Effects of morphogen advection on osteogenic differentiation of hMSCs using a microfluidic device with 2D micro patterned cell colonies

It has been established that primary stability of femoral stems is a determinant of the clinical success of cementless total hip arthroplasty. Excessive interface micromotions may lead to a peri-implant fibrous tissue formation resulting in aseptic loosening of the implant. The effect of micromotion on the tissue outcome remains still unclear. However, it is becoming increasingly clear that interstitial fluid flow is the primary mechanism by which bone cells perceive changes in their mechanical environment. However, prior to bone formation by osteoblasts there is a mesenchymal stromal cell (MSC) recruitment to the site which, in a favorable environment, will differentiate to these bone apposing osteoblasts. Therefore the aim of this study is determine the effects of flow advected  morphogens on the osteogenic differentiation of human MSCs. In order to tackle this question a PDMS and polystyrene microfuidic device with a constant unidirectional fluid flow on a symmetric striped pattern of 2D cell colonies was developed and implemented. The pattern of cell colonies was created by micro contact printing fibronectin and backfiling with PLL-g-PEG on an oxygen plasma treated polystyrene surface prior cell seeding. The flow, the dimensions of the flow chamber and size of the cell patterns were designed so as to have an advective dominated dynamics for morphogens. Thus cell patterns of same size up stream and down stream are affected differently as morphogens can only be advected downstream. The degree of osteogenic differentiation for the different cell colonies was evaluated using semi-quantitative immunofluorescence using the following hMSCs markers, CD90, CD105 and CD73. DAPI staining was used for measuring the cell density. And the morphology of the cells was analyzed at fixed time intervals during the flow experiment with a confocal inverse microscope. The flow rates values used were between 5 and 100microns/s, the shear stress undergone by cells was of a maximum of 1 Pa, the cell pattern sizes calculated and used in the experiment varied between 50 to 1000 microns. The Flow chamber height was of 100 microns and width of 0.8 and 1 mm. The micro-contact printing method used allowed a stable pattern of cells for at least on week of cell culture, allowing an experimental duration of at least 7 days. The immunofluorescence and confocal imaging was successfully achieved and its data analysis is currently ongoing. Nevertheless, according to preliminary results, different patterns showed different degrees of differentiation and a dependance on fluid flow. Final results could emphasize the importance of fluid flow in the prei-implant site for proper hMSCs differentiation into osteoblasts, stressing the importance of post-operative micormotion values.