Accurate physical representation of the tissue microenvironment is essential for implant development. In this study, we applied cyclic hydrostatic pressure (HP) to mimic the effect of cyclic hydrostatic pressure (HP) in a bone-like environment, using a custom-made, remote controlled bioreactor. In recent years, plant-derived cellulosic biomaterials have become a popular way to create scaffolds for a variety of tissue engineering applications. Moreover, such scaffolds possess similar physical properties (porosity, stiffness) that resemble bone tissues and have been explored as potential biomaterials for tissue engineering applications. Here, plant-derived cellulose scaffolds (derived from apple hypanthium tissue) were seeded with MC3T3-E1 pre-osteoblast cells. After 1 week of proliferation, cell-seeded scaffolds were exposed to HP up to 270 KPa at a frequency of 1Hz, once per day, for up to 2 weeks. Scaffolds were incubated in osteogenic inducing media or regular culture media. The effect of cyclic hydrostatic pressure combined with osteogenic inducing media on cell-seeded scaffolds resulted in an increase of differentiated cells. This corresponded with an upregulation of alkaline phosphatase activity and scaffold mineralization. The results reveal that in vitro, the mechanosensitive pathways which regulate osteogenesis appear to be functional on novel plant-derived cellulosic biomaterials.
Plant-derived cellulose biomaterials have recently been utilized in several tissue engineering applications. These naturally-derived cellulose scaffolds have been shown to be highly biocompatible in vivo, possess structural features of relevance to several tissues, and support mammalian cell invasion and proliferation. Recent work utilizing decellularized apple hypanthium tissue has shown that it possesses a pore size similar to trabecular bone and can successfully host osteogenic differentiation. In the present study, we further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) and analyzed their mechanical properties in vitro and in vivo. MC3T3-E1 pre-osteoblasts were seeded in cellulose scaffolds. Following chemically-induced osteogenic differentiation, scaffolds were evaluated for mineralization and for their mechanical properties. Alkaline phosphatase and Alizarin Red staining confirmed the osteogenic potential of the scaffolds. Histological analysis of the constructs revealed cell invasion and mineralization throughout the constructs. Furthermore, scanning electron microscopy demonstrated the presence of mineral aggregates on the scaffolds after culture in differentiation medium, and energy-dispersive spectroscopy confirmed the presence of phosphate and calcium. However, although the Young′s modulus significantly increased after cell differentiation, it remained lower than that of healthy bone tissue. Interestingly, mechanical assessment of acellular scaffolds implanted in rat calvaria defects for 8 weeks revealed that the force required to push out the scaffolds from the surrounding bone was similar to that of native calvarial bone. In addition, cell infiltration and extracellular matrix deposition were visible within the implanted scaffolds. Overall, our results confirm that plant-derived cellulose is a promising candidate for BTE applications. However, the discrepancy in mechanical properties between the mineralized scaffolds and healthy bone tissue may limit their use to low load-bearing applications. Further structural re-engineering and optimization to improve the mechanical properties may be required for load-bearing applications.
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