Osteoinduction capability of recombinant human bone morphogenetic protein-2 in intramuscular and subcutaneous sites: an experimental study

Journal of Cranio-Maxillofacial Surgery (1998) 26, 112-115 © 1998 European Association for Cranio-Maxillofacial Surgery

Osteoinduction capability of recombinant human bone morphogenetic protein-2 in intramuscular and subcutaneous sites: an experimental study Kazuya Yoshida 1, Kazuhisa Bessho 1, Kazuma Fujimura 1, Kenji Kusumoto 2, Yutaka Ogawa 2, Yoshiaki TanP, Tadahiko Iizuka 1

1Department of Oral and Maxillofacial Surgery (Heath Prof. Dr. T. Iizuka), Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan 2Department of Plastic and Reconstructive Surgery (Head." Prof. Dr. Y. Ogawa), Kansai Medical University, 10-15 Fumizono-cho, Moriguchi-shi, Osaka 570, Japan 3Research Centerfor Biomedical Engineering (Head." Prof. Dr. Y. Tani), Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606, Japan SUMMARY. The osteoinduction capability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in the muscle and in the subcutaneous tissue in Wistar rats (n = 20) was evaluated, using atelopeptide type-I collagen as a carrier. The alkaline phosphatase (ALP) activity and calcium (Ca) content were quantitatively analyzed 1,3,7 and 21 days after the implantation of 5 gg of rhBMP-2. At 3 days, the ALP activity began to increase gradually. The Ca content showed a slow increase until 7 days and was markedly elevated at 21 days. There was no significant difference observed between the intramuscular and subcutaneous sites until 3 days. However, at 7 days, both the ALP activity and Ca content were significantly higher intramuscularly than subcutaneously. Also, at 21 days they were higher in the muscle than in the subcutaneous tissue. These results suggest that the difference in osteoinduction could be related to the partial pressure of oxygen or the blood supply in the intramuscular and subcutaneous sites, and that immature mesenchymal cells in the muscle could more easily differentiate into osteoblasts, leading to osteoinduction. This study clearly demonstrated that even a small amount (5 gg) of rhBMP-2 induces new bone in the subcutaneous tissue, which has a lesser blood flow than the muscle.

INTRODUCTION

regarding this. In the present study in rats, we evaluated the osteoinduction capability of rhBMP-2 in the muscle and subcutaneous tissue.

Urist (1965) demonstrated that extracts from demineratized bone induced new bone formation if implanted into ectopic sites in rodents. Wozney et at (1988) succeeded in molecular cloning of the gene for bone morphogenetic proteins (BMP) 1,2,3 and 4, using peptide sequence information from a group of proteins purified from such an extract. Excellent methods for the isolation and purification of BMP have been reported (Urist et al, 1984; Bessho et al, 1989). An appropriate delivery system and the effective level of BMP were unresolved problems for clinical application. However, Bessho (1990) used atelopeptide type-I collagen derived from fresh porcine skin as a carrier, and detected ectopic osteoinductive activity of purified BMR Kusumoto et al (1995) found, using the atelopeptide type-I collagen as a carrier, that the recombinant human bone morphogenetic protein-2 (rhBMP-2) induced bone matrix and rich marrow, including fatty marrow and angioid tissue, around and among the bone trabeculae. BMP is one of the most promising biomaterials and has wide-ranging potential for clinical use in reconstructive surgery for bone defects and augmentation. Therefore, it is important clinically to elucidate its specific bone inductive property in various regions, particularly as there is a paucity of information

MATERIALS AND METHODS Animals

Forty male 10-week-old Wistar rats, each weighing 230-260 g, were used. They were fed rodent chow (Certified diet MF; Oriental Koubo, Inc., Tokyo, Japan) pre- and postoperatively. Implant materials

The rhBMP-2 (Batch Number 0213J01) was provided by the Genetics Institute, Cambridge, Massachusetts through Yamanouchi, Inc. (Tokyo, Japan). The rhBMP-2 was suspended in a buffer (pH 4.5) of 5 mM glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween 80, and preserved at -80 °C before being thawed at room temperature. Atelopeptide type-I collagen (3 mg/ml, pH 3.0, Cellmatrix LA; Nitta Gelatin, Inc., Osaka, Japan) was used as a carrier. 5 gg of rhBMP-2 mixed with 3 mg of atelopeptide type-I collagen was lyophilized (EYELA FDU-830; Tokyo Rikakikai, Inc., Tokyo, Japan). The material was 112

Osteoinduction capabilityof rhBMP-2 in intramuscular and subcutaneous sites 113

Fig. la - Histologicalview of new bone formation in the muscle21 days after implantation (M: calf muscle of host; NB: newly formed bone; BM: bone marrow; OB: osteoblast;OC: osteoclast; decalcifiedby EDTA; Haematoxylinand Eosin stain, x 200)

Fig. lb - Histologicalviewof new bone formation in the subcutaneous tissue 21 days after implantation (S: skin of host; NB: newlyformed bone; BM: bone marrow; OB: osteoblast;OC: osteoclast; decalcifiedby EDTA; Haematoxylinand Eosin stain, x 200).

compressed in the injection syringe to a discal form (4 mm diameter and 1.5 mm thickness).

RESULTS

Histological analysis Surgical procedures Each rat was anaesthetized with intraperitoneal sodium pentobarbital (5 mg/100 g of body weight). Following disinfection of the operative region, the lyophilized discal specimens were implanted into the right calf muscle (n = 20) and in the subcutaneous tissue over the musculus latissimus dorsi (n = 20). The fascia and skin were sutured closely in both groups.

Analysis One, 3, 7 and 21 days after the operation, the implanted region including the surrounding tissue was excised from five rats in each group. The tissue was divided into two samples: one for quantitative analysis and the other for histological analysis. Surrounding tissue of the samples for quantitative analysis was removed. The samples were weighed and then homogenized in 0.25 M sucrose in a Polytron homogenizer (Bio-Mixer; type ABM, Nissei, Inc., Osaka, Japan). The sediment was demineralized in 0.5 N HCI, and the Calcium (Ca) content of the soluble fraction was determined by the orthocresolphthalein complexone method (Connerty and Briggs, 1966). The alkaline phosphatase (ALP) activity level and total protein in the resultant supernatant were each determined by the 4 NPP method. The Ca content and ALP activity were used as indices of bone formation. The t-test was used to assess the statistical differences. The other samples for histological analysis, including the surrounding tissue, were fixed in 10% formalin neutral buffer solution (pH 7.4), and were decalcified by EDTA and stained with haematoxylin and eosin for microscopic examination.

N o new bone formation was detected in the intramuscular or the subcutaneous sites after 1 and 3 days. At 7 days, immature bone tissue was observed in some areas around the material implanted in the intramuscular region. New bone formation was observed at 21 days in both the muscle (Fig. la) and the subcutaneous tissue (Fig. lb). Trabecular bone, a few osteoblasts and osteoclasts were also observed at 21 days in both sites. Within the newly formed bone, a small area of bone marrow was observed. The bone marrow included partly angioid tissue (Fig. l a). Neither chondrocytes nor cartilaginous tissue were observed in these sections.

Bioassay Figures 2 and 3 show the change in the ALP activity and the Ca content respectively, at each time after implantation in the muscle and in the subcutaneous tissue. At 3 days, the ALP activity began to increase gradually (intramuscular: 0.2 + 0.1 gg/mg, subcutaneous: 0.16 + 0.13 gg/mg). The Ca content of the muscle showed a slow increase until day 7 (0.28 + 0.18 gg/mg) but there was no increase in the subcutaneous tissue. It was markedly elevated at 21 days, 24.1 + 3.1 gg/mg in the intramuscular site and 14.6 + 4.0 gg/mg in the subcutaneous site. There was no significant difference until day 3. At 7 days, A L P activity and Ca content were significantly higher (ALP-intramuscular: 2.98 + 1.01 gg/mg, subcutaneous: 1.14 + 0.48 gg/mg, P<0.007; Ca-intramuscular: 0.28 + 0.18 IU/mg, subcutaneous: 0 IU/mg, P<0.009) in the muscle than in the subcutaneous tissue. At 21 days, ALP and Ca were higher intramuscularly than subcutaneously

114

Journalof Cranio-Maxillofacial Surgery

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Fig.2- Changein alkalinephosphatase(ALP)activityin the muscleandin thesubcutaneoustissueafterimplantation.

Fig.3- Changein calcium(Ca)contentin themuscleandin the subcutaneoustissueafterimplantation.

(ALP-intramuscular: 3•78 + 1.24gg/mg, subcutaneous: 3.3 +_ 0.93 gg/mg, not significant; Ca-intramuscular: 24.1 + 3.1 IU/mg, subcutaneous: 14.6 + 4.0 IU/mg, P<0.003).

formation ectopically in the tissue under the skin. We found little qualitative difference in the histological appearance in the intramuscular and the subcutaneous sites. Bone formation is influenced by many factors, among which the blood supply is an important one. Mainous (1977) concluded that when the partial pressure of oxygen increased, collagen formation and fibroblastic proliferation, capillary budding, osteoblastic and osteoclastic activity, callus formation and mineralization were all increased. The partial pressure of oxygen in the tissue is directly influenced by the blood supply to the site. Therefore, the quantitative difference in the indices of bone formation between the intramuscular and subcutaneous sites might be due to the partial pressure of oxygen, i.e. the blood supply of the tissue around the implanted material. Resting skeletal muscle blood flow is about 2 to 5ml/min/100g tissue in humans (Mellander and Johannson, 1968). Acute elimination of resting vasoconstrictor fibre influence roughly doubles flow and, during complete smooth muscle relaxation in the resistance vessels, flow can increase to 40 to 60 ml/min/100 g (Barcroft, 1963)• Cutaneous blood flow in the range of 3 to 10 ml/min/100 g has been reported for the human hand at local temperatures of 25 to 35°C in comfortably warm subjects (Catchpole and Jepsen, 1955). At a local temperature of 44°C hand blood flow increased to 40ml/min/100 g or more (Peacock, 1958)• In the rat, the blood flow rate in the muscle is postulated to be higher than that in the subcutaneous tissue under the experimental conditions in this study. In man, the vascular supply to the skin is via a segmental-perforator- cutaneous system. The musculocutaneous arteries are the primary vessels, but they are augmented by a limited number of non-essential direct cutaneous arteries. The vascular supply to the skin in the rat is not similar to that in man (Daniel and Williams, 1973), and the blood

DISCUSSION The therapeutic potential of rhBMP-2 has long been recognized in reconstructive surgery. The possible applications might include reconstruction of bone defects and fracture reduction, in addition to oral and maxillofacial surgical applications such as in orthognathic surgery, alveolar ridge augmentation and dental implants. As the potential range of applications of the rhBMP-2 expands, a greater number of regions and conditions must be considered• Osteoblasts, chondrocytes, myocytes and adipocytes are all derived from common progenitor cells known as undifferentiated mesenchymal cells (Taylor and Jones, 1979). These cells are present in both muscle and subcutaneous tissue• Yamaguchi et al (1991) demonstrated that rhBMP-2 is involved not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation• Katagiri et al (1994) reported that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable. Therefore, it is more likely that the rhBMP2 induces the immature mesenchymal cells to differentiate into osteoblasts, leading to osteoinduction, in the muscle and in the subcutaneous tissue. In our previous study (Fujimura et al, 1995), a control group without BMP, using atelopeptide type-I collagen as a carrier, showed very little volume of bone formation in the muscle. This study revealed that even a small amount (5 gg) of rhBMP-2 can induce bone

Osteoinduction capability of rhBMP-2 in intramuscular and subcutaneous sites

l 15

supply to the skin could be more abundant in humans. In this study, there was no significant inter-site difference observed until day 3, and at 7 days both the ALP activity and Ca content were significantly higher (ALP: P<0.007, Ca: P<0.009) in the muscle than in the subcutaneous tissue. At 21 days, the significant difference was still observed for the Ca content. These results suggest that the difference in osteoinduction is related to the partial pressure of oxygen, i.e. the blood supply in the intramuscular and subcutaneous sites. The differences in ALP activity and Ca content were less pronounced at 21 days than at 7 days. The ALP activity can be thought of as an index for osteoblasts, while the Ca content might be related to the bone volume. The comparable elevation of ALP activity at the two sites at 21 days might indicate that the osteoblasts in the subcutaneous tissue had begun to proliferate and that their number was comparable to that in the muscle. As a result, the proliferation of osteoblasts can gradually result in bone formation. Therefore, it is likely that the difference in bone formation might become less marked after longer than 21 days. Further experiments seem to be necessary. Sampath and Reddi (1983) showed that allogenic implantation of rat extracellular demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. Fujimura et al (1995) reported that the degree of bone formation is dependent on the dose of rhBMP-2. From these findings and the results of the present study, it can be postulated that rhBMP-2 induces new bone in the subcutaneous tissue quite similarly to the induction in muscle. The findings obtained in the present study may be helpful for prognostic evaluation in implantation of rhBMP-2, if it is applied clinically in future studies.

Daniel, R. K., H. R Williams: The free transfer of skin flaps by micro vascular anastomosis. Plast. Reconstr. Surg. 52 (1973) 16-31 Fujimura, tL, K. Bessho, K. Kusumoto, I( Ogawa, T. lizuka: Experimental studies on bone inducing activity of composites of atelopeptide type I collagen as a carrier for ectopic osteoinduction by rhBMP-2. Biochem. Biophys. Res. Commun. 208 (1995) 316-322 Katagiri, 7~, A. Yamaguehi, E. Abe, N Takahashi, T. lkeda, F? Rosen J. ~L Wozney, A. Fujisawa-Sehra, T. Suda: Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127 (1994) 1755-1766 Kusumoto, K., K. Bessho, K. Fujimura, Y.. Konishi, Y. Ogawa, T. Iizuka: Comparative study of bone marrow induced by purified BMP and recombinant human BMP-2. Biochem. Biophys. Res. Commun. 215 (1995) 205-211 Mainous, E. G : Hyperbaric oxygen in maxillofacial osteomyelitis, osteoradionecrosis, and osteogenesis enhancement. In: Hyperbaric Oxygen Therapy. Undersea Medical Society Inc, Bethesda 1977 Mellande~ S., B. Johannson: Control of resistance, exchange, and capacitance functions in the peripheral circulation. Pharmacol Rev. 20 (1968) 117-196 Peacock, J. H. : Vasodilatation in the human hand. Observations on primary Raynaud's disease and acrocyanosis of the upper extremities. Clin. Sci. (London). 17 (1958) 575-586 Sampath, T. K., A. H. Reddi: Homology of bone-inductive proteins from human, monkey, bovine, and rat extracellular matrix. Proc. Natl. Acad. Sci. USA. 80 (1983) 6591-6595 Taylor, S. M., P A. Jones: Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell. 17 (1979) 771-779 Urist, M. R. : Bone formation by autoinduction. Science. 150 (1965) 893 899 Urist, M. R., Y. K. Huo, A. G Brownell, W.. M. Hohl, J, Buyske, A. Lietze, P. Tempst, M. Hunkapiller, R. J. Delange: Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc. Natl. Sci. USA. 81 (1984) 371-375 Wozney, J M., V Rosen, A. J Celeste, L. M. Mitsock, M. J Whitters, R. W. Kriz, R. M. Hewick, E. A. Wang: Novel regulators of bone formation: molecular clones and activities. Science. 242 (1988) 1528-1534 Yamaguehi, A., T. Katagiri, T. Ikeda, J. ~ Wozney, V. Rosen, E. A. Wang, A. J Kahn, T. Suda, S. Yoshiki: Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J. Cell Biol. 113 (1991) 681 687

REFERENCES

Dr Kazuya Yoshida Department of Oral and Maxillofacial Surgery Faculty of Medicine Kyoto University 54 Kawahara-cho Shogoin Sakyo-ku Kyoto 606 Japan

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Paper received 17 November 1997 Accepted 15 February 1998