Adhesion, Proliferation, and Osteogenic Differentiation of a Mouse Mesenchymal Stem Cell Line (BMC9) Seeded on Novel Melt-Based Chitosan/Polyester 3D Porous Scaffolds

Abstract

The aim of the present work was to study the biological behavior of a mouse mesenchymal stem cell line when seeded and cultured under osteogenic conditions onto novel processed melt-based chitosan scaffolds. Scaffolds were produced by compression molding, followed by salt leaching. Scanning electron microscopy (SEM) observations and lCT analysis showed the pore sizes ranging between 250 and 500 lm and the interconnectivity of the porous structure. The chitosan-poly(butylenes succinate) scaffolds presented high mechanical properties, similar to the ones of trabecular bone (E1%*75 MPa). Cytotoxicity assays were carried out using standard tests (accordingly to ISO/EN 10993 part 5 guidelines), namely, MTS test with a 24 h extraction period, revealing that L929 cells had similar metabolic activities to that obtained for the negative control. Cell culture studies were conducted using a mouse mesenchymal stem cell line (BMC9). Cells were seeded onto the scaffold and allowed to proliferate for 3 weeks under osteogenic conditions. SEM observations demonstrated that cells were able to proliferate and massively colonize the scaffolds structure. The cell viability assay MTS demonstrated that BMC9 cells were viable after 3 weeks of culture. The cells clearly evidenced a positive differentiation toward the osteogenic lineage, as confirmed by the high ALP activity levels. Moreover, energy dispersive spectroscopy (EDS) analysis revealed the presence of Ca and P in the elaborated extracellular matrix (ECM). These combined results indicate that the novel melt-based chitosan/polyester scaffolds support the adhesion, proliferation, and osteogenic differentiation of the mouse MSCs and shows adequate physicochemical and biological properties for being used as scaffolds in bone tissue engineering-related strategies.

Chitosan-poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model

“…SEM micrographs of the porous scaffolds are presented in Figure 2; Figure 2a shows the morphology of scaffolds 4 mm in diameter and 1 mm thick. Previous studies (Alves da Silva et al , 2010; Costa‐Pinto et al , 2008; Oliveira et al , 2008) demonstrated that these scaffolds had adequate porosity (Figure 2b) to allow extensive cell proliferation.…”

“…For that, we tested several blends with a mouse mesenchymal stem cell line (BMC9), promoting differentiation into the osteogenic lineage. The results evidenced that the chitosan–PBS blend formulation, 50% wt and 60% porosity, showed the best performance in terms of cell behaviour (Costa‐Pinto et al , 2008). Further studies were performed using human bone marrow mesenchymal stem cells (hBMSCs) in fibre‐mesh scaffold morphology, with excellent results in terms of cell adhesion, proliferation and osteogenic differentiation (Costa‐Pinto et al , 2009).…”

“…Ideally this structure should be biodegradable, allowing cells to adhere and proliferate, leading to the formation of ECM (Salgado et al , 2004). Different naturally based polymers have been proposed for this demanding application, such as starch (Gomes et al , 2001; Salgado et al , 2002) and chitosan (Costa‐Pinto et al , 2008, 2009; Malafaya et al , 2005; Martins et al , 2008). Chitosan has shown an excellent combination of properties, including non‐antigenicity and non‐cytotoxicity, making this biomaterial quite attractive for bone tissue‐engineering applications (Di Martino et al , 2005; Zarzycki and Modrzejewska, 2003).…”

Evaluation of cross-linked chitosan microparticles for bone regeneration

“…Although several studies have reported the use of CS scaffolds (Costa‐Pinto et al , 2008; Heinemann et al , 2008; Seol et al , 2004), the main disadvantage of these scaffolds is the fabrication of the scaffold according to the size of the defect, or carving the graft to fit the defect. This could result in additional bone loss and trauma.…”

Gradual pore formation in natural origin scaffolds throughout subcutaneous implantation

“…To prove the concept of in situ pore formation in vitro , previous studies simulating the inflammatory response were performed, and the formation of pores was clearly visible when lysozyme was incorporated in CaP chitosan scaffolds 2. The combination of chitosan with other biodegradable materials has already been shown to be effective for bone‐related applications 11–13. The inclusion of starch in chitosan matrices constitutes an interesting approach towards obtaining scaffolds with enhanced degradation rates since starch is acting as a sacrifice material 1.…”