TABLE 3.
Summary of non-viral gene delivery vector applied to bone tissue engineering.
| Chemical vector | Scaffold/matrice or add-on | Wound type | Animal; Cells | Growth Factor or else | DNA/RNA | Results | References |
| Lipid-based transfection/Lipid-based gene vectors | |||||||
| FuGENE 6 | N.D. | in vitro | Fetal Rat Osteoblasts | TGF-β1 | pDNA | Higher cell proliferation compared recombinant TGF-β1 delivery in the medium. | Macdonald et al., 2007 |
| Lipofectamine 2000 | N.D. | in vitro | BMSCs | antimiR-138 | Oligonucleotide | Massive bone regeneration and with good vascularisation were achieved. | Yan et al., 2014 |
| (DOTAP)-2-dioleoyl-sn-glycero-3-phosphatidylethanolamine | Transferrin | in vitro | MG63 and MC3T3-E1 cells | β-galactosidase | pDNA | High correlation between lipid formulation and transfection activity. | Oliveira et al., 2009 |
| Cationic liposome-based reagent | N.D. | in vitro | Human BMSCs | GFP | pDNA | High viabilities and recoveries of the transfected cells as well as multipotency. | Madeira et al., 2010 |
| Cationic liposome | N.D. | in vitro | AH130 cells | N.D. | pDNA | Efficient transgene expression as well as enhanced nuclear delivery. | Tachibana et al., 2002 |
| DODAP, HSPC, Chol, and DSPE-PEG | Polycaprolactone (PCL) scaffolds | in vitro | Human BMSCs | Runx2 | pDNA | Osteogenic differentiation was achieved with long-term gene expression of RUNX2. | Monteiro et al., 2014 |
| FuGENE 6 | Type-I collagen and poly(lactide-co-glycolide) (PLG) scaffolds | in vitro | BHK cells | N.D. | pDNA | Improvement of the functional stability and release duration. | Winn et al., 2005 |
| Lipofectamine | N.D. | in vitro | Human BMSCs | BMP-2 and VEGF165 | pDNA | Differentiation abilities of BMSCs were enhanced. | Guo-ping et al., 2010 |
| Amaxa Nucleofector- II | N.D. | in vitro | Human primary calvarial suture MSCs | BMP-2 and BMP-3 | pDNA | Efficient, a non-viral alternative method for in vitro applications. | Dwivedi et al., 2012 |
| Synthetic polymer-based transfections/Synthetic polymer-based gene vectors | |||||||
| Polyethylenimine (PEI) | N.D. | Intracerebral transfer | primary rat brain endothelial cells or chicken embryonic neurons. | Luciferase | pDNA | Results comparable or even better than lipopolyamines. | Boussif et al., 1995 |
| Polyethylenimine (PEI) | N.D. | N.D. | COS-7 cells | Luciferase | pDNA | Transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes. | Akinc et al., 2005 |
| Polyethylenimines (PEIs) with F25-LMW Liposome | N.D. | N.D. | SKOV-3 cells | N.D. | pDNA and siRNA | Lipopolyplexes show improved biological properties over PEI complexes | Schafer et al., 2010 |
| Polyethylenimine (PEI)-7K-L | N.D. | N.D. | 293T cells | Luciferase | pDNA | PEI-7K-L is less cytotoxic and more efficient than both PEI-25K and Lipofectamine 2000 in the in vitro gene transfection | Deng et al., 2009 |
| Polyethylenimine (PEI) | N.D. | N.D. | HeLa cells | N.D. | pDNA | PEI cannot induce changes in lysosomal pH. | Benjaminsen et al., 2013 |
| Polyethylenimine (PEI) | N.D. | Adult (eight weeks old) OFl female or male mice central nervous system/neural disorder | Neuronal cultures | Luciferase and bcl2 | pDNA | PEI appears to have potential for fundamental research and genetic therapy of the brain. | Abdallah et al., 1996 |
| Polyethylenimine (PEI) | N.D. | N.D. | Dendritic cells | GM-CSF | pDNA | Results open new approches for novel delivery vectors for in situ vaccination and the treatment of autoimmunity. | Ali and Mooney, 2008 |
| Polyethylenimine (PEI) | Porous poly(lactide-co-glycolide) (PLG) scaffolds | Subcutaneous implantation | Rat | β-galactosidase | pDNA | In vivo long-term and high level of gene expression. | Huang et al., 2005a |
| Polyethylenimine (PEI) | Poly(lactic-co-glycolic acid) (PLGA) scaffolds | Calvarial defects | Rat | BMP-4 | pDNA | PEI scaffold delivery system was able to enhance bone formation. | Huang et al., 2005b |
| Polyethylenimine (PEI) | Collagen, collagen GAG, and collagen nHa scaffolds | N.D. | Rat MSCs | Luciferase | pDNA | PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and 3D environment. | Tierney et al., 2012 |
| Polyethylenimine (PEI) | Collagen scaffolds | Calvarial defects | Rat; Human BMSCs | PDGF-B | pDNA | PDGF-B gene-activated scaffolds are useful for bone regeneration. | Elangovan et al., 2014 |
| Polyethylenimine (PEI) | Poly-(ε-caprolactone) scaffolds | N.D. | C2C12 cells | BMP-2 | pDNA | PEI, as bioactive implant surfaces give rise to promising results. | Reckhenrich et al., 2012 |
| Poly(ethyleneglycol) (PEG) | N.D. | Calvarial defects | Mice; Mouse calvarial cells | caALK6 and Runx2 | pDNA | First, in vivo gene transfer with therapeutic potential using polyplex nanomicelles. | Itaka et al., 2007 |
| Poly(ethyleneglycol) (PEG) | Poly(ethylene glycol) (PEG) hydrogels | N.D. | HEK293 cells and Human MSCs | GFP and Luciferase | siRNA | Delivery of siRNA and miRNA from the hydrogel constructs enhanced the osteogenic differentiation. | Nguyen et al., 2014 |
| Natural polymer-based transfection/Natural polymer-based gene vectors | |||||||
| Chitosan functionalized with imidazole moieties | N.D. | N.D. | 293T and HepG2 cells | β-galactosidase | pDNA | Enhanced β-gal expression. | Moreira et al., 2009 |
| Calcium phosphate | Chitosan | Subcutaneous implantation | Mice; MC3T3-E1 cells | BMP-2 | pDNA | Bone tissue formation in vivo after implantation. | Krebs et al., 2010 |
| Alginate hydrogel | N.D. | ? | Mice; Human MSCs and MG-63 cells | BMP-2 | pDNA | Alginate hydrogel seems to be highly suitable for the delivery of growth factors in bone regeneration. | Wegman et al., 2011 |
| Alginate hydrogel | Ceramic granules | Spinal cassettes | Goat MSCs | BMP-2 | pDNA | Alginate hydrogel led to stable expression of BMP-2 and promoted osteogenic differentiation. | Wegman et al., 2014 |
| Chitosan | N.D. | N.D. | Human MSCs, MG63, and HEK293 cells | β-galactosidase | pDNA | Chitosan-DNA nanoparticles are cell type-dependent and not cytotoxic. | Corsi et al., 2003 |
| Chitosan-alginate | N.D. | Subcutaneous implantation | Mice; HEK 293 cells and Human MSCs | BMP-7 | pDNA | The chitosan-alginate gel used a gene delivery system seems to be an exciting approach for tissue engineering. | Park et al., 2007 |
| Composites of cationized gelatin microspheres (CGMS) | Oligo(poly(ethylene glycol)fumarate) (OPF) | Subcutaneous implantation | Mice | BMP-2 | pDNA | Composites can prolong and control the release of pDNA. | Kasper et al., 2005 |
| Composites of cationized gelatin microspheres (CGMS) | Oligo(poly(ethylene glycol)fumarate) (OPF) | Calvarial defects | Rat | BMP-2 | pDNA | The release of plasmid DNA from the composites was not sufficient to induce bone repair. | Kasper et al., 2006 |
| Branched triacrylate/amine polycationic polymer with gelatin microparticles | Oligo(poly(ethylene glycol)fumarate) (OPF) | Calvarial defects | Rat; CRL 1764 cells | BMP-2 | pDNA | Polycationic polymers with a slow degradation rate can prolong the release of pDNA. | Chew et al., 2011 |
| Alginate hydrogel | Hyaluronic Acid (HA)-based Gel | Tibial defects | Rabbit | TGF-β1 and FGF-2 | proteins | By angiogenesis inhibition and hypoxic environment promotion, cartilage formation can be exclusively promoted. | Stevens et al., 2005 |
| Inorganic nanoparticles transfection/Inorganic nanoparticles gene vectors | |||||||
| Calcium phosphate nanoparticles | N.D. | N.D. | HeLa and MC3T3-E1 cells | Luciferase | pDNA | Transfection efficiencies due to efficient condensation and bound of pDNA. | Olton et al., 2007 |
| Calcium phosphate nanoparticles | Polyelectrolyte multilayer poly-(L-lysine) (PLL) | N.D. | Human osteoblasts | Spp1 for the silencing of osteopontin expression and Bglap-rs1 for silencing of osteocalcin expression | shRNA | A multilayered films-based delivery system containing nanoparticles for gene silencing can specific for bone cells. | Zhang et al., 2010 |
| Hydroxyapatite nanoparticles | Collagen scaffolds | Calvarial defects | Rat; MSCs, HUVECs, MC3T3-E1s | BMP-2 and VEGF-165 | pDNA | Bone regeneration was accelerated. | Curtin et al., 2015 |
| Alginate | Ceramic granules | Spinal cassettes | Goat; Goat MSCs | BMP-2 and VEGF-165 | pDNA | Transfection from this DNA delivery system led to a stable expression of BMP-2 during 16 weeks. | Wegman et al., 2014 |
| Polyethylenimine (PEI)-LA | Gelatin/collagen scaffolds | Subcutaneous implantation | Rat | bFGF and BMP-2 | pDNA | Scaffolds delivering complexes influenced recombinant protein production. | Rose et al., 2012 |
| Lipofectamine 2000 (coprecipitated within apatite) | PLGA films | N.D. | C3H10T1/2 cells | β-galactosidase | pDNA | The hybrid material system integrates conductivity provided by the apatite and inductivity supplied by the DNA. | Luong et al., 2009 |
| Physical transfection methods/Physical gene vectors methods | |||||||
| Electroporation | HA/β-TCP scaffolds | Calvarial and long-bone segmental defects | Rat; ASCs | BMP-2 to VEGF-165 | pDNA | Induction of rapid angiogenesis and osteogenesis. | Lee et al., 2019 |
| TransIT-2020 | Matrigel | Calvarial defects | Rat; Rat BMSCs | BMP-2 | pDNA | BMSCs transfected with BMP-2 provided better osteogenic differentiation than primary BMSCs. | Hsieh et al., 2018 |
| Sonoporation | N.D. | Ectopic implantation - Mice; Orthotropic implantation – Rat | Mice and Rat | BMP-2 and BMP-7 | pDNA | Sonoporation increased callus formation and heterotopic ossification. | Feichtinger et al., 2014 |
| Ex vivo transfections/Ex vivo gene vectors | |||||||
| NucleofectorTM | Fibrin gel | Coccygeal vertebrae | Rat; Porcine ASCs | BMP-6 | pDNA | ASCs modified with BMP-6 can repair vertebral bone defects. | Sheyn et al., 2011 |
| NucleofectorTM | N.D. | Spinal fusion in lumbar paravertebral muscle | Mice; Porcine ASCs | BMP-6 | pDNA | Formation of a large bone mass adjacent to the lumbar area, which produced posterior spinal fusion. | Sheyn et al., 2008b |
| Microporation transfection | Poly(lactic-co-glycolic acid) (PLGA) scaffolds | Dorsal subcutaneous spaces | Mice; Human ASCs | BMP-2 and Runx2 | pDNA | The co-transfection of two osteogenic lineage-determining genes could enhance osteogenic differentiation of ASCs. | Lee et al., 2010 |
| Lipofectamine 2000 | N.D. | Osteodistraction of the mandible | Rabbit; Rabbit BMSCs | Osterix | pDNA | Promotion of bone formation. | Lai et al., 2011 |
| Peptides | |||||||
| protease-degradable (PEG) functionalized with a peptide (GFOGER) | N.D. | Radius defects | Mice; Human MSCs | BMP-2 | protein | GFOGER hydrogels promote bone regeneration with low delivered BMP-2 doses. | Shekaran et al., 2014 |
| (K)16GRGDSPC | Bioactive bone matricesPLGA-[ASP-PEG]n | Segmental bone defects in femoral shafts | Rabbit; Human BMSCs | TGF-β1 | pDNA | The biomimetic bone matrix is a very promising scaffold to increase of bone repair. | Pan et al., 2014 |
| Hybrid for transfections/Hybrid as gene vectors | |||||||
| Polyethylenimine (PEI)-LA | Gelatine and collagen scaffolds | Subcutaneous implantation | Rat; 293T cells | bFGF and BMP-2 | pDNA | PEI-LA was effective in vivo gene delivery carrier. | Rose et al., 2012 |
| Organic/inorganic hybrid co-precipitated within apatite | PLGA films | N.D. | C3H10T1/2 cells | β-galactosidase | pDNA | This hybrid material system integrates inductivity provided by the DNA and conductivity provided by the apatite. | Luong et al., 2009 |
| Cationized gelatin microspheres and OPF | N.D. | N.D. | N.D. | N.D. | pDNA | In vivo prolongation of the availability of pDNA. | Kasper et al., 2005 |
| Cationized gelatin microspheres within a crosslinked OPF | N.D. | Calvarial defects | Rat | BMP-2 | pDNA | The release of plasmid DNA from the composites was not sufficient to elicit a bone regeneration response. | Kasper et al., 2006 |
| TAPP complexed with gelatine microparticles | poly(propylene fumarate) scaffolds | Calvarial defects | Rat | N.D. | pDNA | Slow degradation rate can prolong the release of pDNA from the composite scaffolds. | Chew et al., 2011 |
| Chitosan-disulfide-conjugated low molecular weight PEI | N.D. | N.D. | MG-63 cells and stem cells | BMP-2 | pDNA | Transfection efficiency was significantly higher than PEI and comparable to Lipofectamine. | Zhao et al., 2013 |
| Others | |||||||
| Electrospinning | Non-woven, nano-fibered, PLGA, PLA-PEG | N.D. | MC3T3-E1 cells | β-galactosidase | pDNA | Incorporation of pDNA into a polymer scaffold can be achieved using electrospinning. | Luu et al., 2003 |
| Polymer Matrices | Porous poly(lactide-co-glycolide) (PLG) scaffolds | Subcutaneous implantation | Rat; 293T cells | PDGF | pDNA | Enhanced matrix deposition and blood vessel formation. | Shea et al., 1999 |
| Gene activated matrices | Collagen I scaffolds | Femoral and tibial metaphysis defects | Dog | PTH | pDNA | Induction new bone formation. | Bonadio et al., 1999 |
N.D., non determined.