Calcium phosphate nanoparticles carrying BMP‐7 plasmid DNA induce an osteogenic response in MC3T3‐E1 pre‐osteoblasts

Abstract

Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

Effects of NSAIDs on the osteogenic differentiation of human adipose tissue-derived stromal cells

“…Effect of NSAID s on capacity of hADSC to acquire osteogenic features. (a) hADSC s were cultured for 14 days in the presence of NSAID s (10 −6 m ) in 10 m m β‐glycerol‐2‐phosphate and 50 μg/ ml ascorbic acid osteogenic medium, and alkaline phosphatase ( ALP ) activity was measured as previously described . Results are presented as enzyme units normalized to cell number.…”

“…(b) hADSC s were treated for 7 days in the presence of NSAID s (10 −6 m ) in osteogenic medium, and gene expression of RUNX 2 , ALP and Osteocalcin ( OC ) was examined by multiplex semi‐quantitative PCR . (c) hADSC s were cultured for 21 days in the presence of NSAID s (10 −6 m ) in osteogenic medium, and mineral deposition was measured by Alizarin Red S staining as previously described . Results are shown as OD 550 nm normalized to cell number and represent the mean ± SEM of n = 6 samples; * P ≤ 0.05.…”

“…Calcium‐rich deposits representing the final stage of mineralization were visualized by Alizarin Red S (ARS) (Sigma, St. Louis, MO, USA) staining and quantified by dye extraction, as previously described . Absorbance was measured at 550 nm with a spectrophotometer (SpectraMax M2, Molecular Devices Inc.).…”

Bioactive Polymeric Nanoparticles for Periodontal Therapy

“…It is expected that calcium and phosphate covered NPs will be even less harmful to cells. A low inherent toxicity of other calcium phosphate nanoparticles, have been previously reported [47] and have been proposed for periodontal regeneration [48,49]. Modification of injectable poly(methylmethacrylate)-based bone cement with nano-HAp incorporation did also show an enhanced degree of cells biocompatibility [50].…”

Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly( l -lysine) as additives

“…Traditionally viral vectors are used to ensure high transfection efficiencies; however, risks associated with viral gene delivery include the elicitation of immunogenic responses, toxicity, insertional mutagenesis and the limited amount of DNA transportable by the carrier [2]. To overcome these issues, non-viral methods have been developed to artificially deliver DNA to cells, including direct injection [3], electroporation [4], and the use of particulate carriers, e.g., polymeric particles [5], cationic polymeric micelles [6], ionic lipids [7], and inorganic particles, the most prospective of which have been calcium phosphates (CaP) [8]–[10]. …”