Layer-by-layer Self-assembly Of Bioactive Molecules On Polycaprolactone Honeycomb-like Porous Films For In Vitro Culture Of Mesenchymal Stem Cells

Cellular behaviors can be affected by porous structure and bioactive molecules.However,most studies have paid attention to the effects of individual factors of biomaterials on cellular behaviors.How cells respond to a combination of different factors is rarely understood.In our study,firstly,polycaprolactone(PCL)was selected to fabricate both flat and honeycomb films by direct casting and breath figure method,respectively.Hydrolysis treatment with NaOH was optimized concerning both time and concentration,and then PCL films was further modified with layer-by-layer(LBL)assembly of bioactive molecules.Films were characterized with scanning electron microscopy(SEM),confocal laser scanning microscopy(CLSM),water contact angle measurement,and quantification of bioactive molecules.Finally,rabbit bone marrow mesenchymal stem cells(rMSCs)were seeded on PCL films with and without LBL modification to evaluate both cell adhesion and proliferation.By optimizing different parameters of breath figure method,a uniform honeycomb-like porous structure was obtained under the following condition:tetrahydrofuran(THF)/chloroform(97:3,v/v)as organic solvent,PCL concentration of 40 mg/mL,casting volume of 750 ?L,and the relative humidity of higher than 65%.Pore morphology and size were uniform based on SEM observation,and the pore size was determined to be 18.87±3.25 ?m.Bioactive molecules were deposited onto PCL films including the following 4 different combinations:gelatin/chondroitin sulfate(CS),gelatin/hyaluronic acid(HA),chitosan/CS and chitosan/HA.Water contact angles fluctuated between the odd and even layers.By exploiting fluorescein isothiocyanate(FITC)-labeled gelatin and chitosan,bioactive molecules were proven to be present on the surfaces of PCL films based on both CLSM observation and fluorescent intensity determination using a fluorometer,and the thickness of LBL increased with the number of deposited layers of bioactive molecule pairs.Further,it was found that LBL modification improved the adhesion and proliferation of rMSCs on PCL films,and cell spreading was restricted on the honeycomb-like porous films compared to that on flat films.In conclusion,surface modificaton of bioactive molecules and the porous structure in synergy play a significant impact on adhesion,morphology,spread and proliferation of rMSCs.