3D printing of heart valves.

3D printing technologies have the potential to revolutionize the manufacture of heart valves through the ability to create bespoke, complex constructs. In light of recent technological advances, we review the progress made towards 3D printing of heart valves, focusing on studies that have utilised these technologies beyond manufacturing patient-specific moulds. We first overview the key requirements of a heart valve to assess functionality. We then present the 3D printing technologies used to engineer heart valves. By referencing International Organisation for Standardisation (ISO) Standard 5840 (Cardiovascular implants - Cardiac valve prostheses), we provide insight into the achieved functionality of these valves. Overall, 3D printing promises to have a significant positive impact on the creation of artificial heart valves and potentially unlock full complex functionality.

Silane-modified hydroxyapatite nanoparticles incorporated into polydioxanone/poly(lactide-co-caprolactone) creates a novel toughened nanocomposite with improved material properties and in vivo inflammatory responses.

The interface tissue between bone and soft tissues, such as tendon and ligament (TL), is highly prone to injury. Although different biomaterials have been developed for TL regeneration, few address the challenges of the TL-bone interface. Here, we aim to develop novel hybrid nanocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL), and hydroxyapatite (HA) nanoparticles suitable for TL-bone interface repair. Nanocomposites, containing 3-10% of both unmodified and chemically modified hydroxyapatite (mHA) with a silane coupling agent. We then explored biocompatibility through in vitro and in vivo studies using a subcutaneous mouse model. Through different characterisation tests, we found that mHA increases tensile properties, creates rougher surfaces, and reduces crystallinity and hydrophilicity. Morphological observations indicate that mHA nanoparticles are attracted by PDO rather than LCL phase, resulting in a higher degradation rate for mHA group. We found that adding the 5% of nanoparticles gives a balance between the properties. In vitro experiments show that osteoblasts' activities are more affected by increasing the nanoparticle content compared with fibroblasts. Animal studies indicate that both HA and mHA nanoparticles (10%) can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. In summary, this work highlights the potential of PDO/LCL/HA nanocomposites as an excellent biomaterial for TL-bone interface tissue engineering applications.

An engineered periosteum for efficient delivery of rhBMP-2 and mesenchymal progenitor cells during bone regeneration.

During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 µg/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.

Three-Dimensional Bioprinting in Cardiovascular Disease: Current Status and Future Directions.

Three-dimensional (3D) printing plays an important role in cardiovascular disease through the use of personalised models that replicate the normal anatomy and its pathology with high accuracy and reliability. While 3D printed heart and vascular models have been shown to improve medical education, preoperative planning and simulation of cardiac procedures, as well as to enhance communication with patients, 3D bioprinting represents a potential advancement of 3D printing technology by allowing the printing of cellular or biological components, functional tissues and organs that can be used in a variety of applications in cardiovascular disease. Recent advances in bioprinting technology have shown the ability to support vascularisation of large-scale constructs with enhanced biocompatibility and structural stability, thus creating opportunities to replace damaged tissues or organs. In this review, we provide an overview of the use of 3D bioprinting in cardiovascular disease with a focus on technologies and applications in cardiac tissues, vascular constructs and grafts, heart valves and myocardium. Limitations and future research directions are highlighted.

Novel hybrid biocomposites for tendon grafts: The addition of silk to polydioxanone and poly(lactide-co-caprolactone) enhances material properties, in vitro and in vivo biocompatibility.

Biopolymers play a critical role as scaffolds used in tendon and ligament (TL) regeneration. Although advanced biopolymer materials have been proposed with optimised mechanical properties, biocompatibility, degradation, and processability, it is still challenging to find the right balance between these properties. Here, we aim to develop novel hybrid biocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL) and silk to produce high-performance grafts suitable for TL tissue repair. Biocomposites containing 1-15% of silk were studied through a range of characterisation techniques. We then explored biocompatibility through in vitro and in vivo studies using a mouse model. We found that adding up to 5% silk increases the tensile properties, degradation rate and miscibility between PDO and LCL phases without agglomeration of silk inside the composites. Furthermore, addition of silk increases surface roughness and hydrophilicity. In vitro experiments show that the silk improved attachment of tendon-derived stem cells and proliferation over 72 h, while in vivo studies indicate that the silk can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. Finally, we selected a promising biocomposite and created a prototype TL graft based on extruded fibres. We found that the tensile properties of both individual fibres and braided grafts could be suitable for anterior cruciate ligament (ACL) repair applications.

Natural, synthetic and commercially-available biopolymers used to regenerate tendons and ligaments.

Tendon and ligament (TL) injuries affect millions of people annually. Biopolymers play a significant role in TL tissue repair, whether the treatment relies on tissue engineering strategies or using artificial tendon grafts. The biopolymer governs the mechanical properties, biocompatibility, degradation, and fabrication method of the TL scaffold. Many natural, synthetic and hybrid biopolymers have been studied in TL regeneration, often combined with therapeutic agents and minerals to engineer novel scaffold systems. However, most of the advanced biopolymers have not advanced to clinical use yet. Here, we aim to review recent biopolymers and discuss their features for TL tissue engineering. After introducing the properties of the native tissue, we discuss different types of natural, synthetic and hybrid biopolymers used in TL tissue engineering. Then, we review biopolymers used in commercial absorbable and non-absorbable TL grafts. Finally, we explain the challenges and future directions for the development of novel biopolymers in TL regenerative treatment.

Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold.

PURPOSE: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. METHODS: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. RESULTS: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. CONCLUSION: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.

Engineering Heart Valve Interfaces Using Melt Electrowriting: Biomimetic Design Strategies from Multi-Modal Imaging.

Interfaces within biological tissues not only connect different regions but also contribute to the overall functionality of the tissue. This is especially true in the case of the aortic heart valve. Here, melt electrowriting (MEW) is used to engineer complex, user-defined, interfaces for heart valve scaffolds. First, a multi-modal imaging investigation into the interfacial regions of the valve reveals differences in collagen orientation, density, and recruitment in previously unexplored regions including the commissure and inter-leaflet triangle. Overlapping, suturing, and continuous printing methods for interfacing MEW scaffolds are then investigated for their morphological, tensile, and flexural properties, demonstrating the superior performance of continuous interfaces. G-codes for MEW scaffolds with complex interfaces are designed and generated using a novel software and graphical user interface. Finally, a singular MEW scaffold for the interfacial region of the aortic heart valve is presented incorporating continuous interfaces, gradient porosities, variable layer numbers across regions, and tailored fiber orientations inspired by the collagen distribution and orientation from the multi-modal imaging study. The scaffold exhibits similar yield strain, hysteresis, and relaxation behavior to porcine heart valves. This work demonstrates the ability of a bioinspired approach for MEW scaffold design to address the functional complexity of biological tissues.

Fabrication of human myocardium using multidimensional modelling of engineered tissues.

Biofabrication of human tissues has seen a meteoric growth triggered by recent technical advancements such as human induced pluripotent stem cells (hiPSCs) and additive manufacturing. However, generation of cardiac tissue is still hampered by lack of adequate mechanical properties and crucially by the often unpredictable post-fabrication evolution of biological components. In this study we employ melt electrowriting (MEW) and hiPSC-derived cardiac cells to generate fibre-reinforced human cardiac minitissues. These are thoroughly characterized in order to build computational models and simulations able to predict their post-fabrication evolution. Our results show that MEW-based human minitissues display advanced maturation 28 post-generation, with a significant increase in the expression of cardiac genes such as MYL2, GJA5, SCN5A and the MYH7/MYH6 and MYL2/MYL7 ratios. Human iPSC-cardiomyocytes are significantly more aligned within the MEW-based 3D tissues, as compared to conventional 2D controls, and also display greater expression of C×43. These are also correlated with a more mature functionality in the form of faster conduction velocity. We used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition (cellular alignment) and intercellular connectivity (C×43) allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development ofin silicotools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture.

The Technological Advancement to Engineer Next-Generation Stent-Grafts: Design, Material, and Fabrication Techniques.

Endovascular treatment of aortic disorders has gained wide acceptance due to reduced physiological burden to the patient compared to open surgery, and ongoing stent-graft evolution has made aortic repair an option for patients with more complex anatomies. To date, commercial stent-grafts are typically developed from established production techniques with simple design structures and limited material ranges. Despite the numerous updated versions of stent-grafts by manufacturers, the reoccurrence of device-related complications raises questions about whether the current manfacturing methods are technically able to eliminate these problems. The technology trend to produce efficient medical devices, including stent-grafts and all similar implants, should eventually change direction to advanced manufacturing techniques. It is expected that through recent advancements, especially the emergence of 4D-printing and smart materials, unprecedented features can be defined for cardiovascular medical implants, like shape change and remote battery-free self-monitoring. 4D-printing technology promises adaptive functionality, a highly desirable feature enabling printed cardiovascular implants to physically transform with time to perform a programmed task. This review provides a thorough assessment of the established technologies for existing stent-grafts and provides technical commentaries on known failure modes. They then discuss the future of advanced technologies and the efforts needed to produce next-generation endovascular implants.

Tissue engineering of corneal stroma via melt electrowriting.

The cornea serves as the main refractive component of the eye with the corneal stroma constituting the thickest component in a stratified layered system of epithelia, stroma, and endothelium. Current treatment options for patients suffering from corneal diseases are limited to transplantation of a human donor cornea (keratoplasty) or to implantation of an artificial cornea (keratoprosthesis). Nevertheless, donor shortage and failure of artificial corneas to integrate with local tissue constitute important problems that have not been yet circumvented. Recent advances in biofabrication have made great progress toward the manufacture of tailored biomaterial templates with the potential of guiding partially or totally the regeneration process of the native cornea. However, the role of the corneal stroma on current tissue engineering strategies is often neglected. Here, we achieved a tissue-engineered corneal stroma substitute culturing primary keratocytes on scaffolds prepared via melt electrowriting (MEW). Scaffolds were designed to contain highly organized micrometric fibers to ensure transparency and encourage primary human keratocytes to self-orchestrate their own extracellular matrix deposition and remodeling. Results demonstrated reliable cell attachment and growth over a period of 5 weeks and confirmed the formation of a dense and highly organized de novo tissue containing collagen I, V, and VI as well as Keratocan, which resembled very closely the native corneal stoma. In summary, MEW brings us closer to the biofabrication of a viable corneal stroma substitute.

Visualization of USPIO-labeled melt-electrowritten scaffolds by non-invasive magnetic resonance imaging.

Melt electrowriting (MEW) is a high-resolution fiber-forming technology for the digital fabrication of complex micro-structured scaffolds for tissue engineering, which has convincingly shown its potential in in vitro and in vivo animal studies. The clinical translation of such constructs to the patient requires the capability to visualize them upon implantation with clinically accepted methods such as magnetic resonance imaging (MRI). To this end, this work presents the modification of polycaprolactone (PCL) scaffolds with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to render them visualizable by MRI. Composite scaffolds containing up to 0.3 weight % USPIOs were 3D printed by MEW and could be sensitively detected in vitro using T2- and T2*-weighted MRI. At the same time, USPIO incorporation did not affect the usability of PCL for tissue engineering applications as demonstrated by the mechanical and cytocompatibility evaluation. Concentrations up to 0.2% caused small to no decrease in the ultimate tensile strength and Young's modulus. Cytocompatibility tests resulted in excellent cell viability, with proliferating cells adhering to all the scaffolds. This work contributes to the materials library for MEW and opens the possibility of using MRI for longitudinal monitoring of MEW grafts.

A novel biocompatible polymeric blend for applications requiring high toughness and tailored degradation rate.

Finding the right balance in mechanical properties and degradation rate of biodegradable materials for biomedical applications is challenging, not only at the time of implantation but also during biodegradation. For instance, high elongation at break and toughness with a mid-term degradation rate are required for tendon scaffold or suture application, which cannot be found in each alpha polyester individually. Here, we hypothesise that blending semi-crystalline poly(p-dioxanone) (PDO) and poly(lactide-co-caprolactone) (LCL) in a specific composition will enhance the toughness while also enabling tailored degradation times. Hence, blends of PDO and LCL (PDO/LCL) were prepared in varying concentrations and formed into films by solvent casting. We thoroughly characterised the chemical, thermal, morphological, and mechanical properties of the new blends before and during hydrolytic degradation. Cellular performance was determined by seeding mouse fibroblasts onto the samples and culturing for 72 hours, before using proliferation assays and confocal imaging. We found that an increase in LCL content causes a decrease in hydrolytic degradation rate, as indicated by induced crystallinity, surface and bulk erosions, and tensile properties. Interestingly, the noncytotoxic blend containing 30% PDO and 70% LCL (PDO3LCL7) resulted in small PDO droplets uniformly dispersed within the LCL matrix and demonstrated a tailored degradation rate and toughening behaviour with a notable strain-hardening effect reaching 320% elongation at break; over 3 times the elongation of neat LCL. In summary, this work highlights the potential of PDO3LCL7 as a biomaterial for biomedical applications like tendon tissue engineering or high-performance absorbable sutures.

Biofabrication and Signaling Strategies for Tendon/Ligament Interfacial Tissue Engineering.

Tendons and ligaments (TL) have poor healing capability, and for serious injuries like tears or ruptures, surgical intervention employing autografts or allografts is usually required. Current tissue replacements are nonideal and can lead to future problems such as high retear rates, poor tissue integration, or heterotopic ossification. Alternatively, tissue engineering strategies are being pursued using biodegradable scaffolds. As tendons connect muscle and bone and ligaments attach bones, the interface of TL with other tissues represent complex structures, and this intricacy must be considered in tissue engineered approaches. In this paper, we review recent biofabrication and signaling strategies for biodegradable polymeric scaffolds for TL interfacial tissue engineering. First, we discuss biodegradable polymeric scaffolds based on the fabrication techniques as well as the target tissue application. Next, we consider the effect of signaling factors, including cell culture, growth factors, and biophysical stimulation. Then, we discuss human clinical studies on TL tissue healing using commercial synthetic scaffolds that have occurred over the past decade. Finally, we highlight the challenges and future directions for biodegradable scaffolds in the field of TL and interface tissue engineering.

Convergence of 3D printed biomimetic wound dressings and adult stem cell therapy.

Biomimetically designed medical-grade polycaprolactone (mPCL) dressings are 3D-printed with pore architecture and anisotropic mechanical characteristics that favor skin wound healing with reduced scarring. Melt electrowritten mPCL dressings are seeded with human gingival tissue multipotent mesenchymal stem/stromal cells and cryopreserved using a clinically approved method. The regenerative potential of fresh or frozen cell-seeded mPCL dressing is compared in a splinted full-thickness excisional wound in a rat model over six weeks. The application of 3D-printed mPCL dressings decreased wound contracture and significantly improved skin regeneration through granulation and re-epithelialization compared to control groups. Combining 3D-printed biomimetic wound dressings and precursor cell delivery enhances physiological wound closure with reduced scar tissue formation.

3D Quantification of Vascular-Like Structures in z Stack Confocal Images.

Optical slice microscopy is commonly used to characterize the morphometric features of 3D cellular cultures, such as in vitro vascularization. However, the quantitative analysis of those structures is often performed on a single 2D maximum intensity projection image, limiting the accuracy of data obtained from 3D cultures. Here, we present a protocol for the quantitative analysis of z stack images, utilizing Fiji, Amira, and WinFiber3D. This protocol facilitates the in-depth examination of vascular-like structures within 3D cell culture models. For complete details on the use and execution of this protocol, please refer to Koch et al. (2020).

Engineering a Humanised Niche to Support Human Haematopoiesis in Mice: Novel Opportunities in Modelling Cancer.

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour-microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

Melt Electrowriting of Complex 3D Anatomically Relevant Scaffolds.

The manufacture of fibrous scaffolds with tailored micrometric features and anatomically relevant three-dimensional (3D) geometries for soft tissue engineering applications remains a great challenge. Melt electrowriting (MEW) is an advanced additive manufacturing technique capable of depositing predefined micrometric fibers. However, it has been so far inherently limited to simple planar and tubular scaffold geometries because of the need to avoid polymer jet instabilities. In this work, we surmount the technical boundaries of MEW to enable the manufacture of complex fibrous scaffolds with simultaneous controlled micrometric and patient-specific anatomic features. As an example of complex geometry, aortic root scaffolds featuring the sinuses of Valsalva were realized. By modeling the electric field strength associated with the MEW process for these constructs, we found that the combination of a conductive core mandrel with a non-conductive 3D printed model reproducing the complex geometry minimized the variability of the electric field thus enabling the accurate deposition of fibers. We validated these findings experimentally and leveraged the micrometric resolution of MEW to fabricate unprecedented fibrous aortic root scaffolds with anatomically relevant shapes and biomimetic microstructures and mechanical properties. Furthermore, we demonstrated the fabrication of patient-specific aortic root constructs from the 3D reconstruction of computed tomography clinical data.

Biologically Inspired Scaffolds for Heart Valve Tissue Engineering via Melt Electrowriting.

Heart valves are characterized to be highly flexible yet tough, and exhibit complex deformation characteristics such as nonlinearity, anisotropy, and viscoelasticity, which are, at best, only partially recapitulated in scaffolds for heart valve tissue engineering (HVTE). These biomechanical features are dictated by the structural properties and microarchitecture of the major tissue constituents, in particular collagen fibers. In this study, the unique capabilities of melt electrowriting (MEW) are exploited to create functional scaffolds with highly controlled fibrous microarchitectures mimicking the wavy nature of the collagen fibers and their load-dependent recruitment. Scaffolds with precisely-defined serpentine architectures reproduce the J-shaped strain stiffening, anisotropic and viscoelastic behavior of native heart valve leaflets, as demonstrated by quasistatic and dynamic mechanical characterization. They also support the growth of human vascular smooth muscle cells seeded both directly or encapsulated in fibrin, and promote the deposition of valvular extracellular matrix components. Finally, proof-of-principle MEW trileaflet valves display excellent acute hydrodynamic performance under aortic physiological conditions in a custom-made flow loop. The convergence of MEW and a biomimetic design approach enables a new paradigm for the manufacturing of scaffolds with highly controlled microarchitectures, biocompatibility, and stringent nonlinear and anisotropic mechanical properties required for HVTE.