Ectopic osteogenesis of mouse bone marrow stromal cells transfected with BMP 2/VEGF₁₆₅ genes in vivo

Abstract

Objective:  To evaluate the osteogenic efficacy of mouse bone marrow stromal cells (mBMSC) transfected with bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF₁₆₅) genes.

Methods:  pIRES‐BMP2‐VEGF₁₆₅ plasmid DNA was introduced into the mBMSC using a liposome‐mediated method. The expression of BMP2 and VEGF₁₆₅ genes was assessed by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemical analysis. Transfected cells were injected into the thigh muscle pouches of four nude mice. The osteoinductivity activity of the transfected cells was evaluated by radiographic and histological analysis at 4 weeks after injection.

Results:  The mRNA and proteins of both BMP2 and VEGF₁₆₅ were successfully expressed in mBMSC as confirmed by RT‐PCR and immunohistochemical analysis. Ectopically formed bone tissue was clearly observed at 4 weeks after cell injection in the thigh muscle pouches of the nude mice.

Conclusion:  mBMSC transfected with BMP2 and VEGF₁₆₅ genes can induce ectopic osteogenesis.

Introduction

It has been well documented that bone morphogenetic proteins (BMP), the largest subfamily of the structurally conserved transforming growth factor‐beta (TGF‐β) superfamily, can induce bone formation, both in vivo and in vitro ¹ . BMP2 is a potent osteoinductive factor which has strong bone‐inductive activity and is being evaluated as a bone growth inducer for orthopaedic applications ² .

Recent research shows that vascular endothelial growth factor (VEGF) is one of the best‐characterized angiogenic factors, and that it plays an important role in bone growth via the endochondral ossification pathway ³ , ⁴ . Bone tissue rehabilitation relies on angiogenesis, as do other tissues. As an important angiogenesis factor, VEGF can induce angiogenesis and affect osteogenesis. In this study, a bicistronic vector encoding human BMP2 and VEGF₁₆₅, the pIRES‐BMP2‐VEGF₁₆₅ plasmid DNA, was constructed and then transfected into mouse bone marrow stromal cells (mBMSC) to investigate co‐expression of the two genes. The osteogenic potential of transfected cells was then evaluated by injecting them into the thigh muscle pouches of nude mice.

Material and methods

Cell culture and transfection procedure

mBMSC were used in the experiments. Cells were inoculated into a 100 ml culture bottle and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Eggenstein, Germany) supplemented with 10% fetal bovine serum, 0.1 mg/ml penicillin and streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. When they had reached greater than 70%–80% confluency, the cells were transfected with the pIRES‐BMP2‐VEGF₁₆₅ vector ⁵ (constructed by our laboratory) by lipotransfection according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Cells transfected with empty pIRES vector were used as controls.

Detection of co‐expression of BMP2 and VEGF genes

At 48 h after transfection, mRNA expression of VEGF and BMP2 in the transfected cells was analyzed by RT‐PCR. Total RNA was isolated from the cell pellets using Trizol (BioAsia, Shanghai, China). First strand cDNA synthesis was performed using a First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). PCR was carried out using sense and antisense primers of BMP2 and VEGF ⁵ . The climbing glasses of transfected cells were gathered. The cells were fixed in ice‐cold methanol and identified with anti‐BMP2 antibody (Wuhan Boster, Wuhan, China) and anti‐VEGF antibody (Wuhan Boster) by using the immunohistochemical SABC (strept–avidin–biotin–peroxidase complex) method and imaging analysis (Axiovert 40 CFL, Carl Zeiss, Jena, Germany), respectively.

Ectopically formed bone tissue in thigh muscle pouches of nude mice

When the cell monolayer had reached about 80% confluency, the cells were harvested and washed twice with complete medium, then resuspended in PBS and adjusted to 1.0 × 10⁷/0.2 ml. Four male nude mice of body weight 50 ± 5 g (provided by the Experiment Animal Center of Tongji Medical College) were used in the experiments. Under anesthesia by intraperitoneal injection the mice were fixed in a prone position and both thighs depilated. Then, cells transfected with the pIRES‐BMP2‐VEGF₁₆₅ vector were injected into the muscle pouches of the left thigh (experimental side) and cells transfected with empty pIRES vector into the right thigh (control side). All the mice were sacrificed 4 weeks after injection and examined in the anteroposterior position by DR (Digital Radiography) (GE Revolution [General Electric]× DR, 38 kV, 100 mA, 0.12 s and 60 cm) to observe ectopic bone formation. In addition, the specimens were sequentially fixed by 10% formalin, decalcified by 10% EDTA (ethylenediaminetetraacetic acid), embedded in paraffin, sliced up, and stained with haematoxylin‐eosin and studied microscopically (Axiovert 40 CFL, Carl Zeiss).

Results

Expression of BMP2 and VEGF₁₆₅ mRNA in vitro

RT‐PCR generated 1.2 kbp and 592 bp products in pIRES‐BMP2‐VEGF₁₆₅ transfected samples. Cells transfected with empty pIRES vector produced no RT‐PCR products (Fig. 1). The cytoplasm of cells transfected with pIRES‐BMP2‐VEGF₁₆₅ vector showed brown granules when stained by immunohistochemistry (Fig. 2). However, the control cells were negative. The results suggest that mBMSC transfected with pIRES‐BMP2‐VEGF₁₆₅ vector can co‐express mRNA and the proteins of BMP2 and VEGF₁₆₅.

Figure 1.

Co‐expression of BMP2 and VEGF₁₆₅ in mBMSC detected by RT‐PCR. Lane 1, Marker; lane 2, BMP2 amplification of mBMSC/pIRES‐BMP2‐VEGF₁₆₅; lane 3, BMP2 amplification of mBMSC/pIRES; lane 4, VEGF₁₆₅ amplification of mBMSC / pIRES‐BMP2‐VEGF₁₆₅; lane 5, VEGF₁₆₅ amplification of mBMSC / pIRES.

Figure 2.

Immunohistochemistry analyses of (A) BMP2 and (B) VEGF proteins expressed in transfected mBMSC. The cytoplasm of cells transfected with pIRES‐BMP2‐VEGF₁₆₅ vector was stained with brown granules ×200.

Ectopically formed bone tissue in thigh muscle pouches of nude mice

Osseous images were found in the experimental areas by X‐ray examination at 4 weeks after injection (Fig. 3). On the contrary, no osseous images were observed on the control sides. Moreover, it was found that the neogenetic cartilage integrated mutually to form matured lamellar bone and bone trabeculae (4, 5), while no bone formation occurred on the control side.

Figure 3.

X‐ray image of ectopic bone formation (white arrow) in the experimental side of a mouse at 4 weeks after injection.

Figure 4.

Cartilage was observed in the experimental side at 4 weeks after injection. HE stain, ×200.

Figure 5.

Trabecular‐like new bone was observed in the experimental side at 4 weeks after injection HE stain, ×100.

Discussion

BMP are produced primarily in osteoinductive extracts of bone matrix. Molecular cloning of BMP demonstrates that they are a family of factors related to differentiation; each of them is capable of inducing the formation of new bone tissue. BMP2 is a potent osteoinductive factor which can stimulate differentiation of mesenchymal stem cells toward an osteoblastic lineage, thereby increasing the number of osteoblasts ² . Recombinant human BMP2 (rhBMP2) is manufactured by a recombinant DNA biotechnological process and has been evaluated preclinically and clinically for use in the treatment of bone defects ⁶ , ⁷ .

Angiogenesis is postulated to be a crucial step for new bone formation ⁸ . Studies over the years have confirmed that VEGF is one of most critical secretory growth factors promoting angiogenesis. It can promote vascular endothelial cell division, multiplication and migration; modulate extracellular matrix ⁹ ; and increase microvascular permeability as well. Thus VEGF has been the first choice in using gene therapy technologies to treat bone defects ¹⁰ . It has been found that it can promote the formation of new vessels during endochondral ossification and bone healing ⁴ , ¹¹ . In addition, the recombinant adenovirus vector has been constructed successfully ¹² and research shows that adenovirus mediated VEGF gene can promote osteogenesis of human marrow mesenchymal stem cells ¹³ . Blocking VEGF leads to a decrease in trabecular bone formation at the growth plate, secondary to suppression of blood vessel invasion and impairment of cartilage resorption ⁴ . Besides, VEGF can up‐regulate BMP2 mRNA and protein concentrations in endothelial cells ¹¹ .

Moreover, it has been found that BMP2 can induce angiogenic activity in bone formation by enhancing VEGF expression ¹⁰ . Bluteau et al. found that expression of VEGF was up‐regulated under chondrogenic stimulation ¹⁴ . Otherwise, it was found that the VEGF and BMP2 were significantly greater in patients with bony union compared with those with bone disunion ¹⁵ . These studies suggest that the co‐expression of BMP2 and VEGF may have a synergistic effect in bone formation and healing.

In the present study, osseous images were found in the experimental areas by X‐ray examination at 4 weeks after injection, no osseous images were observed on the control sides. This suggests successful ectopic osteogenesis in vivo. Kakudo et al. found that rhBMP2 could induce differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and that differentiated cells could express VEGF, which creates an advantageous environment for vascularization in bony tissue ¹⁶ . These findings all suggest that VEGF and BMP2 can promote ectopic osteogenesis.

The pIRES vector contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV). This sequence permits two genes of interest (in this study, BMP2 and VEGF₁₆₅) to be translated separately from a single bicistronic mRNA. Additional advantages of bicistronic vectors compared with two monocistronic vectors are the lower cost of preparation and the smaller amount of endotoxins produced during vector preparation. We constructed pIRES‐BMP2‐VEGF₁₆₅ vector and transfected the vector into mBMSC. Radiographic and histological observations suggest that cells transfected with pIRES‐BMP2‐VEGF₁₆₅ can induce ectopic bone formation in thigh muscle pouches of nude mice.