Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future.
Background Toll‐like receptors (TLR) are a group of receptors that play roles in the innate immune system. Human periodontal ligament cells (hPDL cells) express several TLRs, including TLR3, a nucleotide sensing receptor that recognizes double‐stranded RNA from viral infection. However, its role in hPDL cells is unclear. The aim of this study was to investigate the responses of hPDL cells in terms of immunomodulation after TLR3 engagement. Methods HPDL cells were treated with various doses of poly I:C, a TLR3 activator. The expression of interferon‐gamma (IFNγ), indoleamine 2,3 dioxygenase (IDO), and human leukocyte antigen G (HLA‐G) was determined. Chemical inhibitors and small interfering RNA (siRNA) were used to confirm the role of TLR3. Coculture with human peripheral blood mononuclear cells (PBMCs) with poly I:C‐activated hPDL cells was performed. Results Endosomal TLR3 in hPDL cells was observed by immunocytochemistry. Addition of poly I:C significantly enhanced the expression and secretion of IFNγ, IDO, and HLA‐G. Knockdown of TLR3 using siRNA decreased the poly I:C‐induced expression of these three molecules. Bafilomycin‐A, an inhibitor of auto‐phagosome and lysosome fusion, inhibited poly I:C‐induced IDO and HLA‐G expression, whereas cycloheximide and a TLR3‐neutralizing antibody had no effect. In co‐culture experiments, poly I:C‐activated hPDL cells inhibited PBMCs proliferation and increased mRNA expression of forkhead box P3 (FOXP3), a transcription factor which is a marker of regulatory T cells. Conclusion Our findings indicated that TLR3 engagement of hPDL cells induced immunosuppressive properties of these cells. Because immunosuppressive properties play an important role in tissue healing and regeneration, activation of TLR3 may help to attenuate tissue destruction by limiting the inflammatory process and perhaps initiate the healing and regeneration process of the periodontium.
Periodontal ligament stem cells (PDLSCs) have been served as a cell reservoir for tissue regeneration during adulthood. For clinical applications, the challenging steps are to maintain the stem cell properties and to improve the regeneration capacity of PDLSCs during culture. Toll-like receptor 3 (TLR3) signaling has been shown to enhance therapeutic potential in several cell types including mesenchymal stem cells (MSCs) by inducing the secretion of multifunctional trophic factors. However, the role of TLR3 in PDLSCs is still unknown. The aim of this study was to investigate the responses of PDLSCs after TLR3 engagement using TLR3 agonist, poly(I:C). The result indicated that stimulation of TLR3 signifi cantly enhanced pluripotent stem cell gene expression (e.g., REX-1 and SOX2) as well as immunomodulatory molecules (e.g., IFNγ and IDO). Interestingly, inhibition of NF-kB signaling decreased the TLR3-activated IFNγ but increased the TLR3-activated IDO expression, suggesting the multiple pathways in the inductive mechanism. Our fi nding supports the concept that activated TLR3 could encourage the stem cell and immunosuppressive properties of PDLSCs. Since immunosuppressive properties of stem cells could support tissue healing and regeneration, activation of TLR3 in PDL cells may trigger the effective PDL tissue regeneration.
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