# BMP-2 Delivery through Liposomes in Bone Regeneration
## 4 Transfection
conducted a study on pigs where they created cavali bone defects in which they inserted implants with or without BMP-2 transfected liposomes. Previously, they had assessed the transfection efficiency of the liposomal vector by introducing green fluorescent protein. The osteogenic capacity was measured at 7 and 28 days and at three different regions of interest. The bone regeneration was significantly enhanced in the group with the liposomal vector applied to the surface of the implant. The direct application of the vector was sufficient for complete bone healing at the margins of the bone defect, but not in the center [111]. Using collagen for the liposome/BMP-2 carrier allows migrating cells to express the protein even after 28 days [13]. Neo-angiogenesis is one of the main components of bone regeneration and, at the same time, one of the biggest challenges. For the formation of new bone, it is important for nutrients to be provided, the acid-base balance to be maintained, and the metabolic by-products to be eliminated. The combined release of BMP-2 and VEGF could solve this issue [112; 113; 114]. [PERSON] et al. evaluated the efficacy of transfection of mouse bone marrow stromal cells (mBMSC) with BMP-2 and VEGF\({}_{165}\) in order to assess the neoangiogenic and ectopic bone tissue forming capability of these molecules. They found co-expression of BMP-2 and VEGF\({}_{165}\) mRNA in vitro by immunohistochemistry and RT-PCR. In mice, they obtained ectopic trabecular-like bone formation at 4 weeks after injection [115]. [PERSON] et al. researched, in vitro, the transfection efficiency of a vector which co-expressed hBMP-2 and hVEGF\({}_{165}\) and the resulted protein levels. They found that the transcription of hVEGF may be upregulated by hBMP-2 by RT-PCR analysis of the proteins. Western blot did  
Figure 2: Mechanism of liposomal transfection. not show this cooperativity. Osteocalcin mRNA and collagen I were high in the groups transfected with BMP-2, but negligible in the groups with VEGF alone [116]. When comparing liposomes with polyethylene glycol (PEG) as gene carriers, BMP-2 levels in cells and mRNA levels of BMP-2 were double in PEG group than in liposomal group. The liposomal group was only used as control in vitro, but not in vivo [117]. Liposome-loaded DNA have been introduced in multilayer HA coatings deposited on titanium disks using the layer-by-layer technique. The amount of DNA was increased by each additional layer, the plasmids were released, and cells were transfected, with an increased expression of Rmx2, Osx, ALP, and OC, but without calcified nodule formation at 14 days [118]. At implantation in rabbits, the uncoated implants yielded new woven bone, showing a statistically significant difference at 4 weeks (but not at 2 or 8 weeks) in favor of the BMP-2 gene coated implants. However, the bone-to-implant contact was consistently lower than in the control group, which the authors explained by the short persistence of the protein at the site [119]. PEG membranes are biodegradable materials often used in bone tissue engineering. In adult pig experimental model, PEG membranes containing liposomal BMP-2 transfected osteoblasts facilitated a significantly higher new bone regeneration, cell survival, and protein synthesis at 1, 2, 4, and 8 weeks after implantation. The combination of PEG matrixes and osteoblasts transfected with BMP-2 allowed for a good spatial fixation of the implanted cells in the defect [120; 121]. [PERSON] et al. compared the effect of BMPs to other members of the TGF\(\beta\) superfamily such as TGF\(\beta\) and IGF1. They transfected the genes into BMSCs, implanted the cells into mini-pigs, and evaluated the bone formation. Cells transfected with TGF\(\beta\) and IGF1 did not enhance bone formation as compared with the negative control, while those with BMP-2/7 yielded good quality bone tissue with enhanced mineralization and organized architecture [122]. The extracellular matrix of bone consists of 70-90% of hydroxyapatite (HA) and 10-30% organic material, mainly collagen [123].