Original Article

Gene Therapy (2008) 15, 257–266; doi:10.1038/sj.gt.3303070; published online 22 November 2007

Ultrasound-based nonviral gene delivery induces bone formation in vivo

D Sheyn1, N Kimelman-Bleich1, G Pelled1, Y Zilberman1, D Gazit1,2 and Z Gazit1

  1. 1Skeletal Biotech Laboratory, Hebrew University of Jerusalem, Jerusalem, Israel
  2. 2Department of Surgery, International Stem Cell Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA

Correspondence: Dr Z Gazit, Skeletal Biotech Laboratory, Hebrew University of Jerusalem, Hadassah medical campus, Jerusalem 91120, Israel. E-mail: zulmag@ekmd.huji.ac.il

Received 3 April 2007; Revised 16 October 2007; Accepted 16 October 2007; Published online 22 November 2007.

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Abstract

Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.

Keywords:

direct gene delivery, sonoporation, bone morphogenetic proteins, bone formation

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Introduction

Nonviral gene delivery approaches hold great promise for the development of efficient and safe gene therapy for clinical orthopedic applications. Nonviral approaches are considered relatively safe. Unlike their viral counterparts, very little inflammation or disease is associated with nonviral vectors.1 Viral vectors that integrate into the genome carry the risks of oncogenesis and mutagenesis. These risks are significantly reduced in nonviral gene delivery, which results in episomal insertion. Although virus-mediated transfer generally produces higher transfection efficiencies and a longer period of transgene expression,2 the transient expression of the transgene associated with nonviral methods represents a potential advantage when only a short expression period is desired. Common transfection methods, mainly used for in vitro gene delivery, have been shown to be up to 70% effective in well-established cell lines.3 Electrical field-mediated gene transfer, better known as DNA electrotransfer, is one of the most efficient methods for in vivo direct gene transfer.4, 5 The ultrasound-enhanced gene transfer utilized in this pilot study can be related to the same group of physical gene delivery as electrotransfer.

Sonoporation (transient ultrasound-induced increase in cell membrane permeability) has been shown in several studies to enhance gene transfer both in vitro6, 7, 8 and in vivo.9, 10, 11, 12, 13, 14, 15, 16, 17, 18 The increase in cell membrane permeability is probably caused by oscillation of the membrane in response to acoustic cavitation, which results from a nonthermal interaction between a propagating pressure wave induced by ultrasound and a gas included in aqueous medium.19 This oscillation may be amplified by the presence of microbubbles in the medium. The application of ultrasound causes vibration and collapse of the microbubbles. Destruction of the microbubbles causes high-energy microstreams, or microjets, that cause shear stress on the membrane of a cell and increases its permeability. That is one of the proposed mechanisms by which microbubbles can lower the threshold for cavitation.20 In this study we used a suspension of perfluoropropane-filled albumin microspheres (Optison, Amersham Health, Princeton, NJ, USA), which is approved for use in echocardiography by the US Food and Drug Administration as a contrast agent for diagnostic ultrasonography. In numerous recent studies, Optison has been found to noticeably enhance the efficiency of ultrasound-based gene delivery into skeletal muscle,17, 21, 22 tumors,9, 13 and the liver.15 Sonoporation has been shown to promote drug delivery18 and increase viral vector efficiency in vivo,10, 11, 13, 14 in addition to its major contribution to gene therapy: having the capability to transfer naked plasmid DNA locally to target cells. In previous studies, sonoporation-based gene delivery was mainly applied in the setting of cardiovascular tissues10, 11, 12, 13, 17, 18, 23 and cancer.9, 13

DNA electrotransfer or electroporation is the use of short high-voltage pulses to overcome the barrier of the cell membrane. The electrical pulse destabilizes the cell membrane for a short time, and then small molecules can enter the cell using simple diffusion. Bigger molecules (such as DNA or RNA) are being ‘pushed’ by the electrical current, and so can enter the cell. Electroporation is now in use for delivery of ions, drugs, dyes, tracers, antibodies and RNA and DNA into cells.24 The exact entrance mechanism of electroporated DNA into the nucleus is not clear, but it seems that the DNA migrates electrophoretically through the membrane and then diffuses toward the nucleus.24, 25

In the present study, sonoporation was used to transfer osteogenic gene and induce de novo formation of bone tissue in vivo. It was compared to electroporation as the gold standard of nonviral direct gene delivery method.

The increasing disparity between the demand and availability of human donor organs for the replacement of dysfunctional organs has prompted an intensive search for alternative sources of organs. In the field of orthopedics, massive bone defects caused by trauma, cancer, primary joint replacement, revision joint surgery and congenital anomalies continue to pose a great challenge to reconstructive surgery. The present solutions for these skeletal disorders include artificial implants or allograft and autograft transplants, which often fail in the long term, are not always available, are costly and are associated with the risk of immune system rejection. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily and are known for their ability to induce bone formation; they frequently are the focus of therapeutic applications for bone repair and regeneration.26, 27 It was recently shown that the BMP-2, -6, -7 and -9 genes are the most potent inducers of osteogenic differentiation among the BMP family.28, 29, 30 In this study we used the BMP-9 gene, since our previous observations indicated it could lead to more significant osteogenesis than the human BMP-2 plasmid.28

We hypothesized that, in optimized acoustic conditions, the direct in vivo sonoporation of naked DNA encoding an osteogenic gene recombinant human BMP-9 (rhBMP-9) would induce bone formation. As previously shown, viral delivery of the rhBMP-9 gene has therapeutic potential in bone regeneration.31 However, viral gene delivery carries risks and nonviral gene delivery techniques are considered to have greater potential clinically because they are safer and more available. Significant advances have been made by sonoporation in the field of gene delivery during the last decade.32 However, no report has been made of in vivo tissue de novo formation using this delivery method. This is the first report that demonstrates that the sonoporation- and electroporation-mediated delivery of plasmid DNA encoding the osteogenic gene rhBMP-9 induces bone tissue formation, in vivo. Our data demonstrate, though, that sonoporation was significantly less efficient in bone formation than electroporation.

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Results

Sonoporation of luciferase reporter gene in vivo

Delivery of the reporter luciferase (Luc) gene and quantitative in vivo imaging were used to evaluate the efficiency of transfection and to calibrate the sonoporation parameters. Based on preliminary experiments (data not shown) and previous publications,16, 21, 33 an initial protocol was established. The initial acoustic parameters were based on those outlined in previous studies in which similar sonoporation systems had been used.11, 16, 22 The results of in vivo imaging indicated that the expression of the transgene was higher when the power input of the sonoporation device was of high intensity (5Wcm−2). We tested two frequencies enabled by the Sonitron2000 device: 1 and 3MHz. The most conventional frequency used in ultrasound-based gene delivery, 1Mhz,11 was more efficient in the induction of Luc expression than 3MHz. The effect of exposure duration was tested as well and, as expected, transgene expression increased with a longer time of exposure. The injected plasmid amount and the Optison microbubbles concentration were also optimized. The results indicated that the 5% concentration of Optison was more effective than the 10 or 25% concentration, corresponding with previous findings.16 The increase in plasmid DNA amounts did not further improve the efficiency of sonoporation (data not shown).

Based on preliminary findings by us and others,16, 21, 33 the following experimental protocol was established: the ultrasound frequency was set at 1MHz and the intensity of the power output for high transfection efficiency at 5Wcm−2. The exposure duration we used was 10min, and the duty cycle (ratio of run time to total cycle time) was set at 50%. The plasmid DNA solution that we administered contained 50μg DNA and 5% Optison. A time-dependent trend line of Luc expression induced by sonoporation was constructed (Figure 1). The expression profile indicated that sonoporated gene translation has reached detectable level on day 4 post sonoporation. The peak expression was detected on day 7 (Luc expression doubled between days 4 and 7 post sonoporation), and then becomes undetectable by day 19. This trend line was found similar to previously reported transient Luc expression using adenoviral vector.34 Assuming that the expression profile of the sonoporated human rhBMP-9 gene follows the same expression profile as the Luc gene, transient expression of rhBMP-9 will induce ectopic bone formation in vivo in the sonoporated area.28, 35

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Reporter gene Luc expression profile in mice injected with Luc with or without sonoporation, as quantified by in vivo bioluminescence. (a) Representative CCCD images showing Luc expression in the left hindlimb, which received an injection of Luc followed by sonoporation but no expression in the right hindlimb, which received an injection of Luc but no sonoporation. (b) Quantitative analysis of the bioluminescent signal. The results are expressed in relative light units (RLU). Bars indicate s.e., n=5.

Full figure and legend (90K)

Electroporation of Luc reporter gene in vivo

Using the bioluminescence imaging system, we noticed an expression pattern consistent with nonviral gene delivery systems. The cooled charge-coupled device (CCCD) signal depicted in representative images in Figure 2a were quantitatively analyzed and gene expression profile Luc expression peaked on day 6 (which was the first time point checked), and was decreased in later time points (Figure 2). Compairing these results to those achieved by sonoporation, we can conclude that the reporter Luc gene was about one log more efficiently delivered by electroporation, than by means of sonoporation.

Figure 2.
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Reporter gene Luc expression profile in mice injected with Luc or with non-Luc plasmid (rhBMP-9) after electroporation, as quantified by in vivo bioluminescence. (a) Representative CCCD images showing Luc expression in the left hindlimb, which received an injection of Luc followed by electroporation but no expression in the right hindlimb, which received an injection of rhBMP-9. (b) Quantitative analysis of the bioluminescent signal. The results are expressed in relative light units (RLU). Bars indicate s.e., n=5.

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Luc expression: immunohistochemical analysis

To demonstrate Luc expression in a sonoporated skeletal muscle, thigh tissue was embedded in paraffin and stained immunohistochemically to detect Luc expression. The red stain, which indicates positive Luc expression, revealed widespread expression of Luc in the sonoporated skeletal muscle on day 10 after sonoporation (Figure 3a). No staining was seen in control tissues that had received injections of the Luc gene without application of sonoporation (Figure 3b).

Figure 3.
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In situ Luc expression induced by sonoporation of Luc in vivo and detected by immunohistochemical staining. (a) Thigh muscle of a mouse subjected to sonoporation after an injection of Luc DNA. (b) Thigh muscle of a mouse given an injection of Luc DNA without application of sonoporation. Both tissue samples were harvested 10 days after the initiation of sonoporation.

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Osteogenic gene expression in vivo induced by sonoporation

We applied a direct gene delivery paradigm to deliver the rhBMP-9 gene to an ectopic thigh muscle site via sonoporation. Our experimental model included transgenic mice (Oc-Luc), which harbor the Luc gene under the control of the osteogenic tissue-specific promoter, osteocalcin, as described elsewhere,36 and use of a quantitative, noninvasive CCCD camera, which is used as a real-time monitoring system. In this model, osteogenic activity in the transgenic mice is reflected in Luc expression, which is activated by the osteocalcin promoter and is detected by the CCCD as an emitted light following an injection of luciferin.36 In these experiments, we used the contralateral thigh muscles of treated mice as control tissue; muscles of the contralateral thigh were injected with empty plasmid pcDNA3 vector in a phosphate-buffered saline (PBS) and microbubbles solution, and the same sonoporation protocol was applied.

In mice whose thigh muscles were injected with the osteogenic inducer gene rhBMP-9 followed by sonoporation, the expression of osteocalcin increased during the first 2 weeks and peaked on day 10, which is the expected profile in the bone formation process.36 Indeed, as depicted in Figures 4a and b, osteocalcin-triggered Luc expression increased and peaked on day 10 post sonoporation, after which it declined gradually and was no longer detectable by day 24 post sonoporation. The expression of osteocalcin in the mouse pelvis on day 17 (Figure 4a) is probably an artifact originated in the fact that it is transgenic mice that react in Luc expression to every bone formation or remodeling processes. In the contralateral thigh muscles, which were injected with empty pcDNA3 plasmid vector (the same plasmid backbone as rhBMP-9 plasmid used) and sonoporated using the same conditions serving as control group, low expression of Luc was observed. The osteocalcin-induced Luc expression in the contralateral thigh muscles was detected during the first days after sonoporation and then declined. We believe that the short osteocalcin expression was caused by minor tissue injury. Contrary to Luc expression in wild-type C3H mice, which originated in the sonoporated DNA, as described in the ‘Electroporation of Luc reporter gene in vivo’ section (Figure 1), in this instance osteocalcin expression originated in the transgenic mice genome and was triggered by the sonoporated osteogenic gene. The values presented in Figure 3 were normalized to constitutive tail expression of Luc, as previously described,36 so that we could compare different mice that express the transgene with different intensities.

Figure 4.
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Bioluminescence induced by osteocalcin expression in transgenic Oc-luc mice as a result of sonoporation of either plasmid recombinant human bone morphogenetic protein-9 (rhBMP-9) or the control empty plasmid vector pcDNA3. (a) Representative CCCD images of mice injected with the empty vector in the left thigh and rhBMP-9 in the right thigh, and subjected to sonoporation. (b) Quantitative analysis of the bioluminescent signal. Bars indicate s.e., n=5.

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Ectopic bone formation induced by sonoporation and electroporation of rhBMP-9

The osteogenic process resulted in ectopic bone formation in the thigh muscles of six mice out of eight sonoporated mice and in five out five electroporated mice. This was validated by performing qualitative and quantitative micro-computerized tomography (micro-CT) scanning (Figure 5a). The quantitative analysis of bone tissue demonstrated that the volume of newly formed bone after sonoporation reached 0.9±0.17mm3 (mean±s.e., n=6; Figure 5c). The ectopic bone formation after electroporation of the same amount of rhBMP-9 resulted in bone volume of 3.12±0.8mm3 (mean±s.e., n=5; Figure 5c). The bone volume density of ectopic bone formation generated using sonoporation and electroporation was calculated by dividing total tissue volume to bone volume (BV) and was found to be 0.7±0.09 (mean±s.e., n=6) and 0.27±0.02 (mean±s.e., n=5), respectively (Figure 5d). The bone mineral density was found to be 793.51±68.44mg HA per cm3 (HA, hydroxyapatite; mean±s.e., n=6) in sonoporated muscles and 604.47±14.28mg HA per cm3 (mean±s.e., n=6) in electroporated muscles (Figure 5d). Entire sonoporated hindlimbs were dissected and subjected to a histological analysis. Standard hematoxylin and eosin (H&E) and bone matrix-specific (Masson trichrome, MTC) staining of these tissues are shown in Figure 6. Morphology typical of bone marrow was found inside the bone formation, resembling to other approaches inducing ectopic bone formation.37, 38 Micro-CT images and histological slides clearly demonstrated osteogenic tissue formation in mice subjected to sonoporation with rhBMP-9 (Figures 5 and 6). There was no indication of osteogenic tissue formation in the control hindlimbs (injected with empty plasmid vector, pcDNA3 and sonoporated or electroporated under the same conditions used in the experimental group) in the limbs of all mice, as was already indicated by the bioluminescence imaging. As a reference to the gene delivery method we used electroporation of the same plasmid DNA encoding to rhBMP-9, using the same DNA amounts. The electroporation in vivo is a very well-known method and highly efficient. The ectopic bone mass formed in the electroporated thigh muscle is evident in Figure 5b and its volume is significantly higher than the bone that was generated by means of sonoporation (Figure 5c). However, bone volume density of the newly formed tissue generated by sonoporation was found significantly higher than by the electroporation method. The bone mineral density indicates the maturation state of the bone tissue. Both types of bone tissue originated in nonviral gene delivery of rhBMP-9 gene and formed during the same period (5 weeks), therefore the bone mineral density was found not significantly different (Figure 5e).

Figure 5.
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Ectopic bone formation induced by sonoporation and electroporation of rhBMP-9 in vivo. Ectopic bone formation was detected and quantified by high-resolution micro-CT. (a and b, respectively). Three-dimensional image of representative ectopic bone formation in vivo induced by sonoporation of plasmid DNA encoding rhBMP-9. Enlarged two-dimensional image of representative ectopic bone formation is depicted on the left side of panel a. (ce) Quantitative analysis of ectopic bone formation: bone volume (BV) (c), bone volume density (BVD) (d), and bone mineral density (BMD) (e). Bars indicate s.e., n=9.

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Figure 6.
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Ectopic bone formation induced by sonoporation of rhBMP-9 in vivo. Histological analysis was carried out 6 weeks after sonoporation. (a and b) H & E staining. (c and d) Masson trichrome staining. BF, bone formation; BM, bone marrow; M, skeletal muscle; yellow arrow indicates active osteocytes.

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Discussion

Nonviral gene delivery systems offer a safe and versatile alternative to their viral counterparts. However, a major limiting factor for efficient nonviral gene therapy remains the lack of a suitable and efficient vector for gene delivery. In this study we propose a direct, noninvasive, nonviral, safe and efficient delivery system, which is based on therapeutic ultrasound. As we hypothesized, naked plasmid DNA encoding for rhBMP-9 that is delivered by sonoporation into the skeletal muscle induced the osteogenic process and resulted in bone formation in vivo. These results open a new avenue in osteogenic tissue engineering and orthopedic gene therapy. However, it is important to note that sonoporation-mediated bone formation was less efficient than electroporation-driven one. Further research and development of the gene delivery system is needed in order to significantly increase the efficiency of the ultrasound-based transfection. Direct gene delivery involving a safe and noninvasive injectable technique has enormous clinical significance because it enables incremental and controlled tissue formation using repeated treatments.

This study is the first to use sonoporation in vivo for skeletal tissue formation de novo in combination with noninvasive quantitative imaging of the osteogenic process. The sonoporation technique has previously been applied to skeletal muscle, mostly to deliver reporter genes.14, 17, 22 Comparing the proposed gene delivery technique with other direct gene delivery systems, one can conclude that chemical gene delivery agents possess rather low efficiency in vivo, and the most efficient physical gene delivery system described to date is electroporation.39 Electroporation is very well known and widely studied gene delivery method. Our comparison between sonoporation and electroporation methods showed that electroporation is significantly more efficient and potent method in bone tissue formation de novo. Moreover, the sonoporation technique should be further developed and studied in order to reach the efficiency of electroporation. However, application of electroporation is clinically inferior because tissue can be damaged.40, 41 Sonoporation is considered as a safe technique. It can lead to overheating of tissue, which may in turn cause tissue damage, but the overheating can be avoided without decreasing the efficiency of the transfection, however, by lowering the ultrasound duty cycle to 50% or lower and extending the exposure duration. A low duty cycle previously was shown to increase cell survival after sonoporation.33

An essential tool in tissue engineering in vivo lies in imaging systems, which have been used to monitor gene expression and tissue formation. In this study, marker gene expression was monitored noninvasively by using an in vivo bioluminescence system. Although Luc gene delivery was carried out by sonoporation and the expected peak expression was in the first days, and because the nonviral delivery occurs during the sonoporation and the likelihood uptake of the plasmid DNA later on is rather low, our results showed that the peak expression was detected on day 7. This was probably due to the protein accumulation effect in the nondividing sonoporated myocytes that occurred in the first days till the Luc reached the detectable level on day 3 and the peak expression on day 7. One of the most notable advantages of the present study over previous investigations of sonoporation is the quantitative in vivo analysis of gene expression and protein translation in a longitudinal profile. This feature enables us to study bone formation and gene expression in the same animal over time, generating a more reliable insight into those processes.

The exact mechanism of gene delivery by sonoporation is not clearly understood. Recent reports have revealed that a type of ultrasonography contrast agent, known as a microbubble, generates cavitation when exposed to ultrasound. This cavitation results in the bursting of microbubbles, leading to distribution of material over a specific area of interest. Sonoporation and cavitation are thought to produce synergistic effects, yielding increased transfection efficiency.7, 17

In comparison with other tissue-engineering approaches such as cell-based therapy, direct gene delivery is less complicated, less expensive and less time consuming. We believe that therapeutic agents (nucleic acids and contrast agents) have the potential to be developed into products easily stored on the shelf. We also believe that the procedure can be repeated because it is noninvasive, no tissue damage is observed in the histological analysis and no immune response to naked nucleic acids is expected when compared with methods involving the viral vectors currently in use in direct tissue engineering. Direct gene delivery usually generates less bone than the cell-based therapy that was also developed in our laboratory,28 however, its advantage lies in the simplicity and cost effectiveness of the therapeutic approach. We do not have the tools to indicate what cell types expressed the delivered DNA or what cells formed the bone tissue. Nevertheless, we can speculate that the gene is delivered to myocytes, muscle progenitors and satellite cells, and is expressed by them. We believe that the rhBMP-9 protein recruits both bone marrow-derived osteoprogenitor cells circulating in the blood stream and skeletal muscle-derived osteoprogenitor cells within the muscle, which have been shown to respond to secreted BMP-2 in a similar model to produce intramuscular bone.42

Finally, we have shown that rhBMP-9 gene delivery using sonoporation is sufficient to elicit bone formation in an ectopic site. However, its efficiency is still significantly inferior comparing to the electroporation-mediated gene delivery. This is the first pilot study utilizing this technique in tissue formation de novo approach. Compared with the stem cell-mediated approach, the sonoporation-based system offers direct nonviral gene delivery, which is easier to administer, does not require tissue culture, saves time and money. This proposed sonoporation-based bone-tissue engineering approach should be further developed to increase gene delivery efficiency, and would be applied to bone fracture repair models and other tissue regeneration models. Although the candidate cell population in nonunion bone defect site differs from skeletal myocytes that were targeted in this study, the direct gene transfer into bone defect has been shown to be successful before.43, 44

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Materials and methods

Plasmid DNA constructs and microbubbles

Two constructs of plasmid DNA were used: plasmid Luc under the control of the cytomegalovirus (CMV) promoter and plasmid recombinant human BMP-9 (rhBMP-9) based on the pcDNA3 vector and driven by CMV promoter as well. Both constructs were amplified using standard procedures and purified using an EndoFree Kit (Qiagen, Valancia, CA, USA). After purification, both constructs were eluted in a PBS solution for in vivo injection. The plasmid DNA was prepared in aliquots containing 50-μg DNA in 100μl PBS solution with 5% Optison, unless otherwise indicated. For mock transfections we used aliquots of 50-μg pcDNA3 empty vector in PBS solution with 5% Optison.

Animal models

Female wild-type C3H/HeN mice, each between 6 and 9 months old, were used in the experiments for optimization of the sonoporation and electroporation protocols, which was carried out using the reporter gene Luc. For the osteogenesis studies, in which we delivered rhBMP-9 in vivo, we used FVB/N transgenic mice, which had been generated to harbor the Luc gene under the control of the human osteocalcin promoter (hOc), as previously described.45

Sonoporation technique

The mice were anesthetized with a mixture of xylazine and ketamine (0.15% xylazine and 0.85% ketamine), which was injected intraperitoneally (i.p.) at 1μlg−1 body weight. Before the DNA solution was injected, the animals' back limbs were prepared by shaving the skin hair and swabbing it with 70% isopropanol and 0.5% chlorhexidine. The solutions of plasmid DNA with microbubbles in PBS were injected into the thigh muscle close to the femur. The needle penetrated a point in the thigh muscle and was pushed farther in until the femur was reached. Once the needle reached its destination, we released the plasmid–Optison mix slowly into the thigh muscle. Right after the injection, the needle was removed and the skin was covered with Aloe-sound gel (Rich-Mar Corp., Inola, OK, USA) at designated sonoporation sites. Immediately thereafter, transcutaneous, noninvasive sonoporation was applied using the 6-mm probe of a Sonitron2000 device kindly provided by Rich-Mar Corp. at a power output intensity of 5Wcm−2 and various acoustic parameters. According to the manufacturer, the beam of the ultrasound evenly distributes the energy to at least 4mm of tissue depth covering most of the mouse thigh muscle. In the experiment inducing ectopic bone formation, the mice received infiltration injections into the thigh muscle followed by sonoporation, three times, separated by a day of rest, on days 0, 2 and 4.

Electroporation technique

The mice were anesthetized as described above. Designated electroporation sites were prepared in the same manner as for the sonoporation technique, and 50μg of plasmid DNA encoding for Luc or rhBMP-9 in 100μl of 1 × PBS was injected into the left or right thigh muscle of the mice, respectively. Immediately post injection, the skin was covered with 40% glycerol in 1 × PBS and transcutaneous electric pulses were applied using two 1-cm cube electrodes placed 2–4mm apart at both sides of the limb. To generate pulses an ECM 830 electroporator (BTX, San Diego, CA, USA) was used. We applied 4 × 4 pulses of 100V for 20ms per pulse, 1Hz frequency, rotating the electrodes around the limb between the sets of pulses.

Noninvasive bioluminescence imaging

The imaging unit consisted of an intensified Peltier CCCD, Model LN/CCD-1300EB, which was equipped with an ST-133 controller and a 50-mm Nikon lens (Roper Scientific, Princeton Instruments, Trenton, NJ, USA). In this system, a pseudocolor image represents light intensity (with blue representing least intense and red most intense). In all cases, the integrated light is the result of a 2-min exposure and acquisition. The exposure conditions (including time, f/stop, position of the stage, binding ratio and time after the injection of luciferin) were maintained at identical levels so that all measurements would be comparable.36

In all experiments, the mice were anesthetized with a mixture of xylazine and ketamine (0.15ml xylazine and 0.85ml ketamine), which was injected i.p. at 1 μlg−1 body weight. Ten minutes before the light emissions were monitored, each mouse was given an i.p. injection of beetle luciferin (Promega Corp., Madison, WI, USA) in PBS at 126mgkg−1 body weight. The mice were placed in a light-tight chamber (a dark box) and a gray-scale image was first recorded using dimmed light. Photon emission was then integrated over a period of 2min and recorded as pseudocolor images.34, 36, 46 On the basis of our published data, an internal control (at the tail level) was needed for quantification of the results due to differences in basal expression levels of Luc.36

Immunohistochemical analysis of Luc expression

The immunohistochemical analysis was carried out as previously described.47 Briefly, thigh muscles sonoporated with the Luc gene were harvested 10 days after sonoporation. Treated and control organs were fixed in 4% paraformaldehyde for 3 days. Then the samples were decalcified in EDTA (0.5M, pH 8) for 14 days, embedded in paraffin, and cut into 5-mm-thick sections. Immunohistochemical staining was carried out using a Histostein SP kit (Zymed Laboratories, San Francisco, CA, USA). Primary Luc antibody (polyclonal rabbit anti-mouse) and a secondary goat anti-rabbit immunoglobulin G biotin-conjugated antibody (Zymed Laboratories) were diluted in 1:100 in PBS. Slides were counterstained with H&E, washed and mounted.

Evaluation of bone formation in vivo

For a detailed qualitative and quantitative three-dimensional evaluation of the bone that formed, we used a scanning protocol that yielded a high resolution.48 The dissected specimen was mounted on a turntable and 500 projections were obtained. The long axis of the femur was placed perpendicular to the X-ray beam axis. The X-ray tube was operated at 60KeV and 150μA, and the integration time was 200ms. Scans were obtained at a resolution of 20μm in all three spatial dimensions. Two-dimensional CT images were reconstructed in 1024 × 1024 pixel matrices by using a standard convolution back-projection procedure with a Shepp and Logan filter. The bone tissue was segmented from bone marrow and soft tissue by using a global thresholding procedure.49 In addition to the visual assessment of structural images, morphometric indices were determined on the basis of microtomographic data sets by performing direct three-dimensional morphometry.50, 51

Histological analysis

Tissue samples from an intramuscular site were harvested 5 weeks after the application of sonoporation, fixed in 4% formalin overnight, decalcified using Calci-Clear Rapid (National Diagnostics, East Riding of Yorkshire, England) for 48h, passed through increasing concentrations of ethanol and embedded in paraffin. Five-micrometer-thick sections were cut from each paraffin block using a motorized microtome (Leica Microsystems, Nussloch, Germany). The slides were heated at 65°C for 45min and was followed by deparaffinization. H&E and MTC staining procedures were carried out in a manner previously reported.38

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Acknowledgements

We thank Rich-Mar Corp. (Inola) for kindly providing the sonoporation Sonitron2000 device for the experiments described in this study.