TY - JOUR
T1 - Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells
AU - Rattanapisit, Kaewta
AU - Abdulheem, Supaniga
AU - Chaikeawkaew, Daneeya
AU - Kubera, Anchanee
AU - Mason, Hugh S.
AU - Ma, Julian K.C.
AU - Pavasant, Prasit
AU - Phoolcharoen, Waranyoo
N1 - Funding Information:
This study was supported by Thailand Research Fund grant No MRG5980087 and IRN59W0001. KR was supported by the Ratchadaphiseksomphot Fund, Chulalongkorn University for the Postdoctoral Fellowship. PP was supported by the 2012 Research Chair Grant, Thailand National Science and Technology Development Agency (NSTDA). DC was supported by Research grant, Faculty of Dentistry, and IRN59W0001. We would like to thank Professor Cecilia M. Giachelli, University of Washington, USA for providing the osteopontin gene.
Publisher Copyright:
© The Author(s) 2017.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-017-17666-7
DO - 10.1038/s41598-017-17666-7
M3 - Article
C2 - 29229947
AN - SCOPUS:85038620073
SN - 2045-2322
VL - 7
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 17358
ER -