Effects of recombinant human bone morphogenetic protein-2 on differentiation of cells isolated from human bone, muscle, and skin

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Effects of Recombinant Human Bone Morphogenetic Protein-2 on Differentiation of Cells Isolated from Human Bone, Muscle, and Skin K. KAWASAKI,1 M. AIHARA,1 J. HONMO,1 S. SAKURAI,1 Y. FUJIMAKI,1 K. SAKAMOTO,1 E. FUJIMAKI,1 J. M. WOZNEY,2 and A. YAMAGUCHI3 1

Department of Orthopedic Surgery, Showa University School of Medicine, Tokyo, Japan Genetics Institute, Inc., Andover, MA, USA 3 Department of Oral Pathology, Showa University School of Dentistry, Tokyo, Japan 2

Key Words: Osteoblasts; Muscle; Bone morphogenetic protein; Human; Cell culture.

We investigated the effects of recombinant human BMP-2 (rhBMP-2) on differentiation of cells isolated from human bone, muscle, and skin. Cells isolated from bones of six patients (HBM-1 to HBM-6), muscle from five patients (HM-1 to HM-5), and skin from three patients (HF-1 to HF-3) were used. rhBMP-2 had no effects on proliferation of two HBM cells, but had a stimulatory effect on three HM cell samples. rhBMP-2 stimulated both alkaline phosphatase (ALP) activity in all HBM cells and parathyroid hormone (PTH)-dependent cAMP production in three of the four HBM cell samples, although the magnitudes of these stimulatory effects differed among the cells tested. Although none of the HBM cells examined produced detectable amounts of osteocalcin in the absence of 1,25-(OH)2vitamin D3, they synthesized measurable amounts of osteocalcin in its presence. rhBMP-2 inhibited 1,25-(OH)2vitamin D3-dependent osteocalcin production in all of the HBM cell samples. Transplantation of HBM-6 cells with rhBMP-2 using diffusion chambers into the peritoneal cavity of athymic mice induced formation of cartilage and bone in the diffusion chambers, but neither cartilage nor bone was formed in chambers transplanted without rhBMP-2. rhBMP-2 also stimulated ALP activity in all of the HM and HF cell samples examined and PTH-dependent cAMP production in three of four HM cell samples. rhBMP-2 induced no osteocalcin production in any of the HM or HF cells in the presence of 1,25(OH)2vitamin D3. rhBMP-2 markedly inhibited myotube formation by all of the HM cell samples. Transplantation of HM-4 cells with rhBMP-2, using diffusion chambers, into athymic mice induced ALP-positive cells in the chambers, but neither cartilage nor bone was observed. These results suggest that rhBMP-2 is a potent stimulator of osteoblast differentiation and bone formation in human cells. (Bone 23: 223–231; 1998) © 1998 by Elsevier Science Inc. All rights reserved.

Introduction Bone has remarkable regenerative potential during repair of fractures and skeletal defects. In cases associated with large skeletal defects, however, the regenerative ability of bones themselves is often not sufficient for complete repair. Stimulating new bone formation might be an important application for growth factors. Bone morphogenetic protein (BMP), which was first identified as a protein capable of inducing ectopic bone formation at sites of implantation,25 is of great clinical importance to such factors.6,14,24,32 Wozney et al.28 first cloned four cDNAs encoding human BMPs (BMP-1, BMP-2A [BMP-2], BMP-3, and BMP-4 [BMP-2B]), and demonstrated that, with the exception of BMP-1, they belonged to the transforming growth factor-b (TGF-b) superfamily. The BMP subfamily is composed of at least 15 molecules. Among the members of the BMP subfamily, recombinant proteins of several BMPs capable of inducing ectopic bone formation in vivo have been successfully produced.16,19,27,28 Although many experiments using these recombinant proteins have been conducted to explore their effects on osteoblast differentiation and bone formation,10 –12,16,19,21,23,31 most studies have been performed using lower animals or cells derived from rats and mice. Information concerning the effects of BMP on osteoblast differentiation in human cells has accumulated,1,12,13,20,33 but the results are confusing, because rhBMPs exert different effects on osteoblast differentiation among the cell samples tested. Skeletal tissue is composed of various types of mesenchymal cells such as osteoblasts, chondrocytes, muscle cells, and bone marrow stromal cells, including adipocytes. These cell lineages are believed to originate from common pluripotent progenitors, known as skeletal mesenchymal stem cells.17 These progenitors acquire specific phenotypes depending on maturation stage during the differentiation process, but the precise regulatory mechanism that controls the differentiation process of these cell lineages is still unclear. We investigated the roles of BMPs in the differentiation of skeletal mesenchymal cells using several cell lines derived from rats and mice.10,11,29 –31 BMP-2 was shown to induce differentiation of undifferentiated mesenchymal progenitors into osteoblasts and/or chondrocytes.10,26 BMP-2 also stimulated the committed osteoprogenitors to differentiate into more

Address for correspondence and reprints: Akira Yamaguchi, D.D.S., Ph.D., Department of Oral Pathology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.

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Table 1. Clinical characteristics of the patients from whom cells were isolated and the sites from which tissues were obtained Name of cells Bone-derived cells HBM-1 HBM-2 HBM-3 HBM-4 HBM-5 HBM-6 Muscle-derived cells HM-1 HM-2 HM-3 HM-4 HM-5 Skin-dervied cells HF-2 HF-1 HF-3

Age

Gender

Clinical diagnosis

Tissue sites

20 18 17 23 30 24

M F M M M M

Fracture of iliac bone Dysplasia of iliac bone Dysplasia of iliac bone Fracture of iliac bone Fracture of iliac bone Fracture of iliac bone

Iliac Iliac Iliac Iliac Iliac Iliac

21 30 12 36 11

M M M M M

Fracture of clavicle Fracture of iliac bone Wryneck Dysplasia of iliac bone Fracture of spine

M. M. M. M. M.

21 24 18

M M F

Fracture of clavicle Fracture of iliac bone Fracture of iliac bone

Gluteal skin Inginal skin Gluteal skin

bone bone bone bone bone bone

pectoralis major gluteus maximus pectoralis major adductor magnum regio nuchakis

HBM-6 and HF-1 cells, and HN-1 and HF-2 cells, respectively, were isoalted from the same patients. Other cells were isolated from different patients.

mature osteoblasts.31 Bone marrow-derived stromal cells differentiated into bone-forming osteoblasts in response to BMP2.23,30 In contrast to the stimulatory effects on osteoblast differentiation, BMP-2 inhibited differentiation of myogenic cells into myotubes.11,15,29 In some myogenic cells, BMP-2 converted the differentiation pathway of the cells to the osteoblast lineage.11 Thus, the role of BMPs in the regulation of differentiation pathways of mesenchymal cells has been studied extensively using cells derived from rats and mice. However, such effects of BMPs on human mesenchymal cells have not been fully investigated. More extensive basic studies using human cells are required so that BMP can be used appropriately for clinical applications. In this study, we investigated the effects of recombinant human BMP-2 (rhBMP-2) on differentiation of cells isolated from human bone, muscle, and skin using in vitro culture and in vivo transplantation experiments. Our results provide important information in support of clinical application of rhBMP-2 in man. Materials and Methods Recombinant Human Bone Morphogenetic Protein-2 rhBMP-2 was provided by Yamanouchi Pharmaceutical Co. (Tokyo, Japan). This recombinant protein was produced in Chinese hamster ovary cells and purified as described previously.6 Isolation and Culture of Cells From Human Bone, Muscle, and Skin Small biopsy specimens (5 3 5 mm), obtained from iliac bones of six patients, muscle of five patients, and skin of three patients, were used for isolation of cells from each tissue. They were obtained from patients not suffering from systemic dysplasia of bones or metabolic bone diseases. All patients provided written informed consent in accordance with the Helsinki Declaration. The clinical features of these patients and the sites from which the samples were obtained are summarized in Table 1. The patients did not suffer from specific diseases other than those indicated in the table. To obtain bone-derived cells, bone samples were minced and

washed twice with calcium and magnesium-free phosphatebuffered saline [(PBS(2)]. They were incubated for 1 h with gentle shaking in solution containing 0.1% collagenase and 0.2% dispase in PBS(2) at 37°C. This process was performed twice, and the cells thus obtained were combined and cultured separately for each case. They were designated HBM-1 to HBM-6. Muscle-derived cells were isolated by digestion of the muscle specimens with 0.05% trypsin for 1 h at 37°C according to the method of Blau et al.5 (designated HM-1 to HM-5). To allow skin fibroblasts to adhere to the surface of culture dishes, the skin samples were placed on the surface of 35 mm dishes for 1 h without supplement of culture medium at 37°C, then they were cultured for 7 days with supplement of culture medium. The skin fibroblasts that migrated from the explants were collected by digestion with 0.1% trypsin and 0.02% ethylene-diamine tetraacetic acid (EDTA) in PBS(2) (designated HF-1 to HF-3). This cell population contained few keratinocytes after subculture, as judged immunohistochemically using wide-spectrum keratin (data not shown). All experiments were performed using cells within three passages, and they were inoculated at a density of 2 3 104 cells/cm2. HBM cells and HF cells were cultured with a-minimum essential medium (a-MEM; Gibco Laboratories, Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin). HM cells were maintained in a-MEM supplemented with 15% FBS and antibiotics. In some experiments, HM cells were cultured with a-MEM containing 5% FBS to induce myogenic differentiation. These HBM, HF, and HM cells were treated with various concentrations of rhBMP-2 for 3 or 6 days, and the following parameters were measured. Measurement of Cell Proliferation The cells were cultured for 3 days with various concentrations of rhBMP-2, and the cell number in each well was estimated using a cell-counting kit (Dojindo Laboratories, Kumamoto, Japan). This technique employs a tetrazolium salt, WST-1, which produces a highly water-soluble formazan dye.8 The relative cell number was determined by measuring absorbance of the formazan dye product in the cultures at a wavelength of 405 nm

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after incubation with the reagents in the kit according to the manufacturer’s instructions. Measurement of Alkaline Phosphatase Activity To examine ALP-positive cells histochemically, the cells were fixed with 10% neutral buffered formalin in PBS for 20 min. ALP activity in the cells was determined using naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, MO) as a substrate and fast blue BB salt (Sigma) as a coupler, as described previously.11 ALP activity was also determined biochemically by an established technique using p-nitrophenylphosphate as substrate.31 Protein concentration was determined using BCA protein assay reagent (Pierce Chemical Co., Rockford, IL).

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coating were also transplanted into athymic mice as controls. Seven weeks later, the diffusion chambers were removed, fixed in 10% neutral buffered formalin, and embedded in Technovit 8100 (Heraeus Kulzer, Germany). Sections 4 mm thick were prepared, and stained by the von Kossa method to visualize mineralization and with toluidine blue for identification of cartilage. Statistical Analysis The results are expressed as the mean 6 SEM. Statistical analysis between the two groups was evaluated by Student’s t-test or two-way ANOVA. Results

cAMP Production in Response to PTH Cells were preincubated for 20 min with a-MEM containing 0.5% BSA and 1 mmol/L 3-isobutyl-1-methylxanthine. After removal of the preincubation media, cells were incubated for 8 min with 200 ng/mL of human parathyroid hormone [hPTH(134); provided by Dr. Hori, Asahi Chemical Co., Shizuoka, Japan] dissolved in the same culture medium. The cAMP concentrations in the cell layers were determined by RIA using a cAMP assay kit (Yamasa Co., Chiba, Japan). Osteocalcin Production The amount of osteocalcin secreted into the culture medium was determined by immunoradiometric assay (IRMA) using a human osteocalcin assay kit (Mitsubishi Yuka Co., Tsukuba, Japan). Immunohistochemistry Myotubes that appeared in HM cell cultures were examined immunohistochemically for myosin heavy chain (MHC) expression. To detect MHC, cells were fixed for 10 min with a cold acetone/ethanol mixture (1:1), and stained by an indirect immunoperoxidase technique using mouse antihuman MHC monoclonal antibody (Nichirei Co., Tokyo). After incubation with this antibody, the cells were incubated with biotinylated secondary antibody against mouse IgG, then reacted with peroxidase-labeled streptavidin (Nichirei Co.). The reaction products were visualized using an AEC substrate kit (Nichirei Co.). The numbers of MHC-positive myotubes were counted in 0.785 cm2 fields in arbitrarily selected areas of each well after immunohistochemical staining. Double staining for ALP activity and immunohistochemical staining for MHC was also performed. In this case, the cells were stained for ALP as described above just before development of immunohistochemical reaction products with AEC. Transplantation of Cells in Diffusion Chambers Diffusion chambers containing rhBMP-2 were prepared as described previously.30 HBM-6 or HM-4 cells (3–5 3 106 cells in 100 mL of culture medium) were loaded into the diffusion chambers. To allow the cells to adhere to membrane filters precoated with 5 mg of rhBMP-2, each diffusion chamber was incubated for 12 h with a-MEM containing 10% FBS at 37°C in an atmosphere of 5% CO2 in air. Diffusion chambers were transplanted into the peritoneal cavities of 6-week-old athymic male mice (one diffusion chamber per mouse). Three diffusion chambers coated with rhBMP-2 were transplanted for each cell type. The same number of diffusion chambers without rhBMP-2

Characterization of Cells Isolated From Human Bone, Muscle, and Skin Although cells isolated from bone, muscle, and skin exhibited various levels of ALP activity when cultured in the absence rhBMP-2, the cells isolated from bone and muscle showed higher ALP activity than those isolated from skin (see Figure 2). With the exception of HF-3, PTH exposure increased intracellular cAMP levels to varying degrees in all cell samples examined (see Figure 3). No osteocalcin was produced by any of the six HBM cell samples cultured for 6 days in the absence of 1,25(OH)2vitamin D3. However, these cells synthesized measurable levels of osteocalcin when cultured for the last 24 h with 1,25-(OH)2vitamin D3 (see Figure 4A). None of the HM and HF cell samples produced detectable levels of osteocalcin in the presence or absence of 1,25-(OH)2vitamin D3 (data not shown). A substantial number of myotubes appeared in cultures of cells isolated from muscle (HM cells) (see Figure 5). HM cells cultured with 5% FBS generated more myotubes than those cultured with 15% FBS. Effects of rhBMP-2 on Cell Proliferation Cell proliferation was examined in two samples of HBM cells (HBM-2 and HBM-6), three of HM cells (HM-1, HM-2, and HM-5), and one of HF cells (HF-1). The cell numbers estimated using a cell proliferation kit increased 43% in HM cells and 21% in HBM cells during the first 3 days in culture. rhBMP-2 had no effect on proliferation of these HBM (Figure 1A) or HF cells (data not shown) at any concentration tested, compared with control cultures. In contrast, rhBMP-2 stimulated proliferation of the three HM cell samples at concentrations .50 ng/mL (Figure 1B). Effects of rhBMP-2 on ALP Activity rhBMP-2 significantly increased ALP activity in all of the HBM cell samples at a concentration of 500 ng/mL, compared with those in cultures without rhBMP-2 (Figure 2A). The maximum effect of rhBMP-2 at this concentration was observed in HBM-6 cells with a 5.4-fold increase compared with untreated cells. Dose-response experiments showed increased ALP activity in HBM-5 cells treated with rhBMP-2 at concentrations .50 ng/ mL, and in HBM-1, HBM-2, HBM-3, HBM-4, and HBM-6 cells at concentrations .250 ng/mL (Figure 2A). ALP activity in all of the HM cell samples was significantly increased by treatment for 3 days with 500 ng/mL rhBMP-2 (Figure 2B). rhBMP-2 significantly elevated enzyme activity in all HM cell samples at concentrations .250 ng/mL. rhBMP-2 significantly stimulated ALP activity in HF-2 at

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Figure 1. Dose-response effects of rhBMP-2 on cell proliferation in HBM (A) and HM (B) cells. Cells were plated in 96 well plates and cultured for 3 days with graded concentrations of rhBMP-2. The numbers of cells were estimated using a cell proliferation kit as described in Materials and Methods. Data are means 6 SE of three wells. Asterisks: significantly different from cells cultured without rhBMP-2 (p , 0.05), using Student’s t-test.

concentrations .250 ng/mL, and in HF-1 and HF-3 cells at 500 ng/mL (Figure 2C). Effects of rhBMP-2 on PTH Responsiveness We assessed PTH response by measuring cAMP production in four HBM cell samples (HBM-1, -2, -4, and -5) treated for 3 days with or without 500 ng/mL rhBMP-2. rhBMP-2 significantly elevated PTH responses in HBM-1, HBM-2, and HBM-5 cells (Figure 3A) with the maximum effect observed in HBM-5 cells, compared with that in BMP-2-untreated cells. No apparent effect of rhBMP-2 was observed in HBM-4 cells. rhBMP-2 significantly increased PTH responses in three of the four HM cell samples tested (HM-1, -3, and -5) compared with each value in rhBMP-2-untreated cells (Figure 3B). No significant increase was observed in HM-4 cell samples between rhBMP-2-treated and -untreated cells. Among the skin fibroblasts, one of the HF cell samples (HF-1) exhibited a slight increase in cAMP in response to PTH following rhBMP-2 treatment, but it was not significantly different from rhBMP-2-untreated cells (Figure 3C).

Figure 2. Dose-response effects of rhBMP-2 on ALP activity in HBM (A), HM (B), and HF (C) cells. Cells were cultured for 3 days with graded concentrations of rhBMP-2. ALP activity was determined as described in Materials and Methods. Data are means 6 SE of three wells. Asterisks: significantly different from cells cultured without rhBMP-2 (p , 0.05), using Student’s t-test.

Effects of rhBMP-2 on Osteocalcin Production Treatment with rhBMP-2 (500 ng/mL) significantly inhibited 1,25-(OH)2vitamin D3-dependent osteocalcin production in all of the HBM cell samples (Figure 4A). To confirm the inhibitory effects of rhBMP-2 on osteocalcin production, we conducted dose-response experiments using HBM-4 and HBM-6 cells. At concentrations .250 ng/mL, rhBMP-2 significantly inhibited 1,25-(OH)2vitamin D3-dependent osteocalcin production in these cells (Figure 4B). None of the HBM cells treated for 6 days with

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Figure 4. Effects of rhBMP-2 on osteocalcin production of HBM cells. Cells were cultured for 6 days in the absence (open bars) or presence (closed bars) of 500 ng/mL rhBMP-2 (A). Dose-response effects of rhBMP-2 on osteocalcin production of HBM-4 (closed circles) and HBM-6 (open squares) cells (B). Cells were cultured for 6 days with various concentrations of rhBMP-2. They were treated with 1,25(OH)2vitamin D3 (2 3 1028 mol/L) for the last 24 h, and the amounts of osteocalcin secreted into culture media during the last 24 h were determined by RIA as described in Materials and Methods. Data are means 6 SE of three wells. Asterisks: significantly different from controls without BMP-2 (p , 0.05), using Student’s t-test.

Effects of rhBMP-2 on Myogenic Differentiation Figure 3. Effects of rhBMP-2 on cAMP production in response to PTH in HBM (A), HM (B), and HF (C) cells. Cells were cultured for 3 days in the absence (open bars) or presence (closed bars) of 500 ng/mL rhBMP-2, then treated for 8 min with 200 ng/mL hPTH(1-34). The amounts of cAMP produced were determined as described in Materials and Methods. Data are means of three wells. Asterisks: significantly different from corresponding controls without BMP-2 (p , 0.05), by two-way ANOVA.

500 ng/mL rhBMP-2 produced detectable levels of osteocalcin in the absence of 1,25-(OH)2vitamin D3. Treatment of HM and HF cells for 6 days with 500 ng/mL rhBMP-2 induced no osteocalcin production in the absence or presence of 1,25-(OH)2vitamin D3.

In all of the HM cell samples tested (HM-1 to -5), 500 ng/mL rhBMP-2 almost completely inhibited myotube formation as judged by immunohistochemical staining for MHC (Figures 5 and 6). Quantitative analysis of MHC-positive cells demonstrated that rhBMP-2 inhibited myotube formation at concentrations .50 ng/mL in three HM cell samples (HM-2, -3, and -5), and .250 ng/mL in two HM cell samples (HM-1 and HM-4) (Figure 6). Formation of MHC-positive myotubes was almost completely inhibited by treatment with 500 ng/mL of rhBMP-2 in all of the HM cells. Figure 5 shows the results of double staining for ALP and immunohistochemical staining for MHC in HM cells. Numerous MHC-positive cells and several ALP-positive cells were observed in cultures without rhBMP-2. Treatment with 500 ng/mL rhBMP-2 almost completely inhibited the appearance of MHC-

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Figure 5. Effects of rhBMP-2 on muscle cell differentiation. HM-2 cells were cultured for 3 days with a-MEM containing 5% FBS in the absence (A) or presence (B) of 500 ng/mL rhBMP-2. The cells were dual stained for ALP activity and for myosin heavy chain (HMC) expression as described in Materials and Methods. Blue staining represents ALP activity, and red represents HMC-positive myotubes.

positive myotubes, and increased the number of ALP-positive cells.

in the diffusion chambers, but formation of neither bone nor cartilage was observed (data not shown).

Transplantation Experiments

Discussion

HBM-6 cells transplanted for 7 weeks into the peritoneal cavity of athymic mice using diffusion chambers without BMP-2 generated only fibrous connective tissue (Figure 7A). In contrast, two of three diffusion chambers transplanted with BMP-2 for 7 weeks generated mineralized foci as determined by soft X-ray examination. Histological examination revealed that these diffusion chambers contained calcified cartilage and bone (Figure 7B). Large numbers of unmineralized hypertrophic chondrocytes were also observed in the diffusion chambers. Another diffusion chamber transplanted with rhBMP-2 exhibited proliferation of fibrous connective tissues without apparent cartilage or bone formation. HM-4 cells similarly transplanted with rhBMP-2 for 7 weeks into athymic mice showed induction of ALP-positive cells

BMPs affect proliferation of various cell types. In this study, BMP-2 induced no changes in proliferation of two HBM cell samples isolated from human bones and one HF cell sample from human skin, while showing a slight stimulatory effect on proliferation of three HM cell samples isolated from human muscle. Shibano et al.20 also demonstrated that rhBMP-2 induced no significant changes in the doubling time of cultured osteogenic stromal cells. Zheng et al.33 reported that rhBMP-2 stimulated cell proliferation in a human osteoblast-like cell line (HOB1T). The discrepancy in the effects of rhBMP-2 on cell proliferation between our osteoblast-like cells and HOB1T might have been due to the difference in nature of the cells used; that is, primary cells isolated from human bones vs. human cell lines established by transfection with SV40 large T antigen.33 Figure 8 summarizes the relative effects of rhBMP-2 on ALP activity, PTH response, and osteocalcin production in each cell sample tested in the present study. Our results indicate that rhBMP-2 stimulated ALP activity and/or PTH responsiveness, early markers of osteoblast differentiation, in many cells isolated from bone, muscle, and skin. These results raise the possibility that rhBMP-2 stimulated osteoblast differentiation in human cells isolated not only from bones but also from muscle and skin, as demonstrated in rodents.10,11,22,23,27,30,31 However, the magnitudes of these stimulatory effects differed among the cells tested. In addition, the stimulatory effects of rhBMP-2 on ALP activity and PTH responsiveness did not always correlate even in the same cell population. These results suggest that the stimulatory effects of rhBMP-2 on ALP activity and PTH responsiveness were regulated differently by rhBMP-2, because we measured these parameters using the same samples of cells to prevent changes caused by passage. Alternatively, these results might be attributable to heterogeneity of the cells used in the present study. Opposite effects of BMP on ALP activity between cell types used were also reported by Knutsen et al.12; OP-1 (BMP-7) stimulated ALP activity in the human osteosarcoma cell line TE85 but had an inhibitory effect in bone cells derived from the human mandible. BMPs induced or stimulated synthesis of

Figure 6. Dose-response effects of rhBMP-2 on myotube formation in HM cells. HM cells were cultured with a-MEM containing 5% FBS for 3 days in the absence or presence of various concentrations of rhBMP-2. The numbers of HMC-positive myotubes were counted after staining for HMC as described in Materials and Methods.

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Figure 7. Histological analysis of HBM-6 cells transplanted into the peritoneal cavity of athymic mice using diffusion chambers. HBM-6 cells transplanted with BMP-2 formed mineralized cartilage and bone in the diffusion chamber (B), but cells implanted without BMP-2 produced only fibrous connective tissue (A). The sections were stained by the von Kossa method and with hematoxylin and eosin. O: outside of diffusion chamber; M: membrane filter; I: inside of diffusion chamber.

osteocalcin, which is a marker of more mature osteoblasts, in many cells isolated from rats and mice. Lecanda et al.13 recently reported that rhBMP-2 stimulated osteocalcin production in both human bone marrow stromal cells (relatively immature preosteoblastic cells) and osteoblasts (more differentiated osteoblasts); rhBMP-2 more effectively stimulated osteocalcin production in the former than in the latter. Amedee et al.1 also reported that BMP-3 induced osteocalcin synthesis in human bone marrowderived cells as well as type I collagen production, ALP activity, and PTH response. In contrast, in the present study, rhBMP-2 induced no production of osteocalcin in any of the cells tested, but inhibited 1,25-(OH)2vitamin D3-induced osteocalcin synthesis. We also confirmed inhibitory effects of rhBMP-2 on 1,25-(OH)2vitamin D3-induced osteocalcin synthesis in bone cells isolated from human mandibulae at both mRNA and protein levels (manuscript submitted). Recently, Shibano et al.20 reported that rhBMP-2 exerted no stimulatory effect on osteocalcin production in osteogenic stromal cells, whereas 1,25-(OH)2vitamin D3 stimulated its production in the same cell population. Thus, the in vitro effects of BMPs on osteoblast differentiation in human cells are controversial. This might be due to differences in the nature of human osteoblast-like cells depending on methods used for isolation of cells and donor characteristics such as age. Although isolation of homogeneous cell populations from normal human tissues is very difficult at present,18 it will be important to develop more consistent techniques to isolate human cells for further investigation into the effects of BMPs on human cells. Extensive investigation of BMP receptors will also provide insight into the mechanism responsible for the conflicting effects of BMPs on human cells. To confirm further that rhBMP-2 induced osteoblast differentiation in human cells, we transplanted human bone-derived cells into athymic mice using diffusion chambers. The cells isolated from human bones formed bone in the diffusion cham-

Figure 8. Relative effects of rhBMP-2 on ALP activity, PTH responsiveness, and 1,25-(OH)2vitamin D3-induced osteocalcin production in HBM, HM, and HF cells. Values represent percent increase in each cell sample treated for 3 days with rhBMP-2 (500 ng/mL) compared with cells cultured in the absence of rhBMP-2. The horizontal dotted lines in each graph represent values of control cells (100%). Stars: values not determined. No osteocalcin production was detected in HM or HF cells in the presence or absence of rhBMP-2 and 1,25-(OH)2vitamin D3.

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bers when transplanted with rhBMP-2, but not in its absence. This indicates that rhBMP-2 promoted the formation of bone from bone-derived human cells. In our in vitro experiments, rhBMP-2 induced no osteocalcin production in HBM cells, and inhibited 1,25-(OH)2vitamin D3-induced osteocalcin synthesis. Osteocalcin production is believed to be a marker of mature osteoblasts, but our in vitro and in vivo experiments raised the possibility that synthesis of osteocalcin in vitro cannot simply reflect an ability to form bone in vivo. This was supported by recent findings in osteocalcin knockout mice; that is, osteocalcindeficient mice exhibited increased bone formation, compared with wild-type controls.7 We demonstrated, by in vivo transplantation experiments, that the cells isolated from human bones also formed cartilage in the diffusion chambers in response to rhBMP-2. In our in vitro experiments, however, rhBMP-2 induced no chondrocyte differentiation in human bone-derived cells, as judged by Alcian blue staining and immunohistochemical analysis of type II collagen expression (unpublished observation). These results suggest that human bone-derived cells retain the capacity to differentiate into both osteoblasts and chondrocytes in response to rhBMP-2, whereas their capacity might be more easily evoked following in vivo transplantation into athymic mice than in simplified in vitro culture systems. When muscular tissues are injured or exposed to some dystrophic stimuli, the satellite cells, which are mononuclear cells lying along the muscle fibers in the normal muscle, divide and differentiate into myoblasts, which fuse to form muscle fibers.9 The effects of BMPs on muscle cell differentiation have been reported.15,31 We also reported that rhBMP-2 not only inhibited muscle differentiation but also converted the differentiation pathway into the osteoblast lineage in both the C2C12 myoblasts originating from satellite cells and the primary myogenic cells isolated from newborn mice.11 This suggests that the satellite cells were potential progenitors differentiating into osteoblasts in response to BMPs during ectopic bone formation in muscular tissues. To examine whether a similar conversion occurs in human muscle-derived cells, we investigated the effects of rhBMP-2 on differentiation of cells isolated from human muscle. rhBMP-2 strongly inhibited the formation of MHC-positive myotubes and increased osteoblast phenotypic markers such as ALP activity and PTH responsiveness in human muscle-derived cells. However, rhBMP-2 could not induce these cells to differentiate into more mature osteoblasts producing osteocalcin as shown in rodent cells.11 In addition, transplantation of myogenic cells with rhBMP-2 using diffusion chambers into athymic mice induced ALP-positive cells within the chambers, but did not induce the formation of bone or cartilage. Interestingly, several reports have demonstrated that transplantation of BMPs into primate muscle induced no or less ectopic bone formation,2– 4,14 whereas such transplantation effectively induced ectopic bone formation in rodents. There is a possibility that the potential of human muscle cells for differentiation into other cell lineages, including osteoblasts and chondrocytes, might be more restricted when compared with that in lower animals such as rodents. Further experiments are needed to confirm this hypothesis. Transplantation of BMPs into subcutaneous tissues also induced ectopic bone formation in rodents. We confirmed that rhBMP-2 induced osteoblast differentiation in cultured skin fibroblasts isolated from adult rats (unpublished results). Because these observations suggest that multipotent progenitors capable of differentiation into osteogenic cells in response to BMPs are also present in the subcutaneous tissues, we investigated the osteogenic potential of human skin fibroblasts. rhBMP-2 stimulated ALP activity in all of the skin fibroblast samples, but failed to induce osteocalcin production. Thus, the differentiation capac-

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ity into osteogenic lineages of cells from human subcutaneous tissues might also be more restricted than that in rodents. In the present study, we demonstrated that rhBMP-2 promotes osteoblast and chondrocyte differentiation in human bone-derived cells by in vitro and in vivo experiments. These results support the potential clinical application of rhBMP-2, but further studies, including investigation of the molecular aspects of these effects, are necessary to clarify precisely the mechanism of action of rhBMP-2 in human cells. Acknowledgment: The authors thank Dr. Y. Tashiro (Showa University) for assisting in the statistical analysis. This work was supported in part by grants-in-aid from the Ministry of Science, Education and Culture of Japan.

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Date Received: November 18, 1997 Date Revised: May 27, 1998 Date Accepted: May 28, 1998