Periodic Reporting for period 2 - 4D-BOUNDARIES (Printing spatially and temporally defined boundaries to direct the self-organization of cells and cellular aggregates to engineer functional tissues)
Reporting period: 2023-03-01 to 2024-08-31
The architecture of the extracellular matrix (ECM) of biological tissues is integral to their function. This is particularly the case for musculoskeletal tissues such as articular cartilage (AC) and meniscus, which develop exquisite architectures uniquely suited to their biomechanical function during skeletal development and postnatal maturation. Damage to these tissues initiates the debilitating disease of osteoarthritis (OA), motivating the development of tissue engineering (TE) strategies to direct their functional regeneration. Despite decades of research, existing TE strategies are unable to produce structurally complex tissues with biomimetic collagen architectures, mechanical properties and physiological function, dramatically limiting their clinical utility. Radical new approaches are required if the field of TE is to successfully address this problem.
Clues to addressing the challenge of engineering replacement tissues and organs can be found in normal tissue development, which relies upon both the self-organizing potential of stem cells as well as key physical instructions from the local environment to establish final tissue architectures. Recognising this, the goal of 4D-BOUNDARIES is to leverage emerging 3D bioprinting technologies to provide precisely controlled physical and biochemical signals to cells to engineer structurally anisotropic and mechanically functional musculoskeletal tissues. 3D bioprinting is an emerging technology that enables living tissues to be engineered by printing cells with supporting materials and cell-instructive cues. 4D-BOUNDARIES is developing new bioprinting platforms that provide temporary guiding structures to self-organizing tissues. To demonstrate the utility of these bioprinting platforms they will be used to engineer, for the first time, patient-specific cartilage and meniscal grafts that mimic the internal and external anatomy and complex mechanical properties of the native tissues. The impact of 4D-BOUNDARIES will not be limited to the orthopaedic space, as it is envisioned that these new bioprinting platforms will find numerous applications in tissue engineering and regenerative medicine.
Clues to addressing the challenge of engineering replacement tissues and organs can be found in normal tissue development, which relies upon both the self-organizing potential of stem cells as well as key physical instructions from the local environment to establish final tissue architectures. Recognising this, the goal of 4D-BOUNDARIES is to leverage emerging 3D bioprinting technologies to provide precisely controlled physical and biochemical signals to cells to engineer structurally anisotropic and mechanically functional musculoskeletal tissues. 3D bioprinting is an emerging technology that enables living tissues to be engineered by printing cells with supporting materials and cell-instructive cues. 4D-BOUNDARIES is developing new bioprinting platforms that provide temporary guiding structures to self-organizing tissues. To demonstrate the utility of these bioprinting platforms they will be used to engineer, for the first time, patient-specific cartilage and meniscal grafts that mimic the internal and external anatomy and complex mechanical properties of the native tissues. The impact of 4D-BOUNDARIES will not be limited to the orthopaedic space, as it is envisioned that these new bioprinting platforms will find numerous applications in tissue engineering and regenerative medicine.
The first aim of this project is to direct the fusion, growth and remodelling of microtissues to engineer anisotropic soft tissues. To address this aim, we first investigated the influence of the maturation state of stem derived microtissues on their fusion and the phenotype of the resultant engineered tissue. Having identified that less mature microtissues generated superior engineered tissues, we next assessed the feasibility of 3D bioprinting cartilage microtissues within supporting bioinks. To this end, we developed a novel support bath for bioprinting cellular aggregates or microtissues. We investigated the bioprinting of microtissues at various densities to determine the optimal ink-to-microtissues ratio that ensured high printability, minimal shear stress, and effective fusion between the microtissues after printing.
The second aim of the project is to direct cellular condensation and self-organization in 3D space to engineer anisotropic tissues. To address this aim, we have developed a high cell density, oxidised alginate bioink and an oxidised-methacrylated alginate embedded support bath to enable the fabrication of spatially defined cartilaginous tissues physically supported and guided by alginate boundaries.
The third aim of the project is to bioprint structurally organised articular cartilage and assess its regenerative capacity in a pre-clinical large animal model. In an attempt to engineer organised cartilage tissue, we physically constrained high cell density bioinks with external hydrogel boundaries, demonstrating greater tissue organization with increased physical confinement. With this approach it is possible to bioprint cartilage with user defined collagen architecture.
The fourth and final aim of the project is to bioprint structurally organised meniscal fibrocartilage and assess its regenerative capacity in a pre-clinical large animal model. To this end, meniscal progenitor cells (MPCs) were isolated from both inner (iMPCs) and outer (iMPCs) regions of the meniscus, which were then used to generate region specific meniscus microtissues. We were able to bioprint MPC microtissue containing bioinks into support baths with high spatial resolution. It was observed that the microtissues were able to fuse after 24 hours and showed high viability.
The second aim of the project is to direct cellular condensation and self-organization in 3D space to engineer anisotropic tissues. To address this aim, we have developed a high cell density, oxidised alginate bioink and an oxidised-methacrylated alginate embedded support bath to enable the fabrication of spatially defined cartilaginous tissues physically supported and guided by alginate boundaries.
The third aim of the project is to bioprint structurally organised articular cartilage and assess its regenerative capacity in a pre-clinical large animal model. In an attempt to engineer organised cartilage tissue, we physically constrained high cell density bioinks with external hydrogel boundaries, demonstrating greater tissue organization with increased physical confinement. With this approach it is possible to bioprint cartilage with user defined collagen architecture.
The fourth and final aim of the project is to bioprint structurally organised meniscal fibrocartilage and assess its regenerative capacity in a pre-clinical large animal model. To this end, meniscal progenitor cells (MPCs) were isolated from both inner (iMPCs) and outer (iMPCs) regions of the meniscus, which were then used to generate region specific meniscus microtissues. We were able to bioprint MPC microtissue containing bioinks into support baths with high spatial resolution. It was observed that the microtissues were able to fuse after 24 hours and showed high viability.
As part of the 4D Boundaries project, we have developed a novel platform for 3D bioprinting cellular aggregates, microtissues or organoids at high density within a tuneable support bath. We believe this represents important progress beyond the state of the art, as it not only enables the high resolution bioprinting of highly cellular bioinks and microtissues, but also functions to direct cell and tissue organization, thereby enabling the biofabrication of structurally organised soft tissues (e.g. skeletal muscle, meniscus, and articular cartilage).
In the second phase of this project, we aim to leverage this new bioprinting platform to engineer both structurally organised articular cartilage and meniscal fibrocartilage. We will use our bioprinting platform to spatially pattern these constructs with different growth factors, in an attempt to engineer tissues that mimic the zonal composition of their native equivalents. We will also explore the use of different enzymes (e.g. chondroitinase ABC) to improve the levels of collagen deposition and maturation within the engineered tissues. Having optimised both the bioprinting process and subsequent in vitro maturation, we will then assess the efficacy of these new therapies in clinically relevant, large animal models of synovial joint damage.
In the second phase of this project, we aim to leverage this new bioprinting platform to engineer both structurally organised articular cartilage and meniscal fibrocartilage. We will use our bioprinting platform to spatially pattern these constructs with different growth factors, in an attempt to engineer tissues that mimic the zonal composition of their native equivalents. We will also explore the use of different enzymes (e.g. chondroitinase ABC) to improve the levels of collagen deposition and maturation within the engineered tissues. Having optimised both the bioprinting process and subsequent in vitro maturation, we will then assess the efficacy of these new therapies in clinically relevant, large animal models of synovial joint damage.