Date: 9th March 2021
3D bioprinting holds much promise for regenerative medicine, addressing the urgent need for tissues and organs suitable for transplantation. However, a major challenge is the formation of complex-shaped 3D assemblies that retain structural integrity, remain flexible whilst allowing the integration of cells. Now, researchers have developed synthetic, 3D- printable, highly bioactive self-assembly nanofibers which form superstructures that mimic properties of living tissues and enhance neuron growth, opening avenues for regenerative treatments for neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.
The ability to create complex 3D structures that retain structural integrity has been a significant hindrance for the bioprinting field, such has been the scale of the problem that scientists have even explored bioprinting in space, as low gravitational conditions circumvent the problem of suitable scaffolding and support.
Bioprinting has huge potential for regenerative medicine, as the need for transplantable organs far outweighs availability. Its use however extends beyond that, as bioprinted materials can be used for a wide range of applications such as tissue modelling for research, drug discovery and toxicology.
In nature, living organisms have harnessed reversible hierarchical self-assembly of molecules to control the formation of structures such as protein assemblies, cell membranes and cytoskeletal filaments. However, mimicking these highly organised and dynamic structures with synthetic molecules has been challenging.
Now, scientists led by Samuel Stupp from Northwestern University, US, have demonstrated unique self-assembling nanofibers bundle together creating superstructures that can create a brain-like tissue architectures in vitro. They attract and enhance neuron growth, having implications for cell transplantation strategies for neurodegenerative disease such as Alzheimer’s and Parkinson’s disease.
The team had previously discovered the phenomenon of molecular shuffling in 2018, when they created chemically reversible hydrogels based on peptide amphiphiles (PAs) – peptide-based molecules that can self-assemble into supramolecular nanostructures. One peptide-based hydrogel was mixed with another which also contained peptide -DNA conjugates. When mixed together filaments form via Watson-Crick base pairing and could be disassemble upon the addition of molecules or changes in charge density.
Now the team wanted to harness the power of these hydrogels for an application in regenerative medicine. The new hydrogels were created by mixing PAs which became rigid via dynamic molecular exchange based on host-guest complexes that mimic key-lock interactions among proteins. Here, the team synthesised PAs functionalised to display a well-known host–guest complex. The first PA displayed β‐cyclodextrin (as host) whilst the other guest PA contained adamantine, this complex was chosen as it forms a strong, reversible non‐covalent interaction.
Superstructures formed by walking molecules
The resulting superstructure, contained large pores that allowed neuronal cells to integrate and penetrate the scaffold, and could interact with bioactive signals that were incorporated into the biomaterial. Furthermore, the mechanical forces that the biomaterial experienced during 3D printing disrupted the host-guest interactions, temporarily liquefying the hydrogel to allow printing, but which rapidly solidified after, allowing the assembly of any macroscopic shape.
Bioactive hydrogels
Once the system was in place the team wanted to assess the supramolecular system for bioactivity, here they incorporated PA molecules that mimicked brain-derived neurotrophic factor, called BDNF PA. The mimic of this protein has been shown to activate the BDNF specific TrkB receptor. The bioactive PAs were used to treat embryonic primary mouse cortical neurons in vitro, and supported cell survival which remained above 80% indicating a healthy culture conditions. Furthermore, the cells were able to infiltrate the large pores in the gels when prepared in moulds.
3D bioprinting
However, the ultimate aim was to 3D print complex shapes, creating self‐standing objects that don’t require additional materials which could comprise the properties of the superstructures and limit their future applications. So, the team prepared multiple superstructure ink laden with different cells types and bioactive cues, and printed concentric circles which represented the layers of the brain cortex. They fluorescently labelled primary cortical astrocytes and neurons in different coloured dye, and then these were printed with the bioactive host–guest BDNF PA hydrogel.
After 7 days of culture the team found that the layers were maintained by the host-guest hydrogel, and showed no signs of degradation. The cells were evenly distributed through the layers, and had a high survival rate, demonstrating the PA-system could incorporate bioactive signals, support cell survival and could effectively mimic tissue architecture.
Conclusions and future applications
The team have created a novel biomaterial with properties of a living tissue that can be 3D printed to form self-assembling, complex shapes that were self-supporting. The mechanically robust hydrogel, had a highly porous architecture that allowed cell integration, and could be functionalised with biological signals.
This is an important finding and will have implications for a wide range of disease and injury treatments. With this work here using neurons, the team will assess the hydrogels for cell transplant strategies for neurodegenerative disease such as Parkinson’s and Alzheimer’s disease, and for injuries to the spinal cord. The hydrogel could support and enhance growth of healthy neurons that can be transplanted in to patients to help alleviate degenerative diseases or could be used to support regeneration.
From a wider perspective, the team propose the system could be used to prepare other types of tissue such as cartilage and heart tissue from patient-derived cells to stimulate regeneration after a heart attack or post injury. The biomaterial could be used to build organoids, or artificial organs for drug discovery or as a test bed for novel therapies. By changing the chemical sequences in the biomaterial it could allow the hydrogels to provide alternative signals to enhance different cell types, forming a wide range of tissues in the biologically appropriate structure and shape, and for the treatment of a vast number of diseases or injuries.
The development of specialised hydrogels is a crucial milestone for the advancement of bioprinting. We have recently seen self-healing yield-stress gels enabling self-assembly of spheroids for fabrication of tissue, and the development of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) which allows collagen to be bioprinted in a support gel where a pH changes solidifies the collagen. The addition here of hydrogels that self-assemble into supermolecules and that can be functionalised with bioactive molecules, is a very attractive proposition and one that will accelerate the field.
For more information please see the press release from Northwestern Univeristy
Edelbrock, A. N., T. D. Clemons, S. M. Chin, J. J. W. Roan, E. P. Bruckner, Z. Álvarez, J. F. Edelbrock, K. S. Wek and S. I. Stupp “Superstructured Biomaterials Formed by Exchange Dynamics and Host–Guest Interactions in Supramolecular Polymers.” Advanced Science: 2004042.
https://doi.org/10.1002/advs.202004042
