A novel perfusion bioreactor for the culture of large and anatomically accurate tissue scaffolds

摘要

Introduction: The synthesis of biologically relevant tissue in vitro is showing great promise in medical applications from commercially available products such as skin grafts to exciting future possibilities including the treatment of critically sized bone defects. Providing cells with sufficient nutrient supply, removal of cellular waste and appropriate levels of hydrodynamic stimuli has proved to be a considerable challenge when culturing large and anatomically accurate tissue scaffolds. This study demonstrates the development of a versatile perfusion bioreactor system capable of controlling biologically relevant hydrodynamic parameters within scaffolds of varying shapes derived from medical imaging data. Materials and Methods: Pre-existing medical imaging data was used to generate the external shape of large osseous tissue scaffolds including femur, cranium and mandibular sections. A control design was created utilising close fitting cassettes, necessary to ensure the culture media flows through the body of the scaffold. This type of design is typically used in perfusion bioreactor studies. A number of bioreactor-scaffold system concepts were generated using computer aided design software (Solidworks, Dassault Systems) and then evaluated with computational fluid dynamics (Fluent, ANSYS Inc.). Key parameters assessed were the capability for mass transit (fluid velocity distribution and flow paths) and the ability to control hydrodynamic forces throughout the structure of the scaffold. The best performing concept was developed into a functional perfusion bioreactor and tested under fluid flow conditions. Results and Discussion: The control design was found to cause large variations in local fluid velocity and hydrodynamic shear stresses within the body of each scaffold. Scaffold external shape was also found to affect the internal distribution of fluid shear stress, hydrodynamic shear and fluid flow paths. These variations in the fluid velocity and shear stress are likely to result in areas of non-viable cell culture conditions due to mass transit limitations and/or excessive local hydrodynamic shear. Analysis of concepts resulted in the selection and development of a bioreactor-scaffold design that used customised flow normalising geometry to provide a homogenous distribution of fluid velocity and hydrodynamic shear stress throughout the body of the scaffolds. Furthermore, this design reduced fluid flow field variation between scaffolds of significantly different external shape. Prototyping and testing verified the manufacturability and practicality of the design. Conclusions: Customised fluid flow homogenising geometry matched to the shape of the scaffold was found to be an effective method for controlling the fluid flow parameters responsible for viable cell culture for a range of scaffold shapes reconstructed from medical imaging data. Further development of the system is anticipated with testing under live cell culture conditions.