Bone Morphogenetic Protein-2 Plasmid Research Articles

Gene activated scaffolds incorporating star-shaped polypeptide-pDNA nanomedicines accelerate bone tissue regeneration in vivo.

Increasingly, tissue engineering strategies such as the use of biomaterial scaffolds augmented with specific biological cues are being investigated to accelerate the regenerative process. For example, significant clinical challenges still exist in efficiently healing large bone defects which are above a critical size. Herein, we describe a cell-free, biocompatible and bioresorbable scaffold incorporating a novel star-polypeptide biomaterial as a gene vector. This gene-loaded scaffold can accelerate bone tissue repair in vivo in comparison to a scaffold alone at just four weeks post implantation in a critical sized bone defect. This is achieved via the in situ transfection of autologous host cells which migrate into the implanted collagen-based scaffold via gene-loaded, star-shaped poly(l-lysine) polypeptides (star-PLLs). In vitro, we demonstrate that star-PLL nanomaterials designed with 64 short poly(l-lysine) arms can be used to functionalise a range of collagen based scaffolds with a dual therapeutic cargo (pDual) of the bone-morphogenetic protein-2 plasmid (pBMP-2) and vascular endothelial growth factor plasmid (pVEGF). The versatility of this polymeric vector is highlighted in its ability to transfect Mesenchymal Stem Cells (MSCs) with both osteogenic and angiogenic transgenes in a 3D environment from a range of scaffolds with various macromolecular compositions. In vivo, we demonstrate that a bone-mimetic, collagen-hydroxyapatite scaffold functionalized with star-PLLs containing either 32- or 64- poly(l-lysine) arms can be used to successfully deliver this pDual cargo to autologous host cells. At the very early timepoint of just 4 weeks, we demonstrate the 64-star-PLL-pDual functionalised scaffold as a particularly efficient platform to accelerate bone tissue regeneration, with a 6-fold increase in new bone formation compared to a scaffold alone. Overall, this article describes for the first time the incorporation of novel star-polypeptide biomaterials carrying two therapeutic genes into a cell free scaffold which supports accelerated bone tissue formation in vivo.

Nanofibrous Scaffolds Containing Hydroxyapatite and Microfluidic-Prepared Polyamidoamin/BMP-2 Plasmid Dendriplexes for Bone Tissue Engineering Applications.

ObjectiveThe aim of this study is to fabricate functional scaffolds to gene delivery bone morphogenetic protein-2 (BMP-2) plasmid for bone formation in bone tissue engineering.MethodsDendriplexes (DPs) of generation 4 polyamidoamin (G4-PAMAM)/BMP-2 plasmid were prepared through microfluidic (MF) platform. The physiochemical properties and toxicity of DPs were evaluated by DLS, AFM, FESEM and MTT assay. In order to create a suitable environment for stem cell growth and differentiation, poly-l-lactic acid (PLLA) and poly-l-lactic acid/poly (ethylene oxide) (PLLA/PEO) scaffolds containing hydroxyapatite nanoparticles (HA) and DPs were fabricated by the electrospinning method. The osteogenic potency of the scaffolds on human adipose tissue-derived mesenchymal stem cells (hASCs) was investigated.ResultsThe results revealed that tuning the physical properties of DPs by adjusting flow parameters in microfluidic platform can easily improve the cell viability compared to conventional bulk mixing method. Also, the result showed that the presence of HA and DPs in PLLA/PEO scaffold enhanced alkaline phosphatase (ALP) activity and increased the amount of deposited Ca, as well as, related to osteogenesis gen markers.ConclusionThis study indicated that on using the MF platform in preparation of DPs and loading them along with HA in PLLA/PEO scaffold, the osteogenic differentiation of hASCs could be tuned.

Effect of bone morphogenetic protein-4 on proliferation, apoptosis and migration of thyroid cancer cells and its mechanism

Objective To investigate the effects of bone morphogenetic protein-4 (BMP-4) on the proliferation, apoptosis and migration of thyroid cancer cells through extracellular signal-regulated kinase 1/2 (ERK1/2)/p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. Methods Thyroid papillary carcinoma (TPC)-1 cells (thyroid papillary carcinoma cells) were randomly divided into blank control group (BC), negative control group (NC) and interference group (KD). Cells in BC group were not transfected. NC group cells were transfected with negative control plasmids. KD group cells were transfected with the silenced BMP-4 plasmid. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the BMP-4 mRNA levels. Cell counting kit-8 (CCK-8) was used to measure cell proliferation. Flow cytometry was used to determine cell apoptosis. Transwell chamber and scratch assay was used to determine cell migration capacity. The levels of ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), p38MAPK and p-p38MAPK protein were determined by Western blotting. Results The level of BMP-4 mRNA in TPC-1 cells (5.86±0.42) was higher than that in HT-ori3 cells (normal thyroid cells) (1.00±0.03, t=30.537, P 0.05). Conclusion Silencing BMP-4 can inhibit the proliferation and migration of thyroid cancer cells and promote apoptosis, which may be related to down-regulation of ERK1/2/p38MAPK signaling pathway. Key words: Bone morphogenetic protein-4; Extracellular regulated protein kinase; p38 mitogen-activated protein kinase; Thyroid cancer; Proliferation; Apoptosis; Migration

Bone formation promoted by bone morphogenetic protein-2 plasmid-loaded porous silica nanoparticles with the involvement of autophagy.

Gene therapy is one of the most common and effective ways for the regeneration of defective bone tissue, but even highly efficient gene delivery vectors are insufficient. In this study, bone morphogenetic protein-2 plasmid (pBMP-2) was encapsulated by polyethylenimine-modified porous silica nanoparticles (PPSNs), which were synthesized via an ethyl ether emulsion method. Owing to the high specific surface area and high absorption characteristics, low cytotoxicy PPSNs can efficiently load and protect pBMP-2. The resulting PPSN/pBMP-2 can transfect MC3T3-E1 cells effectively to promote osteogenic differentiation and increase calcium deposition in vitro. Interestingly, the mass of calcium deposition nodules decreased dur to the presence of an autophagy inhibitor, demonstrating that PPSNs stimulated the autophagy pathway. Because of their excellent biocompatibility, high transfection efficiency, and ability to stimulate autophagy, the as-prepared PPSN/pBMP-2 could efficiently transfect local cells in a defect area in vivo. Micro-computed tomography and histological images demonstrated that PPSN/pBMP-2 could efficiently promote new bone formation in a 5 mm sized rat calvarial defect model. Taken together, our newly synthesized PPSNs could efficiently carry pBMP-2 and deliver it to the target cells as well as stimulating the autophagy pathway, resulting in significant osteogenic differentiation and bone regeneration.

Innovative Strategy for 3D Transfection of Primary Human Stem Cells with BMP-2 Expressing Plasmid DNA: A Clinically Translatable Strategy for Ex Vivo Gene Therapy

Ex vivo gene therapy offers enormous potential for cell-based therapies, however, cumbersome in vitro cell culture conditions have limited its use in clinical practice. We have optimized an innovative strategy for the transient transfection of bone morphogenetic protein-2 (BMP-2) expressing plasmids in suspended human stem cells within 5-min that enables efficient loading of the transfected cells into a 3D hydrogel system. Such a short incubation time for lipid-based DNA nanoparticles (lipoplexes) reduces cytotoxicity and at the same time reduces the processing time for cells to be transplanted. The encapsulated human mesenchymal stromal/stem cells (hMSCs) transfected with BMP-2 plasmid demonstrated high expression of an osteogenic transcription factor, namely RUNX2, but not the chondrogenic factor (SOX9), within the first three days. This activation was also reflected in the 7-day and 21-day experiment, which clearly indicated the induction of osteogenesis but not chondrogenesis. We believe our transient transfection method demonstrated in primary MSCs can be adapted for other therapeutic genes for different cell-based therapeutic applications.

Accelerated bony defect healing based on chitosan thermosensitive hydrogel scaffolds embedded with chitosan nanoparticles for the delivery of BMP2 plasmid DNA.

Regeneration of alveolar bone is among the main purposes of periodontal treatment. However, it is a challenge for both dentists and patients. An injectable chitosan-based thermosensitive hydrogel scaffold (CS/CSn-GP) was successfully designed and prepared with the incorporation of bone morphogenetic protein-2 plasmid DNA (pDNA-BMP2)-loaded chitosan nanoparticles (CS/CSn(pDNA-BMP2)-GP) in this study. The aim of this study was to evaluate the regenerative effect of this scaffold on the endogenous repair of alveolar bone. Thermosensitive properties, SEM scans and in vivo inflammatory reactions and degradation were evaluated. The CS/CSn-GP solution was initially injected into the muscle pouches of rats, where it changed into a gel, resulting in non-specific inflammation. It effectively enhanced new bone formation in rat calvarial defects and enhanced bony defect healing in beagle dogs. All results indicated that the CS/CSn(pDNA-BMP2)-GP complex system is a potential candidate as a targeted, multi-effect scaffold for the endogenous repair of alveolar bone. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 265-273, 2017.

Characterization and cytocompatibility of thermosensitive hydrogel embedded with chitosan nanoparticles for delivery of bone morphogenetic protein-2 plasmid DNA.

A novel injectable chitosan thermosensitive hydrogel was designed as a target multi-effect scaffold for endogenous repair of the periodontium. The hydrogel complex was designed by embedding chitosan nanoparticles (CSn) loaded with bone morphogenetic protein-2 plasmid DNA (pDNA-BMP2) into a chitosan (CS)-based hydrogel with α,β-glycerophosphate (α,β-GP), termed CS/CSn(pDNA-BMP2)-GP. Characterization, the in vitro release profile for pDNA-BMP2, and cytocompatibility to human periodontal ligament cells (HPDLCs), were then conducted. The average diameter of the CSn(pDNA-BMP2) was 270.1nm with a polydispersity index (PDI) of 0.486 and zeta potential of +27.0mv. A DNase I protection assay showed that CSn could protect the pDNA-BMP2 from nuclease degradation. Encapsulation efficiency and loading capacity of CSn(pDNA-BMP2) were more than 80 and 30%, respectively. The sol-gel transition time was only 3min when CSn(pDNA-BMP2) was added into the CS/α,β-GP system. Scanning electron microscopy showed that CSn(pDNA-BMP2) was randomly dispersed in a network with regular holes and a porous structure. Weighting method showed the swelling ratio and degradation was faster in medium of pH 4.0 than pH 6.8. An in vitro pDNA-BMP2 release test showed that the cumulative release rate of pDNA-BMP2 was much slower from CS/CSn-GP than from CSn in identical release media. In release media with different pH, pDNA-BMP2 release was much slower at pH 6.8 than at pH 4.0. Three-dimensional culture with HPDLCs showed good cell proliferation and the Cell-Counting Kit-8 assay indicated improved cell growth with the addition of CSn(pDNA-BMP2) to CS/α,β-GP. In summary, the CS/CSn(pDNA-BMP2)-GP complex system exhibited excellent biological properties and cytocompatibility, indicating great potential as a gene delivery carrier and tissue regeneration scaffold for endogenous repair of the periodontium.

Non-virally engineered human adipose mesenchymal stem cells produce BMP4, target brain tumors, and extend survival

There is a need for enabling non-viral nanobiotechnology to allow safe and effective gene therapy and cell therapy, which can be utilized to treat devastating diseases such as brain cancer. Human adipose-derived mesenchymal stem cells (hAMSCs) display high anti-glioma tropism and represent a promising delivery vehicle for targeted brain tumor therapy. In this study, we demonstrate that non-viral, biodegradable polymeric nanoparticles (NPs) can be used to engineer hAMSCs with higher efficacy (75% of cells) than leading commercially available reagents and high cell viability. To accomplish this, we engineered a poly(beta-amino ester) (PBAE) polymer structure to transfect hAMSCs with significantly higher efficacy than Lipofectamine™ 2000. We then assessed the ability of NP-engineered hAMSCs to deliver bone morphogenetic protein 4 (BMP4), which has been shown to have a novel therapeutic effect by targeting human brain tumor initiating cells (BTIC), a source of cancer recurrence, in a human primary malignant glioma model. We demonstrated that hAMSCs genetically engineered with polymeric nanoparticles containing BMP4 plasmid DNA (BMP4/NP-hAMSCs) secrete BMP4 growth factor while maintaining their multipotency and preserving their migration and invasion capacities. We also showed that this approach can overcome a central challenge for brain therapeutics, overcoming the blood brain barrier, by demonstrating that NP-engineered hAMSCs can migrate to the brain and penetrate the brain tumor after both intranasal and systemic intravenous administration. Critically, athymic rats bearing human primary BTIC-derived tumors and treated intranasally with BMP4/NP-hAMSCs showed significantly improved survival compared to those treated with control GFP/NP-hAMCSs. This study demonstrates that synthetic polymeric nanoparticles are a safe and effective approach for stem cell-based cancer-targeting therapies.

Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration.

The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ∼73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.

Fabrication of Core-Shell PEI/pBMP2-PLGA Electrospun Scaffold for Gene Delivery to Periodontal Ligament Stem Cells.

Bone tissue engineering is the most promising technology for enhancing bone regeneration. Scaffolds loaded with osteogenic factors improve the therapeutic effect. In this study, the bioactive PEI (polyethylenimine)/pBMP2- (bone morphogenetic protein-2 plasmid-) PLGA (poly(D, L-lactic-co-glycolic acid)) core-shell scaffolds were prepared using coaxial electrospinning for a controlled gene delivery to hPDLSCs (human periodontal ligament stem cells). The pBMP2 was encapsulated in the PEI phase as a core and PLGA was employed to control pBMP2 release as a shell. First, the scaffold characterization and mechanical properties were evaluated. Then the gene release behavior was analyzed. Our results showed that pBMP2 was released at high levels in the first few days, with a continuous release behavior in the next 28 days. At the same time, PEI/pBMP2 showed high transfection efficiency. Moreover, the core-shell electrospun scaffold showed BMP2 expression for a much longer time (more than 28 days) compared with the single axial electrospun scaffold, as evaluated by qRT-PCR and western blot after culturing with hPDLSCs. These results suggested that the core-shell PEI/pBMP2-PLGA scaffold fabricated by coaxial electrospinning had a good gene release behavior and showed a prolonged expression time with a high transfection efficiency.

Combination of bone morphogenetic protein-2 plasmid DNA with chemokine CXCL12 creates an additive effect on bone formation onset and volume

Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.

Bone morphogenetic protein-2 nonviral gene therapy in a goat iliac crest model for bone formation.

Treatment and reconstruction of large bone defects, delayed unions, and nonunions is challenging and has resulted in an ongoing search for novel tissue-engineered therapies. Bone morphogenetic protein-2 (BMP-2) gene therapy is a promising strategy to provide sustained production of BMP-2 locally. Alginate polymer-based nonviral gene therapy with BMP-2 plasmid DNA (pBMP-2) in constructs with multipotent mesenchymal stromal cells (MSCs) has resulted in prolonged gene expression and bone formation in vivo. To further translate this technology toward larger animal models, important issues remain to be investigated, such as the necessity of seeded cells as a target for gene therapy. For that purpose, a large animal-screening model in an orthotopic location, with fully separated chambers, was investigated. Four cylinder-shaped implants were placed in the iliac crests of ten goats. Polycaprolactone tubes around each implant allowed bone ingrowth from the underlying bone and bone marrow and ensured separation of the experimental conditions. An empty tube showed low levels of spontaneous bone ingrowth, and implantation of autologous bone indicated proper bone function with respect to remodeling and resorption. Control ceramic scaffolds were compared to scaffolds containing pBMP-2 either or not combined with seeded MSCs. Fluorochrome incorporation evaluated at 3, 6, and 9 weeks and histomorphometry at 12 weeks after implantation revealed clear differences between the groups, with pBMP-2 combined with MSCs being the most effective. The BMP-2 was demonstrated in a variety of bone-residing cells through immunohistochemistry. Further analysis indicated that multinucleated giant cells might have an important role in transgene expression. Taken together, this work introduces a large animal model for studying bone formation at multiple sites simultaneously in an orthotopic location. The model appeared robust, showed no neighboring effects, and demonstrated effectivity of combined cell and gene therapy.

Bone Morphogenetic Protein 2 Gene Delivery via Polyethylene Imine Grafted Chondroitin Sulfate Non-viral Gene Carrier

Background: Bone morphogenetic protein 2 (BMP-2) has been frequently used for bone regeneration. The purpose of this study was to design a gene vector to deliver the BMP-2 plasmid into cells. Polyethylene imine (PEI) is commonly used for gene transfer, however the high cytotoxicity often leads to decreased transfection efficiency. Solutions to this problem have been widely discussed, one of which being the grafting of a highly biocompatibility polymer such as chondroitin sulfate (CS). Methods: Different molecular weights (1,800 and 25,000 g/mol) of PEI were grafted onto CS (PEI1.8K-g-CS and PEI25K-g-CS, respectively) through the ethyldimethylaminopropyl carbodiimide/ N-hydroxysuccinimide reaction. This modification resulted in a non-viral gene carrier with improved biocompatibility as well as enhanced transfection efficiency compared to PEI alone. Cytotoxicity toward human embryonic kidney cell line (293T) was assayed. Results: Cell viability in the PEI-grafted-CS group was higher than that of the PEI group, and this was particularly evident at high concentrations. The efficiency of transfection and the amount of intracellular plasmid DNA were monitored using green fluorescent protein. The expression of BMP-2 was measured by enzyme-linked immunosorbent assay. A polymer:DNA weight ratio of 4:1 in the PEI25K-g-CS/DNA polyplexes exhibited the highest transfection efficiency. Conclusions: The low toxicity and high transfection efficiency of PEI25K-g-CS make it ideal for application in gene delivery and therapy.

Nanoparticle Delivery of the Bone Morphogenetic Protein 4 Gene to Adipose-Derived Stem Cells Promotes Articular Cartilage Repair In Vitro and In Vivo

To evaluate the effect of poly(lactic-co-glycolic acid) (PLGA) nanoparticles delivering pDC316-BMP4-EGFP plasmid into rabbit adipose-derived stem cells (ADSCs) in vitro and chondrogenesis of the bone morphogenetic protein 4 (BMP-4)--transfected ADSCs seeded onto poly(L-lactic-co-glycolic acid) (PLLGA) scaffold in a rabbit model. Cell viability and transfection efficiency of PLGA nanoparticles were measured by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) and flow cytometry. The BMP-4 and chondrogenesis markers were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Thirty rabbits (60 knees) with full-thickness cylinder articular cartilage defects (diameter, 4.5 mm; depth, 0.8 mm) on the femoral trochlea were divided into a group in which the BMP-4--transfected ADSCs were seeded onto PLLGA scaffold and implanted into the defects (group ABNP), a group with untransfected ADSCs seeded onto scaffold (group ABP), and a group with a scaffold without cells (group P). Outcomes were evaluated by histology, Rudert score, Pineda score, and scanning electronic microscopy by 2 blinded observers at weeks 6 and 12 postoperatively. Statistical analyses were performed with analysis of variance and the Kruskal-Wallis test. The statistical significance level was set at P < .05. The expression of chondrogenesis-related genes and proteins was significantly increased in BMP-4--transfected ADSCs in vitro (P < .05). The cell viability was 79.86% ± 5.04% after 24 hours. The transfection efficiency was 25.86% ± 4.27% after 72 hours. Defects in group ABNP showed the best in vivo cartilage regeneration. At week 12, the Rudert scores in group ABNP (7.00 ± 1.75) were better than those in group ABP (6.00 ± 2.00) or group P (5.00 ± 1.75) (P < .05), as were the Pineda scores (2.50 ± 3.00, 5.00 ± 2.00, and 6.00 ± 1.75, respectively; P < .001). BMP-4 plasmid can be successfully delivered into ADSCs by PLGA nanoparticles and promoted in vitro chondrogenesis. When compared with the control cells, BMP-4--transfected ADSCs seeded onto PLLGA scaffold significantly improve in vivo chondrogenesis in a rabbit articular defect model. PLGA nanoparticles and BMP-4 have potential for gene therapy in the treatment of chondral defects of the knee.

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.

Gene delivery of bone morphogenetic protein-2 plasmid DNA promotes bone formation in a large animal model

In the field of bone regeneration, BMP-2 is considered one of the most important growth factors because of its strong osteogenic activity, and is therefore extensively used in clinical practice. However, the short half-life of BMP-2 protein necessitates the use of supraphysiological doses, leading to severe side-effects. This study investigated the efficiency of bone formation at ectopic and orthotopic sites as a result of a low-cost, prolonged presence of BMP-2 in a large animal model. Constructs consisting of alginate hydrogel and BMP-2 cDNA, together acting as a non-viral gene-activated matrix, were combined with goat multipotent stromal cells (gMSCs) and implanted in spinal cassettes or, together with ceramic granules, intramuscularly in goats, both for 16 weeks. Bone formation occurred in all cell-seeded ectopic constructs, but the constructs containing both gMSCs and BMP-2 plasmid DNA showed higher collagen I and bone levels, indicating an osteogenic effect of the BMP-2 plasmid DNA. This was not seen in unseeded constructs, even though transfected, BMP-2-producing cells were detected in all constructs containing plasmid DNA. Orthotopic constructs showed mainly bone formation in the unseeded groups. Besides bone, calcified alginate was present in these groups, acting as a surface for new bone formation. In conclusion, transfection of seeded or resident cells from this DNA delivery system led to stable expression of BMP-2 during 16 weeks, and promoted osteogenic differentiation and subsequent bone formation in cell-seeded constructs at an ectopic location and in cell-free constructs at an orthotopic location in a large animal model.

162. Polymeric Delivery of a Bone Morphogenetic Protein-2 Plasmid at Rat Subcutaneous Site

162. Polymeric Delivery of a Bone Morphogenetic Protein-2 Plasmid at Rat Subcutaneous Site

Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

Bone regeneration is one of the major 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. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

BMP-2 plasmid loaded PLGA/HAp composite scaffolds for treatment of bone defects in nude mice

We studied three different types of scaffolds, encapsulating bone morphogenetic protein-2 (BMP-2) plasmid, in terms of their performances in bone regeneration in nude mice. The plasmid was loaded into fibrous matrices in three different ways: coating of naked DNA (Group A) or DNA/chitosan nanoparticles (Group B) onto scaffolds after fiber fabrication by dripping, and encapsulation of DNA/chitosan nanoparticles into scaffold by mixing them with PLGA/DCM solution before fiber fabrication (Group C). Their individual performances were examined by soft X-ray observation, histological analysis and immunostaining of bone tissue. In addition, the BMP-2 protein concentration and alkaline phosphatase (ALP) activity in serum were monitored. The results revealed that the bioactivity of BMP-2 plasmid released from all three kinds of scaffolds was well maintained; this eventually helped improve the healing of segmental defects in vivo. Interestingly, the three kinds of scaffolds released DNA or DNA nanoparticles in different modes and their performances in bone healing were diverse. These observations demonstrate that the in vivo performance of these newly developed DNA delivery devices correlates well with their in vitro release profiles.

Modifying the Proliferative State of Target Cells to Control DNA Expression and Identifying Cell Types Transfected In Vivo

Although the majority of current gene transfer techniques have focused on increasing the ability of the DNA to enter the cell, it is possible that changing the proliferative and migratory state of cells will influence the cells ability to take up and express plasmid DNA. This study was designed to test the hypothesis that growth factors (basic fibroblast growth factor (bFGF) and hepatocyte growth factor/scatter factor (HGF/SF)) used to alter the proliferative and migratory state of cells can alter plasmid DNA uptake and expression. In vitro studies indicate that enhancing cell proliferation with growth factor exposure enhances plasmid DNA uptake and expression. Furthermore, dual localized delivery of bFGF and plasmid DNA in vivo increases the expression, 3-6 times over control, as compared to plasmid delivery alone. Dual delivery of a factor promoting cell proliferation and a plasmid led to a further increase in the expression of the plasmid encoding bone morphogenetic protein-2 in a rat cranial defect by specific cell populations. The results of these studies suggest that increasing the proliferative state of target cell populations can enhance non-viral gene transfer.