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    • Also critical to the success of hard tissue engineering stra

    2018-10-20

    Also critical to the success of hard tissue engineering strategies is the selection and use of an appropriate growth factor regimen. Although several growth factors are reported to play an important role in the process of matrix mineralisation, IGF-1 is the most abundant growth factor in bone matrix (Canalis et al., 1993) and several studies have shown that the IGF axis plays an important role in the development and maintenance of skeletal tissues (Delany et al., 2001; Gan et al., n.d.; Wong et al., 1990; Yang et al., n.d.; Zhang et al., 2002). IGF1 also regulates the osteogenic differentiation of mesenchymal stem Bioactive Compound Library (MSCs) via mTOR signalling (Xian et al., 2012), BMP9 (Chen et al., n.d.) and MEK-ERK mediated upregulation of the transcriptional co-activator with PDZ-binding motif (TAZ) protein (Xue et al., 2013). These alterations in cell physiology ultimately lead to expression of characteristic osteoblast marker genes, the phenotypic differentiation of osteoblasts and the process of bone accretion (Koch et al., 2005). While some of the molecular details associated with IGF action on osteogenic differentiation are becoming apparent much less information is available regarding the potential functions of the six high affinity IGF binding proteins (IGFBP 1–6) in this process. Such studies are largely limited to the role of IGFBP-4 and IGFBP-5 in mature osteoblasts or bone tissue (Andress, 1995; Andress & Birnbaum, 1992; Durham et al., 1995; Durham et al., 1994) with little information on the role of IGFBPs on the process of differentiation per se.
    Materials & methods
    Results Three donors were used to isolate dental pulp from healthy third molars. Donor profile is shown in Supplementary Table I. Dental pulp cells were treated with mineralisation medium for 1 and 3wk. In order to confirm differentiation we examined the expression of alkaline phosphatase (ALPL), runt related transcription factor 2 (Runx2) and osteocalcin (OCN) under both basal and mineralising culture conditions. ALPL is an early marker of differentiation and was upregulated approximately 3-fold at 1wk with expression levels returning to basal levels at 3wk (p<0.05 1wk v 3wk). Runx2 is a key transcription factor regulating matrix mineralisation and was upregulated at both time points although there was a tendency toward higher levels of expression at 3wk (3-fold) compared to 1wk (1.5-fold). OCN is a later marker of differentiation and is upregulated approximately 2-fold at 3wk compared to 1wk (p<0.01 1wk v 3wk) Supplementary Fig. 1A. In addition differentiated DPCs showed positive staining for both alkaline phosphatase (Supplementary Fig. 1B) and Alizarin Red (Supplementary Fig. 1C) at both 1 and 3wk time points. This data confirms the differentiation of DPCs toward a mineralising phenotype under our experimental conditions. We next analysed expression of IGF axis genes (IGF-1, IGF-2, IGF-1R, IGF-2R and IGFBP1–6) in DPCs under basal and mineralising conditions. All 10 genes were expressed under both basal and mineralising conditions although in our cultures IGF-1 and IGFBP-1 were expressed at low levels Ct>30. For IGFBPs the level of expression was IGFBP-4>IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-3>IGFBP-1 – (Fig. 1). IGFBP-4, -5 and -6 expression did not alter during differentiation of DPCs. In contrast, in each analysis of pulp cells obtained from all 3 donors we found that IGFBP-2 and IGFBP-3 expression were reciprocally regulated during differentiation at both 1wk and 3wk time points (Fig. 2). Induction of IGFBP-2 varied from around 4–20 fold following differentiation and similar fold reductions in IGFBP-3 expression were also seen. We also measured IGFBP-2 and -3 concentrations using ELISA (see Section 2.2) of conditioned medium (CM) from cells cultured under basal or mineralising conditions (Fig. 3). In agreement with our qRT-PCR data, IGFBP-2 protein levels in CM were elevated following differentiation of cells derived from all donors at both 1 and 3wk time points. In most instances these differences achieved statistical significance (Fig. 3). Similarly, the decrease in IGFBP-3 mRNA expression following differentiation shown in our qRT-PCR experiments was reflected in reduced IGFBP-3 protein concentrations in conditioned cell media. For cultures derived from all donors significant decreases in IGFBP-3 concentrations were seen following differentiation of cells at both 1 and 3wk time points Global analysis of IGFBP-2 and IGFBP-3 protein concentrations is presented in Supplementary Figs. 2 and 3.