Application of microfabrication techniques for tissue engineering a cardiac conductive device

Impairment of the atrioventricular electrical conduction (AV-block) is a major cause for the implantation of an electronic pacemaker device. Even though this is the standard treatment today, it has its disadvantages. One major problem is the implementation of long-term pacing therapy in pediatric patients owing to the restrictions imposed by a child’s small size and their inevitable growth. Thus there is a genuine need for innovative therapies especially for children with cardiac rhythm disorders. The aim of the Biopacer project is to develop an autologous conductive tissue device that will serve as an electrical conduit between the upper and lower chambers of the heart. The idea is to use pediatric cardiomyocytes together with a fibrin-based scaffold to produce a tissue construct that is completely autologous and has the ability to grow with the patient. Mimicking the complexity and highly organized structure of native cardiac tissue sets significant challenges for the engineering process. To address this challenge, bottom-up tissue engineering, in which structural components of different cell types with high-degree organization are fabricated separately and later assembled together, was applied. The aim of this thesis was to utilize different microfabrication techniques for the realization of the fibrin scaffold with different cell types to fabricate building blocks for the assembly of a conductive tissue device with a controllable, spatially organized tissue microarchitecture. Two methods, micromolding in capillaries (MIMIC) and microfabrication of fibrin fibers by extrusion, were established and exploited in the fabrication of cell-seeded 3D fibrin tissue modules. Using the MIMIC technique, cells were spatially confined into a 3D fibrin gel structure and they were shown to align longitudinally in small diameter fibrin gels. In extruded fibrin fibers, endothelial cells were demonstrated to coalesce and bundle the surrounding fibrin into a core around which they arranged themselves resembling the phenomenon of tubulogenesis. The organization of these microfabricated tissue modules together to create a conductive tissue device consisting of separate functional units (e.g. aligned cardiomyocytes, vasculature, extracellular matrix) was additionally displayed. This way, all the structural components of native cardiac tissue are accounted for and highly organized in the engineered construct

Application of microfabrication techniques for tissue engineering a cardiac conductive device

Impairment of the atrioventricular electrical conduction (AV-block) is a major cause for the implantation of an electronic pacemaker device. Even though this is the standard treatment today, it has its disadvantages. One major problem is the implementation of long-term pacing therapy in pediatric patients owing to the restrictions imposed by a child’s small size and their inevitable growth. Thus there is a genuine need for innovative therapies especially for children with cardiac rhythm disorders. The aim of the Biopacer project is to develop an autologous conductive tissue device that will serve as an electrical conduit between the upper and lower chambers of the heart. The idea is to use pediatric cardiomyocytes together with a fibrin-based scaffold to produce a tissue construct that is completely autologous and has the ability to grow with the patient. Mimicking the complexity and highly organized structure of native cardiac tissue sets significant challenges for the engineering process. To address this challenge, bottom-up tissue engineering, in which structural components of different cell types with high-degree organization are fabricated separately and later assembled together, was applied. The aim of this thesis was to utilize different microfabrication techniques for the realization of the fibrin scaffold with different cell types to fabricate building blocks for the assembly of a conductive tissue device with a controllable, spatially organized tissue microarchitecture. Two methods, micromolding in capillaries (MIMIC) and microfabrication of fibrin fibers by extrusion, were established and exploited in the fabrication of cell-seeded 3D fibrin tissue modules. Using the MIMIC technique, cells were spatially confined into a 3D fibrin gel structure and they were shown to align longitudinally in small diameter fibrin gels. In extruded fibrin fibers, endothelial cells were demonstrated to coalesce and bundle the surrounding fibrin into a core around which they arranged themselves resembling the phenomenon of tubulogenesis. The organization of these microfabricated tissue modules together to create a conductive tissue device consisting of separate functional units (e.g. aligned cardiomyocytes, vasculature, extracellular matrix) was additionally displayed. This way, all the structural components of native cardiac tissue are accounted for and highly organized in the engineered construct

Differences in fetal bovine serum affect the responsiveness of cells to mechanical loads

Nowadays, the end-point of a cell culture in bone tissue engineering\u3cbr/\u3e(BTE) is the acquisition of a well mineralized extracellular\u3cbr/\u3ematrix. The biological performance of BTE relies on evaluation of\u3cbr/\u3ethe cell capacity to proliferate and to produce extracellular matrix by\u3cbr/\u3equantification of gene expression and by histology or calcium\u3cbr/\u3equantification assays. Micro-computed tomography (micro-CT) allows\u3cbr/\u3emonitoring of BTE mineral constructs in a non-destructive\u3cbr/\u3emanner. Although fetal bovine serum (FBS) is commonly used as\u3cbr/\u3esupplement in cell cultures, its high composition variability between\u3cbr/\u3edifferent brands and batches leads to differences in the experimental\u3cbr/\u3eoutcomes. Nevertheless, only few studies have focused on a systematic\u3cbr/\u3einvestigation of the differences. While we have recently\u3cbr/\u3ereported the influence of FBS type on matrix mineralization under\u3cbr/\u3estatic culture conditions, it is still unknown how FBS affects cells in\u3cbr/\u3edynamic cultures. Different FBS types were used to differentiate\u3cbr/\u3ehuman mesenchymal stem cells down the osteogenic lineage under\u3cbr/\u3edynamic spinner-flask bioreactors. Opposite to static culture conditions,\u3cbr/\u3edifferences in FBS affected the responsiveness of cells to\u3cbr/\u3edifferentiate under mechanical loads. Although all FBS types upregulated\u3cbr/\u3ethe expression of bone-specific genes, differences in the\u3cbr/\u3eosteogenic differentiation stage were observed among the different\u3cbr/\u3eFBS. Accordingly, micro-CT analysis only showed mineral deposition\u3cbr/\u3efor cultures in an advanced differentiation stage.\u3cbr/\u3eThus the selection of the FBS type is crucial for the success in the\u3cbr/\u3eacquisition of BTE constructs. The combination of micro-CT with\u3cbr/\u3emolecular biology techniqueswill benefit efforts to optimize scaffolds\u3cbr/\u3edesign and cell culture conditions for scaling-up the BTE constructs

Scaffold Pore Geometry Guides Gene Regulation and Bone-like Tissue Formation in Dynamic Cultures

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell–scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. In this study, monodisperse templated scaffolds (MTSC) were fabricated and used as well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow-derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress [WSS] using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cell differentiation, bone-like tissue formation, structure, and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and microcomputed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared with static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, whereas MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared with MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure, and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs

Scaffold Pore Geometry Guides Gene Regulation and Bone-like Tissue Formation in Dynamic Cultures

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. In this study, monodisperse templated scaffolds (MTSC) were fabricated and used as well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow-derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress [WSS] using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cell differentiation, bone-like tissue formation, structure, and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and microcomputed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared with static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, whereas MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared with MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure, and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs. Impact statement Aging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction toward the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization

Mechanical and biochemical mapping of human auricular cartilage for reliable assessment of tissue-engineered constructs

It is key for successful auricular (AUR) cartilage tissue-engineering (TE) to ensure that the engineered cartilage mimics the mechanics of the native tissue. This study provides a spatial map of the mechanical and biochemical properties of human auricular cartilage, thus establishing a benchmark for the evaluation of functional competency in AUR cartilage TE. Stress-relaxation indentation (instantaneous modulus, Ein; maximum stress, σmax; equilibrium modulus, Eeq; relaxation half-life time, t1/2; thickness, h) and biochemical parameters (content of DNA; sulfated-glycosaminoglycan, sGAG; hydroxyproline, HYP; elastin, ELN) of fresh human AUR cartilage were evaluated. Samples were categorized into age groups and according to their harvesting region in the human auricle (for AUR cartilage only). AUR cartilage displayed significantly lower Ein, σmax, Eeq, sGAG content; and significantly higher t1/2, and DNA content than NAS cartilage. Large amounts of ELN were measured in AUR cartilage (>15% ELN content per sample wet mass). No effect of gender was observed for either auricular or nasoseptal samples. For auricular samples, significant differences between age groups for h, sGAG and HYP, and significant regional variations for Ein, σmax, Eeq, t1/2, h, DNA and sGAG were measured. However, only low correlations between mechanical and biochemical parameters were seen (R<0.44). In conclusion, this study established the first comprehensive mechanical and biochemical map of human auricular cartilage. Regional variations in mechanical and biochemical properties were demonstrated in the auricle. This finding highlights the importance of focusing future research on efforts to produce cartilage grafts with spatially tunable mechanics