Gene Therapy For Bone Regeneration Research Articles

Adenovirus-Based Gene Therapy for Bone Regeneration: A Comparative Analysis of In Vivo and Ex Vivo BMP2 Gene Delivery.

Adenovirus-mediated gene therapy is a promising tool in bone regenerative medicine. In this work, gene-activated matrices (GAMs) composed of (1) polylactide granules (PLA), which serve as a depot for genetic constructs or matrices for cell attachment, (2) a PRP-based fibrin clot, which is a source of growth factors and a binding gel, and (3) a BMP2 gene providing osteoinductive properties were studied. The study aims to compare the effectiveness of in vivo and ex vivo gene therapy based on adenoviral constructs with the BMP2 gene, PLA particles, and a fibrin clot for bone defect healing. GAMs with Ad-BMP2 and MSC(Ad-BMP2) show osteoinductive properties both in vitro and in vivo. However, MSCs incubated with GAMs containing transduced cells showed a more significant increase in osteopontin gene expression, protein production, Alpl activity, and matrix mineralization. Implantation of the studied matrices into critical-size calvarial defects after 56 days promotes the formation of young bone. The efficiency of neoosteogenesis and the volume fraction of newly formed bone tissue are higher with PLA/PRP-MSC(Ad-BMP2) implantation (33%) than PLA/PRP-Ad-BMP2 (28%). Thus, ex vivo adenoviral gene therapy with the BMP2 gene has proven to be a more effective approach than the in vivo delivery of gene constructs for bone regeneration.

Ubiquitin-specific protease 53 promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

The ubiquitin protease pathway plays important role in human bone marrow-derived mesenchymal stem cell (hBMSC) differentiation, including osteogenesis. However, the function of deubiquitinating enzymes in osteogenic differentiation of hBMSCs remains poorly understood. In this study, we aimed to investigate the role of ubiquitin-specific protease 53 (USP53) in the osteogenic differentiation of hBMSCs. Based on re-analysis of the Gene Expression Omnibus database, USP53 was selected as a positive regulator of osteogenic differentiation in hBMSCs. Overexpression of USP53 by lentivirus enhanced osteogenesis in hBMSCs, whereas knockdown of USP53 by lentivirus inhibited osteogenesis in hBMSCs. In addition, USP53 overexpression increased the level of active β-catenin and enhanced the osteogenic differentiation of hBMSCs. This effect was reversed by the Wnt/β-catenin inhibitor DKK1. Mass spectrometry showed that USP53 interacted with F-box only protein 31 (FBXO31) to promote proteasomal degradation of β-catenin. Inhibition of the osteogenic differentiation of hBMSCs by FBXO31 was partially rescued by USP53 overexpression. Animal studies showed that hBMSCs with USP53 overexpression significantly promoted bone regeneration in mice with calvarial defects. These results suggested that USP53 may be a target for gene therapy for bone regeneration.

The Toll-like receptor ligand, CpG oligodeoxynucleotides, regulate proliferation and osteogenic differentiation of osteoblast

BackgroundThis study aimed to investigate the regulation of CpG oligodeoxynucleotides (ODNs) on proliferation and osteogenic differentiation of MC3T3 cells.MethodsThe laser co-focusing and flow cytometry assay were employed to detect cell uptake of CpG ODN 2006. Twelve ODNs were sythesized, and their effects on proliferation and differentiation were detected by MTT and alkaline phosphatase (ALP) activity assay. Flow cytometry assay was used to examine the regulation of CpG ODN on cell cycle. Quantitative real-time PCR (qRT-PCR) and western blot were used to evaluate the regulation of CpG ODN on mRNA and protein expression of osteogenic differentiation genes.ResultsThe phosphorothioate CpG ODN 2006 could efficiently enter the MC3T3 cells in 1 h and locate in the cytoplasm. The MTT assay demonstrated CpG ODNs could promote MC3T3 cell proliferation and differentiation in the early stage, and gradually attenuated along with the increase of treating time, except for BW001 and FC001. qRT-PCR assay demonstrated that all the 12 CpG ODNs could promote the relative expression level of osteogenic differentiated genes, SP7 and OCN. In addition, western blot analysis suggested the CpG ODNs of BW001 and FC001 could increase the protein expression of P27Kip1 and Runx2 and decrease the protein expression of cyclin D1.ConclusionThe selected CpGODNs may be a potential gene therapy for bone regeneration of periodontitis.

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.

Recent Advances and Future of Gene Therapy for Bone Regeneration.

The purpose of this review is to discuss the recent advances in gene therapy as a treatment for bone regeneration. While most fractures heal spontaneously, patients who present with fracture nonunion suffer from prolonged pain, disability, and often require additional operations to regain musculoskeletal function. In the last few years, BMP gene delivery by means of electroporation and sonoporation resulted in repair of nonunion bone defects in mice, rats, and minipigs. Ex vivo transfection of porcine mesenchymal stem cells (MSCs) resulted in bone regeneration following implantation in vertebral defects of minipigs. Sustained release of VEGF gene from a collagen-hydroxyapatite scaffold to the mandible of a human patient was shown to be safe and osteoinductive. In conclusion, gene therapy methods for bone regeneration are systematically becoming more efficient and show proof-of-concept in clinically relevant animal models. Yet, on the pathway to clinical use, more investigation is needed to determine the safety aspects of the various techniques in terms of biodistribution, toxicity, and tumorigenicity.

Recent advances in gene-enhanced bone tissue engineering.

The loss of bone tissue represents a critical clinical condition that is frequently faced by surgeons. Substantial progress has been made in the area of bone research, providing insight into the biology of bone under physiological and pathological conditions, as well as tools for the stimulation of bone regeneration. The present review discusses recent advances in the field of gene-enhanced bone tissue engineering. Gene transfer strategies have emerged as highly effective tissue engineering approaches for supporting the repair of the musculoskeletal system. By contrast to treatment with recombinant proteins, genetically engineered cells can release growth factors at the site of injury over extended periods of time. Of particular interest are the expedited technologies that can be applied during a single surgical procedure in a cost-effective manner, allowing translation from bench to bedside. Several promising methods based on the intra-operative genetic manipulation of autologous cells or tissue fragments have been developed in preclinical studies. Moreover, gene therapy for bone regeneration has entered the clinical stage with clinical trials for the repair of alveolar bone. Current trends in gene-enhanced bone engineering are also discussed with respect to the movement of the field towards expedited, translational approaches. It is possible that gene-enhanced bone tissue engineering will become a clinical reality within the next few years.

Enhanced Bone Regeneration by Diabetic Cell-Based Adenoviral BMP-2 Gene Therapy in Diabetic Animals.

The application of bone morphogenetic protein 2 (BMP-2) has been extensively investigated to improve diabetes-impaired bone healing; however, the delivery of BMP-2 by gene therapy for bone regeneration has rarely been investigated in diabetic animals. In this study, we aimed to evaluate which cells induce more new bone formation in diabetic animals when cell-based BMP2 gene therapy is applied. For this purpose, we harvested bone marrow stromal cells (BMSCs) twice in the same animal before (non-diabetic BMSCs; nBMSCs) and after diabetes induction (diabetic BMSCs; dBMSCs) using modified bone marrow ablation methods. And then, cells were transduced by adenoviral vectors carrying the BMP2 gene (AdBMP2). In in vitro, AdBMP2-transfected dBMSCs (B2/dBMSCs) produced higher BMP-2 mRNA levels over 48 h, whereas AdBMP2-transfected nBMSCs (B2/nBMSCs) exhibited a transient increase in BMP-2 mRNA followed by a decrease to the baseline level within 48 h. Both B2/dBMSCs and B2/nBMSCs induced secretion of BMP-2 for 3 weeks. However, B2/dBMSC BMP-2 secretion peaked from day 3 to 10, whereas B2/nBMSC BMP-2 secretion peaked from day 1 to 7. The analysis of osteogenic activity revealed that mineralization nodule formation and the expression levels of osteogenic genes were significantly higher in B2/dBMSCs than B2/nBMSCs and were accompanied by upregulation of canonical Wnt/β-catenin and Smad signaling. AdBMP2-transfected autologous cells were implanted into critical-sized calvarial defects in diabetic animals and induced significantly more bone regeneration than non-AdBMP2-transfected cells. In addition, B2/dBMSCs led to significantly more new bone formation than B2/nBMSCs. Thus, BMP2 gene therapy using diabetic cells effectively supported diabetic bone healing and it was related to the enhanced responses to AdBMP2 of dBMSCs.

3D printing: The cutting edge of digital medicine

Prof. Kerong Dai is a senior member of Chinese Academy of Engineering (CAE), a foreign corresponding member of French National Academy of Medicine, and an expert in orthopedic surgery and biomechanics. He graduated from Shanghai Medical University (i.e. Fudan University Shanghai Medical College) in 1955, and was a visiting fellow in Mayo Clinic in the United States from 1983-1984. He was the Tenured Professor and the former president of Shanghai Ninth People's Hospital Affiliated to Shanghai Second Medical University, and also the director of the Department of Orthopedics. He is currently the director of Shanghai Clinical Medical Center of Joint Surgery, of the Bone and Joint Research Center of Shanghai Jiao Tong University (SJTU) School of Medicine, of the Engineering Research Center of Digital Medicine, Ministry of Education, and of the Stem Cell and Regenerative Medicine Transformation Base of the Institute of Translational Medicine of SJTU. He pioneered to apply shape-memory alloy within human body internationally. He has made breakthrough in quantitative evaluation of gait and body balance, stress-shielding effect of internal fixation, osteoporotic fracture, basic study of artificial joint and customized artificial joints, stem cell transplantation and gene therapy for bone regeneration, and the application of 3D printing technology in musculoskeletal system. He has won more than 45 prizes, among which are the National Invention Prize of China (the second prize), the National Science and Technology Progress Award (the second and third prizes), and a number of municipal and ministerial awards. In addition, he has gained more than 40 patents. He has published over 500 papers, and compiled 59 monographs.

Characterization of scaffold carriers for BMP9‐transduced osteoblastic progenitor cells in bone regeneration

AbstractSuccessful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell‐based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically used scaffold materials, type I collagen sponge, hydroxyapatite‐tricalcium phosphate (HA‐TCP), and demineralized bone matrix (DBM), using BMP9‐expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus‐mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9‐transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9‐expressing C2C12 cells with collagen sponge or HA‐TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA‐TCP scaffold carriers may provide more cell‐friendly environment to support the survival, propagation, and ultimately differentiation of BMP9‐expressing progenitor cells. This line of investigation should provide important experimental evidence for further preclinical studies in BMP9‐mediated cell‐based approach to bone tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3429–3438, 2014.

Characterization of scaffold carriers for BMP9-transduced osteoblastic progenitor cells in bone regeneration

Successful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell-based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically used scaffold materials, type I collagen sponge, hydroxyapatite-tricalcium phosphate (HA-TCP), and demineralized bone matrix (DBM), using BMP9-expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus-mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9-transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9-expressing C2C12 cells with collagen sponge or HA-TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA-TCP scaffold carriers may provide more cell-friendly environment to support the survival, propagation, and ultimately differentiation of BMP9-expressing progenitor cells. This line of investigation should provide important experimental evidence for further preclinical studies in BMP9-mediated cell-based approach to bone tissue engineering.

Non-viral gene therapy for bone tissue engineering

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.

Lentiviral-mediated gene transfer into human adipose-derived stem cells: role of NELL1 versus BMP2 in osteogenesis and adipogenesis <italic>in vitro</italic>

NEL-like molecule 1 (NELL1) is a potent osteogenic factor associated with craniosynostosis. Adenoviruses, the most commonly used viral vectors for gene therapy, have several disadvantages that may restrict osteogenesis. Previous studies have shown that lentiviruses can serve as ideal vectors for gene therapy for bone regeneration. In this study, two lentiviral vectors (LvNELL1 and LvBMP2) that encode human NELL1 and bone morphogenetic protein-2 (BMP2), respectively, were constructed. The effect of LvNELL1 infection on the proliferation, osteogenesis, and adipogenesis of human adipose-derived stem cells (hADSCs) in vitro was assessed and compared with that of LvBMP2. The results showed that hADSCs infected with LvNELL1 could efficiently and stably overexpress the target genes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay results demonstrated that LvBMP2, but not LvNELL1, enhanced the proliferation of hADSCs. Assessment of alkaline phosphatase activity and cellular mineralization indicated that LvNELL1 infection promoted the osteogenic differentiation of hADSCs, and the effect was comparable with that of LvBMP2. Real-time polymerase chain reaction (PCR) revealed that LvNELL1 infection upregulated OSX expression but not RUNX2 expression in hADSCs. In addition, adipogenic markers (lipid droplets, peroxisome proliferator-activating receptor γ, and lipoprotein lipase) analysis showed that LvNELL1 could dramatically inhibit the adipogenic differentiation of hADSCs, but LvBMP2 had no such effect. Taken together, these findings suggested that lentiviral-mediated NELL1 gene transfer in hADSCs may be a novel and promising approach to achieve effective and precise bone regeneration.

Human Bone Morphogenetic Protein 2–Transduced Mesenchymal Stem Cells Improve Bone Regeneration in a Model of Mandible Distraction Surgery

Bone morphogenetic proteins (BMPs) are actively involved in ossification, and BMP-2 participates throughout the entire process. Gene therapy for bone regeneration using adenovirus-expressing BMPs has been successful in small mammals, but it has not been satisfactory in large mammals. We generated a 3-component implant (3C graft) comprising autologous mesenchymal stem cells (MSCs), ex vivo transduced with an adenovirus vector-expressing BMP-2 and embedded in a demineralized human bone matrix (DBM). In vitro studies demonstrated vector-induced osteogenesis; osteoblast population and mineralization of the extracellular matrix were greater in the vector-transduced cultures than in the controls (nontransduced MSCs stimulated with osteogenic media were used as positive controls, and nontransduced MSCs served as a negative control). The 3-component grafts were used to fill osteotomies created by bone distraction surgery in mongrel dogs. Control groups comprised dogs with bone distraction alone and dogs with nontransduced MSC grafts. The radiography follow-up, performed 10 weeks after distraction, demonstrated a remarkable reduction in the consolidation period compared with controls. Postmortem mandibles submitted for anatomic and histologic analyses showed improved remodeling and bone maturation in the 3C-grafted dogs. Inflammatory infiltrates were not observed in any of the treated areas, and no liver toxicity was detected. We demonstrated acceleration of osteogenesis in a dog model for bone distraction by using an implant of BMP-2 modified MSCs. These results are helpful for future clinical trials of mandible bone distraction.

Lentiviral-Mediated Integrin α5 Expression in Human Adult Mesenchymal Stromal Cells Promotes Bone Repair in Mouse Cranial and Long-Bone Defects

Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

Comparative efficacy of dermal fibroblast-mediated and direct adenoviral bone morphogenetic protein-2 gene therapy for bone regeneration in an equine rib model

Cell-mediated and direct adenoviral (Ad) vector gene therapies can induce bone regeneration, including dermal fibroblasts (DFbs). We compared two effective therapies, DFb-mediated and direct Ad vector delivery of bone morphogenetic protein-2 (BMP2), for relative efficacy in bone regeneration. Equine rib drill defects were treated by percutaneous injection of either DFb-BMP2 or an Ad-BMP2 vector. At week 6, both DFb-BMP2- and Ad-BMP2-treated rib defects had greater bone filling volume and mineral density, with DFb-BMP2 inducing greater bone volume and maturity in the cortical bone aspect of the defect than Ad-BMP2. The transplantation of DFb alone induced modest bone formation. Increased mineral density and bone turnover were evident in the cortical and cancellous bone directly adjacent to the healing drill defects treated with either DFb-BMP2 or Ad-BMP2. Using our cell/vector dosage and model, BMP2, whether delivered by the DFb vector or direct Ad vector, induced greater and robust bone regeneration. DFb-mediated BMP2 therapy promoted greater cortical bone regeneration than did direct gene delivery, possibly because of an increased cellularity of the bone healing site. BMP2 delivery, regardless of gene delivery method, increased the mineral density of the neighboring bone, which may be beneficial clinically in repairing or weak bone.

Direct Gene Therapy for Bone Regeneration: Gene Delivery, Animal Models, and Outcome Measures

While various problems with bone healing remain, the greatest clinical change is the absence of an effective approach to manage large segmental defects in limbs and craniofacial bones caused by trauma or cancer. Thus, nontraditional forms of medicine, such as gene therapy, have been investigated as a potential solution. The use of osteogenic genes has shown great potential in bone regeneration and fracture healing. Several methods for gene delivery to the fracture site have been described. The majority of them include a cellular component as the carrying vector, an approach known as cell-mediated gene therapy. Yet, the complexity involved with cell isolation and culture emphasizes the advantages of direct gene delivery as an alternative strategy. Here we review the various approaches of direct gene delivery for bone repair, the choice of animal models, and the various outcome measures required to evaluate the efficiency and safety of each technique. Special emphasis is given to noninvasive, quantitative, in vivo monitoring of gene expression and biodistribution in live animals. Research efforts should aim at inducing a transient, localized osteogenic gene expression within a fracture site to generate an effective therapeutic approach that would eventually lead to clinical use.

Direct Gene Therapy for Bone Regeneration: Gene Delivery, Animal Models, and Outcome Measures

Direct Gene Therapy for Bone Regeneration: Gene Delivery, Animal Models, and Outcome Measures

Combinatorial gene therapy for bone regeneration: Cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7

Bone morphogenetic proteins (BMPs) have demonstrated effectiveness as bone regeneration agents whether delivered as recombinant proteins or via gene therapy. Current gene therapy approaches use vectors expressing single BMPs. In contrast, multiple BMPs are coordinately expressed during bone development and fracture healing. Furthermore, BMPs likely exist in vivo as heterodimeric molecules having enhanced biological activity. In the present study, we test the hypothesis that gene therapy-based bone regeneration can be enhanced by expressing combinations of BMPs. For in vitro studies, mesenchymal cell lines were transduced with individual adenoviruses containing BMP2, 4, or 7 cDNA under control of a CMV promoter (AdBMP2, 4, 7) or virus combinations. Significantly, combined transduction with AdBMP2 plus AdBMP7 or AdBMP4 plus AdBMP7 resulted in a synergistic stimulation of osteoblast differentiation. This synergy is best explained by formation of BMP2/7 and 4/7 heterodimers. To test in vivo biological activity, fibroblasts were transduced with specific virus combinations and implanted into C57BL6 mice. Consistent with in vitro results, strong synergy was observed using combined AdBMP2/BMP7 treatment, which induced twofold to threefold more bone than would be predicted based on the activity of individual AdBMPs. These studies show that dramatic enhancement of osteogenesis can be achieved using gene therapy to express specific combinations of interacting regenerative molecules.

Bone regeneration in rats with BMP-4 and VEGF plasmid DNA

Problem: Gene therapy offers a promising new method of bone regeneration. One technique uses bioresorbable polymer scaffolding at the bony defect site that contains plasmid DNA of bone growth factors. Previous studies have demonstrated bone growth using bone morphogenetic protein-4 (BMP-4) plasmid DNA. Research has suggested a synergistic effect of BMP-4 and vascular endothelial growth factor (VEGF) on bone regeneration. The present study aims to confirm bone regeneration with a polymer scaffold containing BMP-4 plasmid DNA in the rat calvarial defect model and to quantify a synergistic effect of BMP-4 and VEGF plasmid DNA in this model. Methods: Calvarial critical-sized defects were created in 100 male Sprague-Dawley rats. The animals were divided into 5 equal groups by treatment: none (negative control); VEGF plasmid DNA; BMP-4 plasmid DNA; VEGF and BMP-4 plasmid DNA; and autograft (positive control). The animals were euthanized at 4 and 8 weeks; calvaria were radiographed and histologically sectioned. Results: Radiomorphometry measured bone growth within the calvarial defects to quantify the bone regeneration effects of BMP-4 and VEGF plasmid DNA alone and in combination. Compared to control groups, significant bone growth was noted in the BMP-4 and BMP-4/VEGF groups, but no bone growth was noted in the VEGF group. No significant difference in bone growth was noted between the BMP-4 and BMP-4/VEGF groups. Microscopy of histologic sections (1 slide/specimen) confirmed the bone growth noted on radiomorphometry. Conclusion: BMP-4 plasmid DNA demonstrates significant bone growth in the rat calvarial defect model. VEGF plasmid DNA alone demonstrates no bone growth. When BMP-4 and VEGF plasmid DNA are combined, no synergistic effect is noted. Significance: Reconstruction of osseous craniofacial defects such as autografting for cleft palate and osteocutaneous free tissue transfer for mandibular defects often requires bone harvest with an associated donor site morbidity. Tissue engineering employing gene therapy for bone regeneration may offer a less morbid alternative. Support: None reported.

In vivo new bone formation by direct transfer of adenoviral-mediated bone morphogenetic protein-4 gene

Previous studies have demonstrated that bone morphogenetic protein-4 (BMP4) could participate in vivo endochondral ossification and is one of the main local contributing factors in the early stage of fracture healing. To investigate the effectiveness of BMP4 gene transfer, we constructed an adenoviral vector, Ad-BMP4, and evaluated its osteoinduction activity both in vitro and in vivo. In vitro study suggested that this vector could efficiently transduce mouse myoblast C2C12 cells and produce osteogenic BMP4 protein, as confirmed by immunofluorescence analysis and alkaline phosphatase activity assay. For in vivo study, Ad-BMP4 was directly injected into the hind limb muscles of male athymic nude rats. Visible new bone formation under X-ray films could be detected as early as three weeks post-injection. The bone tissue was further analyzed by histological staining and revealed a typical remodeled bone structure. In conclusion, this study is the first to establish the feasibility of adenovirus-based BMP4 gene therapy for bone regeneration.