Enhancement of Bone Morphogenetic Protein-2-Induced Ectopic Bone Formation by Transforming Growth Factor-β1

Abstract

Bone morphogenetic proteins (BMPs) possess osteoinductive activities and are useful for clinical treatments, including bone regeneration. We found that transforming growth factor (TGF)-β1 strongly enhances the osteoinductive activity of BMP-2. Collagen sponges containing 5 μg of BMP-2 were implanted into mouse muscle tissues, after which lump-like masses appeared and grew until day 7. Subsequently, calcification occurred in the lump-like masses by day 14. Addition of 50 ng of TGF-β1 to the BMP-2-containing sponges markedly accelerated the growth of the lump-like masses and resulted in a fivefold increase in total bone volume as compared with BMP-2 alone. The number of osteoblasts in ectopic bone tissues at 14 days after implantation induced by BMP-2+TGF-β1 was twofold greater than that with BMP-2 alone, whereas the number of osteoclasts was decreased by half. On the other hand, TGF-β1 accelerated the differentiation of both osteoblasts and osteoclasts in the early stage (2–7 days after implantation) of ectopic bone formation. We also implanted collagen sponges into bone defects surgically created in mouse calvaria. Sponges containing 2.5 μg of BMP-2 and 25 ng of TGF-β1 caused complete filling of the defects with orthotopic bone, whereas those containing 2.5 μg of BMP-2 alone caused only partial filling. These results suggest that TGF-β1 enhances BMP-2-induced ectopic bone formation by accelerating the growth of lump-like masses, and regulates osteoblast and osteoclast generation. Our findings may contribute to the development of a new treatment method for skeletal disorders.

Introduction

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF)-β superfamily and are essentially involved in embryogenesis,^1–4 skeletal development,^5–8 and fracture healing.^9–12 These proteins promote differentiation of meschenchymal cells into chondrocytes and osteoblasts, which play critical roles in bone development and metabolism.

Among the various BMP members, BMP-2, −4, −6, and −7 have shown potential to induce ectopic bone formation when implanted into muscle tissues.^12–20 This osteoinductive activity of BMPs is considered to be useful for reconstructive surgery of the skeleton, as well as treatment of bone defects associated with periodontal diseases and rheumatoid arthritis. Although use of recombinant BMP-2 protein has been approved for treatment of some skeletal disorders, high doses of BMP proteins are necessary to regenerate an adequate amount of bone mass.^21–24 Thus, identification of factors that strongly enhance the osteoinductive activities of BMPs may lead to more effective treatments for bone regeneration.

To identify factors that enhance the osteoinductive activities of BMPs, a system for induction of ectopic bones in mice has been widely used.^25–34 In this system, ectopic bones are induced by implantation of carriers such as collagen sponges that contain BMPs. After implantation, fibroblastic mesenchymal cells surround the collagen sponges, with bone tissues appearing at around 2 weeks after implantation.³⁵ Thus, factors added to the carriers that enhance osteoinductive activity would cause an increase in the volume of the ectopic bone tissues.

To elucidate a powerful enhancer of BMPs, we examined the effects of various molecules related to bone metabolism (e.g., parathyroid hormone, active vitamin D₃, β-estradiol, activin, and TGF-β1; data not shown) associated with ectopic bone formation induced by BMP-2 and found that TGF-β1 strongly enhances ectopic bone formation induced by BMP-2.

In the present study, we show the effects of TGF-β1 on ectopic bone formation, which will contribute to the development of new method for skeletal treatment using BMP-2.

Materials and Methods

Cytokines and animals

Purified recombinant human BMP-2 was kindly provided by Astellas Pharmaceuticals Co., Ltd. Recombinant human TGF-β1 was purchased from R&D Systems. Bovine serum albumin (BSA) fraction V was purchased from Sigma-Aldrich. Experimental mice (ddY strain, male, 5 weeks old) were purchased from Sankyo Laboratories Animal Center. Mice were housed and acclimated in cages with free access to food and water for 1 week. Before surgery, mice were anesthetized using intraperitoneal sodium pentobarbital (50 mg/kg).

Preparation of collagen sponges

We added 10 μL of HEPES buffer (0.2 M, pH 7.4) to 90 μL of a type I collagen gel solution (3 mg/mL, Cell Matrix^®; Nitta Gelatin, Inc.) in 1.5 mL microtubes, and mixed them by pipetting. Subsequently, 1, 3, 5, or 10 μg of BMP-2 (dissolved in LF6 buffer consisting of 0.074% L-glutamic acid, 0.03% sodium chloride, 2.5% glycine, 0.5% sucrose, and 0.01% Tween 80, pH 4.5), and/or 1, 10, 50, or 150 ng of TGF-β1 (dissolved in phosphate-buffered saline [PBS] containing 0.1% BSA), or the vehicle (PBS containing 0.1% BSA) were added to the microtubes. These collagen gel solutions were frozen in liquid nitrogen and freeze-dried for 6–12 h. Dried collagen in the microtubes was gently pressed with a stick to form disc shapes (4 mm in diameter, 0.5 mm thick, and 0.5 mg).

Measurement of ectopic bone volume

Lump-like masses formed in mice were isolated and processed for microcomputed tomography (μCT: SMX-90CT inspeXio^®, Shimadzu Co.) examinations to quantify bone volume by bone morphometric analysis using the TRI/3D-BONE^® software package (Ratoc System Enginieering Co. Ltd.).

Induction of ectopic bone formation in mice

To examine the effects of TGF-β1 (Fig. 1), 12 mice were divided into four groups and collagen sponges containing the vehicle (control group: n = 3), 50 ng of TGF-β1 (TGF-β1 alone group: n = 3), 5 μg of BMP-2 (BMP-2 alone group: n = 3), or a combination of 5 μg of BMP-2 and 50 ng of TGF-β1 (BMP-2+TGF-β1 group: n = 3) were surgically implanted into their dorsal muscle pouches. At 14 days after surgery, the mice were euthanized, and then the areas where the collagen sponges had been implanted were photographed and subjected to μCT examinations.

FIG. 1.

Effects of BMP-2 and TGF-β1 on formations of lump-like masses and ectopic bones in mice. (A) Representative images of backs of mice implanted with collagen sponges containing the vehicle alone (control), 50 ng of TGF-β1, 5 μg of BMP-2, and 50 ng TGF-β1+5 μg BMP-2 at 14 days after surgery. Arrows indicate the areas where the collagen sponges were implanted. (B) Representative μCT images of bones in mice shown in (A). Magnification bars correspond to 5 mm. BMP, bone morphogenetic proteins; TGF, transforming growth factor; μCT, microcomputed tomography. Color images available online at www.liebertonline.com/ten.

For a dose–response test (Fig. 2), 48 mice were divided into 16 groups and surgically implanted with collagen sponges containing various combination of BMP-2 and TGF-β1 as follows: 0 μg BMP-2+0 ng TGF-β1 (n = 3); 5 μg BMP-2+0 ng TGF-β1 (n = 3); 5 μg BMP-2+1 ng TGF-β1 (n = 3); 5 μg BMP-2+10 ng TGF-β1 (n = 3); 5 μg BMP-2+50 ng TGF-β1 (n = 3); and 5 μg BMP-2+150 ng TGF-β1 (n = 3) (Fig. 2A); and 0 μg BMP-2+0 ng TGF-β1 (n = 3); 1 μg BMP-2+50 ng TGF-β1 (n = 3); 3 μg BMP-2+50 ng TGF-β1 (n = 3); 5 μg BMP-2+50 ng TGF-β1 (n = 3); 10 μg BMP-2+50 ng TGF-β1 (n = 3); 0 μg BMP-2+0 ng TGF-β1 (n = 3); 1 μg BMP-2+0 ng TGF-β1 (n = 3); 3 μg BMP-2+0 ng TGF-β1 (n = 3); 5 μg BMP-2+0 ng TGF-β1 (n = 3); and 10 μg BMP-2+0 ng TGF-β1 (n = 3) (Fig. 2B). At 14 days after surgery, the mice were euthanized and ectopic bone volume was measured by μCT.

FIG. 2.

Effects of TGF-β1 on volume of ectopic bone induced by BMP-2. (A) Total volume of ectopic bone induced by combinations of BMP-2 (0 or 5 μg per sponge) and TGF-β1 (0, 1, 10, 50, or 150 ng per sponge) at 14 days after implantation (n = 3). (B) Total volume of ectopic bone induced by various dosages of BMP-2 (0, 1, 3, 5, or 10 μg per sponge) with (open bars) or without (closed bars) 50 ng of TGF-β1 at 14 days after implantation (n = 3). (C) Representative images of ectopic bones induced by combinations of BMP-2 (1 or 5 μg per sponge) and TGF-β1 (0 or 50 ng per sponge) at 14 days after implantation. *p < 0.05; **p < 0.01; N.S., not significant. Magnification bars correspond to 5 mm.

In a time-course study (Fig. 3), 30 mice were divided into two groups and implanted with collagen sponges containing 5 μg of BMP-2 (BMP-2 alone group: n = 15) or 5 μg of BMP-2+50 ng of TGF-β1 (BMP-2+TGF-β1 group: n = 15). The weights of lump-like masses and ectopic bone volumes were determined at 1, 3, 7, 14, and 21 days after surgery (n = 3 from each group).

FIG. 3.

Growth of lump-like masses and ectopic bones caused by administrations of BMP-2 and TGF-β1. (A) Weights of lump-like masses induced by 5 μg of BMP-2 with (closed circles) or without (open circles) 50 ng of TGF-β1 at 1, 3, 7, 14, and 21 days after surgery (n = 3). (B) Representative images of isolate lump-like masses shown in (A). (C) Volume of ectopic bone in the lump-like masses at 1, 3, 7, 14, and 21 days after surgery (n = 3). (D) Representative μCT images of lump-like masses shown in (B). *p < 0.05; **p < 0.01 (BMP-2+TGF-β1 v.s. BMP-2); N.S., not significant. Magnification bars correspond to 5 mm.

The experiments were approved by and conducted according to the guidelines of the Showa University Animal Care and Use Committee (approval number 19083).

Histological and morphogenetic analysis

For histological analysis (Figs. 4 and 5), lump-like masses induced in mice by implantation of collagen sponges containing 5 μg of BMP-2 (BMP-2 alone group: n = 6) and those with 5 μg of BMP-2+50 ng of TGF-β1 (BMP-2+TGF-β1 group: n = 6) were removed at 7 (n = 3 from each group) or 14 (n = 3 from each group) days after surgery. The samples were fixed with 4% paraformaldehyde, decalcified with ethylenediaminetetraacetic acid, embedded with paraffin, and subjected to thin section analysis after Villanueva staining to determine the number of osteoblasts per total volume (N.Ob/TV), osteoblast surface per bone surface (Ob.S/BS), number of osteoblasts per bone surface (N.Ob/BS), bone volume of total volume (BV/TV), osteoid volume of total volume (OV/TV), osteoid volume of bone volume (OV/BV), number of osteoclasts in total volume (N.Oc/TV), number of multinucleated osteoclasts in total volume (N.Mu.Oc/TV), and erosion surface per bone surface (ES/BS). Osteoblasts and osteoclasts were determined by morphological characteristics. Osteoblasts were observed as mononuclear cells, whereas osteoclasts were observed as multinucleated cells.

FIG. 4.

Histological and morphometric analyses of ectopic bone tissues. (A) Villanueva staining of sections of ectopic bones induced by 5 μg of BMP-2 (left) or 5 μg of BMP-2+50 ng of TGF-β1 (right) at 14 days after implantation. Magnification bars correspond to 1 mm. (B) Representative images of trabecular bones in (A) with higher magnification. Arrows, arrowheads, and lines indicate osteoclasts, osteoblasts, and osteoids, respectively. Magnification bars correspond to 100 μm. (C) Morphometric analyses of ectopic bones shown in A. N.Ob/TV, number of osteoblasts per total volume; Ob.S/BS, osteoblast surface per bone surface; N.Ob/BS, number of osteoblasts per bone surface; BV/TV, bone volume per total volume; OV/TV, osteoid volume per total volume; OV/BV, osteoid volume per bone volume; N.Oc/TV, number of osteoclasts per total volume; N.Mu.Oc/TV, number of multinucleated osteoclasts per total volume; ES/BS, erosion surface per bone surface. *p < 0.05; **p < 0.01 (n = 3). Color images available online at www.liebertonline.com/ten.

FIG. 5.

TGF-β1 accelerates differentiation of osteoblasts and osteoclasts in early stage of ectopic bone formation. (A) Villanueva staining of sections of ectopic bones induced by 5 μg of BMP-2 (left) or 5 μg of BMP-2+50 ng TGF-β1 (right) at 7 days after implantation. Arrows indicate osteoblasts (upper panels) and osteoclasts (lower panels). Magnification bars correspond to 50 μm. (B) mRNA expression levels of alkaline phosphatase, osteocalcin, cathepsin K, and GAPDH at 2, 3, 5, and 7 days after implantation of collagen sponges containing 5 μg of BMP-2 or 5μg of BMP-2+50 ng of TGF-β1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Color images available online at www.liebertonline.com/ten.

Analysis of mRNA expression levels in lump-like masses by reverse transcription–polymerase chain reaction assays

To examine mRNA expression during ectopic bone formation (Fig. 5B), total RNA from the lump-like masses induced by implantation of collagen sponges containing 5 μg of BMP-2 (n = 12) and those with 5 μg of BMP-2+50 ng of TGF-β1 (n = 12) was prepared at 2, 3, 5, and 7 days after implantation using TRIzol solution (Invitrogen Life Technologies) (n = 3 from each group). First-strand cDNA was synthesized for polymerase chain reaction (PCR) using Superscript III (Invitrogen Life Technologies) and subjected to amplification with GoTaq DNA Polymerase (Promega Corporation) using the following specific PCR primers: alkaline phosphatase, 5′-GATCATTCCCACGTTTTCAC-3′ (forward) and 5′-TGCGGGCTTGTGGGACCTGC-3′ (reverse); osteocalcin, 5′-CAAGTCCCACACAGCAGCTT-3′ (forward) and 5′-AAAGCCGAGCTGCCAGAGTT-3′ (reverse); cathepsin K, 5′-AGGCGGCTATATGACCACTG-3′ (forward) and 5′-CCGAGCCAAGAGAGCATATC-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5′-GAAGGTCGGTGTGAACGGATTTGGC-3′ (forward) and 5′-CATGTAGGCCATGAGGTCCAACAC-3′ (reverse). Representative reverse transcription-PCR data are shown (Fig. 5B).

Induction of orthotopic bone formation in mice

To investigate regeneration of calvarial bone defects (Fig. 6), a skin incision was made aseptically along the bilateral line and middle of the forehead, and dissection was continued to the calvarium. The periosteum of the calvarium was ablated and a full-thickness standardized trephine defect, 4 mm in diameter, was made in the calvarium using a Trephine Bar^® (Dentech Co.) under continuous PBS irrigation.³⁶ Subsequently, collagen sponges containing the vehicle (control group: n = 3), 25 ng of TGF-β1 (TGF-β1 alone group: n = 3), 2.5 μg of BMP-2 (BMP-2 alone group: n = 3), or 2.5 μg of BMP-2+25 ng TGF-β1 (BMP-2+TGF-β1 group: n = 3) were implanted into the trephine defects. After 14 days, whole calvaria from mice in each group were dissected and subjected to μCT scanning. Further, another control group (n = 3) and the BMP-2+TGF-β1 group (n = 3) were subjected to the same analysis at 42 days after surgery. The occupancy of new bone in the defect areas was measured using the ImageJ software package freely provided by NIH (http://rsbweb.nih.gov/ij/) (n = 3). The experiments were approved by and conducted according to the guidelines of the Showa University Animal Care and Use Committee (approval number 19080).

FIG. 6.

Occupation of bone defects in calvaria by ectopic bone tissue. (A) Representative μCT images of calvaria with hole, in which collagen sponges were implanted containing the vehicle alone (control), 25 ng of TGF-β1, 2.5 μg of BMP-2, or 25 ng of TGF-β1 + 2.5 μg of BMP-2 at 14 days after implantation. (B) Bone defect areas in calvaria shown in (A). *p < 0.05; **p < 0.01 (n = 3); N.S., not significant. (C) Representative μCT images of calvaria with hole, in which collagen sponges were implanted containing the vehicle alone (control) or 25 ng of TGF-β1 + 2.5 μg of BMP-2 at 42 days after implantation. Magnification bars correspond to 5 mm.

Statistical analysis

All data are expressed as mean ± SD. Significant differences were determined using Student's t-test (*p < 0.05 and **p < 0.01).

Results

Effects of TGF-β1 on ectopic bone formation induced by BMP-2

After implantation of collagen sponges containing BMP-2 and/or TGF-β1 into muscle tissues of mice for 14 days, we found lump-like masses in the BMP-2 (5 μg) alone group and BMP-2 (5 μg)+TGF-β1 (50 ng) group, but not in the control or TGF-β1 (50 ng) alone groups (Fig. 1A). Notably, the lump-like masses that developed in the BMP-2+TGF-β1 group were remarkably larger than those in the BMP-2 alone group (Fig. 1A). We subsequently examined these mice with μCT scanning to determine whether ectopic bone formation had occurred in the lump-like masses (Fig. 1B). Bone formation was detected in the μCT images of both the BMP-2 alone and BMP-2+TGF-β1 groups, whereas none was seen in the control and TGF-β1 alone groups (Fig. 1B). The length of the long axis of the ectopic bones induced in the BMP-2+TGF-β1 group was significantly greater than that of those in the BMP-2 alone group (6.35 ± 0.73 mm [n = 3] vs. 10.0 ± 0.24 mm [n = 3]; p < 0.01).

Effects of dosages of TGF-β1 on ectopic bone volumes

To determine the most efficient dosage of TGF-β1 to enhance ectopic bone formation induced by BMP-2, we implanted collagen sponges containing various amounts of TGF-β1 and BMP-2 into mice (Fig. 2A, B). When 0–150 ng of TGF-β1 was administrated with 5 μg of BMP-2, total ectopic bone volume increased in a dose-dependent manner (Fig. 2A). The bone volume reached a maximum level when >50 ng of TGF-β1 was given, which was fivefold greater than that induced by 5 μg of BMP-2 alone (Fig. 2A). On the other hand, administration of 50 ng of TGF-β1 also significantly increased ectopic bone volume induced by 1–10 μg of BMP-2 (Fig. 2B, C).

Time-course analysis of ectopic bone formation

Next, we determined the weights of the lump-like masses at 1, 3, 7, 14, and 21 days after surgery (Fig. 3A, B). In both the BMP-2 alone group and BMP-2+TGF-β1 group, the lump-like masses appeared on day 1 and grew until day 7, after which significant growth was not seen up to day 21 after implantation. The growth of the lump-like masses in the BMP-2+TGF-β1 group was markedly greater than that in the BMP-2 alone group (Fig. 3A, B). In both groups, calcification occurred from day around 7–14 (Fig. 3C, D), a subsequent event after the period of significant growth of the lump-like masses (Fig. 3A, B). Thereafter, bone volumes in these groups did not significantly change from day 14 to 21 after implantation (Fig. 3C, D).

TGF-β1 increased osteoblasts and decreased osteoclasts in ectopic bones

For histological analysis of the ectopic bones, we isolated the lump-like masses at 14 days after implantation (Fig. 4A–C). Inside the masses from the BMP-2 alone and BMP-2+TGF-β1 groups, bone matrix and cells were observed (Fig. 4B). The trabecular bone thickness (Tb.Th) and bone density (BV/TV) in the BMP-2+TGF-β1 group were significantly greater than those in the BMP-2 alone group (Fig. 4C). In addition, magnified images of ectopic bone sections (Fig. 4B) showed that osteoid volume (OV/BV) in the BMP-2+TGF-β1 group was threefold greater, whereas the bone formation indexes osteoblast number (N.Ob/TV and N.Ob/BS) and osteoblast surface (Ob.S/BS) were increased by twofold in the BMP-2+TGF-β1 group as compared with the BMP-2 alone group (Fig. 4C). In contrast, the bone resorption indexes osteoclast number (N.Oc/TV and N.Mu.Oc/TV) and eroded surface (ES/BS) in the BMP-2+TGF-β1 group were decreased to less than half of those in the BMP-2 alone group (Fig. 4C).

TGF-β1 controls differentiation of osteoblasts and osteoclasts in early stage of ectopic bone formation

Next, we observed thin sections of ectopic bones isolated from the BMP-2 alone and BMP-2+TGF-β1 groups at 7 days after implantation of collagen sponges. In both groups, we found osteoblasts and osteoclasts existing in the ectopic bones (Fig. 5A). On the basis of these observations, we analyzed the mRNA expression levels of osteoblast markers such as alkaline phosphatase and osteocalcin, and that of an osteoclast marker, cathepsin K, in the early stage (2–7 days after implantation) in each group (Fig. 5B). Expression of each differentiation marker was accelerated in the BMP-2+TGF-β1 group as compared with that in the BMP-2 alone group (Fig. 5B).

TGF-β1 promotes regeneration of bone defects in calvaria

To determine whether TGF-β1 promotes bone regeneration in a clinical situation, we implanted collagen sponges into bone defects surgically created in mouse calvaria (Fig. 5). Since 5 μg of BMP-2+50 ng of TGF-β1 induced orthotopic bones that were too large to cover the trephine defects (data not shown), the amounts of BMP-2 and TGF-β1 were reduced to half of those used in the ectopic bone experiments (2.5 μg of BMP-2 and 25 ng of TGF-β1). On day 14 after implantation, the calvarial defects were nearly completely filled with orthotopic bone tissue in the BMP-2+TGF-β1 group, whereas they were only partially filled in the BMP-2 alone group (Fig. 6A, B). No orthotopic bone tissue was induced in the calvarial defects in the control or TGF-β1 alone groups (Fig. 6A, B). Further, the thickness of the orthotopic bone tissues that filled the defects induced by BMP-2+TGF-β1 was apparently thicker than normal calvarial bone on day 14 (Fig. 6A). In addition, the thickness of ectopic bone tissues on day 42 after implantation was similar to that of normal bones (Fig. 6C).

Discussion

The present findings clearly demonstrated that TGF-β1 profoundly enhances ectopic bone formation induced by BMP-2 in muscle tissue and calvarial bone defects. We know of no previously reported factor that can increase total ectopic bone to fivefold greater than that of BMP-2 alone, as seen in the present study. In addition to total bone volume, bone mineral density (BV/TV) was significantly increased by supplementation of TGF-β1 with BMP-2. Together, these results suggest that TGF-β1 is a powerful enhancer of BMP osteoinductive activity and may be useful for treatment of bone disorders in the future.

It has been reported that various factors, such as pleiotrophin, fibroblast growth factor-2, −4, activin, heparin, prostaglandin E₂ receptor agonist, parathyroid hormone, and an inhibitor of phosphodiesterase, enhance ectopic bone formation induced by BMP-2.^{29,34,35,37–41} In addition, Ripamonti et al. and Duneas et al. reported that implantation of TGF-β1 alone induced endochondral bone formation in extraskeletal sites of adult baboons, whereas implantation of both TGF-β1 and BMP-7 functioned in a synergistic manner to induce that formation.^42,43 Further, Simmons et al. showed that transplantation of bone marrow stromal cells with both BMP-2 and TGF-β3 in SCID mice induced significant bone formation as compared with those added individually.⁴⁴ Although the experimental conditions differed from ours, those reports support our finding that TGF-β1 accelerates BMP-2 function in vivo.

Although TGF-β1 strongly enhanced ectopic bone formation by BMP-2, it is well known that this cytokine inhibits osteoblast differentiation in vitro.^45–49 Our histological analysis of ectopic bones isolated at 14 days after implantation revealed a significant increase in the number of osteoblasts in ectopic bones induced by BMP-2+TGF-β1 as compared with BMP-2 alone. Further, osteoblast differentiation was accelerated by TGF-β1 in the early stage of ectopic bone formation. These results suggest that TGF-β1 promotes osteoblast differentiation in the early and late stages of ectopic bone formation despite its inhibitory effects in vitro. If TGF-β1 were to act on osteoblast precursors directly, expression of Runx2 (also called Cbfa1), a transcription factor involved in osteoblast differentiation, would be suppressed by Smad3-dependent intracellular signals.⁴⁵ Thus, we consider that TGF-β1 acts indirectly on osteoblast differentiation during ectopic bone formation.

Our histological analysis of ectopic bones isolated at 14 days after implantation showed that the number of osteoclasts was decreased in the presence of TGF-β1. On the other hand, the expression level of the osteoclast marker cathepsin K was increased in the early stage of ectopic bone formation. These results suggest that TGF-β1 promotes osteoclast differentiation in the early stage, whereas it suppresses that in the later stage. In previously reported in vitro cultures of osteoclast precursors, TGF-β1 promoted osteoclast differentiation induced by recombinant RANKL protein in the absence of osteoblasts.⁵⁰ However, TGF-β1 inhibited osteoclast differentiation induced in the presence of osteoblasts, which produce RANKL protein in response to osteoporotic hormones such as active vitamin D₃ by an indirect action, indicating that TGF-β1 possesses contrasting effects on osteoclast differentiation that are dependent on culture condition.^51,52 Thus, the inhibitory and acceleratory effects of TGF-β1 on osteoclast formation in ectopic bone is possibly mediated by both direct and indirect actions toward osteoclast precursors. Further investigation is necessary to explore the action of TGF-β1 on the differentiation of osteoblasts and osteoclasts in ectopic bone formation.

During the early stage, in which lump-like masses appeared and grew, TGF-β1 markedly accelerated the growth of the masses induced by BMP-2. Since these lump-like masses undergo calcification during a later stage (days 7–14), their volume on day 7 may be a restricting factor that determines total ectopic bone volume. Thus, acceleration of the growth of the lump-like masses might be a central function of TGF-β1 to increase total ectopic bone volume. In support of this notion, TGF-β1 is known to regulate recruitment and proliferation of various types of cells, including chondrocytes, osteoblasts, and mesenchymal cells.^45–48,53 Further, TGF-β1 also supports blood vessel development required for the growth of tissues.^54–57 Determination of cells targeted by TGF-β1 by analyzing the lump-like masses in the early stage will help to elucidate the precise roles of TGF-β1 in ectopic bone formation.

We successfully promoted regeneration of bone defects surgically created in mouse calvaria using TGF-β1. Although the ectopic bone tissues induced by BMP-2+TGF-β1 were much thicker than normal calvaria on day 14, they were similar to normal calvarial thickness by day 42, which may have been due to regulation of bone homeostasis controlled by osteoblasts and osteoclasts in the ectopic bone. This suggests a possible use of TGF-β1 in clinical treatment for hard tissue disorders. However, we did not analyze side effects related to the combination of BMP-2+TGF-β1 on other organs and tissues. Since TGF-β1 is known to possess wide range of biological activities toward various kinds of cells including tumors, it will be important to carefully examine its safety and efficacy before clinical use.

Conclusion

In the present study, we found that TGF-β1 strongly enhances ectopic bone formation induced by BMP-2, with the resulting bone volume fivefold greater than that induced by BMP-2 alone. In addition, TGF-β1 enhanced the early stage of ectopic bone formation, during which lump-like masses are formed and grow, whereas it also increased the number of osteoblasts and decreased the number of osteoclasts in the late stage, during which bone matrices become calcified. Further, TGF-β1 markedly enhanced the filling of bone defects in calvaria with new ectopic bone induced by BMP-2. Together, our results suggest that TGF-β1 is a strong enhancer of ectopic bone formation and may contribute to development of an effective treatment strategy for bone defects.

Footnotes

Acknowledgments

The authors appreciate Dr. Mariko Ikeda, Dr. Yukikatsu Iwasaki, Dr. Hirotada Otsuka, and Dr. Masanori Nakamura (Showa University) for technical assistance. The authors also thank Ito Bone Histomorphometry Institute for analyzing histological sections of ectopic bones and Astellas Pharmaceuticals Co., Ltd., for supplying BMP-2 proteins. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and the High-Tech Research Center Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, 2005–2009.

Disclosure Statement

The authors state that they have no conflicts of interest.

References

Hogan

B.L.

Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev, 10:1580. 1996.

Newfeld

S.J.

, Wisotzkey

R.G.

, Kumar

Molecular evolution of a developmental pathway: phylogenetic analyses of transforming growth factor-β family ligands, receptors and Smad signal transducers. Genetics, 152:783. 1999.

Chang

, ten Dijke

, Wu

D.K.

BMP pathways are involved in otic capsule formation and epithelial-mesenchymal signaling in the developing chicken inner ear. Dev Biol, 251:380. 2002.

Zhao

G.Q.

Consequences of knocking out BMP signaling in the mouse. Genesis, 35:43. 2003.

Katagiri

, Takahashi

Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis, 8:147. 2002.

Canalis

, Economides

A.N.

, Gazzerro

Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev, 24:218. 2003.

Waite

K.A.

, Eng

From developmental disorder to heritable cancer: it's all in the BMP/TGF-β family. Nat Rev Genet, 4:763. 2003.

Wan

, Cao

BMP signaling in skeletal development. Biochem Biophys Res Commun, 328:651. 2005.

Yasko

A.W.

, Lane

J.M.

, Fellinger

E.J.

, Rosen

, Wozney

J.M.

, Wang

E.A.

The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg Am, 74:659. 1992.

10.

Marukawa

, Asahina

, Oda

, Seto

, Alam

M.I.

, Enomoto

Bone regeneration using recombinant human bone morphogenetic protein-2 (rhBMP-2) in alveolar defects of primate mandibles. Br J Oral Maxillofac Surg, 39:452. 2001.

11.

Matin

, Nakamura

, Irie

, Ozawa

, Ejiri

Impact of recombinant human bone morphogenetic protein-2 on residual ridge resorption after tooth extraction: an experimental study in the rat. Int J Oral Maxillofac Implants, 16:400. 2001.

12.

Urist

M.R.

Bone: formation by autoinduction. Science, 150:893. 1965.

13.

Thies

R.S.

, Bauduy

, Ashton

B.A.

, Kurtzberg

, Wozney

J.M.

, Rosen

Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology, 130:1318. 1992.

14.

Wozney

J.M.

, Rosen

, Celeste

A.J.

, Mitsock

L.M.

, Whitters

M.J.

, Kriz

R.W.

, Hewick

R.M.

, Wang

E.A.

Novel regulators of bone formation: molecular clones and activities. Science, 242:1528. 1988.

15.

Wang

E.A.

, Rosen

, D'Alessandro

J.S.

, Bauduy

, Cordes

, Harada

, Israel

D.I.

, Hewick

R.M.

, Kerns

K.M.

, LaPan

Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A, 87:2220. 1990.

16.

Sampath

T.K.

, Maliakal

J.C.

, Hauschka

P.V.

, Jones

W.K.

, Sasak

, Tucker

R.F.

, White

K.H.

, Coughlin

J.E.

, Tucker

M.M.

, Pang

R.H.

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem, 267:20352. 1992.

17.

Hughes

F.J.

, Collyer

, Stanfield

, Goodman

S.A.

The effects of bone morphogenetic protein-2, −4, and −6 on differentiation of rat osteoblast cells in vitro. Endocrinology, 136:2671. 1995.

18.

Boden

S.D.

, Hair

, Titus

, Racine

, McCuaig

, Wozney

J.M.

, Nanes

M.S.

Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6. Endocrinology, 138:2820. 1997.

19.

Yamaguchi

, Komori

, Suda

Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev, 21:393. 2000.

20.

Bentz

, Thompson

A.Y.

, Armstrong

, Chang

R.J.

, Piez

K.A.

, Rosen

D.M.

Transforming growth factor-β2 enhances the osteoinductive activity of a bovine bone-derived fraction containing bone morphogenetic protein-2 and 3. Matrix, 11:269. 1991.

21.

Urist

M.R.

, Kovacs

, Yates

K.A.

Regeneration of an enchondroma defect under the influence of an implant of human bone morphogenetic protein. J Hand Surg Am, 11:417. 1986.

22.

Boden

S.D.

, Zdeblick

T.A.

, Sandhu

H.S.

, Heim

S.E.

The use of rhBMP-2 in interbody fusion cages. Definitive evidence of osteoinduction in humans: a preliminary report. Spine (Phila Pa 1976), 25:376. 2000.

23.

Boden

S.D.

, Kang

, Sandhu

, Heller

J.G.

Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: a prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies. Spine (Phila Pa 1976), 27:2662. 2002.

24.

Burkus

J.K.

, Gornet

M.F.

, Dickman

C.A.

, Zdeblick

T.A.

Anterior lumbar interbody fusion using rhBMP-2 with tapered interbody cages. J Spinal Disord Tech, 15:337. 2002.

25.

Friess

, Uludag

, Foskett

, Biron

, Sargeant

Characterization of absorbable collagen sponges as recombinant human bone morphogenetic protein-2 carriers. Int J Pharm, 185:51. 1999.

26.

Winn

S.R.

, Uludag

, Hollinger

J.O.

Carrier systems for bone morphogenetic proteins. Clin Orthop Relat Res, 367,Suppl:S95. 1999.

27.

Horiuchi

, Saito

, Kinoshita

, Wakabayashi

, Yotsumoto

, Takaoka

Effect of phosphodiesterase inhibitor-4, rolipram, on new bone formations by recombinant human bone morphogenetic protein-2. Bone, 30:589. 2002.

28.

Yamamoto

, Takahashi

, Tabata

Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein. Biomaterials, 24:4375. 2003.

29.

Horiuchi

, Saito

, Kinoshita

, Wakabayashi

, Tsutsumimoto

, Otsuru

, Takaoka

Enhancement of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced new bone formation by concurrent treatment with parathyroid hormone and a phosphodiesterase inhibitor, pentoxifylline. J Bone Miner Metab, 22:329. 2004.

30.

Kim

C.S.

, Kim

J.I.

, Kim

, Choi

S.H.

, Chai

J.K.

, Kim

C.K.

, Cho

K.S.

Ectopic bone formation associated with recombinant human bone morphogenetic proteins-2 using absorbable collagen sponge and beta tricalcium phosphate as carriers. Biomaterials, 26:2501. 2005.

31.

Hosseinkhani

, Yamamoto

, Inatsugu

, Hiraoka

, Inoue

, Shimokawa

, Tabata

Enhanced ectopic bone formation using a combination of plasmid DNA impregnation into 3-D scaffold and bioreactor perfusion culture. Biomaterials, 27:1387. 2006.

32.

Hsu

H.P.

, Zanella

J.M.

, Peckham

S.M.

, Spector

Comparing ectopic bone growth induced by rhBMP-2 on an absorbable collagen sponge in rat and rabbit models. J Orthop Res, 24:1660. 2006.

33.

Majid

, Tseng

M.D.

, Baker

K.C.

, Reyes-Trocchia

, Herkowitz

H.N.

Biomimetic calcium phosphate coatings as bone morphogenetic protein delivery systems in spinal fusion. Spine J, 2010 Jan 22[Epub ahead of print].

34.

Zhao

, Katagiri

, Toyoda

, Takada

, Yanai

, Fukuda

, Chung

U.I.

, Koike

, Takaoka

, Kamijo

Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem, 281:23246. 2006.

35.

Kubota

, Iseki

, Kuroda

, Oida

, Iimura

, Duarte

W.R.

, Ohya

, Ishikawa

, Kasugai

Synergistic effect of fibroblast growth factor-4 in ectopic bone formation induced by bone morphogenetic protein-2. Bone, 31:465. 2002.

36.

Suzuki

, Kamakura

, Katagiri

, Nakamura

, Zhao

, Honda

, Kamijo

Bone formation enhanced by implanted octacalcium phosphate involving conversion into Ca-deficient hydroxyapatite. Biomaterials, 27:2671. 2006.

37.

Sato

, Takita

, Ohata

, Tamura

, Kuboki

Pleiotrophin regulates bone morphogenetic protein (BMP)-induced ectopic osteogenesis. J Biochem, 131:877. 2002.

38.

Nakamura

, Tensho

, Nakaya

, Nawata

, Okabe

, Wakitani

Low dose fibroblast growth factor-2 (FGF-2) enhances bone morphogenetic protein-2 (BMP-2)-induced ectopic bone formation in mice. Bone, 36:399. 2005.

39.

Ogawa

, Schmidt

D.K.

, Nathan

R.M.

, Armstrong

R.M.

, Miller

K.L.

, Sawamura

S.J.

, Ziman

J.M.

, Erickson

K.L.

, de Leon

E.R.

, Rosen

D.M.

Bovine bone activin enhances bone morphogenetic protein-induced ectopic bone formation. J Biol Chem, 267:14233. 1992.

40.

Toyoda

, Terai

, Sasaoka

, Oda

, Takaoka

Augmentation of bone morphogenetic protein-induced bone mass by local delivery of a prostaglandin E EP4 receptor agonist. Bone, 37:555. 2005.

41.

Horiuchi

, Saito

, Kinoshita

, Wakabayashi

, Tsutsumimoto

, Takaoka

Enhancement of bone morphogenetic protein-2-induced new bone formation in mice by the phosphodiesterase inhibitor pentoxifylline. Bone, 28:290. 2001.

42.

Ripamonti

, Duneas

, Van Den Heever

, Bosch

, Crooks

Recombinant transforming growth factor-β1 induces endochondral bone in the baboon and synergizes with recombinant osteogenic protein-1 (bone morphogenetic protein-7) to initiate rapid bone formation. J Bone Miner Res, 12:1584. 1997.

43.

Duneas

, Crooks

, Ripamonti

Transforming growth factor-β1: induction of bone morphogenetic protein genes expression during endochondral bone formation in the baboon, and synergistic interaction with osteogenic protein-1 (BMP-7)

Growth Factors, 15:259. 1998.

44.

Simmons

C.A.

, Alsberg

, Hsiong

, Kim

W.J.

, Mooney

D.J.

Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells. Bone, 35:562. 2004.

45.

Alliston

, Choy

, Ducy

, Karsenty

, Derynck

TGF-β-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation. EMBO J, 20:2254. 2001.

46.

Spinella-Jaegle

, Roman-Roman

, Faucheu

, Dunn

F.W.

, Kawai

, Gallea

, Stiot

, Blanchet

A.M.

, Courtois

, Baron

, Rawadi

Opposite effects of bone morphogenetic protein-2 and transforming growth factor-β1 on osteoblast differentiation. Bone, 29:323. 2001.

47.

Centrella

, Horowitz

M.C.

, Wozney

J.M.

, McCarthy

T.L.

Transforming growth factor-β gene family members and bone. Endocr Rev, 15:27. 1994.

48.

Centrella

, Casinghino

, Kim

, Pham

, Rosen

, Wozney

, McCarthy

T.L.

Independent changes in type I and type II receptors for transforming growth factor β induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype. Mol Cell Biol, 15:3273. 1995.

49.

Maeda

, Hayashi

, Komiya

, Imamura

, Miyazono

Endogenous TGF-β signaling suppresses maturation of osteoblastic mesenchymal cells. EMBO J, 23:552. 2004.

50.

Sells Galvin

R.J.

, Gatlin

C.L.

, Horn

J.W.

, Fuson

T.R.

TGF-β enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF. Biochem Biophys Res Commun, 265:233. 1999.

51.

Chenu

, Pfeilschifter

, Mundy

G.R.

, Roodman

G.D.

Transforming growth factor β inhibits formation of osteoclast-like cells in long-term human marrow cultures. Proc Natl Acad Sci U S A, 85:5683. 1988.

52.

Chenu

, Serre

C.M.

, Raynal

, Burt-Pichat

, Delmas

P.D.

Glutamate receptors are expressed by bone cells and are involved in bone resorption. Bone, 22:295. 1998.

53.

Tang

, Wu

, Lei

, Pang

, Wan

, Shi

, Zhao

, Nagy

T.R.

, Peng

, Hu

, Feng

, Van Hul

, Wan

, Cao

TGF-β1-induced migration of bone mesenchymal stem cells couples bone resorption with formation. Nat Med, 15:757. 2009.

54.

Roman

A.C.

, Carvajal-Gonzalez

J.M.

, Rico-Leo

E.M.

, Fernandez-Salguero

P.M.

Dioxin receptor deficiency impairs angiogenesis by a mechanism involving VEGF-A depletion in the endothelium and transforming growth factor-β overexpression in the stroma. J Biol Chem, 284:25135. 2009.

55.

Ferrari

, Cook

B.D.

, Terushkin

, Pintucci

, Mignatti

Transforming growth factor-β 1 (TGF-β1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis. J Cell Physiol, 219:449. 2009.

56.

Merwin

J.R.

, Anderson

J.M.

, Kocher

, Van Itallie

C.M.

, Madri

J.A.

Transforming growth factor β 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis. J Cell Physiol, 142:117. 1990.

57.

Tuxhorn

J.A.

, McAlhany

S.J.

, Yang

, Dang

T.D.

, Rowley

D.R.

Inhibition of transforming growth factor-β activity decreases angiogenesis in a human prostate cancer-reactive stroma xenograft model. Cancer Res, 62:6021. 2002.