The osteogenic differentiation of hPMSC was promoted by HAG
Compared with autograft or allograft, biomaterials have become an indispensable bone material source in the field of bone regeneration due to their non-immunogenicity and accessibility. Among various bone materials, hydroxyapatite (HA) is the most commonly used one because of the following advantages, the same mineral composition as natural bone, good biocompatibility, bone conductivity and mechanical stability. However, the interaction between scaffolds and cells is influenced by various characteristics and morphology of scaffold surface, including chemical composition, electric charge, microstructure, and so on4,5. In previous work, on the basis of porous hydroxyapatite scaffold, we prepared a new scaffold material HAG through integrates microchannel structure. Moreover, the HAG promoted the osteogenic response of hPMSCs than HA18. Consistently, in this study, we repeated the osteogenic effect of HAG in hPMSCs (Fig. 1). The properties of hPMSCs were detected by flow cytometry. The positive surface marker CD44 and CD90 were highly expressed (97.08%), while CD45 (0.10%) was hardly expressed in the cells (Fig. 1A). The porous structure (as the arrow shown, left) and a 25–30 µm groove structure (as the arrow shown, right) on HAG scaffolds surface as shown in Fig. 1B. After osteogenesis induced for 7, 14 days, the expression of ALP, BMP2, Ocn mRNA were quantified by qPCR, and the protein levels of ALP, OCN were detected at 7d (Fig. 1C,D) Alizarin Red staning was used to detect the mineralized nodules 3 weeks after induction (Fig. 1E). The results reproduced the positive effect of HAG scaffolds osteogenic differentiation in hPMSC.
The miRNA expression in HAG-hPMSCs and control hPMSCs were differential
In recent years, several studies revealed that miRNAs participated the process of osteogenic differentiation through targeting mRNA sequences of different genes related to osteogenesis19–22. However, roles of miRNAs in bone formation by HAG scaffolds mediated remain unclear. Thus, in order to uncover that how HAG scaffolds promoted osteogenic differentiation of hPMSCs, microarray assay was used to analyse the miRNA expression profiles between HAG-hPMSCs and hPMSCs. We observed 45 mature miRNAs significantly differentially expressed in HAG-hPMSCs group (FC ≥ 2, Fig. 2A). Red represented highly expressed genes, while green represents the down-regulated genes, and the darker the color, the more distinct the expression difference. Compared with differentiated hPMSCs, the expressions of 16 miRNAs in HAG-hPMSCs were up-regulated and 29 were down-regulated, as shown in the volcano plot (FC ≥ 2, Fig. 2B).
Pathway and functional analysis of differential miRNAs
The differential expression of miRNAs in HAG-hPMSCs group may indicate that miRNA plays an important role in the process of osteogenic differentiation promoted by HAG. Therefore, the gene ontology enrichment analyses, including BP (biological process), CC (cellular component), and MF (molecular function) by cluster Profiler in R software, were performed to explore the biological functions may be regulated by differential miRNAs. The statistically significant results (p values < 0.05) showed that these miRNAs participated in the regulation of cell metabolic (p value = 2.58E-12), cell junction (p value = 6.45E-08), cell development (p value = 1.82E-11), cell differentiation (p value = 4.04E-07), and signal transduction (p value = 8.12E-12) (Fig. 3A). Importantly, the process of osteogenic differentiation is closely related to these functions.
Furthermore, KEGG pathway analysis was used to understand the signaling pathways in which these differentially expressed miRNAs were involved in regulation. The results indicated that genes in multiple signaling pathways, including axon guidance (p value = 5.02E-05), MAPK signaling (p value = 6.65E-07), and the TGF-β signaling pathway (p value = 5.7E-04), highly associated with osteogenic differentiation are potential target genes for these differentially expressed miRNAs (Fig. 3B). It suggested that HAG may achieve its osteogenic effect by promoting differential expression of these miRNAs.
qRT-PCR validation of miRNA expression and miRNA-mRNA network analysis
According to the results of microarray analyses, the 6 miRNAs (miR-210-3p;miR-146a-5p; miR-483-5p; miR-3615༛miR-125b-2-3p༛miR-145-5p), with the most significant expression differences were verified by qPCR. Consistently, the expression of miR-210-3p, miR-146a-5p, miR-483-5p were obviously increased, while miR-3615, miR-125b-2-3p and miR-145-5p were significantly decreased (FC > 2, p < 0.05) (Figu.4A). As known, miRAN achieve its regulatory effect by pairing with the complementary sequence of the target gene and degrading it or preventing its translation. More importantly, a miRNA can target multiple mRNA and the same mRNA can be regulated by multiple miRNAs23. Thus, TargetScan7.1 and mirdbV6 were used to predict the target genes of the above 6 miRNAs. Moreover, the miRNA-mRNA networks were constructed by Cytoscape (Fig. 4B). As shown in Fig. 4B, the miRNAs can target hundreds of mRNA, while the mRNA of BRCC3 can be recognized by different miRNA. More importantly, a great quantity of target mRNA were to be involved in osteogenic differentiation related pathways. For instance, the Brain-Derived Neurotrophic Factor (BDNF), a well-known growth factor of the neurotrophin family, is involved in regulating the growth, survival of neurons and angiogenesis24. Interestedly, Yamashiro et al. found that the mRNA and protein of BDNF were expressed in osteoblasts25. More importantly, further studies showed that the osteogenic differentiation ability of hPMSCs were enhanced with the expression of BDNF26,27.
Overexpression of miR146a-5p promoted the osteogenic differentiation of hPMSCs
To verify the effect of differentially expressed miRNA on osteoblastic differentiation, we transfected the miR146a-5p mimics into the hPMSCs. As shown in Fig. 5A and B, the mimics were transfected into cells successfully. Furthermore, we observed that the expression of osteogenic genes were increased when miR146a-5p were overexpressed (Fig. 5C). Meanwhile, following osteogenic differentiation, the hPMSCs transfected with miR-146a-5p mimics showed a higher intensity of ALP staining and formed a larger number of calcified nodules (Fig. 6D, E). Therefore, the differentially expressed miRNAs may play an important role in the process of HAG promoting osteogenic differentiation.