Overview and concept
In February 2013, American Skylar Tibbits introduced the concept of 4D printing, and five months later, academic Lu Bingheng from Xi'an Jiaotong University proposed the concept of 5D printing.
In an article titled “Development Roadmap of 3D Printing Technology” published in China Information Week on July 29, 2013, Academician Lu Bingheng was the first to suggest that 5D printing is the current form of cellular printing, where The living tissues and organs we need can be created through printing.
He described 5D printing on several occasions, explaining that as time passes, not only does the form change, but also the functionality evolves. For example, in printing human organs, after printing a scaffold, human cells are embedded in it and in the right environment, they transform into different tissues, finally becoming an organ.
Of course, 5D printing is much more than just a simple concept: if 4D printing adds the dimension of time to 3D printing, using intelligent materials for self-assembly, then 5D printing introduces the ability to self-grow, which does not it's just adding another dimension, but expanding into multiple dimensions.
It's important to note: First, although 5D printing still uses 3D printing technology equipment, the printed materials are living cells and biologically active factors that have vitality. These biomaterials must undergo functional changes during their subsequent development; therefore, a full lifecycle project must be considered from the beginning.
Secondly, part of the current 5D free-form manufacturing refers to five-axis machining at the manufacturing technology level, which is still within the domain of 3D manufacturing and is totally different from the concept of 5D printing without a paper leadership in scientific and technological innovation.
Clearly, 5D printing will transform traditional manufacturing, which is characterized by static structures and fixed performances, into dynamic and changeable functionalities, breaking conventional manufacturing paradigms towards structural intelligence and functional genesis.
This will bring disruptive changes to manufacturing technology and artificial intelligence, evolving production from non-living entities to life-like entities with the ability to change shape and properties.
In the short term, this technology could revolutionize organ transplants and healthcare services for humans, and in the long term, it has the potential to create a new direction for manufacturing science and life sciences, driving innovative development in artificial intelligence.
Background of 5D Printing
The essence of 5D printing lies in manufacturing tissues with vital functions, offering humans the ability to custom-fabricate functional organs. Technology for manufacturing artificial tissues and organs is a key area supported by global industrial powers.
For example, the United States’ “Manufacturing Challenges Outlook 2020” identifies biological tissue manufacturing as one of the key directions for high technology; the European Commission’s “Strategic Report on the Future of Industry: 2015-2020” suggests a focus on the development of biomaterials and artificial prosthetics, positioning biotechnology as one of the four main disciplines underpinning the future of the industry;
The Japanese Society of Mechanical Engineers' technology roadmap highlights microbiomechanics to promote tissue regeneration as one of ten research directions. The international and domestic sectors have achieved partial clinical applications and industrialization in manufacturing personalized human substitutes and membrane-like active tissues.
However, manufacturing complex active tissues and organs still presents many challenges. Currently, there are more than 300 institutions and companies around the world dedicated to the research and development of 3D biological technology.
Among them, the Wake Forest Institute for Regenerative Medicine, in the United States, achieved a series of pioneering results in the field of biological 3D: they were the first to successfully print stem cells and induce the differentiation of functional bone tissue; in collaboration with the US Army Institute of Regenerative Medicine, they developed a 3D skin printer; they also 3D printed structures similar to “artificial kidneys”.
Internationally, heterogeneous integrated vascular network structures and heterogeneous integrated cell printing devices have been developed, producing heterogeneous cellular structures such as human cranial bone patches and ear cartilage.
In China, printing of bones, teeth, ear cartilage structures and vascular structures has been carried out, with preliminary clinical applications; Glioblastoma stem cell models and multicellular heterogeneous brain tumor fiber models have also been fabricated. Renowned Chinese universities, including Tsinghua University, Xi'an Jiaotong University, Zhejiang University, South China University of Technology, Sichuan University and Jilin University, have carried out in-depth research in this field.
The gap between some national organic production areas and the international advanced level is closing, with some even reaching a global leadership position.
Key issues in 5D printing
5D printing represents the convergence of manufacturing technology and life sciences technology, where intentional design, manufacturing and regulation are at the center. The main key issues include the following five aspects.
(1) Structural Design and Function-Based Manufacturing for Living Entities
Based on understanding the self-growth properties of living entities, it is necessary to develop theories for the structural and functional design of cells and genes in the elementary phase and throughout the growth process.
Key challenges include: first, disrupting existing mechanical design theories focused on structural design and mechanical function to develop design methods that co-evolve structure, actuation, and function; second, understand the laws that govern the replication and self-replication of cells and genes to design the composition and structure of the initial state of cells that grow according to their own rules;
and third, conduct research on materials, manufacturing processes, and engineering control methods for living entities that are degradable, have adequate engineering strength, and can be activated and grown in certain environments.
(2) 5D Printing Techniques for Regulating Living Units and Maintaining Viability
In 5D printing, living units serve as the basis for tissue growth and development, with single cells or genes constituting the core of subsequent functional manifestation. The micro- and nanoscale accumulation of these living units requires the study of their stacking principles and interrelationships.
By adjusting intercellular relationships, we can control three-dimensional spatial structure and functions, thereby facilitating tissue growth and functional regeneration. The hallmark of 5D printing is the functional regeneration of living entities, with the preservation of their viability being essential.
Therefore, the manufacturing of living entities needs to provide a corresponding cultivation environment, including controlling nutrients, oxygen, carbon dioxide and other atmospheric conditions in the culture medium, to create a synergy between the biological environment and the printing process.
(3) Mechanisms of Functional Formation and Component Function Development
It is vital to study the mechanisms and process innovation that allow different materials and structures to grow into various tissues and functions in certain environments. Initial structures and functions in 5D printing need to evolve into final functionalities in specific environments.
This requires an understanding of the relationship between function formation and design fabrication, as well as the laws of functional change over time in multicellular systems.
This includes interconnectivity relationships and cellular interactions, which, through their effects, build functions of energy release (muscle cells) or information transmission (neurons), providing a technical basis for the development of multifunctional devices.
(4) Information Carriers and Conduction Tissue Construction
Living entities are functional organizations controllable by information, similar to the role of neurons in animals and humans. In 5D printing, it is crucial to explore which materials and structures can replace neural functions, how to correctly transmit electrical or chemical signals, and how to drive the formation of diverse functionalities in tissues.
Research into neural and brain-like tissues will help establish information transmission organizations based on natural human characteristics, further advancing artificial intelligence with natural brain-like organization.
Current deep learning in artificial intelligence relies on model conjecture, data training, continuous accumulation of learning, and even uses biological genetic algorithms to perform artificial intelligence functions, in the same way that planes have replaced birds.
In the future, brain-like entities could use 5D printing to implant chips into recreated or artificial organs, learn from the random interconnectivity of human brain neurons to create powerful biological chips, or use genes to entirely replicate a biologically active brain.
The collection of information, the control of decision-making and the interaction between the artificial brain, the original human organs and various artificial organs are areas that await further research and innovation.
(5) Manufacturing and Functional Assessment of Multifunctional Devices or Fabrics
When implementing 5D printing technology, it is essential to understand the principles of design and manufacturing. Targeting specific organs or biological devices, it is necessary to engage in a systematic design of structural and functional growth.
This involves understanding how to regulate cellular or genetic combinations in 5D printing, how to control process-induced damage to the living organism during printing and how to manage the functions of the organs or devices formed, as well as interventions and guidance in cell growth. .
It is necessary to understand the relationship between 5D printing and functional formation, evaluate and measure the functions of multifunctional devices or fabrics, and establish a research system that integrates living unit design, damage-free printing and function creation. This provides the technical support necessary for the development of organs and devices with biological properties.
The development direction of 5D printing
5D printing will shift manufacturing from materials such as wood, metal and silicon to biological materials, transitioning from immutable structures to devices capable of functional regeneration.
To achieve this, it is crucial to establish transformative, functionality-driven design and manufacturing techniques and advance manufacturing technology through interdisciplinary integration. The State Laboratory of Mechanical Manufacturing Systems Engineering of Xi'an Jiaotong University has conducted promising explorations towards the development of 5D printing.
(1) Manufacturing of cardiac tissue
Myocardial infarction is a serious disease that poses a significant threat to human health. Existing engineered cardiac patches lack electrophysiological properties and fail to establish electrical signal conduction with the host myocardium, thus failing to achieve synchronous contraction and severely impairing the functional recovery of the infarcted myocardium.
Therefore, research is needed on the integration of conductive sensing functions into traditional cardiac tissues. This involves the use of multi-material micro/nano 3D printing technology to achieve integrated and controllable fabrication of cardiac structures with conductive sensing, offering new means to explore the pathogenesis and treatment of myocardial infarction.
This research will advance biofabrication from traditional scaffold manufacturing to the development of smart conductive sensing scaffolds. By simulating the micro/nanofiber structure of the natural cardiac extracellular matrix, research has been carried out on micro/submicroscale composite conductive fiber multimaterial electrostatic printing techniques.
Using electrostatic fusion printing, poly(caprolactone) (PCL) microfibers with a diameter of 9.5μm±1.5μm were manufactured; Using electrostatic solution printing, conductive poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)-polyethylene oxide (PEDOT:PSS-PEO) fibers with a diameter of 470nm±76nm were produced.
The PEDOT:PSS-PEO submicroscale conductive fibers exhibited excellent conductivity, with a conductivity of 1.72×10 3 S/m. By employing a layer-by-layer accumulation method, multilayer composite scaffolds were created, consisting of multilayer microfiber scaffolds with various orientations and micro/submicroscale conductive scaffolds, as shown in Figure 8-17.
The multilayer composite scaffold showed favorable mechanical properties in the fiber direction, with an elastic modulus of approximately 13.0MPa. Scaffold conductivity measurements demonstrated that the addition of PEDOT:PSS-PEO submicroscale conductive fibers significantly improved the conductivity of the scaffold.
Furthermore, the micro/submicroscale conductive scaffolds maintained stable conductivity in an aqueous environment, laying the foundation for subsequent cellular experiments.
Primary cardiomyocytes, the most important cells in cardiac tissue, provide the force for the heart's contraction and blood flow.
The influence of the above-mentioned multilayer composite structure on the oriented growth and synchronous beating of rat primary cardiomyocytes was studied. After eight days of co-cultivation, we observed that primary cardiomyocytes were capable of growing along micrometer-scale PCL fibers and forming complex, oriented cellular networks on submicrometer-scale PEDOT:PSS-PEO conductive fibers.
The cells also expressed substantial amounts of the cardiac-specific proteins α-actinin and CX43. Fluorescence quantification analysis revealed that the amount of these proteins expressed in submicrometer PEDOT:PSS-PEO conductive fibers was significantly higher compared to micrometer PCL fibers.
This demonstrates that PEDOT:PSS-PEO submicrometer conductive fibers improved the conductivity of the scaffold, improved intercellular electrical signal transmission, protein expression and beating capacity of cardiomyocytes. Furthermore, the oriented and layered design of the multilayer conductive structure further facilitated the synchronous beating of primary cardiomyocytes.
(2) Manufacture of brain-like tissues
Neuroscience is one of the most significant directions of scientific research today and a pinnacle of scientific competition between nations. In 2013, President Obama of the United States announced the Brain Initiative, which was soon followed by the European Union and Japan with the launch of the Human Brain Project and the Brain/Minds Project, respectively.
In China's “13th Five-Year Plan”, brain science and brain-like research ranks fourth among the 100 major projects. According to the World Health Organization, brain diseases such as Parkinson's, Alzheimer's, autism and depression have become a greater social burden than cardiovascular diseases and cancer. Due to limited understanding of its pathogenesis, almost all cases lack effective treatments.
In brain science and brain disease research, the lack of human brain tissue donors has become a major bottleneck. Animal brain tissues cannot fully represent the characteristics of the human brain; Therefore, the construction of in vitro models that closely mimic natural human brain tissue is an inevitable requirement for the advancement of neuroscience.
The functionality of neurons in brain tissue and their signaling are fundamental to cognitive function. The arrangement of these cells, their types and densities in the cortical layers support the functional zones of the cerebral cortex. Progressing from understanding the brain to creating it marks the direction for the development of brain-like computers.
The in vitro morphological and functional construction of brain tissue depends on biomimetic design and precise fabrication of neuron types, building structures, and neuron combinations corresponding to target functional areas. This is a forward-looking direction that 5D printing with brain-like biological function should follow.
In developing equipment for in vitro construction of brain-like tissue, an integrated cell printing/culture system was designed and assembled. It can simultaneously print multiple cells and matrix components, with a printhead speed of 100 to 1000 mL/min and a worktable XY movement accuracy of no more than 20μm.
It can print fabric layers from 100 to 300 μm thick while maintaining a printing chamber temperature of 37°C±1°C. Oxygen and carbon dioxide concentrations are adjustable, with concentration deviations within ±1%, providing an equipment platform for in vitro printing of brain-like multicellular tissues, as shown in Figure 8-18.
Based on existing printing equipment, printing process parameters were optimized to accommodate the requirements of printing neuronal cells, achieving the preparation of viable three-dimensional neural tissues encapsulating rat primary neuronal cells with a post-printing cell viability. greater than 94%.
Natural brain tissue consists mainly of two types of neural cells: neurons and neuroglia. Using the aforementioned platform, we construct models of pure neuronal tissue, mixed tissue of neurons and glial cells, and complex tissue structures with neurons and glial cells coexisting in a predefined three-dimensional spatial arrangement.
This setup allowed the co-culture of active brain-like tissue neurons and glial cells in vitro under various spatial structural relationships. Research indicates that neurons, positioned adjacent to but stratified from glial cells, can exhibit morphologies and biochemical expressions that are more reminiscent of natural brain tissues compared to neurons cultured alone in vitro.
This model provides a more accurate representation and research basis for the coexistence of neuroglial cells and neurons from a three-dimensional perspective, laying the foundation for subsequent brain science efforts and pathological pharmacological studies using in vitro models.
(3) Biomechanical Symbiotic Entities
Existing machines are limited by low energy conversion efficiency and flexibility. Bioinspired flexible multidirectional robots, powered by living cells or muscle tissue, represent the future of biosymbiotic machinery with high energy conversion efficiency, intrinsic safety, and agile movement. To this end, research into fabrication methods of multicellular/multimaterial composites for bioinspired robots is needed.
This research aims to provide a rapid, repeatable and customizable manufacturing approach based on the functional requirements of real robot locomotives that integrate biological and mechanical systems.
① For biological entity design, we developed a negative Poisson's ratio scaffold microstructure to cultivate and differentiate muscle cells. This design increases the degree of differentiation of muscle cells and the strength of contraction of muscle tissue, while providing the protection and nutrients necessary to maintain the long-term activity of the biological entity.
② Regarding the manufacturing of the biological entity, 3D printing was used to manufacture biocomponents. Experimental research into the growth and differentiation of skeletal muscle cells has revealed that these cells can differentiate into mature muscle fibers, laying the foundation for the construction of functional biological entities. Furthermore, we built a crawling biomechanical hybrid robot inspired by the sea slug.
③ In terms of regulating the functionality of the biological entity, a multifield coupling stimulation platform has been established. Studies were carried out on the regulatory mechanisms of bionic environmental enrichment stimuli (such as electrical and mechanical stimuli) on the driving performance of the biological entity.
④ Regarding the driving performance of bioinspired robots, a kinematic and dynamic model based on a second-order spring-damper system was developed for the robot. Using a kinematic and dynamic experimental platform, robot driving performance tests were carried out. The results showed that under a square wave pulse stimulation of 50 Hz frequency and 1 V voltage, the robot could crawl forward at a speed of 2 mm/s.
The aforementioned research explores possible future directions for robots with living bodies.
