Periodic Reporting for period 3 - ReCaP (Regeneration of Articular Cartilage using Advanced Biomaterials and Printing Technology)
Reporting period: 2021-08-01 to 2023-01-31
Damaged articular cartilage joints (e.g. knee) are associated with loss of function and joint degeneration which can lead to osteoarthritis (OA) and the need for total joint replacement. For the treatment of small cartilage defects, conventional therapies e.g. microfracture and osteochondral autograft transplantation are used, but with limited success. An advanced biomaterial capable of restoring healthy joint function remains something of a ‘Holy Grail’.
Developing a biomaterial-based solution for the repair of large joint damage presents a particularly complex challenge due to: (i) the complex zonal structure of the tissue (i.e. articular cartilage on top, an intermediate calcified cartilage layer, and underlying subchondral bone); (ii) difficulty in keeping a biomaterial in place in the joint; and (iii) challenges in directing stem cells to promote the formation of stable cartilage.
The ReCaP research program aims to develop an advanced biomaterial-based approach to repair large, clinically relevant areas of damaged articular tissue that typically progresses to OA. To date, we have successfully developed a promising scaffold that can withstand the load-bearing forces of the knee, while also promoting layer specific differentiation, driving stem cells to form different tissue types, mimicking native tissue.
Developing a biomaterial-based solution for the repair of large joint damage presents a particularly complex challenge due to: (i) the complex zonal structure of the tissue (i.e. articular cartilage on top, an intermediate calcified cartilage layer, and underlying subchondral bone); (ii) difficulty in keeping a biomaterial in place in the joint; and (iii) challenges in directing stem cells to promote the formation of stable cartilage.
The ReCaP research program aims to develop an advanced biomaterial-based approach to repair large, clinically relevant areas of damaged articular tissue that typically progresses to OA. To date, we have successfully developed a promising scaffold that can withstand the load-bearing forces of the knee, while also promoting layer specific differentiation, driving stem cells to form different tissue types, mimicking native tissue.
WP1: We successfully fabricated a 3D-Printed Multi-Layered (PML) scaffold that mimics the biochemical/biophysical features of native joint tissue; the scaffold is able to withstand load-bearing forces and it drives layer-specific stem cell differentiation. In order to achieve this, a single 3D printed structure with 3 finely tailored designs was fabricated: stiff at the bottom and soft at the top. This structure was then combined with a layered collagen-based matrix comprising collagen/nanohydroxyapatite (bone), an intermediate collagen/hyaluronic acid matrix, and another collagen/hyaluronic acid matrix on top (cartilage). The 3D printed structure and the layered scaffold matrix were combined to form a regenerative scaffold that enables cell infiltration/growth, while directing stem cells to differentiate into cartilage or bone forming cells in specific layers to mimick the structure of healthy human cartilage. Subsequent to the promising in vitro findings described, the scaffold is now being evaluated in vivo.
WP2: genetically activating collagen-based 3D matrices with small "nanoparticles" (NPs) carrying different genetic cargoes that are taken up by stem cells. The optimal NP was determined by assessing it's physicochemical features (size/charge/shape) as well as % internalization and transfection efficiency (capacity to increase the expression of genes of interest). The optimal NP formulation identified comprises a novel cell-penetrating peptide, known as the GAG-binding Enhanced Transduction system (GET) obtained through collaborations w/ University of Nottingham. To date, we have successfully developed and validated GASPs capable of delivering microRNAs, silencingRNAs and plasmid DNAs to cartilage forming cells, enhancing cartilage formation while reducing hypertrophy and inflammation.
WP3: aims to refine/further validate our technology. We have designed a PCL porous framework sheet with similar mechanical properties to human cartilage (capable of withstanding loadbearing forces while maintaining appropriate flexibility), which was successfully used as a supporting framework to reinforce previously optimised cartilage scaffolds. This 3D sheet was proven to retain the mechanical properties of our plugs while still retaining the desired microstructure of our collagen-based matrix. Furthermore, the reinforced 3D cartilage sheet was found to improve (i) MSC chondrogenic differentiation and (ii) sulphated glycosaminoglycan-rich cartilage matrix deposition compared to non-reinforced collagen-based scaffolds. This is very positive as our target was to achieve equivalence to the non-reinforced scaffolds.
WP2: genetically activating collagen-based 3D matrices with small "nanoparticles" (NPs) carrying different genetic cargoes that are taken up by stem cells. The optimal NP was determined by assessing it's physicochemical features (size/charge/shape) as well as % internalization and transfection efficiency (capacity to increase the expression of genes of interest). The optimal NP formulation identified comprises a novel cell-penetrating peptide, known as the GAG-binding Enhanced Transduction system (GET) obtained through collaborations w/ University of Nottingham. To date, we have successfully developed and validated GASPs capable of delivering microRNAs, silencingRNAs and plasmid DNAs to cartilage forming cells, enhancing cartilage formation while reducing hypertrophy and inflammation.
WP3: aims to refine/further validate our technology. We have designed a PCL porous framework sheet with similar mechanical properties to human cartilage (capable of withstanding loadbearing forces while maintaining appropriate flexibility), which was successfully used as a supporting framework to reinforce previously optimised cartilage scaffolds. This 3D sheet was proven to retain the mechanical properties of our plugs while still retaining the desired microstructure of our collagen-based matrix. Furthermore, the reinforced 3D cartilage sheet was found to improve (i) MSC chondrogenic differentiation and (ii) sulphated glycosaminoglycan-rich cartilage matrix deposition compared to non-reinforced collagen-based scaffolds. This is very positive as our target was to achieve equivalence to the non-reinforced scaffolds.
To date, ReCaP’s team has broadened the scope of the project moving beyond large articular tissue defects into treatments for early stage OA and controlling the effects of chronic joint inflammation.
The design and fabrication of multilayer PML scaffolds (WP1) involved using an innovative 3D printing technique to print a single structure with tailored mechanical properties in each layer, to be utilized as a skeleton to reinforce our collagen-based scaffolds. This innovative methodology has applications beyond bone and cartilage repair. An interesting example of the wide applicability of our technology is its recent use in the development of a new reinforced scaffold with dual capacity for bone regeneration and antimicrobial activity (i.e. to repair bone defects caused by infections e.g. osteomyelitis). The technology is also being tested within our research group to develop constructs for tracheal repair, glaucoma therapies, and scaffolds for the treatment of bladder prolapse.
The design of novel gene activated scaffold platforms (WP2) is probably the most challenging aspect of ReCaP involving: 1) fabrication of optimal gene activated NPs that can be internalised by cells and safely control gene expression; 2) loading NPs into collagen-based 3D scaffolds; and 3) identifying novel therapeutic molecules that regulate bone/cartilage repair. The ReCaP team has investigated many strategies in an attempt to solve these complex research challenges. Primarily, we have investigated the suitability of several different non-viral vectors designed in-house and obtained through scientific collaborations, including nano-hydroxyapatite, layered double hydroxide, GET and RALA NPs. Despite the selection of GET as the optimal vector for ReCaP, the other vectors we investigated have proven optimal for other applications, thus expanding the scope of ReCaP beyond osteochondral repair.
ReCaP has also benefited from a number of concurrent projects in Prof. O’Brien’s group including some focused on the design of in vitro models of healthy/diseased cartilage. Further, a fruitful collaboration involving knowledge exchange between Prof Jos Malda (UMC Utrecht) and the ReCaP team has developed. Specifically, ReCaP Fellow Dr Gonzalez Vazquez, has received hands-on training in the isolation of articular chondrocytes/chondroprogenitor cells from human knee joints. The availability of clinically relevant cartilage-forming cells has helped our team design accurate 3D models to investigate gene therapies to modulate (i) molecular mechanisms that govern cartilage repair, (ii) cartilage breakdown associated with chronic inflammation and (iii) signalling pathways that control hypertrophy.
Prof O’Brien and Dr Gonzalez Vazquez have also been leading an innovative research programme aiming to design novel biomaterials that recapitulate the extraordinary regenerative capacity of children, in adults. We have partnered with surgeons in the Children’s Health Ireland (CHI) hospital at Temple Street and Crumlin. These collaborations have been particularly successful in providing us with tissue samples that we have subsequently used to investigate the properties of juvenile stem cells from healthy tissue, plus tissue affected by craniosysnostosis (a rare condition causing premature ossification of calvarial sutures). This knowledge is now being used to design child-inspired biomaterials that restore a child-like bone or cartilage regenerative capacity in adults.
The design and fabrication of multilayer PML scaffolds (WP1) involved using an innovative 3D printing technique to print a single structure with tailored mechanical properties in each layer, to be utilized as a skeleton to reinforce our collagen-based scaffolds. This innovative methodology has applications beyond bone and cartilage repair. An interesting example of the wide applicability of our technology is its recent use in the development of a new reinforced scaffold with dual capacity for bone regeneration and antimicrobial activity (i.e. to repair bone defects caused by infections e.g. osteomyelitis). The technology is also being tested within our research group to develop constructs for tracheal repair, glaucoma therapies, and scaffolds for the treatment of bladder prolapse.
The design of novel gene activated scaffold platforms (WP2) is probably the most challenging aspect of ReCaP involving: 1) fabrication of optimal gene activated NPs that can be internalised by cells and safely control gene expression; 2) loading NPs into collagen-based 3D scaffolds; and 3) identifying novel therapeutic molecules that regulate bone/cartilage repair. The ReCaP team has investigated many strategies in an attempt to solve these complex research challenges. Primarily, we have investigated the suitability of several different non-viral vectors designed in-house and obtained through scientific collaborations, including nano-hydroxyapatite, layered double hydroxide, GET and RALA NPs. Despite the selection of GET as the optimal vector for ReCaP, the other vectors we investigated have proven optimal for other applications, thus expanding the scope of ReCaP beyond osteochondral repair.
ReCaP has also benefited from a number of concurrent projects in Prof. O’Brien’s group including some focused on the design of in vitro models of healthy/diseased cartilage. Further, a fruitful collaboration involving knowledge exchange between Prof Jos Malda (UMC Utrecht) and the ReCaP team has developed. Specifically, ReCaP Fellow Dr Gonzalez Vazquez, has received hands-on training in the isolation of articular chondrocytes/chondroprogenitor cells from human knee joints. The availability of clinically relevant cartilage-forming cells has helped our team design accurate 3D models to investigate gene therapies to modulate (i) molecular mechanisms that govern cartilage repair, (ii) cartilage breakdown associated with chronic inflammation and (iii) signalling pathways that control hypertrophy.
Prof O’Brien and Dr Gonzalez Vazquez have also been leading an innovative research programme aiming to design novel biomaterials that recapitulate the extraordinary regenerative capacity of children, in adults. We have partnered with surgeons in the Children’s Health Ireland (CHI) hospital at Temple Street and Crumlin. These collaborations have been particularly successful in providing us with tissue samples that we have subsequently used to investigate the properties of juvenile stem cells from healthy tissue, plus tissue affected by craniosysnostosis (a rare condition causing premature ossification of calvarial sutures). This knowledge is now being used to design child-inspired biomaterials that restore a child-like bone or cartilage regenerative capacity in adults.