4D Printing for Biomedical Applications.

4D (bio-)printing endows 3D printed (bio-)materials with multiple functionalities and dynamic properties. 4D printed materials have been recently used in biomedical engineering for the design and fabrication of biomedical devices, such as stents, occluders, microneedles, smart 3D-cell engineered microenvironments, drug delivery systems, wound closures, and implantable medical devices. However, the success of 4D printing relies on the rational design of 4D printed objects, the selection of smart materials, and the availability of appropriate types of external (multi-)stimuli. Here, this work first highlights the different types of smart materials, external stimuli, and design strategies used in 4D (bio-)printing. Then, it presents a critical review of the biomedical applications of 4D printing and discusses the future directions of biomedical research in this exciting area, including in vivo tissue regeneration studies, the implementation of multiple materials with reversible shape memory behaviors, the creation of fast shape-transformation responses, the ability to operate at the microscale, untethered activation and control, and the application of (machine learning-based) modeling approaches to predict the structure-property and design-shape transformation relationships of 4D (bio)printed constructs.

Bone cell response to additively manufactured 3D micro-architectures with controlled Poisson's ratio: Auxetic vs. non-auxetic meta-biomaterials.

The Poisson's ratio and elastic modulus are two parameters determining the elastic behavior of biomaterials. While the effects of elastic modulus on the cell response is widely studied, very little is known regarding the effects of the Poisson's ratio. The micro-architecture of meta-biomaterials determines not only the Poisson's ratio but also several other parameters that also influence cell response, such as porosity, pore size, and effective elastic modulus. It is, therefore, very challenging to isolate the effects of the Poisson's ratio from those of other micro-architectural parameters. Here, we computationally design meta-biomaterials with controlled Poisson's ratios, ranging between -0.74 and +0.74, while maintaining consistent porosity, pore size, and effective elastic modulus. The 3D meta-biomaterials were additively manufactured at the micro-scale using two-photon polymerization (2PP), and were mechanically evaluated at the meso­scale. The response of murine preosteoblasts to these meta-biomaterials was then studied using in vitro cell culture models. Meta-biomaterials with positive Poisson's ratios resulted in higher metabolic activity than those with negative values. The cells could attach and infiltrate all meta-biomaterials from the bottom to the top, fully covering the scaffolds after 17 days of culture. Interestingly, the meta-biomaterials exhibited different cell-induced deformations (e.g., shrinkage or local bending) as observed via scanning electron microscopy. The outcomes of osteogenic differentiation (i.e., Runx2 immunofluorescent staining) and matrix mineralization (i.e., Alizarin red staining) assays indicated the significant potential impact of these meta-biomaterials in the field of bone tissue engineering, paving the way for the development of advanced bone meta-implants. STATEMENT OF SIGNIFICANCE: We studied the influence of Poisson's ratio on bone cell response in meta-biomaterials. While elastic modulus effects are well-studied, the impact of Poisson's ratio, especially negative values found in architected biomaterials, remains largely unexplored. The complexity arises from intertwined micro-architectural parameters, such as porosity and elastic modulus, making it challenging to isolate the Poisson's ratio. To overcome this limitation, this study employed rational computational design to create meta-biomaterials with controlled Poisson's ratios, alongside consistent effective elastic modulus, porosity, and pore size. The study reveals that two-photon polymerized 3D meta-biomaterials with positive Poisson's ratios displayed higher metabolic activity, while all the developed meta-biomaterials supported osteogenic differentiation of preosteoblasts as well as matrix mineralization. The outcomes pave the way for the development of advanced 3D bone tissue models and meta-implants.

Orthopedic meta-implants.

Meta-biomaterials, engineered materials with distinctive combinations of mechanical, physical, and biological properties stemming from their micro-architecture, have emerged as a promising domain within biomedical engineering. Correspondingly, meta-implants, which serve as the device counterparts of meta-biomaterials, offer exceptional functionalities, holding great potential for addressing complex skeletal diseases. This paper presents a comprehensive overview of the various types of meta-implants, including hybrid, shape-morphing, metallic clay, and deployable meta-implants, highlighting their unprecedented properties and recent achievement in the field. This paper also delves into the potential future developments of meta-implants, addressing the exploration of multi-functionalities in meta-biomaterials and their applications in diverse biomedical fields.

4D printed shape-shifting biomaterials for tissue engineering and regenerative medicine applications.

The existing 3D printing methods exhibit certain fabrication-dependent limitations for printing curved constructs that are relevant for many tissues. Four-dimensional (4D) printing is an emerging technology that is expected to revolutionize the field of tissue engineering and regenerative medicine (TERM). 4D printing is based on 3D printing, featuring the introduction of time as the fourth dimension, in which there is a transition from a 3D printed scaffold to a new, distinct, and stable state, upon the application of one or more stimuli. Here, we present an overview of the current developments of the 4D printing technology for TERM, with a focus on approaches to achieve temporal changes of the shape of the printed constructs that would enable biofabrication of highly complex structures. To this aim, the printing methods, types of stimuli, shape-shifting mechanisms, and cell-incorporation strategies are critically reviewed. Furthermore, the challenges of this very recent biofabrication technology as well as the future research directions are discussed. Our findings show that the most common printing methods so far are stereolithography (SLA) and extrusion bioprinting, followed by fused deposition modelling, while the shape-shifting mechanisms used for TERM applications are shape-memory and differential swelling for 4D printing and 4D bioprinting, respectively. For shape-memory mechanism, there is a high prevalence of synthetic materials, such as polylactic acid (PLA), poly(glycerol dodecanoate) acrylate (PGDA), or polyurethanes. On the other hand, different acrylate combinations of alginate, hyaluronan, or gelatin have been used for differential swelling-based 4D transformations. TERM applications include bone, vascular, and cardiac tissues as the main target of the 4D (bio)printing technology. The field has great potential for further development by considering the combination of multiple stimuli, the use of a wider range of 4D techniques, and the implementation of computational-assisted strategies.

Deep Learning for Size-Agnostic Inverse Design of Random-Network 3D Printed Mechanical Metamaterials.

Practical applications of mechanical metamaterials often involve solving inverse problems aimed at finding microarchitectures that give rise to certain properties. The limited resolution of additive manufacturing techniques often requires solving such inverse problems for specific specimen sizes. Moreover, the candidate microarchitectures should be resistant to fatigue and fracture. Such a multi-objective inverse design problem is formidably difficult to solve but its solution is the key to real-world applications of mechanical metamaterials. Here, a modular approach titled "Deep-DRAM" that combines four decoupled models is proposed, including two deep learning (DL) models, a deep generative model based on conditional variational autoencoders, and direct finite element (FE) simulations. Deep-DRAM integrates these models into a framework capable of finding many solutions to the posed multi-objective inverse design problem based on random-network unit cells. Using an extensive set of simulations as well as experiments performed on 3D printed specimens, it is demonstrate that: 1) the predictions of the DL models are in agreement with FE simulations and experimental observations, 2) an enlarged envelope of achievable elastic properties (e.g., rare combinations of double auxeticity and high stiffness) is realized using the proposed approach, and 3) Deep-DRAM can provide many solutions to the considered multi-objective inverse design problem.

Auxeticity as a Mechanobiological Tool to Create Meta-Biomaterials.

Mechanical and morphological design parameters, such as stiffness or porosity, play important roles in creating orthopedic implants and bone substitutes. However, we have only a limited understanding of how the microarchitecture of porous scaffolds contributes to bone regeneration. Meta-biomaterials are increasingly used to precisely engineer the internal geometry of porous scaffolds and independently tailor their mechanical properties (e.g., stiffness and Poisson's ratio). This is motivated by the rare or unprecedented properties of meta-biomaterials, such as negative Poisson's ratios (i.e., auxeticity). It is, however, not clear how these unusual properties can modulate the interactions of meta-biomaterials with living cells and whether they can facilitate bone tissue engineering under static and dynamic cell culture and mechanical loading conditions. Here, we review the recent studies investigating the effects of the Poisson's ratio on the performance of meta-biomaterials with an emphasis on the relevant mechanobiological aspects. We also highlight the state-of-the-art additive manufacturing techniques employed to create meta-biomaterials, particularly at the micrometer scale. Finally, we provide future perspectives, particularly for the design of the next generation of meta-biomaterials featuring dynamic properties (e.g., those made through 4D printing).

Biomimetic Approaches for the Design and Fabrication of Bone-to-Soft Tissue Interfaces.

Bone-to-soft tissue interfaces are responsible for transferring loads between tissues with significantly dissimilar material properties. The examples of connective soft tissues are ligaments, tendons, and cartilages. Such natural tissue interfaces have unique microstructural properties and characteristics which avoid the abrupt transitions between two tissues and prevent formation of stress concentration at their connections. Here, we review some of the important characteristics of these natural interfaces. The native bone-to-soft tissue interfaces consist of several hierarchical levels which are formed in a highly specialized anisotropic fashion and are composed of different types of heterogeneously distributed cells. The characteristics of a natural interface can rely on two main design principles, namely by changing the local microarchitectural features (e.g., complex cell arrangements, and introducing interlocking mechanisms at the interfaces through various geometrical designs) and changing the local chemical compositions (e.g., a smooth and gradual transition in the level of mineralization). Implementing such design principles appears to be a promising approach that can be used in the design, reconstruction, and regeneration of engineered biomimetic tissue interfaces. Furthermore, prominent fabrication techniques such as additive manufacturing (AM) including 3D printing and electrospinning can be used to ease these implementation processes. Biomimetic interfaces have several biological applications, for example, to create synthetic scaffolds for osteochondral tissue repair.

Additive Manufacturing of Biomaterials-Design Principles and Their Implementation.

Additive manufacturing (AM, also known as 3D printing) is an advanced manufacturing technique that has enabled progress in the design and fabrication of customised or patient-specific (meta-)biomaterials and biomedical devices (e.g., implants, prosthetics, and orthotics) with complex internal microstructures and tuneable properties. In the past few decades, several design guidelines have been proposed for creating porous lattice structures, particularly for biomedical applications. Meanwhile, the capabilities of AM to fabricate a wide range of biomaterials, including metals and their alloys, polymers, and ceramics, have been exploited, offering unprecedented benefits to medical professionals and patients alike. In this review article, we provide an overview of the design principles that have been developed and used for the AM of biomaterials as well as those dealing with three major categories of biomaterials, i.e., metals (and their alloys), polymers, and ceramics. The design strategies can be categorised as: library-based design, topology optimisation, bio-inspired design, and meta-biomaterials. Recent developments related to the biomedical applications and fabrication methods of AM aimed at enhancing the quality of final 3D-printed biomaterials and improving their physical, mechanical, and biological characteristics are also highlighted. Finally, examples of 3D-printed biomaterials with tuned properties and functionalities are presented.

3D-Printed Submicron Patterns Reveal the Interrelation between Cell Adhesion, Cell Mechanics, and Osteogenesis.

The surface topography of implantable devices is of crucial importance for guiding the cascade of events that starts from the initial contact of the cells with the surface and continues until the complete integration of the device in its immediate environment. There is, however, limited quantitative information available regarding the relationships between the different stages of such cascade(s) and how the design of surface topography influences them. We, therefore, used direct laser writing to 3D-print submicron pillars with precisely controlled dimensions and spatial arrangements to perform a systematic study of such relationships. Using single-cell force spectroscopy, we measured the adhesion force and the work of adhesion of the preosteoblast cells residing on the different types of surfaces. Not only the adhesion parameters (after 2-60 s) but also the formation of focal adhesions was strongly dependent on the geometry and arrangement of the pillars: sufficiently tall and dense pillars enhanced both adhesion parameters and the formation of focal adhesions. Our morphological study of the cells (after 24 h) showed that those enhancements were associated with a specific way of cell settlement onto the surface (i.e., "top state"). The cells interacting with tall and dense pillars were also characterized by numerous thick actin stress fibers in the perinuclear region and possibly high internal stresses. Furthermore, living cells with highly organized cytoskeletal networks exhibited greater values of the elastic modulus. The early responses of the cells predicted their late response including matrix mineralization: tall and dense submicron pillars significantly upregulated the expression of osteopontin after 21 days of culture under both osteogenic and nonosteogenic conditions. Our findings paint a detailed picture of at least one possible cascade of events that starts from initial cell adhesion and continues to subsequent cellular functions and eventual matrix mineralization. These observations could inform the future developments of instructive surfaces for medical devices based on physical surface cues and early markers.

Curvature Induced by Deflection in Thick Meta-Plates.

The design of advanced functional devices often requires the use of intrinsically curved geometries that belong to the realm of non-Euclidean geometry and remain a challenge for traditional engineering approaches. Here, it is shown how the simple deflection of thick meta-plates based on hexagonal cellular mesostructures can be used to achieve a wide range of intrinsic (i.e., Gaussian) curvatures, including dome-like and saddle-like shapes. Depending on the unit cell structure, non-auxetic (i.e., positive Poisson ratio) or auxetic (i.e., negative Poisson ratio) plates can be obtained, leading to a negative or positive value of the Gaussian curvature upon bending, respectively. It is found that bending such meta-plates along their longitudinal direction induces a curvature along their transverse direction. Experimentally and numerically, it is shown how the amplitude of this induced curvature is related to the longitudinal bending and the geometry of the meta-plate. The approach proposed here constitutes a general route for the rational design of advanced functional devices with intrinsically curved geometries. To demonstrate the merits of this approach, a scaling relationship is presented, and its validity is demonstrated by applying it to 3D-printed microscale meta-plates. Several applications for adaptive optical devices with adjustable focal length and soft wearable robotics are presented.

Spiral Honeycomb Microstructured Bacterial Cellulose for Increased Strength and Toughness.

Natural materials, such as nacre and silk, exhibit both high strength and toughness due to their hierarchical structures highly organized at the nano-, micro-, and macroscales. Bacterial cellulose (BC) presents a hierarchical fibril structure at the nanoscale. At the microscale, however, BC nanofibers are distributed randomly. Here, BC self-assembles into a highly organized spiral honeycomb microstructure giving rise to a high tensile strength (315 MPa) and a high toughness value (17.8 MJ m-3), with pull-out and de-spiral morphologies observed during failure. Both experiments and finite-element simulations indicate improved mechanical properties resulting from the honeycomb structure. The mild fabrication process consists of an in situ fermentation step utilizing poly(vinyl alcohol), followed by a post-treatment including freezing-thawing and boiling. This simple self-assembly production process is highly scalable, does not require any toxic chemicals, and enables the fabrication of light, strong, and tough hierarchical composite materials with tunable shape and size.

Quantitative mechanics of 3D printed nanopillars interacting with bacterial cells.

One of the methods to create sub-10 nm resolution metal-composed 3D nanopillars is electron beam-induced deposition (EBID). Surface nanotopographies (e.g., nanopillars) could play an important role in the design and fabrication of implantable medical devices by preventing the infections that are caused by the bacterial colonization of the implant surface. The mechanical properties of such nanoscale structures can influence their bactericidal efficiency. In addition, these properties are key factors in determining the fate of stem cells. In this study, we quantified the relevant mechanical properties of EBID nanopillars interacting with Staphylococcus aureus (S. aureus) using atomic force microscopy (AFM). We first determined the elastic modulus (17.7 GPa) and the fracture stress (3.0 ± 0.3 GPa) of the nanopillars using the quantitative imaging (QI) mode and contact mode (CM) of AFM. The displacement of the nanopillars interacting with the bacteria cells was measured by scanning electron microscopy (50.3 ± 9.0 nm). Finite element method based simulations were then applied to obtain the force-displacement curve of the nanopillars (considering the specified dimensions and the measured value of the elastic modulus) based on which an interaction force of 88.7 ± 36.1 nN was determined. The maximum von Mises stress of the nanopillars subjected to these forces was also determined (3.2 ± 0.3 GPa). These values were close to the maximum (i.e., fracture) stress of the pillars as measured by AFM, indicating that the nanopillars were close to their breaking point while interacting with S. aureus. These findings reveal unique quantitative data regarding the mechanical properties of nanopillars interacting with bacterial cells and highlight the possibilities of enhancing the bactericidal activity of the investigated EBID nanopillars by adjusting both their geometry and mechanical properties.

Fracture Behavior of Bio-Inspired Functionally Graded Soft-Hard Composites Made by Multi-Material 3D Printing: The Case of Colinear Cracks.

The functional gradient is a concept often occurring in nature. This concept can be implemented in the design and fabrication of advanced materials with specific functionalities and properties. Functionally graded materials (FGMs) can effectively eliminate the interface problems in extremely hard-soft connections, and, thus, have numerous and diverse applications in high-tech industries, such as those in biomedical and aerospace fields. Here, using voxel-based multi-material additive manufacturing (AM, = 3D printing) techniques, which works on the basis of material jetting, we studied the fracture behavior of functionally graded soft-hard composites with a pre-existing crack colinear with the gradient direction. We designed, additively manufactured, and mechanically tested the two main types of functionally graded composites, namely, composites with step-wise and continuous gradients. In addition, we changed the length of the transition zone between the hard and soft materials such that it covered 5%, 25%, 50%, or 100% of the width (W) of the specimens. The results showed that except for the fracture strain, the fracture properties of the graded specimens decreased as the length of the transition zone increased. Additionally, it was found that specimens with abrupt hard-soft transitions have significantly better fracture properties than those with continuous gradients. Among the composites with gradients, those with step-wise gradients showed a slightly better fracture resistance compared to those with continuous gradients. In contrast, FGMs with continuous gradients showed higher values of elastic stiffness and fracture energy, which makes each gradient function suitable for different loading scenarios. Moreover, regardless of the gradient function used in the design of the specimens, decreasing the length of the transition zone from 100%W to 5%W increased the fracture resistance of FGMs. We discuss the important underlying fracture mechanisms using data collected from digital image correlation (DIC), digital image microscopy, and scanning electron microscopy (SEM), which were used to analyze the fracture surface.

Determinants of bone damage: An ex-vivo study on porcine vertebrae.

Bone's resistance to fracture depends on several factors, such as bone mass, microarchitecture, and tissue material properties. The clinical assessment of bone strength is generally performed by Dual-X Ray Photon Absorptiometry (DXA), measuring bone mineral density (BMD) and trabecular bone score (TBS). Although it is considered the major predictor of bone strength, BMD only accounts for about 70% of fragility fractures, while the remaining 30% could be described by bone "quality" impairment parameters, mainly related to tissue microarchitecture. The assessment of bone microarchitecture generally requires more invasive techniques, which are not applicable in routine clinical practice, or X-Ray based imaging techniques, requiring a longer post-processing. Another important aspect is the presence of local damage in the bony tissue that may also affect the prediction of bone strength and fracture risk. To provide a more comprehensive analysis of bone quality and quantity, and to assess the effect of damage, here we adopt a framework that includes clinical, morphological, and mechanical analyses, carried out by means of DXA, µCT and mechanical compressive testing, respectively. This study has been carried out on trabecular bones, taken from porcine trabecular vertebrae, for the similarity with human lumbar spine. This study confirms that no single method can provide a complete characterization of bone tissue, and the combination of complementary characterization techniques is required for an accurate and exhaustive description of bone status. BMD and TBS have shown to be complementary parameters to assess bone strength, the former assessing the bone quantity and resistance to damage, and the latter the bone quality and the presence of damage accumulation without being able to predict the risk of fracture.

Mechanical properties of cortical bone and their relationships with age, gender, composition and microindentation properties in the elderly.

The growing incidence of skeletal fractures poses a significant challenge to ageing societies. Since a major part of physiological loading in the lower limbs is carried by cortical bone, it would be desirable to better understand the structure-mechanical property relationships and scale effects in this tissue. This study aimed at assessing whether microindentation properties combined with chemical and morphological information are usable to predict macroscopic elastic and strength properties in a donor- and site-matched manner. Specimens for quasi-static macroscopic tests in tension, compression, and torsion and microindentation were prepared from a cohort of 19 male and 20 female donors (46 to 99 years). All tests were performed under fully hydrated conditions. The chemical composition of the extra-cellular matrix was investigated with Raman spectroscopy. The results of the micro-mechanical tests were combined with morphological and compositional properties using a power law relationship to predict the macro-mechanical results. Microindentation properties were not gender dependent, remarkably constant over age, and showed an overall small variation with standard deviations of approximately 10 %. Similar results were obtained for chemical tissue composition. Macro-mechanical stiffness and strength were significantly related to porosity for all load cases (p<0.05). In case of macroscopic yield strain and work-to-failure this was only true in torsion and compression, respectively. The correlations of macro-mechanical with micro-mechanical, morphological, and chemical properties showed no significance for cement line density, mineralisation, or variations in the microindentation results and were dominated by porosity with a moderate explanatory power of predominately less than 50 %. The results confirm that age, with minor exceptions gender, and small variations in average mineralisation have negligible effect on the tissue microindentation properties of human lamellar bone in the elderly. Furthermore, our findings suggest that microindentation experiments are suitable to predict macroscopic mechanical properties in the elderly only on average and not on a one to one basis. The presented data may help to form a better understanding of the mechanisms of ageing in bone tissue and of the length scale at which they are active. This may be used for future prediction of fracture risk in the elderly.

Continuum damage interactions between tension and compression in osteonal bone.

Skeletal diseases such as osteoporosis impose a severe socio-economic burden to ageing societies. Decreasing mechanical competence causes a rise in bone fracture incidence and mortality especially after the age of 65 y. The mechanisms of how bone damage is accumulated under different loading modes and its impact on bone strength are unclear. We hypothesise that damage accumulated in one loading mode increases the fracture risk in another. This study aimed at identifying continuum damage interactions between tensile and compressive loading modes. We propose and identify the material constants of a novel piecewise 1D constitutive model capable of describing the mechanical response of bone in combined tensile and compressive loading histories. We performed several sets of loading-reloading experiments to compute stiffness, plastic strains, and stress-strain curves. For tensile overloading, a stiffness reduction (damage) of 60% at 0.65% accumulated plastic strain was detectable as stiffness reduction of 20% under compression. For compressive overloading, 60% damage at 0.75% plastic strain was detectable as a stiffness reduction of 50% in tension. Plastic strain at ultimate stress was the same in tension and compression. Compression showed softening and tension exponential hardening in the post-yield regime. The hardening behaviour in compression is unaffected by a previous overload in tension but the hardening behaviour in tension is affected by a previous overload in compression as tensile reloading strength is significantly reduced. This paper demonstrates how damage accumulated under one loading mode affects the mechanical behaviour in another loading mode. To explain this and to illustrate a possible implementation we proposed a theoretical model. Including such loading mode dependent damage and plasticity behaviour in finite element models will help to improve fracture risk analysis of whole bones and bone implant structures.