Introduction: These past years, marked by the increasing age of the global population, the frequency of bone loss and fractures has considerably increased. The technique most frequently used to overcome bone loss is autograft, but it has multiple post operational complications that arise in 8.5-20% of graft procedures[1]. As an alternative, biomimetic materials have been developed to favour mesenchymal stem cells (MSCs) adhesion[2]. For this purpose, the biomaterial is usually functionalized by peptides containing the sequence Arg-Gly-Asp (RGD) found in several proteins of the extracellular matrix[3]. RGD is recognized by cellular receptor integrins. However, osteoblastic differentiation of MSCs is also influenced by the bone morphogenetic proteins (BMPs). The co-localization of integrin and BMP receptors[4] highlights the potential of functionalizing biomaterials not only with adhesive peptides, but also with peptides derived from BMPs[5]. Saito et al have developed a peptide derived from the knuckle epitope of BMP-2 that promotes MSC differentiation into osteoblasts[6]. Since BMP-9 has a higher osteoinductive potential than BMP-2[7],[8], our research group has developed a peptide derived from BMP-9 that induces osteoblast differentiation in vitro and bone formation in vivo[9]-[11]. We also recently found that a peptide containing the RGD and synergy sequences of fibronectin (pFibro) promotes a higher MSC response to peptides derived from BMP-9 (pBMP-9 and SpBMP-9) in comparison to a peptide derived from the bone sialoprotein. The present study has therefore analysed the effect of polycaprolactone (PCL) films co-functionalized by pFibro and SpBMP-9 on MSC behaviours.
Material and Methods: We first determined the number of murine C3H10T1/2 MSC attached to functionalized PCL after incubation for 0.5 to 4h. The cytoskeletal organization of these attached cells was also studied by labelling F-actin. The activation state of the canonical BMP pathway was determined using Western blots. Finally, by carrying out qPCR, we also analysed the activation of genes encoding osteogenic marker Dlx5 and Runx2 at days 3 and 6.
Results and Discussion: At 4h, all functionalized PCL films promoted the attachment of C3H10 T1/2 cells. These cells organized their actin cytoskeleton. MSCs attached to PCL films functionalized with SpBMP-9 alone or a combination of pFibro with SpBMP-9 also contained a higher level of phosphorylated Smad 1/5/8, demonstrating that grafted SpBMP-9 was able to activate the canonical BMP pathway. At 6 days, the cells attached to PCL functionalized by SpBMP-9 alone or a combination of SpBMP-9 and pFibro contained higher level of Dlx5 and Runx2 transcripts, suggesting the osteoblastic commitment of the MSCs.
Conclusion: A better understanding of cellular response to co-functionalized PCL is required. However, the co-functionalization of PCL by pFibro and SpBMP9 seems a promising approach to develop scaffold for tissue engineering application.
NSERC
References:
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