Jose F. Alvarez-Barreto, Chemical, Biological and Materials Engineering, University of Oklahoma, 100 E. Boyd St. Rm T-335, Norman, OK 73019 and Vassilios I. Sikavitsas, School of Chemical, Biological, and Materials Engineering, The University of Oklahoma, 100 East Boyd, Room T335, Norman, OK 73019.
It has been demonstrated that RGD incorporated on the surface of biomaterials enhances osteoblastic differentiation of mesemchymal stem cells (MSC) in a dose-dependent manner. Previous studies have shown that MSC osteoblastic differentiation can also be enhanced mechanically by the presence of flow perfusion. However, culture of MSC in the presence RGD has only been carried out under static conditions. In order to carry out a dynamic culture in a flow perfusion bioreactor, the seeding of RGD-modified scaffolds must be carefully characterized. In this study, we aim to evaluate the effect of RGD surface concentration on scaffold cellularity and cell adhesion on 3-D scaffolds seeded in a flow perfusion bioreactor. Modification of PLLA foams with RGD peptides and scaffold seeding using oscillating flow perfusion have been previously characterized in our laboratory. PLLA foams were soaked in an acetone-water mixture, and then incubated in a solution of poly (L-lysine) (polyK). RGDC peptides were linked to the polyK through a disulfide bond, using SPDP as the linker. RGDC surface concentration was varied by changing the polyK incubation concentration. MSC were seeded on the modified scaffolds using oscillating flow perfusion. Inoculated cell suspensions, containing 500,000, 750,000 and 1,000,000 cells, were placed on top of scaffolds accommodated in the chambers of a flow perfusion bioreactor and oscillating flow was applied at 0.15 ml/min for 2h. Cells were allowed to condition for 2h without flow; unidirectional flow was incorporated at 0.15 ml/min and increased after 2h to different flow rates. These flow rates included 0.15, 0.5 and 1.0 ml/min. Scaffold cellularity increased with the inoculation cell number and then reached a maximum. Furthermore, cellularity presented a direct dependence on the RGDC surface concentration. However, cell detachment increased at higher flow rates, with a greater extent of detachment at lower RGDC surface concentrations. We were able to produce expressions of scaffold cellularity as a function of unidirectional flow rate and inoculation cell number at a constant RGDC surface concentration using the Design of Experiments (DOE) technique. Thus, the number of cells attached to a scaffold modified with a specific RGD surface concentration can be predicted.