Periodic Reporting for period 2 - BioSMaLL (Biopolymers for the Generation of 3D Tissue Engineering Scaffolds by Solution Mask Liquid Lithography)
Période du rapport: 2021-07-01 au 2022-06-30
Musculoskeletal conditions reduce the quality of life of many adults worldwide, with the WHO citing that up to 1.71 billion people are affected. Lower back injury is a prevailing issue, suffered by nearly 568 million people. As the joint cartilage breaks down, inflammation/pain causes poor mobility and dexterity for patients, sometimes needing surgical intervention. The continued burden on healthcare professionals is to come up with a viable treatment solution, and cartilage regeneration is of particular interest. This demand for a pragmatic solution has prompted the investigation of biomaterials, which are a class of synthetically optimised biocompatible polymers, which promote low to no toxicity within humans. Hydrogels are highly hydrated, soft materials, which are highly explored as biomaterials in biomedicine. Continued research efforts into biomaterial hydrogel development has led to many promising studies on their use as biocompatible 3D implantable or injectable augments for cartilage regeneration, albeit in academic settings. These studies may provide solutions to current healthcare issues surrounding impact or wear and tear injuries that damage cartilage, by replacing existing treatments such as mosaicplasty, an autologous cartilage transplant – which isn’t always successful in patients. However, current biomaterial hydrogels have limitations, including material reproduction issues and poor mechanical strength. Newer tailored made materials with improved properties for cartilage regeneration are of significant interest – with polypeptide hydrogels of note and representing high promise in biomedicine. Coupling tailor made polypeptide materials and cell components with a low-cost technology for 3D printing objects, namely Solution Mask Liquid Lithography (SMaLL) – a digital light processing (DLP) method, 3D biomedical scaffolds may be realised. This project aims to provide new hydrogel material platform(s) for potential clinical translation with 3 overlapping objectives: 1. Development of synthetically simplified polypeptide hydrogels that can be reproduced on demand. 2. The use of these hydrogels as viable growth matrices for mammalian cells with a focus on cartilage regeneration. 3. Enabling 3D object development with this hydrogel/cell blend using a custom DLP 3D printer.
In BioSMaLL, novel water-soluble polypeptides (polymers of amino acids) were developed and optimised using pragmatic chemistries to form hydrogels with the ability to change their physical state through light-based crosslinking (photocrosslinking) for different 3D printing methods. Two distinct light reactive polypeptide systems were prepared via synthetic strategies which used pre (system 1) and post polymerisation modification (system 2). System 1 polypeptides were synthesised from novel monomers with light reactive vinyl bonds, to create 3D printable photocrosslinkable water-based formulations. The polypeptides could successfully undergo a rapid transformation from liquid to solid following exposure to a cell-friendly wavelength of light (405 nm). These polymers were suitable for DLP, stereolithography (SLA) and direct laser writing (DLW) 3D printing techniques, forming 3D structures with tailored mechanical properties and high-resolution features on µm scales. System 2 polypeptides were designed to have high solid-like viscosities, along with reactive vinyl bonds, where peptide secondary structure allowed for assembly into soft hydrogels. The materials were used in direct ink writing (DIW) based 3D bioprinting with embedded genetically engineered bacteria (E. coli), where they could be extruded and photocrosslinked (at 405 nm) into ‘‘living’’ 3D shapes. These 3D printed biocomposites demonstrated high viability with E.coli growth dependent on the mechanical properties of polypeptides over time scales which were indicated by green fluorescent protein (GFP) induction. Further advances on these polypeptides have been made in mammalian cell bioprinting, with 2D cell studies showing good viability with embedded fibroblast cells and human mesenchymal stem cells. Initial studies demonstrated good bioprintability, and final studies are underway to assess their functionality as cartilage scaffolds. The results of BioSMaLL have been dissemination in a number of forms including journal publications and conference presentations. Specifically, 3 research manuscripts have been peer reviewed and published, and 3 others are currently in late stages of manuscript drafting. The progress of the BioSMaLL project has also been disseminated at 3 different conferences via oral presentations; in 2022 at American Chemical Society Spring (ACS, San Diego, USA) and Bordeaux Polymer Conference (BPC, Bordeaux, France), and in 2023 at the Society of Polymer Science Japan International Polymer Conference (SPSJ IPC, Sapporo, Japan).
Hydrogels used in 3D printing applications are inherently limited to biopolymers such as gelatin, hyaluronic acid and alginate, which have several limitations including poor mechanical properties and batch variability. They are primary choices as hydrogel inks or resins in many 3D printing techniques which include extrusion (e.g. DIW) or light-based (e.g. SLA) 3D printing, although their poorly defined chemical structures mean extensive chemical modifications are needed to realise their printability. To circumvent the drawbacks of biopolymers, the BioSMaLL project set out to offer new hydrogel materials with highly defined chemical and mechanical properties, tailor made for light-based 3D printing techniques. Novel amino acid monomers (starting materials to produce polypeptides) and polymers were readily made after extensive synthetic optimisation. Their highly defined chemical structures and batch reproducibility surpass the same properties of conventionally used biopolymers. These polymers were made into hydrogel platforms that have unprecedented, highly tailorable mechanical properties, another significant advancement on biopolymer systems. Although unsuccessful in generating 3D objects via the SMaLL DLP system, materials were developed that could be successfully adapted for several other different techniques (DLP, SLA, DLW, DIW) to readily form 3D objects on mm and µm scale. These new polypeptides are promising additions in the hydrogel field, being chemically modifiable with ease for specific applications – a high necessity in polymer and biomedical science. They are envisaged to inspire future research into hydrogel developed for 3D printing, having highly modular properties and synthetic scalability. The polypeptide hydrogels can also be readily adapted as host matrices for mammalian and bacteria cell bioprinting, the latter of which was not originally a targeted application for the BioSMaLL project but is currently a blossoming area in material science. In collaboration with the US Army Corps Engineer Research and Development Center, BioSMaLL endeavors managed to create a first of its kind study on 3D printed “living” materials with different bacterial growth regimes, achieved through pragmatic material design. As such, these novel hydrogels appear to have a wide range of applicability and impacts outside of biomedicine, for example in agriculture and/or electronics.