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Bone with a Vascular Flap Induced from Fat Tissue with the Use of rhBMP-2 in Rats
1 Oral Surgery, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo, Tokyo 113-8549, Japan; Correspondence: * corresponding author, maruoka.osur{at}tmd.ac.jp
Here we report that successful bone formation with a vascular flap inside a cylindrical mold was induced from fat tissue with the use of recombinant human bone morphogenetic protein-2 in rats. Fat tissue connected to blood vessels was prepared to fit into the mold and implanted intramuscularly into the hind leg in Wistar rats. RhBMP-2 (20 µg) was applied in a collagen sheet previously placed on the inside surface of the mold. Bone formation was confirmed radiologically and morphologically at 2, 4, and 8 weeks after the surgery. In the control group without rhBMP-2 or the group with ligation of the blood vessels before the implantation, bone formation was not observed. Our success in bone formation having a definite size, shape, and blood supply may lead to a therapeutic approach to effective bone reconstitution. The present study is the first report on bone induction from fat tissue by rhBMP-2 in vivo.
Key Words: recombinant human bone morphogenetic protein-2 (rhBMP-2) • bone formation within a mold • vascularized bone flap • bone induction from fat tissue
The vascularized bone graft is expected to be a useful method in reconstructive bone surgery because of its notable osteogenic potential and great resistance against infections (Taylor, 1983). However, there have been limitations in obtaining suitable amounts of bone in desirable shapes (Weiland et al., 1984; DeLuca et al., 1997). In vivo placement and incubation of the muscle tissue with a vascular flap inside a BMP-containing mold resulted in induction of bone formation in the mold (Alam et al., 2001). However, muscle damage causes considerable dysfunction in the body. We describe the preparation of fat tissue with a vascular flap and implantation in a mold containing rhBMP-2 in rats. New bone formation with a vascular flap was clearly demonstrated. The present study is the first report on bone induction from fat tissue by rhBMP-2 in vivo.
Preparation of Implant Materials A silicon rubber impression material (vinyl polysiloxane impression material, GC Dental Products Corp., Tokyo, Japan) was used to make the mold for the induction of bone tissue. A cylindrical plastic bar (4 mm in diameter, 8 mm in length; As One Corporation, Tokyo, Japan) was covered with a thin layer of silicon rubber. After the silicon solidified, the bar was removed by being sectioned in half longitudinally. A groove was made on each half from one end of the mold to preserve space for the vascular flap (Fig. 1A  ). A collagen sheet (Helistat®, Integra Life Sciences Corporation, Plainsboro, NJ, USA) was cut and put on each surface of the mold. Under aseptic conditions, a 20-µg quantity of rhBMP-2 (Genetic Institute, Inc., Cambridge, MA, USA), dissolved in 20 µL of LF6 buffer solution (5 mM sodium glutamate, 2.5% glycine, 0.5% sucrose, and 0.01% Tween 80), was added to the collagen sheets. In the control group, only the buffer solution was applied.
Animals and Surgical Procedures Eight-week-old male Wistar rats, each weighing from 250 to 300 g, were divided into 3 groups: experimental (N = 31), ligated (N = 12), and control (N = 14). Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg per 100 g body weight). All surgeries were performed under sterile conditions and in accordance with the guidance of the Animal Research Committee of Tokyo Medical and Dental University. An incision was made on each side of the rat’s lower abdomen down to the inguinal region. A bundle of superficial inferior epigastric arteries and veins, which branch from femoral vessels, was identified and separated from surrounding tissues. The appropriate amount of subcutaneous fat tissue with vascular flap was placed into the mold, which was then tightened with nylon threads (Figs. 1B  , 1C). The mold with vascular flap was implanted into a pouch prepared between the rat’s thigh muscles, and procedures were performed under an operative microscope.
Harvest of the Mold
Radiographic Examination
Morphological Examinations Specimens were taken 2 wks after surgery and analyzed under transmission electron microscopy. The samples were fixed with 2.5% glutaraldehyde in 0.2 M phosphate buffer at 4°C for 4 hrs and washed in 0.1 M phosphate buffer at 4°C overnight. The specimens were post-fixed in 1% osmium tetroxide in 0.2 M phosphate buffer at 4°C for 2 hrs, dehydrated in graded concentrations of ethanol, infiltrated, and embedded in Epon. Thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and finally examined under an electron microscope (H-600, Hitachi, Tokyo, Japan).
All of the animals survived the surgical procedures with no infection. Macroscopically, all silicon molds were surrounded with scar tissue. In all samples from the experimental group, bone formation was observed inside the silicon mold. Bone was not found outside of the mold. In contrast, no hard-tissue formation was observed in the samples from the control group. In the ligated group, regressive tissue changes occurred in the mold, probably due to poor blood supply.
Radiologically, experimental group specimens showed radiodense images along the outline of induced bone (Fig. 2
Histologically, newly formed bone was observed in all experimental samples. The tissue enveloped remaining fat tissue and was interspersed with small blood vessels (Fig. 3A ). The area of bone tissue increased in a time-dependent manner. In barium-sulfate-injected cases, small droplets were observed in fine vessels in the new bone area (Fig. 3B). Osteoblasts were found to line the bony trabeculae, and osteocytes were seen in the lacunae of the bone. In all of the control samples, the original fat tissue remained in the mold. The samples of the ligated group had few cell components, and necrosis of the tissue was observed (data not shown).
Electron-microscopic analysis of the samples at 2 wks after surgery revealed morphological changes of adipocytes, similar to fibroblasts, attached to the collagen sheet. They showed spindle-shaped figures and produced collagen fibers. The cells were rich in rough endoplasmic reticulum, mitochondria, vacuoles, and lipid droplets (Fig. 4A ). Osteoblast-like cells were found in contact with the bone matrix (Fig. 4B).
Studies for osteoinduction in a muscle flap have been carried out with the use of BMPs. It was reported that rhBMP-2 mixed with atelopeptide type I collagen induced ectopic bone formation in the latissimus dorsi muscle flap (Kusumoto et al., 1998). Mandibular bone reconstruction with prefabricated vascularized bone graft was attempted in miniature pigs (Terheyden et al., 1999; 2001). Recently, a technique of bone formation with a vascularized periosteal flap was reported (Vögelin et al., 2002). However, in this system, a definitive blood supply to newly induced bone tissue has not been verified. It is reported that a preparation of muscle tissue with a vascular flap implanted into a rhBMP-2-containing mold demonstrated new bone formation in the mold, with sufficient blood supply for grafting (Alam et al., 2000). Meanwhile, transplantation of collagen sponge containing bone marrow stromal cells around vessels induced vascularized bone flap without BMP (Mankani et al., 2001). In this study, we used fat tissue instead of muscle because there is less harm to the body. It has already been reported that adipocytes or fat-tissue-derived cells differentiate into osteogenic cells in the presence of specific factors in vitro (Lecoeur and Ouhayoun, 1997; Halvorsen et al., 2001; Zuk et al., 2001; Huang et al., 2002). For the first time, we have accomplished in vivo bone induction from fat tissue. Ectopic osteogenesis induced by BMPs has a tendency toward size reduction and gradual disappearance. However, in the present study, newly formed bone was intact without resorption, even after 48 wks (data not shown). The silicon mold may have prevented excessive vascularization for bone resorption. Oral surgeons occasionally use various kinds of flaps for correction of maxillary defects. The present result, a vascularized bone flap induction from fat tissue, provides a promising and useful method for reconstruction of bone defects in the maxillofacial region.
We are grateful to Genetic Institute, Inc., for kindly providing us with rhBMP-2. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan (No. 13771208). This paper is based on a thesis submitted to the graduate school, Tokyo Medical and Dental University, in fulfillment of the requirement for the PhD degree. Received for publication September 16, 2002. Revision received April 11, 2003. Accepted for publication May 23, 2003.
Journal of Dental Research, Vol. 82, No. 8,
581-584 (2003)
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