A Preclinical Trial Protocol Using an Ovine Model to Assess Scaffold Implant Biomaterials for Repair of Critical-Sized Mandibular Defects.

The present work describes a preclinical trial (in silico, in vivo and in vitro) protocol to assess the biomechanical performance and osteogenic capability of 3D-printed polymeric scaffolds implants used to repair partial defects in a sheep mandible. The protocol spans multiple steps of the medical device development pipeline, including initial concept design of the scaffold implant, digital twin in silico finite element modeling, manufacturing of the device prototype, in vivo device implantation, and in vitro laboratory mechanical testing. First, a patient-specific one-body scaffold implant used for reconstructing a critical-sized defect along the lower border of the sheep mandible ramus was designed using on computed-tomographic (CT) imagery and computer-aided design software. Next, the biomechanical performance of the implant was predicted numerically by simulating physiological load conditions in a digital twin in silico finite element model of the sheep mandible. This allowed for possible redesigning of the implant prior to commencing in vivo experimentation. Then, two types of polymeric biomaterials were used to manufacture the mandibular scaffold implants: poly ether ether ketone (PEEK) and poly ether ketone (PEK) printed with fused deposition modeling (FDM) and selective laser sintering (SLS), respectively. Then, after being implanted for 13 weeks in vivo, the implant and surrounding bone tissue was harvested and microCT scanned to visualize and quantify neo-tissue formation in the porous space of the scaffold. Finally, the implant and local bone tissue was assessed by in vitro laboratory mechanical testing to quantify the osteointegration. The protocol consists of six component procedures: (i) scaffold design and finite element analysis to predict its biomechanical response, (ii) scaffold fabrication with FDM and SLS 3D printing, (iii) surface treatment of the scaffold with plasma immersion ion implantation (PIII) techniques, (iv) ovine mandibular implantation, (v) postoperative sheep recovery, euthanasia, and harvesting of the scaffold and surrounding host bone, microCT scanning, and (vi) in vitro laboratory mechanical tests of the harvested scaffolds. The results of microCT imagery and 3-point mechanical bend testing demonstrate that PIII-SLS-PEK is a promising biomaterial for the manufacturing of scaffold implants to enhance the bone-scaffold contact and bone ingrowth in porous scaffold implants. MicroCT images of the harvested implant and surrounding bone tissue showed encouraging new bone growth at the scaffold-bone interface and inside the porous network of the lattice structure of the SLS-PEK scaffolds.

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.

A Comparison of In Vivo Bone Tissue Generation Using Calcium Phosphate Bone Substitutes in a Novel 3D Printed Four-Chamber Periosteal Bioreactor.

Autologous bone replacement remains the preferred treatment for segmental defects of the mandible; however, it cannot replicate complex facial geometry and causes donor site morbidity. Bone tissue engineering has the potential to overcome these limitations. Various commercially available calcium phosphate-based bone substitutes (Novabone®, BioOss®, and Zengro®) are commonly used in dentistry for small bone defects around teeth and implants. However, their role in ectopic bone formation, which can later be applied as vascularized graft in a bone defect, is yet to be explored. Here, we compare the above-mentioned bone substitutes with autologous bone with the aim of selecting one for future studies of segmental mandibular repair. Six female sheep, aged 7-8 years, were implanted with 40 mm long four-chambered polyether ether ketone (PEEK) bioreactors prepared using additive manufacturing followed by plasma immersion ion implantation (PIII) to improve hydrophilicity and bioactivity. Each bioreactor was wrapped with vascularized scapular periosteum and the chambers were filled with autologous bone graft, Novabone®, BioOss®, and Zengro®, respectively. The bioreactors were implanted within a subscapular muscle pocket for either 8 weeks (two sheep), 10 weeks (two sheep), or 12 weeks (two sheep), after which they were removed and assessed by microCT and routine histology. Moderate bone formation was observed in autologous bone grafts, while low bone formation was observed in the BioOss® and Zengro® chambers. No bone formation was observed in the Novabone® chambers. Although the BioOss® and Zengro® chambers contained relatively small amounts of bone, endochondral ossification and retained hydroxyapatite suggest their potential in new bone formation in an ectopic site if a consistent supply of progenitor cells and/or growth factors can be ensured over a longer duration.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

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.

Development of 3D printable graphene oxide based bio-ink for cell support and tissue engineering.

Tissue engineered constructs can serve as in vitro models for research and replacement of diseased or damaged tissue. As an emerging technology, 3D bioprinting enables tissue engineering through the ability to arrange biomaterials and cells in pre-ordered structures. Hydrogels, such as alginate (Alg), can be formulated as inks for 3D bioprinting. However, Alg has limited cell affinity and lacks the functional groups needed to promote cell growth. In contrast, graphene oxide (GO) can support numerous cell types and has been purported for use in regeneration of bone, neural and cardiac tissues. Here, GO was incorporated with 2% (w/w) Alg and 3% (w/w) gelatin (Gel) to improve 3D printability for extrusion-based 3D bioprinting at room temperature (RT; 25°C) and provide a 3D cellular support platform. GO was more uniformly distributed in the ink with our developed method over a wide concentration range (0.05%-0.5%, w/w) compared to previously reported GO containing bioink. Cell support was confirmed using adipose tissue derived stem cells (ADSCs) either seeded onto 3D printed GO scaffolds or encapsulated within the GO containing ink before direct 3D printing. Added GO was shown to improve cell-affinity of bioinert biomaterials by providing more bioactive moieties on the scaffold surface. 3D cell-laden or cell-seeded constructs showed improved cell viability compared to pristine (without GO) bio-ink-based scaffolds. Our findings support the application of GO for novel bio-ink formulation, with the potential to incorporate other natural and synthetic materials such as chitosan and cellulose for advanced in situ biosensing, drug-loading and release, and with the potential for electrical stimulation of cells to further augment cell function.

Hydrogel: A Potential Material for Bone Tissue Engineering Repairing the Segmental Mandibular Defect.

Free flap surgery is currently the only successful method used by surgeons to reconstruct critical-sized defects of the jaw, and is commonly used in patients who have had bony lesions excised due to oral cancer, trauma, infection or necrosis. However, donor site morbidity remains a significant flaw of this strategy. Various biomaterials have been under investigation in search of a suitable alternative for segmental mandibular defect reconstruction. Hydrogels are group of biomaterials that have shown their potential in various tissue engineering applications, including bone regeneration, both through in vitro and in vivo pre-clinical animal trials. This review discusses different types of hydrogels, their fabrication techniques, 3D printing, their potential for bone regeneration, outcomes, and the limitations of various hydrogels in preclinical models for bone tissue engineering. This review also proposes a modified technique utilizing the potential of hydrogels combined with scaffolds and cells for efficient reconstruction of mandibular segmental defects.

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.

3D Printing of Cytocompatible Graphene/Alginate Scaffolds for Mimetic Tissue Constructs.

Tissue engineering, based on a combination of 3D printing, biomaterials blending and stem cell technology, offers the potential to establish customized, transplantable autologous implants using a patient's own cells. Graphene, as a two-dimensional (2D) version of carbon, has shown great potential for tissue engineering. Here, we describe a novel combination of graphene with 3D printed alginate (Alg)-based scaffolds for human adipose stem cell (ADSC) support and osteogenic induction. Alg printing was enabled through addition of gelatin (Gel) that was removed after printing, and the 3D structure was then coated with graphene oxide (GO). GO was chemically reduced with a biocompatible reductant (ascorbic acid) to provide electrical conductivity and cell affinity sites. The reduced 3D graphene oxide (RGO)/Alg scaffold has good cytocompatibility and can support human ADSC proliferation and osteogenic differentiation. Our finding supports the potential for the printed scaffold's use for in vitro engineering of bone and other tissues using ADSCs and potentially other human stem cells, as well as in vivo regenerative medicine.

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.

Cell Processing for 3D Bioprinting: Quality Requirements for Quality Assurance in Fundamental Research and Translation.

Bioprinting is an additive manufacturing process where biomaterials-based inks are printed layer-by-layer to create three-dimensional (3D) structures that mimic natural tissues. Quality assurance for 3D bioprinting is paramount to undertaking fundamental research and preclinical and clinical product development. It forms part of quality management and is vital to reproducible and safe tissue fabrication, function, and regulatory approval for translational application. This chapter seeks to place the implementation of quality practices in 3D bioprinting front-of-mind, with emphasis on cell processing, although important to all components and procedures of the printing pipeline.

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.