Hierarchical porous materials for tissue engineering

Biological organisms have evolved to produce hierarchical three-dimensional structures with dimensions ranging from nanometres to metres. Replicating these complex living hierarchical structures for the purpose of repair or replacement of degenerating tissues is one of the great challenges of chemistry, physics, biology and materials science. This paper describes how the use of hierarchical porous materials in tissue engineering applications has the potential to shift treatments from tissue replacement to tissue regeneration. The criteria that a porous material must fulfil to be considered ideal for bone tissue engineering applications are listed. Bioactive glass foam scaffolds have the potential to fulfil all the criteria, as they have a hierarchical porous structure similar to that of trabecular bone, they can bond to bone and soft tissue and they release silicon and calcium ions that have been found to up-regulate seven families of genes in osteogenic cells. Their hierarchical structure can be tailored for the required rate of tissue bonding, resorption and delivery of dissolution products. This paper describes how the structure and properties of the scaffolds are being optimized with respect to cell response and that tissue culture techniques must be optimized to enable growth of new bone in vitro.     

The role of nanoscale architecture in supramolecular templating of biomimetic hydroxyapatite mineralization

Understanding and mimicking the hierarchical structure of mineralized tissue is a challenge in the field of biomineralization and is important for the development of scaffolds to guide bone regeneration. Bone is a remarkable tissue with an organic matrix comprised of aligned collagen bundles embedded with nanometer-sized inorganic hydroxyapatite (HAP) crystals that exhibit orientation on the macroscale. Hybrid organic-inorganic structures mimic the composition of mineralized tissue for functional bone scaffolds, but the relationship between morphology of the organic matrix and orientation of mineral is poorly understood. Herein the mineralization of supramolecular peptide amphiphile templates, that are designed to vary in nanoscale morphology by altering the amino acid sequence, is reported. It is found that 1D cylindrical nanostructures direct the growth of oriented HAP crystals, while flatter nanostructures fail to guide the orientation found in biological systems. The geometric constraints associated with the morphology of the nanostructures may effectively control HAP nucleation and growth. Additionally, the mineralization of macroscopically aligned bundles of the nanoscale assemblies to create hierarchically ordered scaffolds is explored. Again, it is found that only aligned gel templates of cylindrical nanostructures lead to hierarchical control over hydroxyapatite orientation across multiple length scales as found in bone.     

Nanostructured polymeric scaffolds for orthopaedic regenerative engineering

Successful regeneration necessitates the development of three-dimensional (3-D) tissue-inducing scaffolds that mimic the hierarchical architecture of native tissue extracellular matrix (ECM). Cells in nature recognize and interact with the surface topography they are exposed to via ECM proteins. The interaction of cells with nanotopographical features such as pores, ridges, groves, fibers, nodes, and their combinations has proven to be an important signaling modality in controlling cellular processes. Integrating nanotopographical cues is especially important in engineering complex tissues that have multiple cell types and require precisely defined cell-cell and cell-matrix interactions on the nanoscale. Thus, in a regenerative engineering approach, nanoscale materials/scaffolds play a paramount role in controlling cell fate and the consequent regenerative capacity. Advances in nanotechnology have generated a new toolbox for the fabrication of tissue-specific nanostructured scaffolds. For example, biodegradable polymers such as polyesters, polyphosphazenes, polymer blends and composites can be electrospun into ECM-mimicking matrices composed of nanofibers, which provide high surface area for cell attachment, growth, and differentiation. This review provides the fundamental guidelines for the design and development of nanostructured scaffolds for the regeneration of various tissue types in human upper and lower extremities such as skin, ligament, tendon, and bone. Examples focusing on the collective work of our laboratory in those areas are discussed to demonstrate the regenerative efficacy of this approach. Furthermore, preliminary strategies and significant challenges to integrate these individual tissues into one complex organ through regenerative engineering-based integrated graft systems are also discussed.     

Advanced biomaterials for skeletal tissue regeneration: Instructive and smart functions

The past half century has seen explosive growth in the use of medical implants. Orthopedic, cardiac, oral, maxillofacial and plastic surgeons are examples of medical specialists treating millions of patients each year by implanting devices varying from pace makers, artificial hip joints, breast and dental implants, to implantable hearing aids. All such medical implants make use of special materials, known as biomaterials, defined as “materials intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ, or function of the body” [D.F. Williams, The Williams Dictionnary of Biomaterials, Liverpool University Press, Liverpool, 1999]. While the priority for the first generation of biomaterials was inertness with living tissues, the field is shifting towards biologically active systems in order to improve their performance and to expand their use. Biomaterials can be combined as scaffolds with cells (i.e. tissue engineering), growth factors or genetic material in order to trigger tissue regeneration. In addition, recent reports have shown the possibility to design biomaterials that can activate cellular processes and tissue formation solely by their intrinsic physicochemical and three dimensional spatial properties. This article reviews the recent developments in the design of biomaterials that integrate our understanding of cellular and molecular mechanisms with materials science. After an overview of the physicochemical and biological processes occurring at the interface between the biomaterials and biological milieu, we will address the biological principles contributing to the design and engineering of advanced biomaterials for application towards recent therapeutic strategies for tissue regeneration. Finally, future directions for the design of advanced biomaterials will be discussed.     

Mechano-biology as an optimization principle for biological tissue engineering

Rapid prototyping and direct fabrication has provided researchers and scientist with a wealth of opportunities to fabricate synthetic tissue replacements, so called scaffolds. The goal is to fill critical size defects with such materials and allow the body to slowly degrade them and build de-novo biological tissue on its place. However, for this process to take place the structural organization levels of these synthetic tissue replacements need to follow design criteria that promote cell attachment, cell proliferation, and maintain the cell's differentiated function. The scaffold's architecture defines the ultimate shape of the newly grown tissue. Furthermore, since most scaffolds are needed for tissue repair in load-bearing applications, the mechano-biological component affects tissue growth long after biochemical factors (e.g., growth hormones) or pre-seeded cells are lasting. This article describes current efforts in identifying mechano-biological principle that are believed to guide tissue formation based on biomechanical loading.     

Fabrication of three-dimensional poly(ε-caprolactone) scaffolds with hierarchical pore structures for tissue engineering

The physical properties of tissue engineering scaffolds such as microstructures play important roles in controlling cellular behaviors and neotissue formation. Among them, the pore size stands out as a key determinant factor. In the present study, we aimed to fabricate porous scaffolds with pre-defined hierarchical pore sizes, followed by examining cell growth in these scaffolds. This hierarchical porous microstructure was implemented via integrating different pore-generating methodologies, including salt leaching and thermal induced phase separation (TIPS). Specifically, large (L, 200–300 μm), medium (M, 40–50 μm) and small (S, < 10 μm) pores were able to be generated. As such, three kinds of porous scaffolds with a similar porosity of ~ 90% creating pores of either two (LS or MS) or three (LMS) different sizes were successfully prepared. The number fractions of different pores in these scaffolds were determined to confirm the hierarchical organization of pores. It was found that the interconnectivity varied due to the different pore structures. Besides, these scaffolds demonstrated similar compressive moduli under dry and hydrated states. The adhesion, proliferation, and spatial distribution of human fibroblasts within the scaffolds during a 14-day culture were evaluated with MTT assay and fluorescence microscopy. While all three scaffolds well supported the cell attachment and proliferation, the best cell spatial distribution inside scaffolds was achieved with LMS, implicating that such a controlled hierarchical microstructure would be advantageous in tissue engineering applications.     

Characterizing the hierarchical structures of bioactive sol-gel silicate glass and hybrid scaffolds for bone regeneration

Bone is the second most widely transplanted tissue after blood. Synthetic alternatives are needed that can reduce the need for transplants and regenerate bone by acting as active temporary templates for bone growth. Bioactive glasses are one of the most promising bone replacement/regeneration materials because they bond to existing bone, are degradable and stimulate new bone growth by the action of their dissolution products on cells. Sol-gel-derived bioactive glasses can be foamed to produce interconnected macropores suitable for tissue ingrowth, particularly cell migration and vascularization and cell penetration. The scaffolds fulfil many of the criteria of an ideal synthetic bone graft, but are not suitable for all bone defect sites because they are brittle. One strategy for improving toughness of the scaffolds without losing their other beneficial properties is to synthesize inorganic/organic hybrids. These hybrids have polymers introduced into the sol-gel process so that the organic and inorganic components interact at the molecular level, providing control over mechanical properties and degradation rates. However, a full understanding of how each feature or property of the glass and hybrid scaffolds affects cellular response is needed to optimize the materials and ensure long-term success and clinical products. This review focuses on the techniques that have been developed for characterizing the hierarchical structures of sol-gel glasses and hybrids, from atomic-scale amorphous networks, through the covalent bonding between components in hybrids and nanoporosity, to quantifying open macroporous networks of the scaffolds. Methods for non-destructive in situ monitoring of degradation and bioactivity mechanisms of the materials are also included.     

Drop-on-demand printing of cells and materials for designer tissue constructs

Adapting bottom-up approaches to tissue engineering is a real challenge. Since the first application of fused deposition modeling for tissue engineering scaffolds, considerable effort has been focused on printing synthetic biodegradable scaffolds. Concurrently a variety of rapid prototyping techniques have been developed to define macroscopically the shapes of deposited biomaterials, including photolithography, syringe-based gel deposition, and solid freeform fabrication. These designed scaffolds have shown promise in regenerating tissues at least equivalent to other scaffolding methods.An exciting advance in scaffold aided tissue regeneration is presented here, that of cell and organ printing, which allows direct printing of cells and proteins within 3D hydrogel structures. Cell printing opens the possibility to programmed deposition of scaffold structure and cell type, thus controlling the type of tissue that can be regenerated within the scaffold. Several examples of printed tissues will be presented including contractile cardiac hybrids. The hybrid materials have properties that can be tailored in 3D to achieve desired porosities, mechanical and chemical properties. The materials include alginate hydrogels with controlled microshell structures that can be built by spraying cross-linkers onto ungelled alginic acid.Endothelial cells were seen to attach to the inside of these microshells. The cells remained viable in constructs as thick as 1 cm due to the programmed porosity. Finite element modeling was used to predict the mechanical properties and to generate CAD models with properties matching cardiac tissue. These results suggest that the printing method could be used for hierarchical design of functional cardiac patches, balanced with porosity for mass transport and structural support.     

Bioinspired nanoscale materials for biomedical and energy applications

The demand for green, affordable and environmentally sustainable materials has encouraged scientists in different fields to draw inspiration from nature in developing materials with unique properties such as miniaturization, hierarchical organization and adaptability. Together with the exceptional properties of nanomaterials, over the past century, the field of bioinspired nanomaterials has taken huge leaps. While on the one hand, the sophistication of hierarchical structures endows biological systems with multi-functionality, the synthetic control on the creation of nanomaterials enables the design of materials with specific functionalities. The aim of this review is to provide a comprehensive, up-to-date overview of the field of bioinspired nanomaterials, which we have broadly categorized into biotemplates and biomimics. We discuss the application of bioinspired nanomaterials as biotemplates in catalysis, nanomedicine, immunoassays and in energy, drawing attention to novel materials such as protein cages. Furthermore, the applications of bioinspired materials in tissue engineering and biomineralization are also discussed.     

New paradigms in hierarchical porous scaffold design for tissue engineering

This paper presented an effective method for the three-dimensional (3D) hierarchical porous scaffold design for tissue engineering. To achieve such a hierarchical porous structure with accurately controlled internal pore architectures, the recursive intersection Boolean operation (RIBO) was proposed in order to satisfy computational efficiency and biological function requirements of a porous scaffold. After generating the distance field (DF) for the given anatomic model and required pore architectures, the recursive DF modifications enable us to design hierarchical porous scaffolds with complex combinations of pore morphologies. A variety of experimental results showed that the proposed hierarchical porous scaffold design method has the potential benefits for accurately controlling both the porosity and the pore architecture gradients while preserving the advantages of triply periodic minimal surface pore geometries.     

Porous nanocellulose gels and foams: Breakthrough status in the development of scaffolds for tissue engineering

We report on the latest scientific advances related to the use of porous foams and gels prepared with cellulose nanofibrils (CNF) and nanocrystals (CNC) as well as bacterial nanocellulose (BNC) – collectively nanocelluloses – as biomedical materials for application in tissue regeneration. Interest in such applications stems from the lightweight and strong structures that can be efficiently produced from these nanocelluloses. Dried nanocellulose foams and gels, including xerogels, cryogels, and aerogels have been synthesized effortlessly using green, scalable, and cost-effective techniques. Methods to control structural features (e.g., porosity, morphology, and mechanical performance) and biological interactions (e.g., biocompatibility and biodegradability) are discussed in light of specific tissues of interest. The state-of-the-art in the field of nanocellulose-based scaffolds for tissue engineering is presented, covering physicochemical and biological properties relevant to these porous systems that promise groundbreaking advances. Specifically, these materials show excellent performance for in vitro cell culturing and in vivo implantation. We report on recent efforts related to BNC scaffolds used in animal and human implants, which furthermore support the viability of CNF- and CNC-based scaffolds in next-generation biomedical materials.     

Inverse Opal Scaffolds and Their Biomedical Applications

Three‐dimensional porous scaffolds play a pivotal role in tissue engineering and regenerative medicine by functioning as biomimetic substrates to manipulate cellular behaviors. While many techniques have been developed to fabricate porous scaffolds, most of them rely on stochastic processes that typically result in scaffolds with pores uncontrolled in terms of size, structure, and interconnectivity, greatly limiting their use in tissue regeneration. Inverse opal scaffolds, in contrast, possess uniform pores inheriting from the template comprised of a closely packed lattice of monodispersed microspheres. The key parameters of such scaffolds, including architecture, pore structure, porosity, and interconnectivity, can all be made uniform across the same sample and among different samples. In conjunction with a tight control over pore sizes, inverse opal scaffolds have found widespread use in biomedical applications. In this review, we provide a detailed discussion on this new class of advanced materials. After a brief introduction to their history and fabrication, we highlight the unique advantages of inverse opal scaffolds over their non‐uniform counterparts. We then showcase their broad applications in tissue engineering and regenerative medicine, followed by a summary and perspective on future directions.     

Modelling the mechanics of partially mineralized collagen fibrils,fibres and tissue

Progressive stiffening of collagen tissue by bioapatite mineral is important physiologically, but the details of this stiffening are uncertain. Unresolved questions about the details of the accommodation of bioapatite within and upon collagen''s hierarchical structure have posed a central hurdle, but recent microscopy data resolve several major questions. These data suggest how collagen accommodates bioapatite at the lowest relevant hierarchical level (collagen fibrils), and suggest several possibilities for the progressive accommodation of bioapatite at higher hierarchical length scales (fibres and tissue). We developed approximations for the stiffening of collagen across spatial hierarchies based upon these data, and connected models across hierarchies levels to estimate mineralization-dependent tissue-level mechanics. In the five possible sequences of mineralization studied, percolation of the bioapatite phase proved to be an important determinant of the degree of stiffening by bioapatite. The models were applied to study one important instance of partially mineralized tissue, which occurs at the attachment of tendon to bone. All sequences of mineralization considered reproduced experimental observations of a region of tissue between tendon and bone that is more compliant than either tendon or bone, but the size and nature of this region depended strongly upon the sequence of mineralization. These models and observations have implications for engineered tissue scaffolds at the attachment of tendon to bone, bone development and graded biomimetic attachment of dissimilar hierarchical materials in general.     

Geometric adaption of biodegradable magnesium alloy scaffolds to stabilise biological myocardial grafts. Part I

Synthetic patch materials currently in use have major limitations, such as high susceptibility to infections and lack of contractility. Biological grafts are a novel approach to overcome these limitations, but do not always offer sufficient mechanical durability in early stages after implantation. Therefore, a stabilising structure based on resorbable magnesium alloys could support the biological graft until its physiologic remodelling. To prevent early breakage in vivo due to stress of non-determined forming, these scaffolds should be preformed according to the geometry of the targeted myocardial region. Thus, the left ventricular geometry of 28 patients was assessed via standard cardiac magnetic resonance imaging (MRI). The resulting data served as a basis for a finite element simulation (FEM). Calculated stresses and strains of flat and preformed scaffolds were evaluated. Afterwards, the structures were manufactured by abrasive waterjet cutting and preformed according to the MRI data. Finally, the mechanical durability of the preformed and flat structures was compared in an in vitro test rig. The FEM predicted higher durability of the preformed scaffolds, which was proven in the in vitro test. In conclusion, preformed scaffolds provide extended durability and will facilitate more widespread use of regenerative biological grafts for surgical left ventricular reconstruction.     

3D Printing of Microgel-Loaded Modular Microcages as Instructive Scaffolds for Tissue Engineering

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based 3D printing strategy is used to fabricate a novel miniaturized modular microcage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional toy interlocking plastic blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-microcage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized microcage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance.     

Effect of electrospinning parameters on the nanofiber diameter and length

Polymer nanofibers exhibit properties that make them a favorable material for the development of tissue engineering scaffolds, filtration devices, sensors, and high strength lightweight materials. Electrospinning is a versatile method commonly used to manufacture polymer nanofibers. Collection of electrospun nanofibers across two parallel plates is a technique useful for creating nanofiber structures because it allows for the collection of linearly oriented individual nanofiber arrays and these arrays can be easily transferred to other substrates or structures. It is of importance to have some understanding of the capabilities of this collection method, such as the maximum length of fibers that can be collected across two parallel plates. The effect of different electrospinning parameters on maximum fiber length, average fiber diameter, diameter uniformity, and fiber quality was explored. It was shown that relatively long continuous polycaprolactone (PCL) nanofibers with average diameters from approximately 350 nm to 1 μm could be collected across parallel plates at lengths up to 35-50 cm. Experimental results lead to the hypothesis that even longer continuous nanofibers over 50 cm could be collected if the size of the parallel plates were increased. Extending the maximum fiber length that can be collected across parallel plates could expand the applications of electrospinning. Polymer solution concentration, plate size, and applied voltage were all shown to have varying effects on maximum fiber length, fiber diameter, and fiber uniformity.     

Theoretical and computational hierarchical nanomechanics of protein materials: Deformation and fracture

Proteins constitute the building blocks of biological materials such as tendon, bone, skin, spider silk or cells. An important trait of these materials is that they display highly characteristic hierarchical structures, across multiple scales, from nano to macro. Protein materials are intriguing examples of materials that balance multiple tasks, representing some of the most sustainable material solutions that integrate structure and function. Here we review progress in understanding the deformation and fracture mechanisms of hierarchical protein materials by using a materials science approach to develop structure-process-property relations, an effort defined as materiomics. Deformation processes begin with an erratic motion of individual atoms around flaws or defects that quickly evolve into formation of macroscopic fractures as chemical bonds rupture rapidly, eventually compromising the integrity of the structure or the biological system leading to failure. The combination of large-scale atomistic simulation, multi-scale modeling methods, theoretical analyses combined with experimental validation provides a powerful approach in studying deformation and failure phenomena in protein materials. Here we review studies focused on the molecular origin of deformation and fracture processes of three types of protein materials. The review includes studies of collagen - Nature’s super-glue; beta-sheet rich protein structures as found in spider silk - a natural fiber that can reach the strength of a steel cable; as well as intermediate filaments - a class of alpha-helix based structural proteins responsible for the mechanical integrity of eukaryotic cells. The article concludes with a discussion of the significance of universally found structural patterns such as the staggered collagen fibril architecture or the alpha-helical protein motif.     

Alternative tissue engineering scaffolds based on starch: processing methodologies, morphology, degradation and mechanical properties

An ideal tissue engineering scaffold must be designed from a polymer with an adequate degradation rate. The processing technique must allow for the preparation of 3-D scaffolds with controlled porosity and adequate pore sizes, as well as tissue matching mechanical properties and an appropriate biological response.

This communication revises recent work that has been developed in our laboratories with the aim of producing 3-D polymeric structures (from starch-based blends) with adequate properties to be used as scaffolds for bone tissue engineering applications. Several processing methodologies were originally developed and optimised. Some of these methodologies were based on conventional melt-based processing routes, such as extrusion using blowing agents (BA) and compression moulding (combined with particulate leaching). Other developed technologies included solvent casting and particle leaching and an innovative in situ polymerization method.

By means of using the described methodologies, it is possible to tailor the properties of the different scaffolds, namely their degradation, morphology and mechanical properties, for several applications in tissue engineering. Furthermore, the processing methodologies (including the blowing agents used in the melt-based technologies) described above do not affect the biocompatible behaviour of starch-based polymers. Therefore, scaffolds obtained from these materials by means of using one of the described methodologies may constitute an important alternative to the materials currently used in tissue engineering.     

25th Anniversary Article: Scalable Multiscale Patterned Structures Inspired by Nature: the Role of Hierarchy

Multiscale, hierarchically patterned surfaces, such as lotus leaves, butterfly wings, adhesion pads of gecko lizards are abundantly found in nature, where microstructures are usually used to strengthen the mechanical stability while nanostructures offer the main functionality, i.e., wettability, structural color, or dry adhesion. To emulate such hierarchical structures in nature, multiscale, multilevel patterning has been extensively utilized for the last few decades towards various applications ranging from wetting control, structural colors, to tissue scaffolds. In this review, we highlight recent advances in scalable multiscale patterning to bring about improved functions that can even surpass those found in nature, with particular focus on the analogy between natural and synthetic architectures in terms of the role of different length scales. This review is organized into four sections. First, the role and importance of multiscale, hierarchical structures is described with four representative examples. Second, recent achievements in multiscale patterning are introduced with their strengths and weaknesses. Third, four application areas of wetting control, dry adhesives, selectively filtrating membranes, and multiscale tissue scaffolds are overviewed by stressing out how and why multiscale structures need to be incorporated to carry out their performances. Finally, we present future directions and challenges for scalable, multiscale patterned surfaces.     

Scaffold design and manufacturing: from concept to clinic

Since Robert Langer and colleagues pioneered the concept of reconstructing tissue using cells transplanted on synthetic polymer matrices in the early 1990s, research in the field of tissue engineering and regenerative medicine has exploded. This is especially true in the development of new materials and structures that serve as scaffolds for tissue reconstruction. The basic tenet of the last two decades holds scaffolds as degradable materials providing temporary function while enhancing tissue regeneration through the delivery of biologics. Although a number of new scaffolding materials and structures have been developed in research laboratories, the application of such materials practice even has been extremely limited. This paper argues that better integration of all these factors is needed to bring scaffolds from "concept to clinic". It reviews current work in all these areas and suggests where future work and funding is needed.