Bone Morphogenetic Protein-2 Gene Delivery Research Articles

Exosome derived from mesenchymal stem cells mediates hypoxia-specific BMP2 gene delivery and enhances bone regeneration

Gene therapy based on non-viral vector has been used to promote the repair of bone damage. However, low transfection efficiency and uncontrollable expression greatly hinder its clinical translation. Exosomes (Exos) have been extensively studied as a promising non-viral delivery carrier recently, while its delivery efficiency for large nucleic acids is low. Here, a novel gene vector was developed by layer-by-layer self-assembly method using polyethyleneimine (PEI) and Exos derived from mesenchymal stem cells (MSCs) to deliver large plasmids efficiently. Moreover, a hypoxia-specific expression plasmid was constructed by combining hypoxia promoter RTP801 and microRNA 877 (miR877) sponge. Experimental results showed that the gene delivery vector based on Exos exhibited high transfection efficiency and low cytotoxicity to MSCs. The hypoxia-specific expression plasmid significantly increased bone morphogenetic protein 2 (BMP2) expression in hypoxic condition and promoted neoangiogenesis. This study may provide a novel method to improve the repair efficiency in bone tissue engineering.

BMP-2 Gene Delivery-Based Bone Regeneration in Dentistry.

Bone morphogenetic protein-2 (BMP-2) is a potent growth factor affecting bone formation. While recombinant human BMP-2 (rhBMP-2) has been commercially available in cases of non-union fracture and spinal fusion in orthopaedics, it has also been applied to improve bone regeneration in challenging cases requiring dental implant treatment. However, complications related to an initially high dosage for maintaining an effective physiological concentration at the defect site have been reported, although an effective and safe rhBMP-2 dosage for bone regeneration has not yet been determined. In contrast to protein delivery, BMP-2 gene transfer into the defect site induces BMP-2 synthesis in vivo and leads to secretion for weeks to months, depending on the vector, at a concentration of nanograms per milliliter. BMP-2 gene delivery is advantageous for bone wound healing process in terms of dosage and duration. However, safety concerns related to viral vectors are one of the hurdles that need to be overcome for gene delivery to be used in clinical practice. Recently, commercially available gene therapy has been introduced in orthopedics, and clinical trials in dentistry have been ongoing. This review examines the application of BMP-2 gene therapy for bone regeneration in the oral and maxillofacial regions and discusses future perspectives of BMP-2 gene therapy in dentistry.

BMP-2 restoration aids in recovery from liver fibrosis by attenuating TGF-β1 signaling

Transforming growth factor-β (TGF-β) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-β1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-β1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-β1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-β and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-β1-stimulated α-SMA and fibronectin expression, and reversed the TGF-β1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-β1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.The authors identified BMP-2 downregulation in human and experimental fibrotic livers, and found that adenovirus-mediated BMP-2 gene delivery ameliorates liver fibrosis and biliary injury in mice. Mechanistically, BMP-2 suppresses proliferation and migration of hepatic stellate cells by antagonizing TGF-β signaling and epithelial-to-mesenchymal transition. BMP-2 may therefore constitute a therapeutic target for liver fibrosis treatment.

A doxycycline inducible, adenoviral bone morphogenetic protein-2 gene delivery system to bone.

We report the novel use of a tuneable, non-integrating viral gene delivery system to bone that can be combined with clinically approved biomaterials in an 'off-the-shelf' manner. Specifically, a doxycycline inducible Tet-on adenoviral vector (AdTetBMP-2) in combination with mesenchymal stromal cells (MSCs), fibrin and a biphasic calcium phosphate ceramic (MBCP®) was used to repair large bone defects in nude rats. Bone morphogenetic protein-2 (BMP-2) transgene expression could be effectively tuned by modification of the doxycycline concentration. The effect of adenoviral BMP-2 gene delivery upon bone healing was investigated in vivo in 4mm critically sized, internally fixated, femoral defects. MSCs were transduced either by direct application of AdTetBMP-2 or by pre-coating MBCP granules with the virus. Radiological assessment scores post-mortem were significantly improved upon delivery of AdTetBMP-2. In AdTetBMP-2 groups, histological analysis revealed significantly more newly formed bone at the defect site compared with controls. Newly formed bone was vascularized and fully integrated with nascent tissue and implanted biomaterial. Improvement in healing outcome was achieved using both methods of vector delivery (direct application vs. pre-coating MCBP). Adenoviral delivery of BMP-2 enhanced bone regeneration achieved by the transplantation of MSCs, fibrin and MBCP in vivo. Importantly, our in vitro and in vivo data suggest that this can be achieved with relatively low (ng/ml) levels of the growth factor. Our model and novel gene delivery system may provide a powerful standardized tool for the optimization of growth factor delivery and release for the healing of large bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Osteoblast differentiation of rabbit adipose-derived stem cells by polyethylenimine-mediated BMP-2 gene transfection in vitro

The aim of this study was to examine the efficiency of polyethylenimine-mediated transfection of the human bone morphogenetic protein-2 (BMP-2) gene into rabbit adipose-derived stem cells (ADSCs), and its effect on osteoblast differentiation. Adipose tissue was isolated from the necks of adult Japanese white rabbits and cultured in vitro to obtain ADSCs. Gene delivery of BMP-2 was mediated by polyethylenimine and stable transformants were selected by G-418. The expression of BMP-2 mRNA was confirmed by reverse transcription-polymerase chain reaction, and of the BMP-2 protein by ELISA. Osteocalcin and collagen type I were detected by western blot and by an alkaline phosphatase kit. Alizarin red S stain was also utilized to examine osteogenesis. The non-transfected group was considered as a control. In this study, we successfully derived ADSCs from rabbit adipose tissue. Through passages 3-6, the expression of CD29 and CD44 gradually increased, whereas the expression of CD34 and CD45 gradually decreased. Both mRNA and protein expression of BMP-2 were confirmed following polyethylenimine-mediated BMP-2 gene delivery. In addition, the expression of alkaline phosphatase, osteocalcin, and collagen type I was found to be upregulated and alizarin red S staining was positive in transfected ADSCs, indicating BMP-2-induced osteogenesis. Therefore, this study determined that polyethylenimine was able to mediate BMP-2 gene delivery and induce osteogenic differentiation of ADSCs.

Reosseointegration Following Regenerative Therapy of Tissue-Engineered Bone in a Canine Model of Experimental Peri-Implantitis.

Due to the existence of inflammation and limited osteogenesis on the precontaminated implant surface, reosseointegration is difficult to realize by current therapies. Tissue-engineering strategy has been proved quite effective in intractable bone defect situation. This study was designed to see whether the adoption of tissue-engineered bone complex of adipose-derived stem cells (ASCs) and bone morphogenetic protein-2 (BMP-2) gene delivery would work efficiently in the correction of experimental peri-implantitis. All premolars in both side of mandibular were removed from six beagle canines three months before implant placement. Typical peri-implantitis were then induced by three month ligature placement. After the implementation of identical anti-bacterial and mechanical debridement therapy, the shaped peri-implant defect were stuffed with four groups of constructs, as A: beta tricalcium phosphate (β-TCP); B: β-TCP with ASCs; C: β-TCP with enhanced green fluorescent protein gene transduced ASCs (AdGFP-ASCs); and D: β-TCP with bone morphogenetic protein-2 gene-modified ASCs (AdBMP-2-ASCs). Systematic radiographic, micro-CT, and histomorphometrical assessments were performed. After six months of healing, more bone formation and reosseointegration was found around the implant of groups B and C than group A. And group D further promoted the new bone height and reosseointegration percentage. Moreover, sequential fluorescence labeling tells that group D exhibited the quickest and strongest bone formation on the cleaned implant surface during the entire observation period as compared to the other three groups. These data demonstrated that tissue engineered bone of ASCs, BMP-2 gene delivery, and β-TCP could exert powerful therapeutic effect on peri-implantitis as expected, which may suggest a feasible way to maintain the stability and masticatory function of dental implant.

Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced re-osseointegration in the regenerative treatment of peri-implantitis

Peri-implantitis is a chronic inflammatory process with advanced bone loss and impaired healing potential. For peri-implantitis treatment, tissue engineering can be applied to enhance bone regeneration of peri-implant defects. This study aimed to evaluate ex vivo bone morphogenetic protein 2 (BMP2) gene delivery using canine periodontal ligament stem cells (PDLSCs) for regeneration of peri-implantitis defects. Canine PDLSCs were transduced with adenoviral vectors containing BMP2 (BMP2/PDLSCs). After peri-implantitis was induced by ligature placement in six beagle dogs, regenerative procedures were performed; hydroxyapatite (HA) particles and collagen gel with autologous canine PDLSCs (PDLSC group) or BMP2/PDLSCs (BMP/PDLSC group) or without cells (control group) were grafted into the defects and covered by an absorbable membrane. Three months later, the animals were sacrificed. In vitro, BMP2/PDLSCs showed similar levels of stem cell properties to PDLSCs, such as colony-forming efficiency and expression of MSC markers STRO-1 and CD 146. BMP2/PDLSCs produced BMP-2 until day 21 at a concentration of 4-8 ng/mL. In vivo, the BMP2/PDLSC group showed significantly more new bone formation and re-osseointegration in peri-implantitis defects compared to the other groups. In conclusion, ex vivo BMP2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects.

Bone Morphogenetic Protein-2 Plasmid DNA as a Substitute for Bone Morphogenetic Protein-2 Protein in Bone Tissue Engineering

Bone regeneration is one of the focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. However, due to a short half-life, supraphysiological doses are applied resulting in severe side effects such as ectopic bone formation or even loss of bone. We compared the effectivity of transient BMP-2 gene delivery with the BMP-2 protein at clinical (high) and physiological (low) doses by subcutaneous implantation of alginate-based constructs in mice. After 6 weeks of implantation, both the protein laden constructs and BMP-2 plasmid DNA-based constructs showed similar early bone onset and elevated bone formation compared to controls without any BMP-2 added. We found no differences in efficiency by using BMP-2 plasmid DNA or any of the BMP-2 protein dosages. Therefore, we conclude that BMP-2 plasmid DNA-based gene therapy in alginate is a promising new strategy for BMP-2 administration for bone (re)generation.

Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

Ex vivo bone morphogenetic protein‐2 gene delivery using gingival fibroblasts promotes bone regeneration in rats

The aim of the present study was to investigate bone regeneration following ex vivo bone morphogenetic protein-2 (BMP-2) gene delivery using human gingival fibroblasts (HGFs) in rat calvarial defects. An 8 mm craniotomy defect was created in Sprague-Dawley rats. The animals were divided into four groups: (1) non-grafted group, the defect was left empty; (2) collagen matrix group, the defect was filled with collagen matrix only; (3) HGF group, the defect was filled with non-transduced HGFs on collagen matrix; (4) BMP-2/HGF group, the defect was filled with BMP-2 gene-transduced HGFs on collagen matrix. Animals were sacrificed at 2 and 4 weeks after surgery, and micro-computed tomographic and histologic observations were performed. The BMP-2/HGF group showed promoted osseous healing of calvarial defects, as compared with the other groups. At both 2 and 4 weeks, regenerated bone area was significantly greater in the BMP-2/HGF group than the other three groups. Quite a few number of transplanted HGFs were observed within the regenerated bone tissues. The results of this study suggest that ex vivo BMP-2 gene delivery induces prominent bone regeneration in vivo and HGFs may be useful as target cells for ex vivo gene therapy.

Gene Therapy Platform for Bone Regeneration Using an Exogenously Regulated, AAV-2-Based Gene Expression System

Viral delivery of the therapeutic gene bone morphogenetic protein-2 (BMP-2) is a promising approach for bone regeneration. The human parvovirus adeno-associated virus (AAV) type 2 is considered one of the most encouraging viral vector systems because of its high transduction rates and biosafety ratings. Bone morphogenetic protein-2 is a highly potent osteoinductive protein, which induces bone formation in vivo and osteogenic differentiation in vitro. The exogenous regulation of BMP-2 expression in bone-regenerating sites is required to control BMP-2 protein secretion, thus promoting safe and controlled bone formation and regeneration. We have therefore constructed a dual-construct vector for the recombinant AAV (rAAV)-based recombinant human BMP-2 (rhBMP-2) gene delivery system, which is regulated by the tetracycline-sensitive promoter (TetON). Each vector was encapsidated separately, yielding two recombinant viruses. We evaluated the efficiency of rAAV-hBMP-2 to induce bone formation in ectopic and orthotopic sites. Doxycycline (Dox), an analogue of tetracycline, was orally administered to mice via their drinking water to induce rhBMP-2 expression. Bone formation was measured using quantitative imaging—microcomputerized tomography and cooled charge-coupled device imaging—to detect osteogenic activity at the cellular level, detecting osteocalcin expression. The rAAV-hBMP-2-treated mice that were given Dox demonstrated bone formation in both in vivo models compared to none in mice prevented from receiving Dox. Thus, the Tet-regulated rAAV-hBMP-2 vector is an effective means of induction and regulation of bone regeneration and repair.