Down to the Bare Bones of Regenerative Medicine

Toys, computer parts, prostheses, pencil holders… 3D printers bring life to imaginary and everyday concepts. And now also to human organs! Using processes that may have filled Dr. Frankenstein with enthusiasm, tissue engineering makes it possible to accurately print and recreate living biological tissues in three dimensions, with the aim of growing customized grafts.

Pierre Layrolle
Pierre Layrolle is getting ready to use one of the bio-printers at the ToNIC laboratory in Toulouse ©Inserm/François Guénet

While technology has made significant progress in recent years, bio-printing is still in its infancy: although it is now possible to reproduce the appearance of organs in a very convincing way, their long-term integration in a host organism is not something that we have achieved yet. For the most part, these organoids are restricted to the role of research tool and provide increasingly in-depth knowledge of the functioning of the human body and diseases. They also serve as a support for therapeutic tests for medical evaluation. However, printed tissues have already been successfully implanted in animals and humans: the Toulouse Neuroimaging Center (ToNIC) is developing various techniques for the bio-manufacturing of human organs ex vivo. Among other things, the researchers have developed, printed and transplanted, in collaboration with surgeons, an alternative to autologous bone grafting.

A researcher presents images preparatory to the grafting of a bioprinted bone fragment
At ToNIC, the researchers are printing customized pieces of bone that are perfectly adapted to the patients’ morphology for the reconstruction of major bone defects ©Inserm/François Guénet

At present, when a bone is damaged by trauma or a tumor, and requires ablation, surgeons replace it with a bone fragment taken from the hip or the fibula. However, this technique is not optimal, and often comes with disabling consequences that reduce the movements of the patient, who is deprived of a bone. Layrolle and his team have developed a less invasive alternative. They custom print pieces of bone that are perfectly suited to the morphology of the patient for the reconstruction of large bone defects.

ToNIC is able to segment and convert medical images of any organ into stereolithography files, i.e. prototyped for 3D printing. This means that the researchers can manufacture tailor-made guides for surgeries, and anatomical biomaterials that are virtually identical to the body part to be reconstructed.

The unit is equipped with three 3D printing technologies: conventional fused deposition modeling (FDM) printers used to print plastic organs to scale, and two types of bio-printer. The first uses blue light to polymerize a biocompatible hydrogel, which contains human cells and becomes rigid once printed. The other is used to form a complex organ, layer per layer.

a bioprinted bone fragment
This bio-printed object will serve as a support for stem cells to lead to the reconstitution of a bone fragment ©Inserm/François Guénet

To finalize the transformation into bone, different stem cells are inoculated on the biomaterial to which they adhere. They are essential for the reconstruction of human tissue and the regeneration into bone: without them, the 3D biomaterial remains inert. For each type of stem cell used, different human tissues can be bio-manufactured. For example, hematopoietic stem cells differentiate into osteoblasts, which produce the bone matrix, whereas vascularization is ensured by endothelial cells. The ink used to print bone fragments is composed of a calcium phosphate which is similar to the bone mineral; it resembles toothpaste and hardens by hydrolysis, like plaster. The 3D biomaterials are built with internal porosity which makes it possible to « plant » stem cells and guide healing, ensuring optimal bone regrowth.

The different steps involved in implanting a bioprinted bone fragment
Implantation of a bio-printed bone fragment in a sheep metatarsus. © Inserm/JRU1214/ToNIC

The printed biomaterial is surgically implanted at the desired location; above, the metatarsus of a sheep. In order to vitalize and accelerate tissue regeneration, a vessel is diverted and placed inside the biomaterial. Within a few weeks, the 3D bio-print turns into bone tissue and replaces the resected bone. For the time being, this work remains at the preclinical stage, but studies to evaluate the safety and efficacy of this procedure in humans are ongoing.

Pierre Layrolle
Layrolle and his team also use medical images taken using MRI to make realistic and personalized 3D polymer printouts. Although these objects are not living tissues, they can be used as surgical models to validate surgical techniques and verify, for example, the diffusion of a contrast medium within an organ’s vascular network. ©Inserm/François Guénet

In addition to printing organs, the major challenge of medical bio-printing is to provide a more realistic support for science in order to better understand living organisms. Conventional 2D cell cultures do not take cell organization into account: part of the information is lost. 3D cell culture makes it possible to recreate the conditions of the various cell microenvironments encountered in the body, and to make better predictions thanks to in vitro models.

It is not yet known how to print cells one by one. However, it is possible to cultivate gels based on living cells that will be used to construct specific tissues. Layrolle is currently developing an ink that is super-concentrated in neural stem cells, which are encapsulated in beads. It is used to manufacture brain organoids on the millimetric scale.

A mini-brain
A mini-brain manufactured at the ToNIC laboratory ©Inserm/JRU1214/ToNIC

The mini-brain model presented above, ultra-realistic and functional, has specific markers of neurons, axons – nerve cell extensions – and vascularization. It even emits electrical activity equivalent to that of a few-weeks-old fetus! Unlike the organoids produced so far, their spatial organization is well structured, they can be identically reproduced, and they can be maintained in culture for several weeks. Far from being human brains, they will make it possible to better understand the brain’s development and functional mechanisms, and also to test the action of drug candidates. Midway between in vivo models and in vitro cell cultures, these are promising tools that will play a crucial role in the research of tomorrow.

Pierre Layrolle is leader of the 3D CHIP team at the ToNIC laboratory (unit 1214 Inserm/Université Toulouse III – Paul-Sabatier, Toulouse Neuroimaging Center), in Toulouse.

Learn more about the team’s activities related to: