Immunohistochemical observations of cellular differentiation and proliferation in endochondral bone formation from grafted periosteum:

ARTICLE IN PRESS Journal of Cranio-Maxillofacial Surgery (2003) 31, 356–361 r 2003 European Association for Cranio-Maxillofacial Surgery. doi:10.1016/S1010-5182(03)00081-7, available online at http://www.sciencedirect.com

Immunohistochemical observations of cellular differentiation and proliferation in endochondral bone formation from grafted periosteum: expression and localization of BMP-2 and -4 in the grafted periosteum Takaaki Ueno1, Toshimasa Kagawa1, Miwa Kanou1, Takashi Fujii1, Joji Fukunaga1, Nobuyoshi Mizukawa1, Toshio Sugahara1, Toshio Yamamoto2 1

Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University, Graduate School of Medicine and Dentistry, Okayama, Japan; 2 Department of Oral Morphology, Okayama University, Graduate School of Medicine and Dentistry, Okayama, Japan SUMMARY. Purpose: To clarify the involvement of bone morphogenetic proteins (BMPs) in the proliferation and

differentiation of osteo/chondrogenic cells during the process of bone formation from grafted periosteum. Material and Methods: Tibial periosteum of young Japanese white rabbits was grafted into suprahyoid muscles and removed after 7, 9, 14 or 21 days. BMP-2, -4, proliferative cell nucleus antigen (PCNA) immunoreaction and Alcian blue staining in grafted periosteum was then sought microscopically. Results: PCNA positive cells in the grafted periosteum expressed BMP-2 at 7 days. These cells differentiated into chondroblasts that expressed BMP-2 and Alcian blue at 9 days. After 14 days, cartilage formation was seen, and BMP-2 and -4 expressions were observed in mature and hypertrophic chondrocytes. Endochondral ossification was observed at 21 days and osteoblasts showed both BMP-2 and -4 expression. Conclusion: Both BMP-2 and -4 appear to play regulatory roles in the process of endochondral ossification from grafted periosteum, due to their involvement in the proliferation and differentiation into chondrogenic and osteogenic cells. r 2003 European Association for Cranio-Maxillofacial Surgery. Keywords: Bone morphogenetic protein-2 and -4 (BMP-2, and -4); Endochondral ossification; Periosteal graft

elucidated. Clarifying BMP involvement in the proliferation and differentiation of osteo- and chondrogenic cells may offer insight into whether BMP administration enhances new bone formation from grafted periosteum. Some investigators reported that local administration of BMP-2 induces ectopic bone formation and improves the healing of fractures (Sailer and Kolb, 1994; Takahashi, 2000; Murata et al., 2000). BMP-4 expression has also been demonstrated during fracture healing (Yaoita et al., 2000). Thus, BMP-2 and -4 are important growth factors in bone formation and fracture healing and were expected to be clinically useful in bone repairs. In the present study, the roles of BMP-2 and -4 were analysed in the process of bone formation from grafted periosteum. The proliferation and differentiation of osteo/chondrogenic cells in the grafted periosteum were observed histologically and ultrastructurally by looking for proliferative cell nuclear antigen (PCNA) and Alcian blue staining, respectively, and detecting BMP-2 and -4 immunoexpression.

INTRODUCTION Periosteum is known to induce heterotopic bone formation when implanted into muscle as a free graft (Burman and Umansky, 1930; Eyre-Brook, 1984). Considerable attention has been focused on its rich osteogenic potential as a tissue engineering material for the repair of bone defects (Nakahara et al., 1989; Vinzenz et al., 1996; Takushima et al., 1998; Reynders et al., 1999; Ueno et al., 2001). Recent molecular biological research has confirmed that bone development and bone fracture repair is regulated by bone growth factors, such as bone morphogenetic proteins (BMPs, Yaoita et al., 2000), insulin-like growth factors (IGFs, Zhao, 2000) and fibroblast growth factors (FGFs, Kuroda, 1999). In particular, BMPs can induce heterotopic bone formation when inserted subcutaneously into rats and mice (Wang et al., 1990; Sampath et al., 1992). Taken together, these findings raise the question whether BMP administration might enhance bone formation from grafted periosteum and thereby extend the application of periosteal grafting in the repair of bone defects. To date, only one report has focused on BMP-2 expression in this process of bone formation from grafted periosteum (Nishimura et al., 1999) and the role of BMPs in this process remains to be fully

MATERIAL AND METHODS Twenty-four Japanese white rabbits (1.5–2.0 kg) purchased from Charles River (Osaka, Japan) were 356

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housed at 251C and fed a standard animal diet (Oriental Co., Osaka, Japan) with water provided ad libitum for 1 week prior to the grafting operation. All surgical procedures were performed under anaesthesia with sodium pentobarbital (10 mg/ kg, b.w.). The grafting procedure employed in the present study was described previously (Ueno et al., 1999). Briefly, a section of periosteum of the tibia was carefully stripped and prepared (7
15 mm) before being folded and then sutured with 5-0 nylon to form a cylinder with the osteogenic layer facing inward. The harvested periosteum was then immediately grafted into the suprahyoid muscles, and each end of the grafted periosteum was secured to the periosteum of the mandible. The grafted periostea were removed at 7, 9, 14, and 21 days (n ¼ 5; respectively) after grafting and analysed together with four harvested non-grafted periosteum specimens. Animals were cared for in line with the Guidelines for Animal Research of Okayama University Dental School according to the principles of the Declaration of Helsinki. Observation by light microscopy All specimens were removed and fixed in 10% neutral buffered formalin before decalcification in 5% trichloroacetic acid for 7 days. They were subsequently dehydrated using a graded ethanol series and embedded in paraffin. Sections cut to a thickness of 6 mm were stained with haematoxylin–eosin and Alcian blue. Ultrastructural observation Five specimens were immersed in fixative, 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 for 7 days at 41C, then decalcified in 5% ethylenediaminetetraacetic acid (EDTA) buffered with 0.1 M cacodylate (pH 7.4) for 7 and 14 days. After decalcification, the specimens were post-fixed in 1% osmium tetroxide buffered with 0.1 M cacodylate (pH 7.4) for 60 min, dehydrated using a graded acetone series and embedded in Epon 812. Semi-thin sections cut to a thickness of 1 mm were stained with toluidine blue for observation under a light microscope. Ultrathin sections were stained with uranyl acetate and lead citrate for fine structural observation. The sections were observed under a Hitachi H-800 electron microscope at an accelerating voltage of 100 kV. Immunohistochemical observation Ten specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 41C for 24 h and prepared for immunohistochemical observation as previously described (Ueno et al., 2001). The slides were exposed to mouse monoclonal anti-proliferating

cell nuclear antigen (PCNA; DAKO, Glostrup, Denmark) for cell proliferation, goat polyclonal anti-BMP-2 antibody, and BMP-4 antibody (Santa Cruz Biotech, Inc. USA), to detect BMPs immunoexpression. The slides were incubated in a medium containing 0.05% 3,30 -diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide to visualize immunoreactivity. The sections were counter-stained with methyl green. For a negative control, some slides were incubated with 3% BSA in PBS instead of primary antibody. X-ray observation Grafted tissues were examined radiographically (Softex, 35 mA, 5 eV, 20 s) after fixation.

RESULTS Harvested periosteum ðn ¼ 4Þ Most cells in the periosteum were fibroblasts in a fibrous layer. Only a few osteoblastic cells were noted in the grafted tissue. Fibroblasts exhibited neither PCNA nor BMP immunoreactions (Fig. 1). Day 7 after grafting ðn ¼ 5Þ The number of fibroblasts had markedly increased in the grafted periosteum (Fig. 2A). These cells each had a large nucleus (Fig. 2B). Most of the cells were PCNA positive, indicating proliferating potential (Fig. 2C), and strong BMP-2 expression (Fig. 2D). Alcian blue staining and BMP-4 expressions were not detected in any cell at this stage. Day 9 after grafting ðn ¼ 5Þ Some fibroblasts differentiated into chondroblasts (Fig. 3A and B) showing Alcian blue staining in the extracellular matrix (Fig. 3C). These chondroblastic cells kept expressing BMP-2 (Fig. 3D). Day 14 after grafting ðn ¼ 5Þ Cartilage formation was apparent. Hypertrophic or mature chondrocytes were observed in the central part of the graft (Fig. 4A–C). The cartilagenous matrix was intensively stained with Alcian blue, indicating the presence of abundant proteoglycans (Fig. 4D). BMP-2 was continuously expressed in the mature and hypertrophic chondrocytes as shown in the early stage (Fig. 4E). BMP-4 began to be expressed in the mature chondrocytes and hypertrophic chondrocytes (Fig. 4F). At this stage no calcification was observed in the grafted periosteum.

ARTICLE IN PRESS 358 Journal of Cranio-Maxillofacial Surgery

Fig. 1 – Harvested periosteum. (A) Fibroblastic cells in the harvested periosteum. Haematoxylin–eosin,
60 (B) Electron microscopic findings of harvested periosteum,
3000. (C) No immunoreaction to BMP-2. (D) No immunoreaction to PCNA,
60. Counter-stained with methyl green.

Fig. 2 – Grafted periosteum 7 days after grafting. (A) Proliferation of cells in the grafted periosteum. Toluidine blue,
120. (B) Electron micrograph of proliferating cells in grafted periosteum. Fibroblastic cell showing well-developed cell organellae and rounded nucleus,
8000. (C) PCNA-immunoreaction of cells in the grafted periosteum,
100. (D) PCNA-positive fibroblastic cells show BMP-2 expression,
120. Counter-stained with methyl green.

Day 21 after grafting ðn ¼ 5Þ Endochondral ossification was observed, and chondrocytes were regularly arranged, i.e. hypertrophic chondrocytes adjacent to the bone trabeculae, and mature, oriented, proliferative chondrocytes and

located organized in distal direction (Fig. 5A). Low kV radiographs revealed calcification of grafted tissue (Fig. 5B). Active osteoblasts and osteoclasts were observed around newly formed trabeculae with osteoblasts expressing both BMP-2 and -4 (Fig. 5C and D).

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Fig. 3 – Grafted periosteum at 9 days. (A) Photomicrograph of cells differentiating into chondroblasts in grafted periosteum. Cell number is increased when compared with 7 days after grafting. T.B.: Toluidine blue,
100. (B) Electron microscopic finding of chondroblasts (Ch),
8000. (C) Cells start to show Alcian blue staining in extracellular matrix,
120. (D) Chondroblasts in grafted periosteum exhibit BMP-2 positive immunoreaction,
80.

DISCUSSION In the present study, BMP-2 and -4 expression were seen in osteo- and chondrogenic cells in the grafted periosteum, indicating that they are related to endochondral ossification. Some investigators have demonstrated BMP in the periosteum. Abe et al. (2000) also reported that BMP-2 and -4 are expressed in primitive mesenchymal cells and chondrocytes at the site of callus formation in the periosteum during fracture healing. Ohnishi et al. (1998) reported that BMPs were expressed in regenerating cartilage and bone cells in the periosteum during bone repair. Yaoita et al. (2000) reported that BMP-4 gene expression peaked early in the soft tissue surrounding bone during fracture healing. Although all of these reports clearly indicate that BMPs are associated with osteo- and chondrogenic cell proliferation and differentiation in the periosteum, little is known about the roles BMPs play in periosteal graft bone formation. We previously reported that grafted periosteum induces characteristic bone formation (Ueno et al., 2001). In this process, fibroblastic cells of the harvested periosteum proliferated and differentiated into chondrocytes to form cartilage preceding bone formation. In the present study, BMP-2 expression was observed in proliferating fibroblastic cells, chondroblasts, mature and hypertrophic chondrocytes, and osteoblasts. These findings suggest that BMP-2 is related to the whole process of endochondral ossification in the grafted periosteum. Such a finding

lends support to the conclusion by De Luca et al. (2001) that BMP-2 accelerates chondrocyte proliferation in cultured rat foetal bone. These authors observed proliferation of BMP-2 stimulated chondrogenic cells by incorporation of [(3)H] thymidine. The results of other studies also suggest that BMP-2 plays a role in chondrogenic differentiation of fibroblastic cells in the grafted periosteum: Murata et al. (2000) reported that recombinant human BMP2 (rhBMP-2) caused chondrogenic differentiation of myofibroblasts in their experimental study of heterotopic bone induction, and Takahashi (2000) demonstrated that rhBMP-2 induced differentiation of mesenchymal cells into osteoblasts and chondroblasts in an in vitro study. BMP-4 expression was identified in the mature and hypertrophic chondrocytes at the stage of cartilage formation; a finding that suggests BMP4 may be involved in hypertrophic differentiation of chondrocytes in grafted periosteum following cartilage formation. Anderson et al. (2000) also demonstrated immunoexpression of BMP-4 in a mature and hypertrophic chondrocyte zone in growth plates. CONCLUSION BMP-2 and -4 were involved in cell proliferation and differentiation in the grafted periosteum. BMP-2 was observed in fibroblasts, chondroblasts, mature chondrocytes, hypertrophic chondrocytes, and osteoblasts, suggesting its involvement in the entire

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Fig. 4 – Grafted periosteum at 14 days. (A) Cartilage formation from grafted periosteum. Haematoxylin–eosin,
45. (B) Electron microscopic finding of mature chondrocyte,
8000. (C) Electron microscopic finding of hypertropic chondrocytes,
8000. (D) Chondrocytes produce proteoglycan stained with strong Alcian blue,
50. (E) BMP-2 expression in mature and hypertrophic chondrocytes,
80. Counter-stained with methyl green. (F) BMP-4 expression first appears in mature and hypertrophic chondrocytes,
80. Counter-stained with methyl green.

Fig. 5 – Grafted periosteum at 21 days. (A) Cartilage replaced by newly formed trabecular bone endochondral ossification, Haematoxylin and Eosin,
25. (B) Soft X-ray finding of grafted periosteum depicts calcification. (C) BMP-2 expression in osteoblasts. (D) BMP-4 expression in osteoblasts,
120. Counter-stained with methyl green.

ARTICLE IN PRESS Expression and localization of BMP-2 and -4 in the grafted periosteum 361

process of endochondral ossification from grafted periosteum. BMP-4 was expressed at a later stage of cartilage formation and may play a role in differentiation of hypertrophic chondrocytes and involved in bone formation from osteoblasts.

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