Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1
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- Hou, H., Zhang, X., Tang, T. et al. Biotechnol Lett (2009) 31: 1183. doi:10.1007/s10529-009-0007-4
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To explore the potential of combined delivery of osteogenic and angiogenic factors to bone marrow stromal cells (BMSCs) for repair of critical-size bone defects, we followed the formation of bone and vessels in tissue-engineered constructs in nude mice and rabbit bone defects upon introducing different combinations of BMP-2, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) to BMSCs with adenoviral vectors. Better osteogenesis and angiogenesis were found in co-delivery group of BMP-2, VEGF and angiopoietin-1 than any other combination of these factors in both animal models, indicating combined gene delivery of angiopoietin-1 and VEGF165 into a tissue-engineered construct produces an additive effect on BMP-2-induced osteogenesis.
KeywordsAngiopoietin-1Bone morphogenic protein 2Bone tissue engineeringCritical-size bone defectVascular endothelial growth factor
Tissue-engineered bone is playing an increasingly more important role in critical-size bone defect repair. Bone healing can be distilled into two major synergistic and interactive processes: osteogenesis and angiogenesis. Thus, integration of these involved aspects may be more effective in achieving better bone defect repair (Murphy and Mooney 1999). Bone morphogenetic proteins (BMPs) and vascular endothelial growth factors (VEGFs) are widely used as osteogenic and angiogenic factors (Levy et al. 1998; Gerber et al. 1999). However, VEGF induced new vessels are immature and leaky due to increased permeability (Schwartz et al. 2000). On-the-other-hand, angiopoietin-1 (Ang-1) is required in the latter stage of angiogenesis for the stabilization and maturation of blood vessels and can counteract VEGF-induced inflammation in endothelial cells (Thurston et al. 1999). Therefore, the combined application of Ang-1 and VEGF may be more effective than application of either factor alone for enhancing genesis of functional vessels in tissue engineering (Chae et al. 2000). Despite this, until now there have been no reported studies on the combined delivery of BMP, VEGF, and angiopoietin in bone-tissue engineering. In the current study we investigated the potential, feasibility and possible risks of introducing these three factors to bone marrow stromal cells (BMSCs) with adenoviral vectors for repair of critical-size bone defects.
Materials and methods
Adenoviral vectors adopted
Four adenoviral vectors were used in this study, all of which were plasmid-based systems with different gene inserts. The vectors were Ad-BMP-2, Ad-VEGF165, Ad-Null, and a bicistronic vector carrying hVEGF165 and Ang-1 genes (Ad-Bic) separated by an internal ribosome entry site region (kindly provided by Ruowen Ge). The vectors were E1-deficient recombinant adenovirus propagated in 293 cells and purified by cesium chloride gradient ultracentrifugation.
Preparation of the cells and scaffold constructs
Bone marrow was harvested from rabbit under general anaesthesia. BMSCs were then isolated and amplified to the third passage when transfection was carried out. Twenty-four hours later, the equivalent number of cells were trypsinized and seeded in a hypobaric environment onto the β-TCP scaffolds, which had a volume porosity of 70%. We used cubic scaffolds with an edge length of 5 mm in nude mice and 4 mm diameter, 15 mm long cylindric scaffolds in rabbits. The cells and scaffold constructs were subsequently cultured for 6 days in vitro before implantation. RT-PCR, western blotting and enzyme-linked immunosorbent assay (ELISA) of target genes were performed after transfection.
Reverse transcription polymerase chain reaction (RT-PCR) assay
Primer sequences for RT-PCR
Sense primer (5′–3′)
Antisense primer (5′–3′)
Western blot analysis
Bone marrow stromal cells were grown in 6-well tissue culture plates and transfected with vectors as previously described. Cell samples from each group were obtained at 7 days post-transfection. Western blotting was then performed according to conventional protocol. Antibodies adopted were: goat anti-hBMP2 monoclonal antibody (R&D system), mouse anti-hVEGF monoclonal antibody (R&D system) and rabbit anti-hAngiopoietin-1 monoclonal antibody (Chemicon).
ELISA for hBMP2 and hVEGF165 expression
The detection of hBMP2 and hVEGF165 secreted from a transfected BMSCs was done with a hBMP2 ELISA kit (R&D system) and hVEGF165 ELISA kits (Chemicon). The transfected BMSCs were grown in 6-well tissue culture plates at 2 × 105 cells/well. Ad-LacZ transfected and non-transfected BMSCs were used as control. The supernatant from each well was collected at 7, 14, 21 and 28 days and the samples were kept frozen at −20°C until they were used for assay. The assay was performed according to the supplier’s instructions.
In vivo osteogenesis and angiogenesis
Co-administration of Ad-BMP-2 and Ad-Bic: During seeding of the transfected cells to the scaffolds, the suspensions of Ad-BMP-2-transfected cells and of Adv-Bic-transfected cells were combined in a ratio of 4:1.
Co-administration of Ad-BMP-2 and Ad-VEGF165: During seeding of the transfected cells to the scaffolds, the suspensions of Ad-BMP-2-transfected cells and of Adv-VEGF165-transfected cells were combined in a ratio of 4:1.
Administration of Ad-BMP-2 alone: All seeded cells were Ad-BMP-2-transfected.
Administration of Ad-Bic alone: All seeded cells were Ad-Bic-transfected.
Administration of Ad-VEGF165 alone: All seeded cells were Ad-VEGF165-transfected.
Administration of Ad-Null alone: All seeded cells were Ad-Null-transfected.
Administration of rat bone marrow stromal cells (rBMSCs) alone: Only non-transfected cells were seeded.
At 4, 8, and 12 weeks after surgery, four implants were retrieved in each group and fixed in 10% (v/v) neutral formalin for histologic analysis [hematoxylin and eosin (H&E) staining] and immunohistochemical analysis.
Ex vivo critical-size bone defect repair
Co-administration of Ad-BMP-2 and Ad-Bic
Administration of Ad-BMP-2 alone
Administration of rBMSCs alone
No implantation of construct into defects
At 2, 6, and 12 weeks after surgery, laterolateral X-rays of the forearms were obtained for all rabbits while they were under general anesthesia. After the animals were sacrificed, the forearms were harvested for biomechanical tests (maximal loading) and histologic analysis of in vivo bone formation.
Histology and histomorphometry
All samples were processed for histologic examination. Tissue sections of 6 μm thick were obtained for H&E staining. The bone-tissue area for each section was determined by dividing the total number of bone pixels by the number of pixels for the total implant in nude mice or the trabecula area percentage in rabbits using Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). Three sections per side were analyzed in a blinded fashion, and the mean area of these three sections was used for statistical analysis.
To determine the extent of blood vessel in-growth, 4-, 8-, and 12-week tissue sections from nude mice were immunostained for CD31. Blood vessels, indicated by CD31 staining, were counted manually at 100× magnification. Only circular CD31 staining was interpreted as indicating a blood vessel.
Statistical analysis was performed using SPSS software (SPSS, Chicago, IL, USA). Statistically significant differences in histomorphometric analysis were determined using one-way ANOVA.
Results and discussion
To our knowledge, this is the first report exploring the orchestration of BMP, VEGF and angiopoietin in tissue engineering. A new bicistronic vector carrying the genes of VEGF165 and Ang-1 was developed and used in this study. The ratio (4:1) of cells when they were combined after transfection was chosen on the basis of other reports (Peng et al. 2002) and our earlier work (data not shown). We also used two animal models rather than one because they are not interchangeable for our purposes. Angiogenesis and osteogenesis cannot be properly studied in New Zealand rabbits because of the genotypic diversity among individual rabbits, and it is difficult to make a critical-size bone defect in nude mice.
Compared with amplification autologous endothelial cells or in situ administration of angiogenic factors, gene transduction to autologous stromal cells is simpler and more cost-effective for delivery of angiogenic factors in vivo. Angiogenic factors are crucial for angiogenesis especially in the early period after the engineered construct is implanted, and several reports have indicated that prolonged administration of VEGF, no matter what vector is used for transduction, will lead to an increased risk of formation of endothelial cell-derived vascular tumors (Lee et al. 2000; Masaki et al. 2002). The transient release of angiogenic factors caused by adenovirus transfection (erased within 2–4 weeks) is therefore potentially safer than the prolonged release by retrovirus or lentivirus. It is for this reason that we used replication-deficient adenoviral vectors in this study.
We note that there are some concerns about the safety of adenoviral vectors. We carried out the gene therapy method ex vivo, which is simpler and more controllable than in vivo because viral vector particles would hardly be conveyed into the body and tests for safety could be run before seeding or implantation if necessary. To date we have detected no evidence of oncogenesis in our studies.
In conclusion, this study confirmed the importance of combining multiple factors in bone regeneration. Combined factors can also be used in engineering a variety of other tissue types because regeneration of all tissues is dependent on the interplay of various growth factors and cell types. Our results show that combined gene delivery of Angiopoietin-1 and VEGF165 into a tissue-engineered construct produces an additive effect on BMP-2-induced osteogenesis through enhanced angiogenesis compared with either therapy alone, even better than that produced by the coadministration of VEGF and BMP-2. To our knowledge, this is the first study showing the efficacy of treatment combining Ang-1, VEGF, and BMP-2 in the field of bone-tissue engineering. We expect that combined growth-factor gene therapy will become an accepted curative modality in critical-size bone defects.