat surfaces. Extrusion bioprinting has an issue where edge-to-edge printing is not possible. The nozzle itself interferes with reaching the periphery of a wound. Vacuum-based wound closure can mitigate this, wherein negative air pressure is applied to lock the bioprinted construct in place.

Conventional methods such as UV-based crosslinking of bioinks cannot be used in a human body without significant device modifications. Printing technologies such as LIFT(Laser-induced forward transfer) have yet to be translated to the operating theatre owing to large parts.

Advances in material science such as the use of nanoparticles, nanocrystals, nanofibrils, nanosheets, nanotubes, stimulus-responsive polymers reacting to changes in temperature, pH and magnetic field are finding their way into the mainstream bioprinting field. Novel cytocompatible crosslinking strategies such as visible-light and innovative enzymatic methods are pushing the boundaries of bioprinting. The addition of nanostructures can be used to improve the mechanical properties as they contribute to reinforcement of the material. Metal nanoparticles are incorporated to make conducting bioinks, which could enhance electrically driven tissues such as skeletal muscle and cardiac muscle.

According to researchers, graphene oxide nanoparticles could find its use to alter the viscosity of bioinks. Graphene oxide also serves many useful biological functions such as driving differentiation of stem cells, supporting neurons and improving oxygenation. While biomaterials strategies can form these interlinked networks, it is crucial to enable oxygen transport into the core of solid tissues for weeks – a timeframe relevant to vascularization.

For this purpose, novel chemical approaches such as loading of Sodium percarbonate and Calcium Peroxide to generate nascent oxygen is under investigation. Shapeshifting bio inks too are in development, wherein Origami based 3D architectures such as hollow structures and branched networks can be made. The flat structures printed, gradually with time, fold to generate the desired shape – this is one of the principles of the nascent field of 4D bioprinting. In this way, biomaterials science is pushing the envelope of engineerable tissues and organs.

Classical issues such as cell sourcing have primarily been addressed, with the advent of Induced Pluripotent Stem Cell (iPSC) based methods and extensively studied directed differentiation of autologous cells, paving the way for personalized medicine. The classical approach to bioprinting of complex tissues has until now relied on the researcher. Manual deposition of various cell types in their anatomically ‘correct’ locations, and allowing the tissue to form and mature.

Recently, however, the phenomenon of self-assembly and spontaneous patterning of cells, reminiscent of developmental biology, has received recognition and interest. In this approach, multiple cell types are printed together, and the cells based on inherent nature assemble into meaningful structures.Thus, forming the ‘biology’ arm of 4D bioprinting.

Researchers have shown spontaneous assembly of skin from heterogenous organoids and periosteum derived ‘soft callus’ organoids for healing significant bone defects. This stark paradigm-shift increased reliance on inherent cellular properties is evident in more recent literature, wherein bioinks have higher cellular densities, with bio ink being the minor component.

For example, reconstituted skin constructs were prepared from 6 human skin cell types, patterned by bioprinting with high cell density in an anatomically consistent pattern, and promptly implanted in a mouse model for full-thickness skin wound healing. Superior wound remodeling and closure was observed compared to controls. Such high cell-density approaches have given rise to ‘scaffold-free’ bioprinting, reliant on droplet-on-demand technologies more than extrusion-based bioprinting. 

Despite facing complications, 3D Bioprinting has come a long way. Recent translational work such as FRESH(freeform reversible embedding of suspended hydrogels) bioprinted heart parts,patient-specific miniature hearts, innervated skeletal muscles and the evolution of 3D kidney, push the frontiers of tissue engineering. Many more tissues have been produced in the microscale and serve as ‘disease in a dish’ and ‘organ on a chip’ platforms for drug discovery and screening, as well as model systems to understand fundamental biology.

In this way, a transdisciplinary approach is indispensable for bioprinting to find its way into mainstream medical practice. Science apart, extensive collaboration across academia and industry, streamlined government and funding in this direction is the need of the hour. Improved regulatory and policy guidelines for development and evaluation of bioengineered tissues, and fostering deep-tech initiatives in 3D bioprinting promises to pave the way for rapid innovation and expands horizons of bioprinting to perhaps one day make engineered tissues a reality.

Author - Preethem Srinath

Preethem is a Senior Scientist at Next Big Innovation Labs®. He comes from a research background of Synthetic Organic Chemistry and Biomaterials for Regenerative Medicine and applies these concepts to NBIL’s Biotech initiatives.

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