A microphysiological model of bone development and regeneration

Endochondral ossification (EO) is an essential biological process than underpins how human bones develop, grow, and heal in the event of a fracture. So much is unknown about this process, thus clinical manifestations of dysregulated EO cannot be adequately treated. This can be partially attributed to the absence of predictive in vitro models of musculoskeletal tissue development and healing, which are integral to the development and preclinical evaluation of novel therapeutics. Microphysiological systems, or organ-on-chip devices, are advanced in vitro models designed for improved biological relevance compared to traditional in vitro culture models. Here we develop a microphysiological model of vascular invasion into developing/regenerating bone, thereby mimicking the process of EO. This is achieved by integrating endothelial cells and organoids mimicking different stages of endochondral bone development within a microfluidic chip. This microphysiological model is able to recreate key events in EO, such as the changing angiogenic profile of a maturing cartilage analogue, and vascular induced expression of the pluripotent transcription factors SOX2 and OCT4 in the cartilage analogue. This system represents an advanced in vitro platform to further EO research, and may also serve as a modular unit to monitor drug responses on such processes as part of a multi-organ system

Temporal enzymatic treatment to enhance the remodeling of multiple cartilage microtissues into a structurally organized tissue

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development. </p

Temporal enzymatic treatment to enhance the remodeling of multiple cartilage microtissues into a structurally organized tissue

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development. </p

A microphysiological model of bone development and regeneration

Endochondral ossification (EO) is an essential biological process than underpins how human bones develop, grow, and heal in the event of a fracture. So much is unknown about this process, thus clinical manifestations of dysregulated EO cannot be adequately treated. This can be partially attributed to the absence of predictivein vitromodels of musculoskeletal tissue development and healing, which are integral to the development and preclinical evaluation of novel therapeutics. Microphysiological systems, or organ-on-chip devices, are advancedin vitromodels designed for improved biological relevance compared to traditionalin vitroculture models. Here we develop a microphysiological model of vascular invasion into developing/regenerating bone, thereby mimicking the process of EO. This is achieved by integrating endothelial cells and organoids mimicking different stages of endochondral bone development within a microfluidic chip. This microphysiological model is able to recreate key events in EO, such as the changing angiogenic profile of a maturing cartilage analogue, and vascular induced expression of the pluripotent transcription factors SOX2 and OCT4 in the cartilage analogue. This system represents an advancedin vitroplatform to further EO research, and may also serve as a modular unit to monitor drug responses on such processes as part of a multi-organ system. </p

Integrating melt electrowriting and fused deposition modeling to fabricate hybrid scaffolds supportive of accelerated bone regeneration

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs. </p

Integrating melt electrowriting and fused deposition modeling to fabricate hybrid scaffolds supportive of accelerated bone regeneration

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs. </p