(8) 231 Upsala J Med Sci 111 (2): 231–242, 2006 Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4 Satoshi Kotajima, Koshi N. Kishimoto, Munenori Watanuki, Masahito Hatori and Shoichi Kokubun Department of Orthopaedic surgery, Tohoku University School of Medicine, Sendai, Japan ABSTRACT Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ecto- pic bone formation by using semi-quantitative RT-PCR. A plasmid vector contai- ning mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the pro- cess of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone for- mation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix. INTRODUCTION Bone morphogenetic protein (BMP) induces or stimulates bone formation. Some iso- forms of BMPs (BMP2, 4, 6, and 7) differentiate mesenchymal cells into osteoblastic linage cells (1-3). In classical experiments using BMPs, a carrier is necessary for bone induction because BMPs have a short half-life in vivo (4). Appropriate carrier implan- tation allows gradual BMPs release and works as a scaffold for cells (5). Received 1 September 2005 Accepted 17 January 2006 Gene therapy technique is thought to be a potent option for utilizing BMPs. When a BMP coding gene is introduced into cells, the cells continuously produce and secrete BMP. Adenoviral transfer of a BMP coding gene into rodent muscle showed abundant ectopic bone formation in immunodeficient animals without a carrier (6). We previ- ously reported that electroporatic transfer of a plasmid DNA containing mouse BMP-4 induced bone in immunocompetent mice (7). It is difficult to quantify the amount of protein produced by gene-transferred cells in an in vivo animal model. The pharmacokinetics of BMPs might be different between protein and gene based experiments. BMPs existing in demineralized bone matrix or put in a carrier are abundant at the time of implantation in vivo and decrease gradually. On the other hand, BMP expression, theoretically, increases after gene transfer, contin- ues for weeks and then decreases gradually. The molecular and cellular events involved in endochondral ossification after im- plantation of demineralized bone matrix were described in detail by Reddi (8). By day 3, mesenchymal and inflammatory cells had appeared around the implant. Chondrob- lasts appeared by day 5 and chondrocytes were maximal on day 7. The hypertrophic cartilage matrix began to undergo calcification by day 9. Basophilic osteogenic precur- sors and osteoblasts appeared on day 10. Bone formation was confirmed on days 12 to 18. In this cascade of endochondral ossification, each differentiation step of osteogenic and chondrogenic cells was analyzed by using the following markers: osteocalcin (9), osteopontin (9), bone sialoprotein (10, 11), aggrecan (12, 13), and type X collagen (14). However, these markers of gene expression in this cascade were not assessed after BMP gene transfer but in protein and carrier models (15-17). To the best of our knowledge, this paper is the first to confirm the temporal gene expressions of osteogenic and chondrogenic markers in ectopic bone formation caused by the intro- duction of BMP4 gene into the muscle. MATERIALS AND METHODS Mice Male C57BL/6J mice were purchased from Clea Japan, Inc. Electroporation was per- formed on 8-week-old mice. Mice were maintained under specific pathogen-free condi- tions at the Institute for Animal Experimentation, Tohoku University School of Medicine. The Institutional Animal Care and Use Committee approved all the procedures used in this study. Plasmid DNA The 1.6kb mouse BMP-4 cDNA was kindly provided by Brigid L. M. Hogan (18). It was inserted into multiple cloning sites of a pCAGGS expression vector (19) (pCAGGS- BMP4). pCAGGS-GFP, a GFP-containing plasmid, was used as the control. Both plas- mids were dissolved in distilled water at 2.0�g/�l. 100 microgrms of each was injected into the animals. 232 In Vivo Electroporation Fifty microliters of 0.5% bupivacaine were injected into the left gastrocnemius as a pre- treatment (20). In vivo electroporation was performed as previously described (pulse set- tings: 100V, 50ms, 6pulses, 1Hz) (7, 20). Pulses were applied through percutaneously inserted electrodes (0.4mm diameter: Unique Medical Imada, Natori, Japan) just after injection of pCAGGS-BMP4 or pCAGGS-GFP (50�l each) into the pretreatment site. Mice were euthanized by cervical dislocation 3, 7, 10, or 14 days after electroporation. Soft X-ray assessment All gastrocnemius muscles were excised after sacrifice and underwent soft X-ray exami- nation at 20.0kV and 2.0mA, for 10 sec (SRO-iM50, Sofron, Tokyo) using X-Omat TL film (Kodak). Histology and Immunohistochemistry Frozen non-decalcified sections (thickness 10�m) of the specimens were made with a cryostat (Bright, UK) after the soft X-ray photography. Slides were stained with hema- toxylin-eosin, alcian blue or von-Kossa. BMP4 expression was detected immunohisto- chemically by rabbit polyclonal anti-BMP-4 antibody (1:100 dilution, overnight at 4°C; Santa Cruz Biotechnology, Santa Cruz, CA). HRP labeled goat anti-rabbit antibody was used as the secondary antibody (1:100, 2h at room temperature; Dako Cytomation). RT-PCR analysis A muscular portion of the gastrocnemius with pCAGGS-BMP4 (n=6) or pCAGGS-GFP (n=6), 10 mm in length containing the center of the electroporated site was dissected (0.12g). Total RNA was isolated from the dissected muscles with the RNeasy Fibrous Tissue Mini Kit (QIAGEN, Germany) and reverse transcription-PCR was done with a TaKaRa One Step RNA PCR Kit (Takara, Japan) following the manufacturer's instructions. The examined marker genes and their primers (in 5’ to 3’ direction) were as follows: Transgene marker: mouse BMP4 (accession number: X56848) Fwd. CCCAGAGAATGAGGTGATCTCC Rev. TGGCAGTAGAAGGCCTGGTAG Chondrogenic markers: procollagen type Xalpha 1 (COL10 accession number: Z21610) Fwd. GCCAGGTCTCAATGGTCCTA Rev. GCACCTACTGCTGGGTAAGC aggrecan (accession number: L07049) Fwd. CAGGTTTCCCCACTGTGTCT Rev. ACTCCAGACCCTGGGAAGTT 233 Osteogenic markers: osteocalcin (OCN acession number: X04142) Fwd. CTTGGTGCACACCTAGCAGA Rev. ACCTTATTGCCCTCCTGCTT osteopontin (OPN accession number: AF515708) Fwd. TCTGATGAGACCGTCACT Rev. TCTCCTGGCTCTCTTTGGAA bone sialoprotein (BSP accession number: L20232) Fwd. AAAGTGAAGGAAAGCGACGA Rev. GTTCCTTCTGCACCTGCTTC Internal control: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH accession number: M32599) Fwd. TGTTTGTGATGGGTGTGAA Rev. ATGGGAGTTGCTGTTGAA Total RNA (1�g) was incubated for 30 minutes at 50˚C in a total volume of 50�l and then for 2 min at 94˚C, followed by 25 cycles for 30 seconds at 94˚C, for 30 seconds at 60˚C and then for 30seconds at 72˚C. The PCR products were analyzed on 2% agarose gels and visualized with ethidium bromide. The density of each band was quantified using Scion Image software (http://www.scioncorp.com). We determined the relative gene expression by dividing the densitometry value of the mRNA RT-PCR product by that of the GAPDH product. Statistical analysis All measurements were performed in triplicate for each specimen and the mean value was used for statistical analysis. Results were presented as mean ± standard deviation. The significance of differences between control and pCAGGS-BMP4 electroporated muscles was determined using Mann-Whitney U test. Values less than P=0.01 were con- sidered significant. RESULTS X-ray assessment A radio-opaque area was observed in the gastrocnemius of all animals electroporated with pCAGGS-BMP4 after 14days, but not in those electroporated with pCAGGS- GFP. Histological findings Mesenchymal cell infiltration was observed 3 days after BMP4 electroporation (Figure. 1A). On day 7, the extracellular matrix was found to be stained clearly with alcian blue (Figure. 1B, C). On day 10, when hypertrophic chondrocytes and cartilagenous matrix appeared, the intensity of alcian blue staining decreased (Figure. 1D, E). The extracel- lular matrix was not revealed by von Kossa staining. On day 14, calcified bone matrix was detected with von Kossa staining (Figure. 1F). 234 After electroporation of control pCAGGS-GFP, mesenchymal cell infiltration was also observed on day 3. Calcium deposits between the muscle bundles were sporadical- ly observed on day 7. No cartilage or bone matrix were found at any time point (data not shown). Immunohistologic analysis revealed that BMP4 was expressed in muscle fibers on day 3 (Figure. 2A), and the intensity of BMP4 expression decreased with time (Figure. 2B, C, D). 235 1 A 1 B 1 C 1 D 1 E 1 F Fig 1. Axial sections of pCAGGS-BMP4 electroporated muscles stained by hematoxylin-eosin (A, B, D), alcian-blue (C, E) and von Kossa (F). Infiltrations of mesenchymal cells were found between muscle fibers on day 3 (A). Extracellular matrices stained with alcian blue were identified on day 7 (B, C). Hypertrophic chondrocytes and cartilage matrices were observed on day 10 (D, E). Calcified bone matrices stained with von Kossa were identified on day 14. Bone marrow-like cells were found in cal- cified matrices (F). Semi-quantitative RT-PCR BMP4 mRNA expression was clearly detected 3 days after BMP4 electroporation. It gradually decreased and was hardly detected on day 14. No BMP4 mRNA expression was detected in control (Figure. 2E). Aggrecan mRNA expression was detected in BMP4 electroporated muscles from day 7, and decreased by day 14. No aggrecan mRNA expression was detected in control. COL10 mRNA expression in 236 Fig 2. Localization of BMP4 expression analyzed by in situ immunohistochemistry at 3 (A), 7 (B), 10 (C) and 14 days (D) after electroporation. BMP4 was highly expressed in muscle fibers surrounding infiltrated mesenchymal cells on day 3 (A). The intensity of BMP4 expression and the number of BMP4 positive cells decreased with time. Reverse transcription-polymerase chain reaction (RT-PCR) showed intense mouse BMP4 mRNA expression in pCAGGS-BMP4 electroporated muscles on day 3 and its expression decreased by day 14. BMP4 mRNA expression was not detected in pCAGGS-GFP (control) electroporated muscles (E). BMP4 electrtoporated muscles was significantly higher than in the control on day 10 and 14 (Figure 3A,B). OCN and BSP mRNA expressions were detected in BMP4 electroporated muscles on day 7. They were significantly elevated on day 10 and increased on day 14. No OCN and BSP mRNA expressions were detected in con- trol. OPN mRNA expression was high on day 3, not detected on day 7, detected again on day 10 and increased on day 14 in BMP4 eletroporated muscles. In con- trol, OPN mRNA expression was not detected after day 7 (Figure. 4A, B, C). 237 Fig 3. Time course of chondrogenic marker genes expression. Aggrecan mRNA expression was initially detected on day 7, and decreased by day 14, whereas it was not detected in the control group throughout this entire period (A). Procollagen type X alpha 1 mRNA expression was significant higher than in the control group on day 10 and 14 (B). The I-bars represent the standard deviation and the asterisk (*) indica- tes significant differences in mRNA expression at each time point (p < 0.01). DISCUSSION Gene transfer using electroporation is safe and inexpensive. In 2002, the present authors reported bone induction by using electroporatic transfer of BMP4, and the rate of bone formation in BALB/cA mice was 67% at 14 days after electroporation (7). It is noteworthy that the present experiment demonstrated 100 % bone formation at 14 238 Fig 4. Time course of osteogenic marker gene expression. Osteocalcin and bone sialoprotein mRNA expressions were detected in the pCAGGS-BMP4 group on day 7, were significantly elevated on day 10 and further increased on day 14. Osteocalcin and Bone sialoprotein mRNA expressions were not detected in the control group (A,B). Osteopontin mRNA expression was high in both groups on day 3. Its expres- sion in pCAGGS-BMP4 electroporated muscles was decreased on day 7 and gradually increased from day 10 and to day 14. Osteopontin mRNA expression in control group was not detectable after day 7 (C). The I-bars represent the standard deviation and the asterisk (*) indicates significant differences in mRNA expression at each time point (p < 0.01). days. In this study, we could induce bone formation in all BMP4 transferred C57BL/6J mice. There are three possible reasons why the bone formation rate improved. The first is that the C57BL/6J strain, is reportedly a better responder for BMPs than the BALB/cA strain (21). The second reason may be that the pCAGGS expression vector containing the CAG promoter has higher activity in muscle (19). The third is that pre- treatment with bupivacaine, which induces muscle necrosis, may have enhanced the efficiency of gene transfer by direct intramuscular plasmid injection (22-24). Addition- ally, the muscle regeneration process after the pretreatment could provide abundant mesenchymal cells, which are essential for ectopic bone formation (25). However, at 14 days, bone induction was also observed in all mice even without Bupivacaine pre- treatment (data not shown), which lead us to believe that the strain of the mouse and the change of the expression plasmid contributed to stable bone formation. It is well known that transplantation of BMP protein or gene transfer of BMP into the muscles induce bone formation. However, there have been no reports describing in detail the cell differentiation in the process of bone formation. It is thought to be diffi- cult to clearly distinguish each stage of differentiation of osteoblastic or chondroblastic cells under microscopic observation alone. However, we can recognize differentiation stages by serially examining the gene expression changes of osteogenic and chondro- genic markers. Aggrecan is an extracellular matrix of cartilage and its gene is reported to be expressed in proliferating chondrocytes (13). COL10 gene is reported to be expressed specifically in hypertrophic chondrocytes (26). Aggrecan and COL10 mRNA expres- sions and histological examination in our experiment indicated that proliferating chon- drocytes appeared on day 7 and had differentiated into hypertrophic chondrocytes by day 10. The gene expressions of OCN and OPN were reported to correlate with the appear- ance of mature osteoblasts (27,28). BSP is extracellular matrix protein produced at the phase of osteoblastic differentiation (29). The OCN and OPN mRNAs expressions in our experiment indicated that differentiation into osteoblasts and bone matrix forma- tion had started by day 10. OPN mRNA expression was strongly detected on day 3 in both BMP4 electroporated and control muscles. 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