Abstract
Inductive molecules are critical components for successful bone tissue engineering. Dentin matrix protein-1 (DMP1), a non-collagenous protein in the bone matrix, has been shown to play roles in osteogenic differentiation and phosphate homeostasis. This study aimed to produce recombinant human dentin matrix protein-1 (hDMP1) in Nicotiana benthamiana and investigated the ability of this plant-produced DMP1 to induce osteogenesis in human periodontal ligament stem cells (hPDLSCs). The hDMP1 gene was cloned into the geminiviral vector for transient expression in N. benthamiana. We found that hDMP1 was transiently expressed in N. benthamiana leaves and could be purified by ammonium sulphate precipitation followed by nickel affinity chromatography. The effects of hDMP1 on the induction of cell proliferation and osteogenic differentiation were investigated. The results indicated that plant-produced hDMP1 could induce the cell proliferation of hPDLSCs and increase the expression levels of osteogenic genes, including osterix (OSX), type I collagen (COL1), bone morphogenetic protein-2 (BMP2), and Wnt3a. Moreover, the plant-produced hDMP1 promoted calcium deposition in hPDLSCs as determined by alizarin red S staining. In conclusion, our results indicated that plant-produced hDMP1 could induce osteogenic differentiation in hPDLSCs and could potentially be used as a bone inducer in bone tissue engineering.
Highlights
Tissue engineering is one of the clinical therapeutic strategies used to address bone defects.Currently, methods to promote regeneration after bone damage involve the use of bone grafts or polymeric bone scaffolds [1]
The human dentin matrix protein-1 (hDMP1) gene was optimized in silico with an N. benthamiana-optimized codon, cloned into geminiviral vectors (Figure 1) and expressed in N. benthamiana leaves
The enzyme-linked immunosorbent assay (ELISA) result showed that the highest expression level of hDMP1 in N. benthamiana leaves occurred on day two post-infiltration with OD600
Summary
Methods to promote regeneration after bone damage involve the use of bone grafts (autologous or allogeneic) or polymeric bone scaffolds [1]. These approaches still have some limitations, such as disease transmission, high cost, and the inability to incorporate into the surrounding host tissue [2]. Plants 2019, 8, 566 biomolecules, such as matrix proteins or growth factors that show osteogenic differentiation potential, can induce the appropriate signaling pathways to promote bone regeneration [3]. In scaffold-based tissue engineering, a suitable surface to promote bone cell adhesion and differentiation could enhance the success of bone regeneration. Dentin matrix protein 1 (DMP1) is a good candidate for inducing bone differentiation
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