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Rheumatology 2001; 40: 1163-1168
© 2001 British Society for Rheumatology

Original Papers

Involvement of bone morphogenic protein-2 (BMP-2) in the pathological ossification process of the spinal ligament

H. Tanaka, E. Nagai, H. Murata, T. Tsubone, Y. Shirakura, T. Sugiyama, T. Taguchi and S. Kawai

Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To investigate the function of bone morphogenic protein-2 (BMP-2) in the ossification of the spinal ligament (OSL).

Methods. Total RNA was prepared from the cultured spinal ligament cells from patients with OSL and analysed by reverse transcription–polymerase chain reaction using specific primers for BMP-2. BMP-2 mRNA expression in ligament tissues was examined by in situ hybridization. Spinal ligament cells from patients without OSL were treated with BMP-2 and examined for alkaline phosphatase activity.

Results. Expression of the BMP-2 gene was detected in cultured spinal ligament cells. In ligament tissues, BMP-2 mRNA was present in the chondrocyte-like cells in the fibrocartilage zone. Exogenous BMP-2 increased alkaline phosphatase activity in spinal ligament cells from patients without OSL.

Conclusion. The BMP-2 gene is expressed in the spinal ligaments of OSL patients, and exogenous BMP-2 stimulates osteogenic differentiation of spinal ligament cells. The expression of BMP-2 in the spinal ligaments could be a clue in elucidating how heterotrophic osteogenesis develops in ligament tissue.

KEY WORDS: BMP, Ossification, Spinal ligament, RT-PCR, In situ hybridization, Alkaline phosphatase.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Pathological ectopic ossification of the spinal ligaments (OSL) has been well described. Forestier and Rotes-Querol first reported ankylosing spinal hyperostosis as a disease characterized by ossification of the anterior longitudinal ligament of the spine [1]. Diffuse idiopathic skeletal hyperostosis, described by Resnick et al. [2], represents a disease concept for systemic ligament ossification, including the spinal ligament. Ossification of the posterior longitudinal ligament (OPLL) of the spine has received attention in a number of reports [3–5]. As in some cases of OPLL, OSL can involve compression of the spinal cord and myelopathy. Many reports have described the aetiology of OSL and the systemic and local factors thought to be involved in the pathogenesis of OSL, but the exact mechanism remains unclear.

Bone morphogenic proteins (BMPs) have been shown previously to have the ability to stimulate ectopic bone formation in vivo, probably by initiating the differentiation of mesenchymal stem cells into mature osteoblasts and chondroblasts [6–8]. The in vitro effects of BMPs on osteoblastic cells also suggest that BMPs stimulate cell proliferation, alkaline phosphatase (ALP) activity and collagen synthesis [9–11]. BMP gene expression has been demonstrated in immature cells in the proliferating periosteum and marrow cavity in the early phases of fracture healing [12], and immunohistochemical studies have demonstrated the accumulation of BMP proteins in fracture haematoma [13]. The BMPs are also produced during limb-bud development and act as morphogens during embryogenesis [14, 15]. These findings suggest that BMPs play an important role in the initiation of the endochondral bone-formation cascade.

The aim of this study was to investigate the function of BMP-2 in OSL. We detected the expression of the BMP-2 gene using the reverse transcriptase–polymerase chain reaction and in situ hybridization in ligament tissues with heterotrophic ossification in patients with OSL. We also examined the direct effects of BMP-2 on osteoblast differentiation in cultured spinal ligament cells.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
Five patients with OSL, comprising two with ossification of the ligamentum flavum (OLF), two with OPLL and one with ossification of the anterior longitudinal ligament (OALL), and four patients with lumbar canal stenosis (LCS) underwent spinal surgery (Table 1Go). OSL was diagnosed by X-radiography or CT scan. The patients with LCS did not have OSL in X-radiography. No patients exhibited any abnormalities in bone metabolism. Informed consent from each patient and institutional permission were obtained before starting this study.


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  		TABLE 1. Profile of patients with and without OSL

 

Samples and tissue preparation
Human specimens of spinal ligamentum were obtained from patients at the time of spinal surgery. For in situ hybridization analysis, small tissue samples were washed in cold phosphate-buffered saline (PBS), fixed immediately in a cold, sterile solution of 4% p-formaldehyde in PBS and kept on ice. The specimens were transferred to a sterile decalcifying solution of ethylenediamine tetraacetic acid (EDTA). The tissues were rinsed with distilled water several times, dehydrated through graded ethanol, infiltrated, and embedded in paraffin. Slide glasses were cleaned in advance with acetone and acid, and coated with poly-L-lysine (Sigma, St Louis, MO, USA). Sections (4 µm thick) were cut and mounted on the treated slides and then kept at 4°C until use.

Cell culture
After removal of the ossified tissue, the fibrous tissue of the ligament was washed in cold PBS and minced into small pieces. Cells were placed on 60-mm diameter plates and cultured in {alpha}-Minimum Essential Medium ({alpha}-MEM) with 10% heat-inactivated fetal bovine serum (FBS), 50 µg/ml L-ascorbic acid, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% carbon dioxide in a humidified incubator. Media were changed twice weekly until the cultures were confluent. Confluent cells were detached with 0.05% (w/v) trypsin–EDTA and used for subsequent experiments to examine gene expression or ALP activity. {alpha}-MEM, FBS, penicillin, streptomycin, trypsin and EDTA were all purchased from Gibco BRL (Grand Island, NY, USA).

SaOS-2 cells (ATCC No. HTB-85) were propagated in McCoy 5A medium+10% FBS. U2OS cells (ATCC No. HTB-96) were cultured in Dulbecco's MEM+10% fetal calf serum. Media were changed twice weekly, and the cells were harvested after confluence.

Messenger RNA extraction and RT-PCR
Total RNA from ligament cells was prepared by the guanidinium thiocyanate–phenol–chloroform extraction method (TRIzol Reagent) (Gibco BRL) following the instructions provided by the supplier. First-strand cDNA was synthesized from 5 µg of total RNA using oligo-dT as a primer and AMV reverse transcriptase (cDNA Synthesis Kit; Boehringer, Mannheim, Germany). PCR was carried out using specific primer pairs. The sense and antisense primers for BMP-2 were designed from the coding region [7] to amplify a product that crossed introns to avoid confusion between the mRNA transcript and genomic DNA: sense, 5'-AACGGACATTCGGTCCTTGC-3'; antisense, 5'-CGCAACTCGAACTCGCTCAG-3'. The reaction mixtures were denatured at 94°C for 45 s, annealed at 65°C for 45 s and extended at 72°C for 90 s for 30 cycles in a GeneAmp PCR System Model 9600 (Perkin-Elmer, Norwalk, CT, USA). The amplification products were checked for predicted size by 2% agarose gel electrophoresis with molecular weight markers. To ensure the amplification of the right message, the PCR products were subjected to DNA sequence analysis.

Preparation and sulphonation of probe DNA
A 1.3-kilobase (kb) DNA fragment encoding the Xenopus BMP-2 cDNA [16] was a gift from Dr N. Ueno. The clone was released from the pUC19 vector (3.1 kb) and digested with EcoRI. Each fragment was separated by electrophoresis in agarose gel and purified. As a negative control, we used a fragment of plasmid vector prepared by suitable digestions [17], although a more usual and preferred approach might have been to use sense/antisense single-stranded probes. Sulphonation of probe DNA was carried out using the New DNA Chemiprobe Kit (Takara, Kyoto, Japan) according to the manufacturer's instructions.

In situ hybridization
In situ hybridization was performed as described [17], with some modifications. The sections were dewaxed in xylene and then rehydrated in ethanol with sequential concentrations from 100% to 30%. Endogenous peroxidase activity was blocked in absolute methanol with 1% hydrogen peroxide. After treatment with 0.2 N HCl for 20 min at room temperature, the sections were digested with 5 mg/ml of proteinase K (Boehringer) in PBS at 37°C for 15 min. The sections were immersed in PBS containing 0.2% glycine, postfixed with 4% p-formaldehyde solution in PBS for 10 min, and washed in distilled water. To prevent non-specific binding, sections were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer for 10 min. Before hybridization, the sections were prehybridized for 2 h at 37°C in a mixture containing 50% deionized formamide (Wako Pure Chemical Industries, Osaka, Japan), 2xSSPE [SSPE contains 0.6 M NaC1, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA], 1xDenhardt's solution (Wako Pure Chemical Industries), 25% dextran and 100 mg/ml of salmon sperm DNA. The heat-denatured probe was added to the hybridization mixture to give a final concentration of 100 ng/ml, and 30 ml was applied to each slide. Hybridization was performed in a humidified chamber at 37°C for 18 h. The non-specifically bound probe was washed off with 50% deionized formamide and decreasing concentrations of SSC (1xSSC=0.15 M NaCl, 0.015 M sodium citrate), starting at 2xSSC and ending with a final stringency of 0.2xSSC at room temperature.

Immunohistochemistry was performed with the New DNA Chemiprobe Kit (Takara) according to the manufacturer's instructions. After the colour reaction, the sections were counterstained with eosin.

Determination of ALP activity
Primary culture cells from patients without OSL were plated at a density of 5x104 cells/cm2 in the same medium as the primary culture. Recombinant human BMP-2 (rhBMP-2) (Yamanouchi Pharmaceutical, Tokyo, Japan) was added to selected flasks to final concentrations of 0, 50, 100, 250, 500 and 1000 ng/ml. The medium was changed twice weekly until confluence was achieved (8–10 days). The cultured osteoblastic cells were sonicated in 0.1 M Tris buffer, pH 7.2, containing 0.1% Triton X-100 for 30 s with a sonicator (ultrasonic disrupter UD-201; Tomy, Tokyo, Japan). ALP activity was determined using p-nitrophenylphosphate as a substrate in 0.05 M 2-amino-2-methylpropanol and 2 mM MgCl2, pH 10.5. The amount of p-nitrophenol released was estimated by measuring the absorbance at 410 nm. Protein concentrations were determined using a BCA protein assay reagent (Pierce Chemical, Rockford, IL, USA) according to the manufacturer's instructions.

Staining for ALP
Control or BMP-2 (250 ng/ml)-treated cells were fixed with 10% formalin for 30 min and washed three times with 10 mM Tris buffer, pH 7.2. Fixed cells were subjected to staining for ALP. ALP was stained with naphthol AS-MX phosphate and fast blue BB salt (Sigma).

Statistical analysis
The Mann–Whitney U-test was done to test differences in ALP activity between cells with or without BMP-2 stimulation at various concentrations. Data are expressed as mean±S.E. for four cultures. P<0.05 was taken to indicate statistical significance in all analyses.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Detection of BMP-2 mRNA by RT-PCR
To initiate the present study, we attempted to detect BMP-2 mRNA by RT-PCR analysis using specific primers. As expected, high levels of BMP-2 expression were found in the human osteoblast-like osteosarcoma cell lines SaOS-2 and U2OS. BMP-2 mRNA was detected at lower levels in all spinal ligament cells obtained from patients with OSL (cases 1–3) (Fig. 1Go). It was not possible to identify BMP-2 transcripts under the PCR condition used in this experiment in any of the ligament cells from patients without OSL (cases 7–9) (data not shown).



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  		FIG. 1. Detection of BMP-2 mRNA by RT-PCR. Total RNA was prepared from cultured cells and analysed by RT-PCR using primers specific for BMP-2. The samples are from the human osteoblast-like osteosarcoma cell lines SaOS-2 (lane 1) and U2OS (lane 2) and spinal ligament cells obtained from patients with OSL (cases 1–3) (lanes 3–5). M, molecular marker.

 

In situ hybridization
The ossified spinal ligaments from two patients with OSL, including one OLF (case 4) and one OALL (case 5), were examined by in situ hybridization (Fig. 2Go). Histological observation of the ossified spinal ligament showed that fibrous ligaments were inserted into the bone structure through fibrocartilage. In the fibrocartilage zone, chondrocyte-like cells exist between rough collagen fibres, and the pericellular matrix of these cells was stained diffusely by Alcian blue (data not shown). BMP-2 mRNA was expressed in the chondrocyte-like cells in this fibrocartilage zone. Close to the fibrocartilage zone, ligament cells similar in shape to fibroblasts also expressed BMP-2 mRNA at moderate levels. The specimens from cases of OALL showed similar patterns of gene expression. The control probe showed no specific hybridization signal. In addition, spinal ligaments from patients without OSL (case 6) did not show any mRNA expression for BMP-2 (data not shown).



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  		FIG. 2. In situ hybridization. Ossified spinal ligaments from cases 4 (OSL) (A–C) and 5 (OALL) (D, E) were probed with BMP-2 cDNA. BMP-2 mRNA was expressed in the chondrocyte-like cells in the fibrocartilage zone (A, D). The arrows indicate the regions shown at higher magnification in B and E. The higher-power light-field photomicrographs show that specific signals were detected in chondrocyte-like cells and ligament cells similar in shape to fibroblasts in the fibrocartilage zone (B, E). No specific hybridization signal was detected with a control probe (C). Bars: A, C, D, 200 µm; B, 40 µm; E, 50 µm. L, ligament; B, bone (ossified tissue).

 

Effects of BMP-2 on ALP activity in spinal ligament cells
To investigate the possibility that BMP-2 induces ossification of the spinal ligament, we examined the direct effects of BMP-2 on ALP activity in spinal ligament cells from patients without OSL (cases 7–9). Histochemical examination of confluent cultures for ALP revealed that continuous treatment with BMP-2 caused a significant increase in the number of ALP-positive cells (Fig. 3AGo). ALP activity was determined at confluence. The enzyme activity was very low at 0–100 ng/ml of BMP-2, whereas it dramatically increased at 250–1000 ng/ml of BMP-2 in a concentration-dependent manner (Fig. 3BGo). Similar results were obtained in three separate experiments.



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  		FIG. 3. Effects of BMP-2 on ALP activity in the spinal ligament cells. (A) Histochemical staining for ALP. The confluent spinal ligament cells were stained for ALP as described in the text (Patients and methods). (B) Concentration-dependent stimulation of ALP activity by BMP-2. The spinal ligament cells were exposed to BMP-2 at the indicated concentrations until confluence, and ALP activity was determined as described in the text (Patients and methods). Data are mean±S.E. for four cultures and are representative of results from three experiments. *P<0.05 compared with control.

 


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
The present study provides the first demonstration of the expression of the BMP gene in ligament tissues with heterotrophic ossification in patients with OSL. BMP-2 mRNA was detected by RT-PCR analysis in cDNA prepared from spinal ligament cell cultures obtained from patients with OSL. The ossified spinal ligaments from two patients with OSL were examined by in situ hybridization. BMP-2 mRNA was expressed in the chondrocyte-like cells in the fibrocartilage zone around the ossified tissues. Close to the fibrocartilage zone, ligament cells similar in shape to fibroblasts also expressed moderate levels of BMP-2 mRNA. These observations are in accordance with those of previous immunohistochemical studies of ossified ligament tissues of patients with OPLL [18, 19] and OLF [20]. Previous evidence has also shown that BMP mRNA appears in chondrocyte-like cells in the process of endochondral bone formation during embryonic development [14] and fracture repair [12], suggesting that BMPs play an important role in the initiation of bone formation. In addition, it has been reported recently that BMP receptors are expressed at high levels in ossified ligament tissues of patients with OPLL [19] and OLF [20]. Thus, BMPs seem to participate in the development of OSL in an autocrine or paracrine manner.

In physiological events in bone, such as healing and remodelling, BMPs are thought primarily to stimulate differentiation of mesenchymal stem cells towards an osteoblast lineage. Osteoprogenitors from the bone marrow have been shown previously to respond to BMP-2 stimulation with accelerated differentiation, as evaluated by early ALP activity [21, 22]. It is tempting to suggest that spinal ligament cells may contain osteoblast progenitor cells that can be induced towards the osteoblast phenotype by BMP-2. To address this hypothesis, we examined the direct effects of BMP-2 on osteoblast differentiation in cultured spinal ligament cells. We found that continuous exposure to BMP-2 stimulated ALP activity in cultured spinal ligament cells from the non-ossification group. This effect was dose-responsive, significant stimulation beginning at 250 ng/ml and maximal levels of stimulation being achieved at 1000 ng/ml. Kon et al. [23] have reported that treatment with BMP-2 after confluence in non-OPLL cells does not influence ALP activity. This discrepancy can be explained in part by evidence that the effects of BMP-2 on osteoblastic differentiation are obtained by continuous exposure or only at the initiation of culture, not at the late stages [11]. Indeed, in our experiments no effect was seen when cells were exposed to BMP-2 after confluence (data not shown).

As the induction of ALP activity has been recognized as one marker of osteoblast differentiation, it has been postulated that spinal ligament cells contain osteoblast progenitor cells that can be induced towards the osteoblast phenotype by BMP-2. A previous in vivo study has shown that ossification of the ligamentum flavum can be produced experimentally in mice by implanting partially purified BMP in the lumbar extradural space [24], confirming the above hypothesis. We have also found that normal spinal ligament cells express ALP, type I collagen and osteocalcin mRNAs in response to treatment with dexamethasone (H. Murata, H. Tanaka and S. Kawai, submitted for publication). In an analysis of BMP receptor distribution in the spinal ligaments, BMP receptor was found to be weakly expressed at the site of ligament attachments to bone in normal tissue [19]. Thus, it is possible that spinal ligament tissue has the potential to induce heterotrophic ossification.

In conclusion, the BMP-2 gene is expressed in the spinal ligaments of OSL patients, and exogenous BMP-2 stimulates osteogenic differentiation of spinal ligament cells. The expression of BMP-2 in the spinal ligaments could be relevant to the process of heterotrophic osteogenesis in ligament tissue. Although it may be clear that BMP-2 participates in the ossification of the spinal ligament in an autocrine or paracrine manner, it remains unknown which factors induce ligament cells to produce BMP.


   Acknowledgments
 
We would like to thank Dr N. Ueno for providing us with the cDNA clone for Xenopus BMP-2. This study was supported (in part) by a Research Grant for Specific Disease, from the Ministry of Health and Welfare, Japan.


   Notes
 
Correspondence to: H. Tanaka. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Forestier J, Rotes-Querol J. Senile ankylosing hyperostosis of the spine. Ann Rheum Dis1950;9:321–30.
  2. Resnick D, Shaul SR, Robins JM. Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations. Radiology1975;115:513–24.[Abstract]
  3. Tsukimoto H. An autopsy case with syndrome of spinal cord compression due to ossification within the canal of the cervical spine [Japanese]. Arch Jpn Chir1960;29:1003–7.
  4. Onji Y, Akiyama H, Shimomura Y, Ono K, Hukuda S, Mizuno S. Posterior paravertebral ossification causing cervical myelopathy. A report of eighteen cases. J Bone Joint Surg Am Vol1967;49:1314–28.[Abstract/Free Full Text]
  5. Tsuyama N. Ossification of the posterior longitudinal ligament of the spine. Clin Orthop1984;184:71–84.
  6. Urist MR, Mikulski A, Lietze A. Solubilized and insolubilized bone morphogenetic protein. Proc Natl Acad Sci USA1979;76:1828–32.[Abstract/Free Full Text]
  7. Wozney JM, Rosen V, Celeste AJ et al. Novel regulators of bone formation: molecular clones and activities. Science1988;242:1528–34.[Abstract/Free Full Text]
  8. Reddi AH, Cunningham NS. Bone induction by osteogenin and bone morphogenetic proteins. Biomaterials1990;11:33–4.[Medline]
  9. Yamaguchi A, Katagiri T, Ikeda T et al. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol1991;113:681–7.[Abstract/Free Full Text]
  10. Katagiri T, Yamaguchi A, Ikeda T et al. The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun1990;172:295–9.[Web of Science][Medline]
  11. Hughes FJ, Collyer J, Stanfield M, Goodman SA. The effects of bone morphogenetic protein-2, -4 and -6 on differentiation of rat osteoblast cells in vitro. Endocrinology1995;136:2671–7.[Abstract]
  12. Nakase T, Nomura S, Yoshikawa H et al. Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing. J Bone Miner Res1994;9:651–9.[Web of Science][Medline]
  13. Jin Y, Yang L, White FH. An immunocytochemical study of bone morphogenetic protein in experimental fracture healing of the rabbit mandible. Chin Med Sci J1994;9:91–5.[Medline]
  14. Lyons KM, Pelton RW, Hogan BL. Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development. Genes Dev1989;3:1657–68.[Abstract/Free Full Text]
  15. Jones CM, Lyons KM, Hogan BL. Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development1991;111:531–42.[Abstract]
  16. Nishimatsu S, Suzuki A, Shoda A, Murakami K, Ueno N. Genes for bone morphogenetic proteins are differentially transcribed in early amphibian embryos. Biochem Biophys Res Commun1992;186:1487–95.[Web of Science][Medline]
  17. Morimoto H, Monden T, Shimano T et al. Use of sulfonated probes for in situ detection of amylase mRNA in formalin-fixed paraffin sections of human pancreas and submaxillary gland. Lab Invest1987;57:737–41.[Web of Science][Medline]
  18. Kawaguchi H, Kurokawa T, Hoshino Y, Kawahara H, Ogata E, Matsumoto T. Immunohistochemical demonstration of bone morphogenetic protein-2 and transforming growth factor-beta in the ossification of the posterior longitudinal ligament of the cervical spine. Spine1992;17:S33–6.[Medline]
  19. Yonemori K, Imamura T, Ishidou Y et al. Bone morphogenetic protein receptors and activin receptors are highly expressed in ossified ligament tissues of patients with ossification of the posterior longitudinal ligament. Spinal Cord1997;35:550–3.[Medline]
  20. Hayashi K, Ishidou Y, Yonemori K et al. Expression and localization of bone morphogenetic proteins (BMPs) and BMP receptors in ossification of the ligamentum flavum. Bone1997;21:23–30.[Medline]
  21. Thies RS, Bauduy M, Ashton BA, Kurtzberg L, Wozney JM, Rosen V. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology1992;130:1318–24.[Abstract/Free Full Text]
  22. Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev Biol1994;161:218–28.[Web of Science][Medline]
  23. Kon T, Yamazaki M, Tagawa M et al. Bone morphogenetic protein-2 stimulates differentiation of cultured spinal ligament cells from patients with ossification of the posterior longitudinal ligament. Am J Pathol1997;150:1335–47.[Abstract]
  24. Miyamoto S, Takaoka K, Yonenobu K, Ono K. Ossification of the ligamentum flavum induced by bone morphogenetic protein. An experimental study in mice. J Bone Joint Surg Br Vol1992;74:279–83.[Web of Science][Medline]
Submitted 8 March 2001; Accepted 9 May 2001


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