Introduction: Bone fracture healing has become a serious problem in the last decades in part due to the increase in life expectancy[1]. The use of strategies that help body to restore bone are needed to increase the quality life of patients. Among this strategies there is the use of natural sources such as; bone, growth factors and other biomolecules. They are very effective but present some limitations like money cost, amount limitation, extra surgeries, rejection and disease transmission[1]. The use of synthetic materials may avoid this nonetheless they need to be improved to be as efficient. Hybrid materials are and interesting alternative. Their organic phase, normally a biopolymer, holds the mechanical stress while their inorganic phase, a glass or ceramic, provides the needed signals to activate specific cell responses and produce bone. However the usual masking of the inorganic phase inside the organic matrix and undesired phase-detachments should be solved to increase their effectiveness[2]. Another limitation is the poor vascularization that synthetic materials induce per se. Previous studies in our group demonstrate that Ca2+ release may help to improve this issue[3]. Here we present a novel protocol consisting of polylactic acid (PLA) nanofibers covalently coated with SiCaP degradable ormoglass nanoparticles2 (Fig.1)[4] which release Ca2+ while keep the ormoglass well attached and totally exposed to the cells.
Materials and Methods: PLA nanofibers were produced by electrospinning and SiCaP Ormoglass nanoparticles by sol-gel method. Three different calcium contents (Ca50, Ca60, Ca80 %) were assayed in order to find the best calcium concentration. The ormoglass nanoparticles were linked on the fibers through the EDC/NHS chemistry and using APTES as coupling agent. Experiments included FE-SEM images, EDS analysis, DLS particle size, Ca2+ release and in vitro biological characterizatoin with rMSCs and rEPCs.
Results: FE-SEM images showed fiber diameters between 1-2 µm and a successful attachment of the nanoparticles (Fig.14). EDS analysis showed the desired composition for the ormoglass. DLS showed a particle size around 100 nm with significant bigger size for Ca50. Ca2+ release measurements in cell-like osmotic media showed a sustained release even after two weeks. The highest values were for Ca50. In vitro experiments showed a significant improvement in cell proliferation for ormoglass coated fibers (Fig.2), specially Ca50 composition.
Discussion and Conclusions: Ormoglass nanoparticles were successfully attached on the fibers. The ormoglass composition, degradability and particle size could be easily modified. The lower the calcium content, the bigger the particle size and lower the degradability. However particle size and therefore coating thickness also affected in ion release in terms of enviromental concentration. Thanks to the high surface area and the degradability of materials there was a significant and sustained Ca2+ release by coated fibers. Ca2+ release seemed to improve cell proliferation. Next steps include studies with EPCs and MSCs co-cultures to check angiogenesis and osteogenesis. This novel protocol solves masking and detachment of the ormoglass while increases the effectiveness of the device in an easy and low-cost way. Important step to make synthetic materials as competitive as biological ones.
European Commision (European ERANET project PI11/03030, NANGIOFRAC); Spanish Ministry of Economy and Competitiveness (project MAT2011-29778-C02-01)
References:
[1] M. Navarro et al. J. R. Soc. Interface (2008) 5, 1137-1158.
[2] N. Sachot et al. J. R. Soc. Interface (2013) 10, 20130684.
[3] A. Aguirre et al. European Cells and Materials Vol. 24 2012 (pages 90-106).
[4] N. Sachot et al. Nanoscale, 2015, 7 15349-15361.