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Keywords:

  • BMP-2;
  • electroporation;
  • ossification;
  • gene delivery;
  • bone

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, × 8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification. © 2005 Wiley-Liss, Inc.

Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily. Its osteogenic capacity has been proven by implanting recombinant BMP-2 (Wozney et al.,1988; Yasko et al.,1992) or BMP-2 gene expressing viral vectors (Alden et al.,1999; Musgrave et al.,1999; Engstrand et al.,2000) into skeletal muscles. Previous reports showed that the ectopic bone formation in the skeletal muscles by BMP-2 gene transfer using adenoviral-, retroviral-, or adenovirus-associated viral vectors underwent an endochondral ossification pattern (Alden et al., 1999; Musgrave et al.,1999; Chen et al.,2003). In time-course studies of osteoinduction by BMP-2 adenoviral vector, cartilage was initially observed in the muscle fibers on day 7 after gene transfer, with bone tissue appearing at 14 days after treatment (Okubo et al., 2001). It indicates that BMP-2 initiates the cascade of biological events known as endochondral bone formation, in which cartilaginous tissue is initially induced, then resorbed and replaced by bone. On the other hand, intramembranous ossification together with endochondral ossification occurred by recombinant human BMP-2 (rh BMP-2) with type I collagen as a carrier (Stoeger et al.,2002). Thus, the ossification pattern induced by BMP-2 may depend on the methods of BMP-2 application, that is, the concentrations of BMP-2, carriers, or tissue environments may affect the pattern.

We previously introduced a new method for BMP-2 gene transfer, that is, transcutaneous in vivo electroporation was applied for the induction of ectopic bone formation in the skeletal muscles of rats (Kawai et al.,2003). A plasmid-based human BMP-2 construct (pCAGGS-BMP-2) was injected into skeletal muscles and electroporated with 100 V, 50 msec, × 8. On day 21 after electroporation, ectopic bone formation, including osteoblasts, osteoclasts, osteocytes, and bone marrow, was observed in the muscles. However, it was still unclear how the ossification process occurred in the skeletal muscles after the BMP-2 gene transfer using the method mentioned above. Therefore, the present study was undertaken to examine the ossification process by BMP-2 gene transfer to skeletal muscles using transcutaneous in vivo electroporation in rats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Plasmid Vector

The details of the construction have been previously described (Kawai et al.,2003). Briefly, we constructed plasmid pCAGGS-BMP-2 by inserting human BMP-2 cDNA into an EcoRI cloning site of the pCAGGS expression vector that had the CAG (cytomegalovirus immediate-early enhancer/chicken β-actin hybrid promoter) (Niwa et al.,1991) and prepared the plasmid using a Qiagen EndoFree plasmid Giga kit (Qiagen, Hilden, Germany). Empty pCAGGS plasmid was employed as a control.

Animals

Nine-week-old male Wistar rats were purchased from Kurea (Osaka, Japan) and maintained under a specific pathogen-free condition and were allowed free access to diet and water in the animal facility. Rats were divided into experimental groups and control groups and were sacrificed under excess anesthesia with pentobarbital at 1, 3, 5, 7, and 10 days after treatments. Each group consisted of six rats.

Intramuscular DNA Injection and Transcutaneous Electroporation

Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (5.0 mg/100 g of body weight). Fur on the gastrocnemius muscle of tibiae was removed with clippers. Plate electrodes (Nepa Gene, Chiba, Japan) consisting of pairs of stainless steel plates with a fixed length of 5 mm were attached to the skin at the target site after being coated with keratin cream (Fukuda Denshi, Tokyo, Japan). The accuracy of the electric current applied was confirmed by measuring the resistance between the electrodes (usually below 800 Ω), which surrounded the middle of the gastrocnemius muscle without a skin incision. Next, 50 μl of plasmid DNA (25 μg) was injected with a 30-gauge needle into the center of the gastrocnemius muscle between the electrodes. Electroporation was started immediately after the injection by applying eight electrical pulses (100 V, 50 msec). Four pulses were followed by four more pulses of the opposite polarity and were administered to the injection site at 1-sec intervals using a square electroporator (CUY21EDIT; Nepa Gene). As a control, empty pCAGGS plasmid was injected and electroporated.

RT-PCR

At 10 days after gene transfer, rats that had been injected with pCAGGS-BMP-2 or pCAGGS alone were sacrificed under general anesthesia and the target muscles were resected (0.15 g). Total RNA was isolated from the muscle tissue using Isogen (Nippon Gene, Tokyo, Japan). Osteocalcin mRNA and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA were detected by RT-PCR using the following primers: osteocalcin backward primer, 5′-TTGGAGCAGCTGTGCCGTCCATA-3′; osteocalcin forward primer, 5′-ATGAGGACCCTCTCTCTGCTCA-3′; G3PDH backward primer, 5′-TCCACCACCCTGTTGCTGTA-3′; G3PDH forward primer, 5′-ACTGGCGTCTTCACCACCAT-3′. RNA (1 μg) was incubated at 42°C for 1 hr in a total volume of 20 μl, then at 94°C for 3 min, followed by 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The PCR products were analyzed by 2% agarose gel electrophoresis to detect the 414 bp osteocalcin mRNA and 682 bp G3PDH mRNA.

Histologic, Immunohistochemical, and Radiographic Analyses

For light microscopic observation, rats were perfused with saline via the left ventricle for 3–5 min; saline was then replaced by a fixative composed of 4% paraformaldehyde-0.05 M phosphate buffer (pH 7.4). For electron microscopic observation, a fixative composed of 2% glutaraldehyde, 2% paraformaldehyde, and 0.05 M phosphate buffer (pH 7.4) was utilized. After 10-min fixation, the target gastrocnemius muscles were excised and immersed in the same fixatives for 30 hr at 4°C. After fixation, all the specimens were analyzed with a soft X-ray to detect calcified areas before histologic studies were carried out (SRO-M50; Sofron, Tokyo, Japan). The specimens fixed with 4% paraformaldehyde were embedded in paraffin without decalcification, cut into 7 μm thick sections, then stained with hematoxylin and eosin stain (H&E) and von Kossa stain. For fine structural analysis, half of the specimens fixed with the glutaraldehyde and paraformaldehyde mixture was embedded without decalcification. The remaining specimens were decalcified with 0.05 M phosphate-buffered 5% ethylenediaminetetra-acetic acid (EDTA) overnight. Both sets of specimens were postfixed with 0.05 M phosphate-buffered 1% osmium, dehydrated with a graded acetone series, then embedded with Epon 812. Semithin sections (1–2 μm) cut from specimen blocks were stained with 1% toluidine blue. Von Kossa stain was also applied for light microscopy. For electron microscopic analysis, ultrathin sections were cut, stained with uranyl acetate, tannic acid, and lead citrate, then observed with an electron microscope (Hitachi model H 800) operated at 100 KV.

Immunohistochemical analysis was performed with the following procedures. After perfusion fixation with 4% paraformaldehyde-0.05 M phosphate buffer (pH 7.4), gastrocnemius muscle was removed and immersed with the same fixative at 4°C overnight. Then the specimens were embedded in paraffin prior to dehydration with a graded alcohol series. Paraffin sections cut 7 μm thick were mounted on silan-coated slides, rehydrated, and immersed in 0.5% periodic acid solution for 10 min to eliminate the nonspecific reaction by endogenous peroxidase and then treated with 2.5% hyaluronidase (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS) containing 0.025% Triton X for 60 min at room temperature. After these procedures, the sections were incubated in PBS containing 10% bovine serum albumin for 15 min and washed with PBS. They were then incubated either in an anti-BMP-2 monoclonal antibody (Sigma), an antitype I collagen antibody (LSL, Tokyo, Japan), or an antitype II collagen (LSL). Each antibody was diluted to 1:400. Antibody incubation was carried out for 12 hr at 4°C and sections were rinsed several times before incubating with peroxidase-labeled secondary antibody (Sigma) diluted 1:200 for 1 hr at room temperature. The incubation was terminated by washing with PBS, then the sections were immersed in a medium that consisted of 3,3′-diaminobenzidine tetrahydrochloride (20 mg), 30% H2O2 (10 μl), and 0.05 M Tris-HCl buffer (pH 7.6; 100 ml) for 10 min at room temperature.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Radiographic Findings

On day 10 after electroporation with pCAGGS-BMP-2, radiographs revealed X-ray opaque areas that had well-defined margins in the target muscles (Fig. 1). In contrast, X-ray opaque areas were not detected on 1, 3, 5, and 7 days after treatment. X-ray opaque areas were not observed in the muscle-injected pCAGGS as a control.

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Figure 1. Soft X-ray of gastrocnemius muscle after the gene transfer of pCAGGS-BMP-2 with electroporation. X-ray opaque areas (arrows) are identified after 10 days of the treatment. Muscle was electroporated transcutaneously with 25 μg of pCAGGS-BMP-2. Scale bar = 10 mm.

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Histologic and Immunohistochemical Analyses

Time-course findings on histologic analysis were carried out on day 1, 3, 5, 7, and 10 after electroporation with pCAGGS-BMP-2. On day 1, each muscle fiber at the plasmid injection site ran randomly in direction with expanded spaces between muscles fibers. Additionally, disrupted or degenerated muscle fibers existed (Fig. 2a and b). In contrast to these fibers, evenly distributed muscle fibers were also observed, indicating normal or intact areas near the affected muscle fibers (Fig. 2a, asterisk). By 3 days posttreatment, the interspaces between muscle fibers had enlarged and filled with lymphocyte-like cells showing condensed nuclei (Fig. 2c). By 5–7 days after electroporation, spindle-shaped fibroblast-like cells had appeared, accompanied by a fibrous matrix (Fig. 2d), and these fibroblast-like cells were BMP-2-positive by immunohistochemistry (Fig. 2e). On day 10 posttreatment, cartilaginous tissues were identified within the fibrous tissues that had formed between muscle fibers (Fig. 3a, arrow). Depending on the developmental stage of the cartilage, some cartilaginous tissues were observed as being completely encircled by perichondrium, while other cartilaginous tissue was only partially encircled (Fig. 3b). In the central area of the cartilage, hypertrophic chondrocytes were seen embedded in conspicuous lacunae (Fig. 3b). The cartilaginous tissues, including the hypertrophic chondrocytes, had tended to calcify as revealed by von Kossa stain (Fig. 3c). Blood vessels were rarely observed near the cartilage. Endochondral ossification was identified in further advanced stage (Fig. 3d and e). Cartilaginous matrix showed the positive immunoreactivity for type II collagen as expected (Fig. 3e, arrows). In addition to endochondral ossification, another ossification pattern, namely, intramembranous ossification, was observed as shown in Figure 4. In these areas, osteocytic cells were embedded sporadically within the matrix and the matrix was intensively stained for type I collagen, but was negative for type II collagen by immunohistochemistry (Fig. 4a and b). Undecalcified sections stained with von Kossa showed that the matrix was calcified in various degree (data were not shown). At the surface of the bone matrix, densely distributed cells conspicuously existed (Fig. 4a). Osteoclasts were not well identified on day 10 after the treatment. Electron microscopic observation revealed that at this stage osteocytes embedded in osteocytic lacunae had moderately developed cell organellae such as rough endoplasmic reticulum and Golgi apparatus, and the matrix was composed of densely distributed collagen fibrils (Fig. 4c). These collagen fibrils showed the cross-striated band that is characteristically observed in type I collagen fibrils. Neither cartilage nor bone was observed in control groups.

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Figure 2. Time-course findings of the muscles electroporated with pCAGGS-BMP-2. a: Muscle fibers are observed as distributed randomly at the injection site, and the spaces between the fibers are expanded. In contrast to these fibers, intact muscle fibers run regularly (asterisk). b: An enlarged micrograph of a. Degenerated fibers (arrows) are frequently seen 1 day after the treatment. c: On day 3 after injection, the expanded spaces between muscle fibers are filled with lymphocyte-like cells. d: By 7 days, spindle-shaped fibroblast-like cells (arrows) appear among lymphocyte-like cells. Asterisks indicate muscle fibers. e: Spindle-shaped fibroblast-like cells are positively stained for BMP-2 immunohistochemistry (arrows)

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Figure 3. Cartilaginous tissue formed between muscle fibers after 10 days of the treatment. a: Cartilage can be identified among muscle fibers (arrow). b: An enlarged micrograph of a. Hypertrophic chondrocytes are identified at the center area of cartilage matrix (arrow) and perichondrium covers the cartilage (arrowhead). c: An adjacent section stained with von Kossa staining reveals that the cartilage is calcified. d: Endochondral ossification appeared at 10 days after treatment. Cartilaginous tissue (arrowhead) and bone (arrows) are consecutively observed. e: A similar area to d. Cartilaginous matrix is positively stained for type II collagen immunohistochemistry (arrows). Counterstained with hematoxylin.

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Figure 4. Intramembranous ossification observed at 10 days after treatment. a: Osteocytes are buried in the matrix and densely gathered cells including osteoblasts exist at bone surface. Bone matrix is intensively stained for type I collagen immunohistochemistry (arrow) than that of tissue around. Counterstained with hematoxylin. b: An adjacent section stained for type II collagen immunohistochemistry. The bone matrix is negatively stained (arrow). Counterstained with hematoxylin. c: An electron micrograph of bone. Osteocytes (arrows) are easily identified in the densely distributed collagen fibers.

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Osteocalcin mRNA Expression

Osteocalcin, a marker of osteoblasts, was detected in muscles resected 10 days after a single transcutaneous electroporation with pCAGGS-BMP-2 (25 μg). The presence of osteocalcin mRNA in the electroporated muscles was confirmed by RT-PCR (Fig. 5). Osteocalcin mRNA was not detected in muscles transfected with pCAGGS. The control G3PDH mRNA was detected in both groups (Fig. 5). Thus, RT-PCR revealed that 10 days after electroporation with pCAGGS-BMP-2, osteocalcin mRNA was expressed in the target muscles.

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Figure 5. Detection by RT-PCR of osteocalcin mRNA in muscle after transcutaneous electroporation with pCAGGS-BMP-2 or pCAGGS on day 10 after treatment.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

We previously reported that human BMP-2 gene transfer using transcutaneous in vivo electroporation induced ectopic bone formation in the skeletal muscles (Kawai et al.,2003), but the processes of bone formation had not been clarified. No report is currently available with respect to ossification patterns of BMP-2 gene transfer using transcutaneous in vivo electroporation-induced bone formation. In the present study, the existence of cartilaginous tissues was identified by immunohistochemical detection of type II collagen in the matrix and histologic appearances, and calcified cartilage leading to endochondral ossification was also found. The bone that had undergone intramembranous ossification was intensively stained for type I collagen, but negative for type II collagen by immunohistochemistry, and light and electron microscopic observations revealed characteristic appearances for bone, was identified also at 10 days after treatment. RT-PCR analysis detected osteocalcin mRNA at 10 days after treatment, indicating the occurrence of bone formation. Regarding the ossification patterns with use of BMP-2 gene transfer, an endochondral ossification pattern was solely observed in a previous study using adenovirus vector (Okubo et al., 2001). In the present study, both endochondral and intramembranous ossification was determined. Therefore, it is strongly suggested that transcutaneous in vivo electroporation for BMP-2 gene transfer induces simultaneously both endochondral ossification and intramembranous ossification in the skeletal muscles of rats. However, the origin of chondrogenic cells and osteogenic cell was not clarified in the present study. It should be mentioned that spindle-shaped fibroblast-like cells showing BMP-2 by immunohistochemistry appeared between muscle fibers, and the areas where such cells existed expanded toward 10 days of treatment. These BMP-2-positive cells may relate to chondrogenic cell and/or osteogenic cell differentiation since it has been reported that the expression of BMPs including BMP-2 is involved in the differentiation process of both chondrogenic cells and osteogenic cells (Yamaguchi et al.,2000). Further study would be necessary to clarify the origin of chondrocytes or osteoblasts in the experimental system used in the present study.

It has been well known that BMPs induce bone by endochondral ossification (Rosen and Wozney.,2002). Intramembranous ossification induced by BMPs with carriers has also been reported (Sasano et al.,1993; Aono et al.,1995). Moreover, it has been reported that rh BMP-2 with type I collagen as a carrier induced endochondral ossification and intramembranous ossification (Stoeger et al.,2002). The ossification patterns observed in the present study using the gene transfer of BMP-2 are in concert with a previous report employing rh BMP-2 (Stoeger et al.,2002). However, these studies (Sasano et al.,1993; Aono et al.,1995; Stoeger et al.,2002) were not carried out under the same conditions as the present study. For example, intramembranous ossification was observed when BMPs were employed with carriers such as fibrous collagen membrane or collagen in these studies. The authors suggested that the different ossification patterns after BMP treatments appeared to depend on the character of the carriers (Sasano et al.,1993). In our study, electronic stimulation, but not carriers or immunosuppressants, was utilized to transfer BMP-2 gene. It may be possible that the electronic stimulation affects the ossification patterns, since it has been reported that dystrophic calcification and bone formation without cartilage formation were observed in ectopic osteoinduction by BMP-4 gene transfer in vivo electroporation (Kishimoto et al.,2002). But simultaneous formations of both cartilage and direct bone was not observed (Kishimoto et al.,2002). Our findings differ from this point, and in addition, dystrophic calcification was not observed in our study. We consider the difference between these findings may be due to whether a direct method (electrodes were inserted into the muscle) or an indirect method (transcutaneous electroporation employed in our study) of in vivo electroporation was applied. Moreover, we postulate that the difference with respect to ossification manners, that is, endochondral ossification and/or intramembranous ossification, may be due to different expression timing and/or amount of BMP-2. With in vivo studies using rh BMP-2 and carrier, microgram quantities of exogenously added rh BMP-2 were released from carrier for 3 days, with endogenous BMP-2 secreted subsequently (Wang et al.,1990; Yamamoto et al.,1998; Nakagawa et al.,2001). While in the case of adenovirus vectors used, nanogram quantities of BMP-2 were secreted over 72 hr (Musgrave et al.,2000). In our previous study using in vivo electroporation, semiquantitative RT-PCR revealed that the expression of BMP-2 derived from pCAGGS-BMP-2 continued over 10 days (Kawai et al.,2003). This finding elucidates that the exogenously induced pCAGGS-BMP-2 works to produce BMP-2 for a longer period than that of protein delivery or adenovirus vectors systems.

In conclusion, we showed that in vivo transcutaneous electroporation with BMP-2 gene expressing plasmid could induce not only endochondral ossification but also intramembranous ossification. The turning point(s) of the two different bone formation modes should be examined in future studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Nancy Kief for editorial assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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