1 Endocrinology, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU2 KMEB, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU3 Medicinsk Bioteknologisk Center, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU4 unknown5 Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, SDU6 ESRF7 Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, SDU8 Endocrinology, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU
Introduction Cell response is closely related to substrate stiffness. Successful induced tissue repair from bioengineered constructs must possess both optimal bioactivity and mechanical strength. This is because cell interaction with the extracellular matrix (ECM) produces two different but concurrent signaling mechanisms: ligation-induced signaling, which depends on ECM biological stimuli, and traction-induced signaling, which depends on ECM mechanical stimuli, . Different substrate stiffness have contrasting effects on migration and proliferation, where cells migrate faster on softer substrates while proliferating preferentially on the stiffer ones. This implicates that substrate rigidity is a critical design parameter in the development of scaffolds aimed at eliciting maximal cell and tissue function. From mechanics it is known that the stiffness of a porous structures scales with the relative density of the porous material, . Hence, variations of substrate rigidity can be controlled through changes in relative density of the substrate itself. In three dimensional porous scaffolds, the substrate is equivalent to struts or beams randomly orientated in space making an interconnected network. These beams are called Plateau borders and are typically solid structures. Thus their stiffness depends solely on the stiffness of the selected biopolymer and the method of production. In this study we demonstrate that it is possible to control the porosity not only of the macroscopic porous scaffold but also of the Plateau borders constituting the scaffold. Materials and Methods Polycaprolactone scaffolds were prepared by thermal induced phase separation followed by lyophilization. Processing conditions were chosen to range the relative density of the obtained scaffolds and its Plateau borders. Naked scaffolds and scaffolds cultivated statically with human bone marrow stromal cells, , for 24 hours, 14 days, and 21 days and prepared for holo-tomography. Synchrotron generated hard X-rays were used to perform quantitative phase sensitive holo-tomography at the ID19 beamline to obtain three-dimensional images of the processed and cultivated scaffolds, . Results and Discussion We have demonstrated that a double graded microstructure can be synthesised in this polycaprolactone system. It is possible to obtain specimens with solid Plateau borders, intermediate structures as shown in the figure and fully inversed microstructures in which the Plateau borders is demished and converted into a three dimensional nano sized mesh. Results from specimens containing human stem cells show the attachement of cells to Plateau borders for the specimens cultivated for 24 hours. Specimens cultivated for 2 and 3 weeks shown the formations of extracellular matrix. Conclusions We have demonstrated that it is possible to control the microstructure of polycaprolactone based scaffolds. Microstructures can evolve into single and double graded structures, but also three dimensional fibrous nano meshes is realized. The morphology of the scaffold with and without human stem cells was investigated using tomography and numerical models were prepared for micromechanical modeling of cell scaffold interaction.