Amiloride-enhanced gene transfection of octa-arginine functionalized calcium phosphate nanoparticles.

Nanoparticles represent promising gene delivery systems in biomedicine to facilitate prolonged gene expression with low toxicity compared to viral vectors. Specifically, nanoparticles of calcium phosphate (nCaP), the main inorganic component of human bone, exhibit high biocompatibility and good biodegradability and have been reported to have high affinity for protein or DNA, having thus been used as gene transfer vectors. On the other hand, Octa-arginine (R8), which has a high permeability to cell membrane, has been reported to improve intracellular delivery systems. Here, we present an optimized method for nCaP-mediated gene delivery using an octa-arginine (R8)-functionalized nCaP vector containing a marker or functional gene construct. nCaP particle size was between 220-580 nm in diameter and all R8-functionalized nCaPs carried a positive charge. R8 concentration significantly improved nCaP transfection efficiency with high cell compatibility in human mesenchymal stem cells (hMSC) and human osteoblasts (hOB) in particular, suggesting nCaPs as a good option for non-viral vector gene delivery. Furthermore, pre-treatment with different endocytosis inhibitors identified that the endocytic pathway differed among cell lines and functionalized nanoparticles, with amiloride increasing transfection efficiency of R8-functionalized nCaPs in hMSC and hOB

Introduction of tenomodulin by gene transfection vectors for rat bone tissue regeneration

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

Gene transfection with octa-arginine functionalized DNA-loaded CaP nanoparticles.

<p><b>a)</b> Schematic concept of fabrication of the octa-arginine-functionalized calcium phosphate nanoparticles. <b>b)</b> Scanning electron micrograph (SEM) and transmission electron micrograph (TEM) of CaP nanoparticles functionalized with octa-arginine (CaP/DNA/CaP/R8). <b>c)</b> Transmission light microscopy (TLM) and fluorescence microscopy (FM) of transfected cells. Representative images of HeLa, Saos-2, hMSC, and hOB cells transfected with the pAcGFP1 plasmid within CaP nanoparticles functionalized with octa-arginine at a concentration of 50 mg ml⁻¹. Transfected cells appear green under fluorescence microscopy. Magnification: x20. Bar = 100 μm.</p

Hydroxyl radicals generated by hydrogen peroxide photolysis recondition biofilm-contaminated titanium surfaces for subsequent osteoblastic cell proliferation

Titanium dental implants have been successfully used for decades; however, some implants are affected by peri-implantitis due to bacterial infection, resulting in loss of supporting bone. This study aimed to evaluate the effect of an antimicrobial chemotherapy employing H2O2 photolysis—developed to treat peri-implantitis—on biofilm-contaminated titanium surfaces in association with osteoblastic cell proliferation on the treated surface. Titanium discs were sandblasted and acid-etched, followed by contamination with a three-species biofilm composed of Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mitis. This biofilm model was used as a simplified model of clinical peri-implantitis biofilm. The discs were subjected to ultrasound scaling, followed by H2O2 photolysis, wherein 365-nm LED irradiation of the disc immersed in 3% H2O2 was performed for 5 min. We analysed proliferation of mouse osteoblastic cells (MC3T3-E1) cultured on the treated discs. Compared with intact discs, biofilm contamination lowered cell proliferation on the specimen surface, whereas H2O2 photolysis recovered cell proliferation. Thus, H2O2 photolysis can recover the degraded biocompatibility of biofilm-contaminated titanium surfaces and can potentially be utilised for peri-implantitis treatment. However, to verify the findings of this study in relation to clinical settings, assessment using a more clinically relevant multi-species biofilm model is necessary

Characterization of functionalized calcium phosphate nanoparticles.

<p>Characterization of functionalized calcium phosphate nanoparticles.</p

Development of triple-functionalized calcium phosphate nanoparticles as an advanced drug delivery system for bone tissue repair

Introduction: During tissue repair or regeneration, several bioactive molecules are released and interact with each other and act as complex additives or inhibitors for tissue reconstruction. In this study, the bone-healing effects of the combination treatment with tumor necrosis factor-α (TNF-α) inhibition, vascular endothelial growth factor A (VEGF-A) and bone morphogenetic protein-7 (BMP-7) release by gene silencing, and gene transfection with calcium phosphate nanoparticles (CaP) in the rat femoral head was histologically, morphologically, and biochemically evaluated. Methods: A triple-functionalized paste of CaP carrying plasmid DNA encoding for BMP-7 and for VEGF), and siRNA against TNF-α was developed and denoted as CaP3mix. To compare the effects of 3mixCaP, CaP with plasmid DNA encoding BMP-7, VEGF, or siRNA encoding TNF-α was prepared and denoted as CaP/PEI/pBMP-7/SiO2, CaP/PEI/pVEGF/SiO2, or CaP/PEI/siRNA-TNF-α/SiO2, respectively. The bone healing in bone defects in the rat femoral head was investigated after 10 and 21 days of implantation. Results: The levels of bone formation-related markers OCN, Runx2, and SP7 increased at the protein and gene levels in 3mixCaP after 10 days, and 3mixCaP significantly accelerated bone healing compared with the other treatments after 21 days of implantation. Conclusion: The triple-functionalized CaP paste loading plasmid DNA encoding BMP-7 and VEGF and siRNA encoding TNF-α is a promising bioactive material for bone tissue repair

Final concentrations of calcium phosphate (CaP), DNA- AcGFP1 or pUC57, octa-arginine, PEI, and protamine in nanoparticle dispersions.

<p>Final concentrations of calcium phosphate (CaP), DNA- AcGFP1 or pUC57, octa-arginine, PEI, and protamine in nanoparticle dispersions.</p

Transfection efficiency (TE) and cell viability (CV) in HeLa, Saos-2, hMSC, and hOB cells.

<p>Transfection efficiency (TE) and cell viability (CV) in HeLa, Saos-2, hMSC, and hOB cells.</p

BMP-2 concentration percentages in the cell culture medium of cells pre-treated with endocytosis inhibitors.

<p><b>A)</b> HeLa, <b>b)</b> Saos-2, <b>c)</b> hMSC, and <b>d)</b> hOB cells transfected with CaP nanoparticles loaded with pUC57 plasmid and functionalized with different concentrated solutions of R8 (0.1, 1, 5, 10, 50, 100 mg ml⁻¹), PEI, and protamine. pUC57 group are single shell non-functionalized CaP nanoparticles. The bars represent the mean ± standard deviation. *p < 0.05 compared to transfection of non-pre-treated cells (NT).</p