3D bioprinting for potential use in nasal cartilage reconstruction

3D printing is an additive manufacturing technique that is rapidly gaining traction in health and medical applications. This technique could potentially benefit plastic and reconstructive surgeries by fabricating patient-specific tissue replacements with tissue-like functions and mechanical properties. One specific example in the field of plastic and constructive surgery is nose reconstruction. Current gold standard for nasal reconstruction after rhinectomy or severe trauma involves a three stage surgery that requires a minimum of three and maximum of seven operations to achieve an acceptable result. The surgical procedure require transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvest of autologous rib cartilage requires a major additional procedure which creates donor site morbidity. Additionally, major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly-skill demanding and time-consuming. These drawbacks of the current approach for nasal reconstruction are some of the reasons why facial plastic and reconstructive surgeons are interested in the application of tissue engineering and 3D printing for reconstructive surgeries. To address these clinical challenges, the aim of the work presented in this thesis was to fabricate a personalised 3D bioprinted composite scaffold for nasal reconstruction mimicking the mechanical properties and architecture of nasal cartilage. The composite consists of biodegradable thermoplastic polycaprolactone (PCL) to provide structural support, and cell-laden thermoresponsive and UV crosslinkable gelatin methacrylate (GelMA) to act as a cell carrier. We first investigated the appropriate cell source to use for cartilage tissue engineering and 3D bioprinting. Primary sheep articular chondrocytes (ShCh) and sheep bone marrow derived Mesenchymal Stem Cells (ShMSCs) were isolated, expanded and differentiated; followed by an assessment of the effects of the 3D printing process on cell viability and functionality. From these studies it was observed that ShCh were easier to isolate and expand than ShMSCs because less steps are required and the doubling time is 50% shorter. Additionally, 80% of the ShCh survived the printing process compared to a 50% of the ShMSCs, suggesting that chondrocytes were able to tolerate higher stress caused by the 3D printing process. PCL and poly (lactic-co-glycolic acid) (PLGA) scaffolds were printed and seeded with chondrocytes post-printing. The printing process and the 3D printed structures of these polymers were characterised before and after printing by measuring their molecular weight, thermal and mechanical properties. It was found that the printing process reduced the molecular weight of PLGA by 50% percent due to thermal degradation. Consequently, its glass transition temperature and young’s modulus decreased post printing. On the contrary, PCL’s molecular weight remain unchanged after printing. Characterisation of the chondrocytes showed that whilst both scaffold materials supported cell attachment the ECM secreted deformed the PLGA whilst the PCL scaffolds were unaffected. Due to superior mechanical properties PCL was selected to 3D print the personalised nose scaffolds. Additional studies on the 3D printed scaffolds showed that controlling the surface pores of scaffolds was important for cell infiltration and proliferation Scaffolds with larger surface pores were 3D printed and these resulted in increased cell seeding and proliferation demonstrated by DNA quantification. Moreover, the printing process of the cell carrier GelMA was optimised by utilising its thermoresponsive properties. A rheological study of three different concentrations of GelMA was performed in order to identify the most suitable for bioprinting. GelMA 15% and 20% at 15 °C and 18 °C respectively were found the appropriate ones. Finally, multi-material 3D bioprinting of PCL and chondrocyte-laden GelMA was utilised for making cartilage constructs. The 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was evidenced by the presence of glycosaminoglycans and collagen type II after cultivation. The findings in this thesis therefore support the feasibility of using 3D bioprinted composite constructs for nasal reconstruction

Feasibility of spatially-offset Raman spectroscopy for in-vitro and in-vivo monitoring mineralisation of bone tissue-engineering scaffolds

We investigated the feasibility of using spatially-offset Raman spectroscopy (SORS) for non-destructive characterisation of bone tissue engineering scaffolds. The deep regions of these scaffolds, or scaffolds implanted subcutaneously in live animals, are typically difficult to measure by confocal Raman spectroscopy techniques because of the limited depth penetration of light caused by the high level of light scattering. Layered samples consisting of bioactive glass foams (IEIC16), 3D-printed biodegradable poly-(lactic-co-glycolic acid) scaffolds (PLGA) and hydroxyapatite powder (HA) were used to mimic non-destructive detection of bio-mineralisation for intact real-size 3D tissue engineering constructs. SORS spectra were measured with a new SORS instrument using a digital micro-mirror device (DMD) to allow software selection of the spatial offsets. The results show that HA can be reliably detected at depths of 0-2.3 mm, which corresponds to the maximum accessible spatial offset of the current instrument. The intensity ratio of Raman bands associated to the scaffolds and HA with the spatial offset depended on the depth at which HA was located. Furthermore, we show the feasibility for in-vivo monitoring mineralisation of scaffold implanted subcutaneously by demonstrating the ability to measure transcutaneously Raman signals of the scaffolds and HA (fresh chicken skin used as a top layer). The ability to measure spectral depth profiles at high speed (5 s acquisition time), and the ease of implementation, make SORS a promising approach for non-invasive characterisation of cell/tissue development in-vitro, and for long-term in-vivo monitoring the mineralisation in 3D scaffolds subcutaneously implanted in small animals

Multi-material 3D bioprinting of porous constructs for cartilage regeneration

© 2020 Elsevier B.V. The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction

Synthesis of methacrylate-terminated block copolymers with reduced transesterification by controlled ring-opening polymerization

This work presents a robust method to achieve the synthesis of low molecular weight polyesters 26 via ring-opening polymerization (ROP) initiated by 2-hydroxyethyl-methacrylate (HEMA) 27 when using triazabicyclodecene (TBD) as catalyst. The effect that the HEMA:TBD ratio has 28 upon the final reaction rate and final polymer molecular architecture is discussed. The optimum 29 HEMA:TBD ratio and reaction conditions required to minimize competing transesterification 30 reactions were determined, in order to synthesize successfully the target ROP macromonomer 31 species containing only a single 2-methacryloyloxyethyl end-group. Additionally, to confirm 32 the terminal end-group fidelity of the product macromonomers and confirm TBD utility for 33 block copolymer manufacture, a small series of di-block polyesters were synthesized using 34 TBD and shown to exhibit good control over the final polymer structure whilst negating the 35 side transesterification reactions, irrespective of the monomers used

Human-scale tissues with patterned vascular networks by additive manufacturing of sacrificial sugar-protein composites

© 2020 Combating necrosis, by supplying nutrients and removing waste, presents the major challenge for engineering large three-dimensional (3D) tissues. Previous elegant work used 3D printing with carbohydrate glass as a cytocompatible sacrificial template to create complex engineered tissues with vascular networks (Miller et al. 2012, Nature Materials). The fragile nature of this material compounded with the technical complexity needed to create high-resolution structures led us to create a flexible sugar-protein composite, termed Gelatin-sucrose matrix (GSM), to achieve a more robust and applicable material. Here we developed a low-range (25–37˚C) temperature sensitive formulation that can be moulded with micron-resolution features or cast during 3D printing to produce complex flexible filament networks forming sacrificial vessels. Using the temperature-sensitivity, we could control filament degeneration meaning GSM can be used with a variety of matrices and crosslinking strategies. Furthermore by incorporation of biocompatible crosslinkers into GSM directly, we could create thin endothelialized vessel walls and generate patterned tissues containing multiple matrices and cell-types. We also demonstrated that perfused vascular channels sustain metabolic function of a variety of cell-types including primary human cells. Importantly, we were able to construct vascularized human noses which otherwise would have been necrotic. Our material can now be exploited to create human-scale tissues for regenerative medicine applications. Statement of Significance: Authentic and engineered tissues have demands for mass transport, exchanging nutrients and oxygen, and therefore require vascularization to retain viability and inhibit necrosis. Basic vascular networks must be included within engineered tissues intrinsically. Yet, this has been unachievable in physiologically-sized constructs with tissue-like cell densities until recently. Sacrificial moulding is an alternative in which networks of rigid lattices of filaments are created to prevent subsequent matrix ingress. Our study describes a biocompatible sacrificial sugar-protein formulation; GSM, made from mixtures of inexpensive and readily available bio-grade materials. GSM can be cast/moulded or bioprinted as sacrificial filaments that can rapidly dissolve in an aqueous environment temperature-sensitively. GSM material can be used to engineer viable and vascularized human-scale tissues for regenerative medicine applications

A Reactive Prodrug Ink Formulation Strategy for Inkjet 3D Printing of Controlled Release Dosage Forms and Implants

We propose a strategy for creating tuneable 3D printed drug delivery devices. 3D printing offers the opportunity for improved compliance and patient treatment outcomes through personalisation, but bottlenecks include finding formulations that provide a choice of drug loading and release rate, are tuneable and avoid the need for surgical removal. Our solution is to exploit 3D inkjet printing freedoms. We use a reactive prodrug that can polymerize into drug-attached macromolecules during 3D printing, and by tuning the hydrophilicity we can facilitate or hinder hydrolysis, which in turn controls the drug release. To demonstrate this approach, we attach ibuprofen to 2-hydroxyethyl acrylate through a cleavable ester bond, formulate it for inkjet 3D printing, and then print to produce a solid dosage form. This allows a much higher loading than is usually achievable-in our case up to 58 wt%. Of equal importance, the 3D inkjet printing freedoms mean that our drug delivery device is highly tuneable: by selection of spacer monomers to adjust the hydrophilicity; through geometry; by spatially varying the components. Consequently, we create bespoke, hierarchical release systems, from the molecular to macro. This approach represents a new paradigm for the formulation of printable inks for drug-loaded medical devices

Characterisation of the surface structure of 3D printed scaffolds for cell infiltration and surgical suturing

3D printing is of great interest for tissue engineering scaffolds due to the ability to form complex geometries and control internal structures, including porosity and pore size. The porous structure of scaffolds plays an important role in cell ingrowth and nutrition infusion. Although the internal porosity and pore size of 3D printed scaffolds have been frequently studied, the surface porosity and pore size, which are critical for cell infiltration and mass transport, have not been investigated. The surface geometry can differ considerably from the internal scaffold structure depending on the 3D printing process. It is vital to be able to control the surface geometry of scaffolds as well as the internal structure to fabricate optimal architectures. This work presents a method to control the surface porosity and pore size of 3D printed scaffolds. Six scaffold designs have been printed with surface porosities ranging from 3% to 21%. We have characterised the overall scaffold porosity and surface porosity using optical microscopy and microCT. It has been found that surface porosity has a significant impact on cell infiltration and proliferation. In addition, the porosity of the surface has been found to have an effect on mechanical properties and on the forces required to penetrate the scaffold with a surgical suturing needle. To the authors' knowledge, this study is the first to investigate the surface geometry of extrusion-based 3D printed scaffolds and demonstrates the importance of surface geometry in cell infiltration and clinical manipulation

Bespoke 3D-Printed Polydrug Implants Created via Microstructural Control of Oligomers

Controlling the microstructure of materials by means of phase separation is a versatile tool for optimizing material properties. Phase separation has been exploited to fabricate intricate microstructures in many fields including cell biology, tissue engineering, optics, and electronics. The aim of this study was to use phase separation to tailor the spatial location of drugs and thereby generate release profiles of drug payload over periods ranging from 1 week to months by exploiting different mechanisms: polymer degradation, polymer diluent dissolution, and control of microstructure. To achieve this, we used drop-on-demand inkjet three-dimensional (3D) printing. We predicted the microstructure resulting from phase separation using high-throughput screening combined with a model based on the Flory-Huggins interaction parameter and were able to show that drug release from 3D-printed objects can be predicted from observations based on single drops of mixtures. We demonstrated for the first time that inkjet 3D printing yields controllable phase separation using picoliter droplets of blended photoreactive oligomers/monomers. This new understanding gives us hierarchical compositional control, from droplet to device, allowing release to be "dialled up"without manipulation of device geometry. We exemplify this approach by fabricating a biodegradable, long-term, multiactive drug delivery subdermal implant ("polyimplant") for combination therapy and personalized treatment of coronary heart disease. This is an important advance for implants that need to be delivered by cannula, where the shape is highly constrained and thus the usual geometrical freedoms associated with 3D printing cannot be easily exploited, which brings a hitherto unseen level of understanding to emergent material properties of 3D printing

Identification of novel ‘inks’ for 3D printing using high throughput screening: bioresorbable photocurable polymers for controlled drug delivery

A robust discovery methodology is presented to identify novel biomaterials suitable for 3D printing. Currently the application of Additive Manufacturing is limited by the availability of functional inks, especially in the area of biomaterials-this method tackles this problem for the first time allowing hundreds of formulations to be readily assessed. Several functional properties, including the release of an antidepressive drug (paroxetine), cytotoxicity and printability are screened for 253 new ink formulations in a high-throughput format as well as mechanical properties. The selected candidates with the desirable properties are successfully scaled up using 3D printing into a range of objects architectures. A full drug release study, degradability and tensile modulus experiments are presented on a simple architecture to validating the suitability of this methodology to identify printable inks for 3D printing devices with bespoke properties

Poly (glycerol adipate) (PGA) backbone modifications with a library of functional diols: Chemical and physical effects

Enzymatically synthesised poly(glycerol adipate) (PGA) has shown a palette of key desirable properties required for a biomaterial to be considered a ‘versatile polymeric tool’ in the field of drug delivery. PGA and its variations can self-assemble into nanoparticles (NPs) and interact at different levels with small active molecules. PGA derivatives are usually obtained by functionalising the glyceryl side hydroxyl group present along the main polymer scaffold. However, if the synthetic pathways are not finely tuned, the self-assembling ability of these new polymeric modifications might be hampered by the poor amphiphilic balance. For this reason, we have designed a straightforward one-pot synthetic modification, using a small library of diols in combination with glycerol, aimed at altering the backbone of the polymer without affecting the hydrophilic glyceryl portion. The diols introduce additional functionality into the backbone of PGA alongside the secondary hydroxyl group already present. We have investigated how extra functionalities along the polymer backbone alter the final polymer reactivity as well the chemical and biological properties of the nanoparticles. In addition, with the intent to further improve the green credentials of the enzymatic synthesis, a solvent derived from renewable resources, (2-methyl tetrahydrofuran, 2-MeTHF) was employed for the synthesis of all the PGA-variants as a replacement for the more traditionally used and fossil-based tetrahydrofuran (THF). In vitro assays carried out to evaluate the potential of these novel materials for drug delivery applications demonstrated very low cytotoxicity characteristic against NIH 3T3 model cell line