Introduction: In the biomedical field, it is growing the request of alternative routes to synthesize new materials with biologically recognized sequences (i.e., peptides, growth factors). Traditionally, adsorption or protein immobilization is an attractive method to improve biomaterial–cell interactions. However, they are generally subjected to denaturation and/or degradation which often create a mismatch with biocompatibility and fluid transport properties of polymer matrices used for a molecular release[1]. In this context, ECM-like molecules (i.e., glycosaminoglycan) may be successfully used to sequester growth factors, so guiding cell function during the regeneration process. In particular, highly negative charge of heparin (HEP) may allow for the formation of weak bonds with many growth factors and positively charged aminoacids, thus affecting the mechanism of molecular release in composite systems[2]. Hence, we propose to investigate two different strategies of Hep conjugation to gelatin (Gel) by amide group linkage to design electrospun fibrous composites as ECM analogues.
Materials and Methods: Poly-ε-caprolactone (PCL) fibers were electrospun by dissolving the polymer in chloroform (0.33 g/ml). Composite was fabricated by intruding gelatin solution in PCL fibers following two different strategies. In “two step” strategy, gelatin B (0.16 g) was dissolved in 5.37 ml of 0.05 M MES buffer at 37°C. EDC (0.0433 g) and NHS (0.010 g) were added until the end of the reaction. After Na2HPO4 solution washes to stop the reaction, the composite was placed in MES solution (1 ml) containing heparin (2 mg/ml) and EDC (50 mM) to trigger the conjugation reaction. In the case of “one step” strategy, heparin was added to the gelatin solution prior to the protein cross-linking. SEM/EDS analysis was performed to qualitative evaluate the morphology, BCA test was performed to evaluate gelatin release whereas toluidine blue (TB) staining was used to verify and quantify the conjugated heparin molecules.
Results and Discussion: EDS detected the presence of heparin on the different scaffolds surface. Toluidine blue assay confirmed the presence of Hep in both cases, highlighting a higher binding - over 30% - in the case of “one step” samples than “two step” ones, due to a preferential interaction between EDC and heparin. All samples have shown an initial burst followed by a slower release. After 28 days, samples by “one step” showed a loss about of 50% less than samples by “two step”, thus indicating a lower exposure of heparin ascribable to a lower availability of amides during gelatin cross-linking.
Conclusion: Hep/Gel conjugation is a promising strategy to sequester/release active molecules in electrospun fibrous composites. The ability to locally deliver growth factors in combination with peculiar morphological patterns makes fibrous composites suitable to mimic main ECM functions of native tissues.
PON2-00029_3203241 (POLIFARMA) and REPAIR (PON01-02342).
References:
[1] Benoit et al. Acta Biomat 1, 461. 2005
[2] Nakamura et al. JBiosci Bioeng 115, 562, 2013