Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation

摘要

Recently, our group has proposed a combinatorial strategy in tissue engineering principles employingcarboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellularrelease and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenicdifferentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if theDex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation ofosteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrowstromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml−1 DexloadedCMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cellsexpanded in the presence and absence of 10−8 M Dex were used as controls. The effect of initial cell numberseeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitativeand quantitative new bone formation was evaluated in a non-destructive manner using micro-computedtomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used forthe histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis ofproteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explantswere also quanti!ed, since these markers denote osteogenic differentiation. At 4 weeks post-implantationresults have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimernanoparticles may be bene!cial as an intracellular nanocarrier, supplying Dex in a regimented mannerand promoting superior ectopic de novo bone formation.