214 IN VITRO MIGRATION OF ADIPOSE-DERIVED STEM CELLS FROM GFP PIGS INTO POLYCAPROLACTONE SCAFFOLDS TREATED WITH FGF OR BMP2

Abstract

We have previously demonstrated that adipose-derived stem cells (ASC) enhance healing of craniofacial bone in vivo. The use of scaffolds in large bone defects can further enhance the healing capacity of ASC. In addition, the presence of fully differentiated cells might affect ASC migration/proliferation into scaffolds. In this study, we have evaluated the in vitro capacity of ASC isolated from fat of GFP transgenic pigs to migrate into polycaprolactone (PCL) scaffolds conjugated with FGF or BMP2 with or without combinations of ASC with pig endothelial and/or neuronal cells. The ASC were extracted from the back fat of a GFP boar while the endothelial cells (ENDO) were extracted from the aorta a newborn piglet and the neurons (NEU) from the brain of a 37 days fetus. All cells were used at passage 3. In a 24-well plate, approximately 10 000 cells (only ASC, ENDO, or NEU, or equal combination of ASC+ENDO, ASC+NEU, or ASC+ENDO+NEU) were plated. The cells were grown for 3 days before adding the scaffolds in the wells. The scaffolds were treated with basic human FGF (25 ng mg^–1 of scaffold), porcine BMP2 (400 ng mg^–1 of scaffold), or no treatment (CTR). The scaffolds were then added to the plate and the medium was changed every 3 days. Using an Olympus IX71 microscope with automatic stage several pictures at 100× and 200× magnifications were taken and time-lapse visualisation of the movement of cells into the scaffold was performed during the first week. At 24 days the scaffolds were fixed with formalin. In order to estimate cell size and visualise non-GFP cells all scaffolds were treated with DAPI. Final photographs of the whole scaffold using a multiple alignment images features at 40× magnification were performed using GFP and DAPI filter plus brightfield. The figures were analysed with ImageJ to quantify the area of GFP and DAPI in the scaffolds. Percentage GFP and DAPI over scaffold area were calculated. Data were analysed using a Proc ANOVA of SAS, with cells and treatment as fixed effects and well as random. At 1 week from the beginning of the trial, we observed ASC cells on the scaffolds. The time-lapse showed ASC actively moving on the scaffold surface and entering into its micropores. The quantification of GFP and DAPI showed a significant larger percentage area covered by GFP cells in scaffolds treated with FGF (P < 0.05) compared to CTR or BMP2 (32 ± 4.6 vs 12.8 ± 9.1 and 3.8 ± 0.1). We observed a numerical lower area covered by GFP in scaffolds treated with BMP2 compared to CTR. The same trends were observed for the area covered by DAPI with a numerically lower colonization of the scaffolds by ENDO when treated with BMP2 vs CTR. Data showed that ASC, as well ENDO and NEU, are able to home into PCL scaffolds. The conjugation of the scaffolds with FGF has a positive effect on ASC migration, with no effects of BMP2. Data support the use of PCL scaffolds to enhance the bone healing capacity in vivo of ASC with a likely further improvement if scaffolds are treated with FGF before implantation.