Novel Sheep Model to Assess Critical-Sized Bone Regeneration with Periosteum for In Vivo Bioreactors.

Considerable research is being undertaken to develop novel biomaterials-based approaches for surgical reconstruction of bone defects. This extends to three-dimensional (3D) printed materials that provide stable, structural, and functional support in vivo. However, few preclinical models can simulate in vivo human biological conditions for clinically relevant testing. In this study we describe a novel ovine model that allows evaluation of in vivo osteogenesis via contact with bone and/or periosteum interfaced with printed polymer bioreactors loaded with biomaterial bone substitutes. The infraspinous scapular region of 14 Dorset cross sheep was exposed. Vascularized periosteum was elevated either attached to the infraspinatus muscle or separately. In both cases, the periosteum was supplied by the periosteal branch of the circumflex scapular vessels. In eight sheep, a 3D printed 4-chambered polyetheretherketone bioreactor was wrapped circumferentially in vascularized periosteum. In 6 sheep, 12 double-sided 3D printed 2-chambered polyetherketone bioreactors were secured to the underlying bone allowing direct contact with the bone on one side and periosteum on the other. Our model enabled simultaneous testing of up to 24 (12 double-sided) 10 × 10 × 5 mm bioreactors per scapula in the flat contact approach or a single 40 × 10 mm four-chambered bioreactor per scapula using the periosteal wrap. De novo bone growth was evaluated using histological and radiological analysis. Of importance, the experimental model was well tolerated by the animals and provides a versatile approach for comparing the osteogenic potential of cambium on the bone surface and elevated with periosteum. Furthermore, the periosteal flaps were sufficiently large for encasing bioreactors containing biomaterial bone substitutes for applications such as segmental mandibular reconstruction.

From Free Tissue Transfer to Hydrogels: A Brief Review of the Application of the Periosteum in Bone Regeneration.

The periosteum is a thin layer of connective tissue covering bone. It is an essential component for bone development and fracture healing. There has been considerable research exploring the application of the periosteum in bone regeneration since the 19th century. An increasing number of studies are focusing on periosteal progenitor cells found within the periosteum and the use of hydrogels as scaffold materials for periosteum engineering and guided bone development. Here, we provide an overview of the research investigating the use of the periosteum for bone repair, with consideration given to the anatomy and function of the periosteum, the importance of the cambium layer, the culture of periosteal progenitor cells, periosteum-induced ossification, periosteal perfusion, periosteum engineering, scaffold vascularization, and hydrogel-based synthetic periostea.

Ex Vivo Preservation of Ovine Periosteum Using a Perfusion Bioreactor System.

Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.

Considerations for modelling diffuse high-grade gliomas and developing clinically relevant therapies.

Diffuse high-grade gliomas contain some of the most dangerous human cancers that lack curative treatment options. The recent molecular stratification of gliomas by the World Health Organisation in 2021 is expected to improve outcomes for patients in neuro-oncology through the development of treatments targeted to specific tumour types. Despite this promise, research is hindered by the lack of preclinical modelling platforms capable of recapitulating the heterogeneity and cellular phenotypes of tumours residing in their native human brain microenvironment. The microenvironment provides cues to subsets of glioma cells that influence proliferation, survival, and gene expression, thus altering susceptibility to therapeutic intervention. As such, conventional in vitro cellular models poorly reflect the varied responses to chemotherapy and radiotherapy seen in these diverse cellular states that differ in transcriptional profile and differentiation status. In an effort to improve the relevance of traditional modelling platforms, recent attention has focused on human pluripotent stem cell-based and tissue engineering techniques, such as three-dimensional (3D) bioprinting and microfluidic devices. The proper application of these exciting new technologies with consideration of tumour heterogeneity and microenvironmental interactions holds potential to develop more applicable models and clinically relevant therapies. In doing so, we will have a better chance of translating preclinical research findings to patient populations, thereby addressing the current derisory oncology clinical trial success rate.

Electro-compacted collagen for corneal epithelial tissue engineering.

Bioengineered corneal substitutes offer a solution to the shortage of donor corneal tissue worldwide. As one of the major structural components of the cornea, collagen has shown great potential for tissue-engineered cornea substitutes. Herein, free-standing collagen membranes fabricated using electro-compaction were assessed in corneal bioengineering application by comparing them with nonelectro-compacted collagen (NECC). The well-organized and biomimetic fibril structure resulted in a significant improvement in mechanical properties. A 10-fold increase in tensile and compressive modulus was recorded when compared with NECC membranes. In addition to comparable transparency in the visible light range, the glucose permeability of the electro-compacted collagen (ECC) membrane is higher than that of the native human cornea. Human corneal epithelial cells adhere and proliferate well on the ECC membrane, with a large cell contact area observed. The as-described ECC has appropriate structural, topographic, mechanical, optical, glucose permeable, and cell support properties to provide a platform for a bioengineered cornea; including the outer corneal epithelium and potentially deeper corneal tissues.

Induced pluripotent stem cells for 2D and 3D modelling the biological basis of schizophrenia and screening possible therapeutics.

Induced pluripotent stem cells (iPSCs) are providing unprecedented insight into complex neuropsychiatric disorders such as schizophrenia (SZ). Here we review the use of iPSCs for investigating the etiopathology and treatment of SZ, beginning with conventional in vitro two-dimensional (2D; monolayer) cell modelling, through to more advanced 3D tissue studies. With the advent of 3D modelling, utilising advanced differentiation paradigms and additive manufacturing technologies, inclusive of patient-specific cerebral/neural organoids and bioprinted neural tissues, such live disease-relevant tissue systems better recapitulate "within-body" tissue function and pathobiology. We posit that by enabling better understanding of biological causality, these evolving strategies will yield novel therapeutic targets and accordingly, drug candidates.

Bioengineering Clinically Relevant Cardiomyocytes and Cardiac Tissues from Pluripotent Stem Cells.

The regenerative capacity of cardiomyocytes is insufficient to functionally recover damaged tissue, and as such, ischaemic heart disease forms the largest proportion of cardiovascular associated deaths. Human-induced pluripotent stem cells (hiPSCs) have enormous potential for developing patient specific cardiomyocytes for modelling heart disease, patient-based cardiac toxicity testing and potentially replacement therapy. However, traditional protocols for hiPSC-derived cardiomyocytes yield mixed populations of atrial, ventricular and nodal-like cells with immature cardiac properties. New insights gleaned from embryonic heart development have progressed the precise production of subtype-specific hiPSC-derived cardiomyocytes; however, their physiological immaturity severely limits their utility as model systems and their use for drug screening and cell therapy. The long-entrenched challenges in this field are being addressed by innovative bioengingeering technologies that incorporate biophysical, biochemical and more recently biomimetic electrical cues, with the latter having the potential to be used to both direct hiPSC differentiation and augment maturation and the function of derived cardiomyocytes and cardiac tissues by mimicking endogenous electric fields.

Engineering in vitro human neural tissue analogs by 3D bioprinting and electrostimulation.

There is a fundamental need for clinically relevant, reproducible, and standardized in vitro human neural tissue models, not least of all to study heterogenic and complex human-specific neurological (such as neuropsychiatric) disorders. Construction of three-dimensional (3D) bioprinted neural tissues from native human-derived stem cells (e.g., neural stem cells) and human pluripotent stem cells (e.g., induced pluripotent) in particular is appreciably impacting research and conceivably clinical translation. Given the ability to artificially and favorably regulate a cell's survival and behavior by manipulating its biophysical environment, careful consideration of the printing technique, supporting biomaterial and specific exogenously delivered stimuli, is both required and advantageous. By doing so, there exists an opportunity, more than ever before, to engineer advanced and precise tissue analogs that closely recapitulate the morphological and functional elements of natural tissues (healthy or diseased). Importantly, the application of electrical stimulation as a method of enhancing printed tissue development in vitro, including neuritogenesis, synaptogenesis, and cellular maturation, has the added advantage of modeling both traditional and new stimulation platforms, toward improved understanding of efficacy and innovative electroceutical development and application.

Biomimetic corneal stroma using electro-compacted collagen.

Engineering substantia propria (or stroma of cornea) that mimics the function and anatomy of natural tissue is vital for in vitro modelling and in vivo regeneration. There are, however, few examples of bioengineered biomimetic corneal stroma. Here we describe the construction of an orthogonally oriented 3D corneal stroma model (3D-CSM) using pure electro-compacted collagen (EC). EC films comprise aligned collagen fibrils and support primary human corneal stromal cells (hCSCs). Cell-laden constructs are analogous to the anatomical structure of native human cornea. The hCSCs are guided by the topographical cues provided by the aligned collagen fibrils of the EC films. Importantly, the 3D-CSM are biodegradable, highly transparent, glucose-permeable and comprise quiescent hCSCs. Gene expression analysis indicated the presence of aligned collagen fibrils is strongly coupled to downregulation of active fibroblast/myofibroblast markers α-SMA and Thy-1, with a concomitant upregulation of the dormant keratocyte marker ALDH3. The 3D-CSM represents the first example of an optimally robust biomimetic engineered corneal stroma that is constructed from pure electro-compacted collagen for cell and tissue support. The 3D-CSM is a significant advance for synthetic corneal stroma engineering, with the potential to be used for full-thickness and functional cornea replacement, as well as informing in vivo tissue regeneration. STATEMENT OF SIGNIFICANCE: This manuscript represents the first example of a robust, transparent, glucose permeable and pure collagen-based biomimetic 3D corneal stromal model (3D-CSM) constructed from pure electro-compacted collagen. The collagen fibrils of 3D-CSM are aligned and orthogonally arranged, mimicking native human corneal stroma. The alignment of collagen fibrils correlates with the direction of current applied for electro-compaction and influences human corneal stromal cell (hCSC) orientation. Moreover, 3D-CSM constructs support a corneal keratocyte phenotype; an essential requirement for modelling healthy corneal stroma. As-prepared 3D-CSM hold great promise as corneal stromal substitutes for research and translation, with the potential to be used for full-thickness cornea replacement.

3D Bioprinting Electrically Conductive Bioink with Human Neural Stem Cells for Human Neural Tissues.

Bioprinting cells with an electrically conductive bioink provides an opportunity to produce three-dimensional (3D) cell-laden constructs with the option of electrically stimulating cells in situ during and after tissue development. We and others have demonstrated the use of electrical stimulation (ES) to influence cell behavior and function for a more biomimetic approach to tissue engineering. Here, we detail a previously published method for 3D printing an electrically conductive bioink with human neural stem cells (hNSCs) that are subsequently differentiated. The differentiated tissue constructs comprise functional neurons and supporting neuroglia and are amenable to ES for the purposeful modulation of neural activity. Importantly, the method could be adapted to fabricate and stimulate neural and nonneural tissues from other cell types, with the potential to be applied for both research- and clinical-product development.

Bioprinting 3D Human Induced Pluripotent Stem Cell Constructs for Multilineage Tissue Engineering and Modeling.

Bioprinting human pluripotent stem cells (PSCs) provides an opportunity to produce three-dimensional (3D) cell-laden constructs with the potential to be differentiated in vitro to all tissue types of the human body. Here, we detail a previously published method for 3D printing human induced pluripotent stem cells (iPSCs; also applicable to human embryonic stem cells) within a clinically amenable bioink (also described in Chapter 10 ) that is cross-linked to a 3D construct. The printed iPSCs continue to have self-replicating and multilineage cell induction potential in situ, and the constructs are robust and amenable to different differentiation protocols for fabricating diverse tissue types, with the potential to be applied for both research- and clinical-product development.

Conducting Polymer Mediated Electrical Stimulation Induces Multilineage Differentiation with Robust Neuronal Fate Determination of Human Induced Pluripotent Stem Cells.

Electrical stimulation is increasingly being used to modulate human cell behaviour for biotechnological research and therapeutics. Electrically conductive polymers (CPs) such as polypyrrole (PPy) are amenable to in vitro and in vivo cell stimulation, being easy to synthesise with different counter ions (dopants) to augment biocompatibility and cell-effects. Extending our earlier work, which showed that CP-mediated electrical stimulation promotes human neural stem cell differentiation, here we report using electroactive PPy containing the anionic dopant dodecylbenzenesulfonate (DBS) to modulate the fate determination of human induced pluripotent stem cells (iPSCs). Remarkably, the stimulation without conventional chemical inducers resulted in the iPSCs differentiating to cells of the three germ lineages-endoderm, ectoderm, and mesoderm. The unstimulated iPSC controls remained undifferentiated. Phenotypic characterisation further showed a robust induction to neuronal fate with electrical stimulation, again without customary chemical inducers. Our findings add to the growing body of evidence supporting the use of electrical stimulation to augment stem cell differentiation, more specifically, pluripotent stem cell differentiation, and especially neuronal induction. Moreover, we have shown the versatility of electroactive PPy as a cell-compatible platform for advanced stem cell research and translation, including identifying novel mechanisms of fate regulation, tissue development, electroceuticals, and regenerative medicine.

Molecular interactions and forces of adhesion between single human neural stem cells and gelatin methacrylate hydrogels of varying stiffness.

Single Cell Force Spectroscopy was applied to measure the single cell de-adhesion between human neural stem cells (hNSC) and gelatin methacrylate (GelMA) hydrogel with varying modulus in the range equivalent to brain tissue. The cell de-adhesion force and energy were predominately generated via unbinding of complexes formed between RGD groups of the GelMA and cell surface integrin receptors and the de-adhesion force/energy were found to increase with decreasing modulus of the GelMA hydrogel. For the softer GelMA hydrogels (160 Pa and 450 Pa) it was proposed that a lower degree of cross-linking enables a greater number of polymer chains to bind and freely extend to increase the force and energy of the hNSC-GelMA de-adhesion. In this case, the multiple polymer chains are believed to act together in parallel like 'molecular tensors' to generate tensile forces on the bound receptors until the cell detaches. Counterintuitively for softer substrates, this type of interaction gave rise to higher force loading rates, including the appearance of high and low dynamic force regimes in de-adhesion rupture force versus loading rate analysis. For the stiffer GelMA hydrogel (900 Pa) it was observed that the extension and elastic restoring forces of the polymer chains contributed less to the cell de-adhesion. Due to the apparent lower extent of freely interacting chains on the stiffer GelMA hydrogel the intrinsic RGD groups are presumed to be "more fixed" to the substrate. Hence, the cell de-adhesion is suggested to be mainly governed by the discrete unbinding of integrin-RGD complexes as opposed to elastic restoring forces of polymer chains, leading to smaller piconewton rupture forces and only a single lower dynamic force regime. Intriguingly, when integrin antibodies were introduced for binding integrin α5ß1, ß1- and αv-subunits it was revealed that the cell modifies the de-adhesion force depending on the substrate stiffness. The antibody binding supressed the de-adhesion on the softer GelMA hydrogel while on the stiffer GelMA hydrogel caused an opposing reinforcement in the de-adhesion. STATEMENT OF SIGNIFICANCE: Conceptual models on cell mechanosensing have provided molecular-level insight to rationalize the effects of substrate stiffness. However most experimental studies evaluate the cell adhesion by analysing the bulk material properties. As such there is a discrepancy in the scale between the bulk properties versus the nano- and micro-scale cell interactions. Furthermore there is a paucity of experimental studies on directly measuring the molecular-level forces of cell-material interactions. Here we apply Single Cell Force Spectroscopy to directly measure the adhesion forces between human neural stem cells and gelatin-methacrylate hydrogel. We elucidate the mechanisms by which single cells bind and physically interact with hydrogels of varying stiffness. The study highlights the use of single cell analysis tools to probe molecular-level interactions at the cell-material interface which is of importance in designing material cues for regulating cell function.

Human Neural Tissues from Neural Stem Cells Using Conductive Biogel and Printed Polymer Microelectrode Arrays for 3D Electrical Stimulation.

Electricity is important in the physiology and development of human tissues such as embryonic and fetal development, and tissue regeneration for wound healing. Accordingly, electrical stimulation (ES) is increasingly being applied to influence cell behavior and function for a biomimetic approach to in vitro cell culture and tissue engineering. Here, the application of conductive polymer (CP) poly(3,4-ethylenedioxythiophene)-polystyrenesulfonate (PEDOT:PSS) pillars is described, direct-write printed in an array format, for 3D ES of maturing neural tissues that are derived from human neural stem cells (NSCs). NSCs are initially encapsulated within a conductive polysaccharide-based biogel interfaced with the CP pillar microelectrode arrays (MEAs), followed by differentiation in situ to neurons and supporting neuroglia during stimulation. Electrochemical properties of the pillar electrodes and the biogel support their electrical performance. Remarkably, stimulated constructs are characterized by widespread tracts of high-density mature neurons and enhanced maturation of functional neural networks. Formation of tissues using the 3D MEAs substantiates the platform for advanced clinically relevant neural tissue induction, with the system likely amendable to diverse cell types to create other neural and non-neural tissues. The platform may be useful for both research and translation, including modeling tissue development, function and dysfunction, electroceuticals, drug screening, and regenerative medicine.

Clinically Amendable, Defined, and Rapid Induction of Human Brain Organoids from Induced Pluripotent Stem Cells.

Human brain organoids provide opportunities to produce three-dimensional (3D) brain-like tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a protocol for rapid and defined induction of brain organoids from human induced pluripotent stem cells (iPSCs), using commercially available culture and differentiation media and a cheap, easy to handle and clinically approved semisynthetic hydrogel. Importantly, the methodology is uncomplicated, well-defined, and reliable for reproducible and scalable organoid generation, and amendable to principles of current good laboratory practice (cGLP), with the potential for prospective adaptation to current good manufacturing practice (cGMP) toward clinical compliance.

Smart graphene-cellulose paper for 2D or 3D "origami-inspired" human stem cell support and differentiation.

Graphene-based materials represent advanced platforms for tissue engineering and implantable medical devices. From a clinical standpoint, it is essential that these materials are produced using non-toxic and non-hazardous methods, and have predictable properties and reliable performance under variable physiological conditions; especially when used with a cellular component. Here we describe such a biomaterial, namely smart graphene-cellulose (G-C) paper, and its suitability for traditional planar two-dimensional (2D) or three-dimensional (3D) human cell support, verified by adipose-derived stem cell (ADSC) culture and osteogenic differentiation. G-C paper is prepared using commercially available cellulose tissue paper as a substrate that is coated by immersion-deposition with graphene oxide (GO) followed by reduction to reduced graphene oxide (RGO) without the use of toxic organic solvents. The fabrication process is amenable to large scale production and the resultant papers have low electrical resistivity (up to ∼300 Ω/sq). Importantly, G-C papers can be configured to 3D constructs by lamination with alginate and further modified by folding and rolling for 3D "origami-inspired" cell-laden structures.

Engineering Human Neural Tissue by 3D Bioprinting.

Bioprinting provides an opportunity to produce three-dimensional (3D) tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a method for fabricating human neural tissue by 3D printing human neural stem cells with a bioink, and subsequent gelation of the bioink for cell encapsulation, support, and differentiation to functional neurons and supporting neuroglia. The bioink uniquely comprises the polysaccharides alginate, water-soluble carboxymethyl-chitosan, and agarose. Importantly, the method could be adapted to fabricate neural and nonneural tissues from other cell types, with the potential to be applied for both research and clinical product development.

Electro-mechano responsive properties of gelatin methacrylate (GelMA) hydrogel on conducting polymer electrodes quantified using atomic force microscopy.

Electrical stimulation of hydrogels has been performed to enable micro-actuation or controlled movement of ions and biomolecules such as in drug release applications. Hydrogels are also increasingly used as low modulus, biocompatible coatings on electrode devices and thus are exposed to the effects of electrical stimulation. As such, there is growing interest in the latter, especially on the dynamic and nanoscale physical properties of hydrogels. Here, we report on the electro-mechano properties of photocrosslinkable gelatin methacrylate (GelMA) hydrogel applied as coatings on conducting polymer polypyrrole-dodecylbenze sulfonate (PPy-DBSA) electrodes. In particular, Electrochemical-Atomic Force Microscopy (EC-AFM) was used to quantify the nanoscale actuation and dynamic changes in Young's modulus as the GelMA coating was electrically stimulated via the underlying PPy-DBSA electrode. Pulsed electrical stimulation was shown to induce dynamic expansion and contraction, or nanoscale actuation, of the GelMA hydrogel due to the reversible ingress of electrolyte ions and associated changes in osmotic pressure during oxidation and reduction of the PPy-DBSA film. In addition, dynamic changes in the Young's modulus of up to 50% were observed in the hydrogel and correlated with the actuation process and ion diffusion during oxidation and reduction of the underlying PPy-DBSA film. These dynamic properties were investigated for hydrogels with varying degrees of cross-linking, porosity and modulus, the latter ranging from ≈0.2-1 kPa. The study demonstrates an AFM-based approach to quantify the dynamic physical properties of hydrogels, which are shown to be modulated via electrical stimulation. This can enable a better understanding of the electro-mechano mechanisms that are important for the controlled release of drugs or controlling cell interactions at the hydrogel-cell interface.

3D Bioprinting Human Induced Pluripotent Stem Cell Constructs for In Situ Cell Proliferation and Successive Multilineage Differentiation.

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine, including individualized, patient-specific stem cell-based treatments. There are, however, few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues, ideally comprising direct-write printing of cells for encapsulation, proliferation, and differentiation. Here, such a method, employing a clinically amenable polysaccharide-based bioink, is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically, we have extrusion printed the bioink including iPSCs, alginate (Al; 5% weight/volume [w/v]), carboxymethyl-chitosan (5% w/v), and agarose (Ag; 1.5% w/v), crosslinked the bioink in calcium chloride for a stable and porous construct, proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm, ectoderm, and mesoderm, or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined, scalable, and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine.

Cryobanking Pluripotent Stem Cells.

Cryobanking human pluripotent stem cells (hPSCs), be they human embryonic (hESCs) or induced pluripotent stem cells (iPSCs), is essential for their use in research and cell-based therapeutics. Working and master cell banks can be generated with a desired level of quality assurance applied during cell freezing and storage. Conventional vitrification has evolved to more advanced control rate freezing, culminating in a myriad of published protocols with variable proficiencies and clinical efficacies. Notwithstanding, standardized and reliable protocols are necessary for basic science through to applied research and clinical product development. This chapter details several methods for hPSC cryopreservation, suitable for routine application, high-quality research, and adaptable for clinical compliance.