CaP nanoparticles and a cationic polymer-based reagent (JetPEI® or in vivo-JetPEI™; Polyplus, Shanghai, China) were used as non-viral gene transfection vectors in this study. For the fabrication of a cationic polymer-based reagent, JetPEI was used according to the manufacturer's protocol. Briefly, 0.5 μg of an aqueous solution of pcDNA3.1+C-HA-Tenomodulin (1 mg/mL), 0.5 μg of an aqueous solution of pcDNA3.1 (+)-C-HA mCherry or 1 μL of JetPEI reagent was mixed with 24.5 or 24 μL of NaCl (150 mM), respectively. The obtained 25 μL of JetPEI solution was added to 25 μL of the DNA solution. After incubation for 20 min at room temperature (20–27 °C), the reacted solution was denoted as JetPEI (Tnmd) or JetPEI (mCherry). For animal experiments, an aqueous solution of pcDNA3.1+ C-HA-Tenomodulin (1 mg/mL) was mixed with 10% glucose solution. Next, in vivo JetPEI was mixed with 10% glucose solution. The obtained in vivo JetPEI solution was added to DNA solution and incubated for 15 min at 20–27 °C. Then, 56 μL of the in vivo-JetPEI dispersion was injected into cut collagen sponge within 30 min before the implantation into the rat and denoted as JetPEI (Tnmd).

Introduction: Periodontal ligament is regenerated in association with hard tissue regeneration. Tenomodulin (Tnmd) expression has been confirmed in periodontal ligament and it reportedly inhibits angiogenesis or is involved in collagen fibril maturation. The introduction of Tnmd by gene transfection in bone tissue regeneration therapy might inhibit topical hard tissue formation and induce the formation of dense fibrous tissue. Therefore, the effect of Tnmd introduction by gene transfection technique in vitro and in vivo was investigated in this study. Methods: Osteogenesis- and chondrogenesis-related gene expression levels in osteoblastic cells (MC3T3E1) and rat bone marrow derived cells were detected using qPCR three days after gene transfection with plasmid DNA (Tnmd) using non-viral gene transfection vectors: a calcium phosphate-based gene transfection vector (CaP(Tnmd)) or a cationic polymer-based reagent (JetPEI (Tnmd)). Next, an atelocollagen scaffold with or without CaP (Tnmd) or JetPEI (Tnmd) was implanted into a rat calvaria bone defect, and the remaining bone defect volume and the tissue reaction at 28 days after surgery were evaluated. Results: Runx 2 and SP7 mRNA was reduced by JetPEI (Tnmd) in both cells, but not in CaP(Tnmd). The volume of expressed Tnmd was at 9 ng/mL in both gene transfection vector. The remaining bone defect volume of JetPEI (Tnmd) was significantly bigger than that of the other groups and CaP (EGFP), and that of CaP (Tnmd) was significantly bigger than that of CaP (EGFP). Conclusions: Tnmd introduction treatment inhibits bone formation in artificial bone defect, however, the effect of that was dependent on non-viral gene transfection vector.