Experimental Murine Model of Ossification of Spinal Ligaments Induced by Bone Morphogenetic Protein-2

Experimental Murine Model of Ossification of Spinal Ligaments Induced by Bone

Morphogenetic Protein-2

Kazuto Hoshi ¹

Introduction

When the spinal ligaments, including the posterior longitudinal ligament and the ligamentum fl avum, ectopically ossify, the spinal cord gradually becomes compressed, which results in severe progressive paraly- sis. This pathological ossifi cation of the posterior lon- gitudinal ligament (OPLL) usually occurs in the cervical spine, whereas that of the ligamentum fl avum (OLF) is often seen in the thoracic spine. Because the incidence of OPLL in Japan is reported to be rather higher than that in other countries, it is the focus of intensive study in orthopedic or neurosurgical fi elds in Japan. Although several hypotheses that this disease may be caused by microdamage to the ligaments following disc degenera- tion [1], hyperparathyroidism [2], high intake of vege- tables and salt with a decrease in the serum levels of sex hormones [3], abnormal glucose tolerance and hyper- insulinemia [4], up-regulation of local factors including bone morphogenetic protein-2 (BMP-2) and transform- ing growth factor- β (TGFβ) [5], changes in growth hormone (GH) action mediated by GP-binding protein [6], or genetic backgrounds related to TGF β [7], colla- gen type XI [8], estrogen receptor, or interleukin-1 β [9]

have been proposed, the detailed molecular mecha- nisms remain unknown.

Previous clinicopathological fi ndings reported by Ono et al. suggested that ossifi cation occurred during an endochondral process because cartilage-like tissue has been observed between the ossifi cation site and a normal ligament [10]. Okada et al. also reported that cartilage-like tissues formed at both ends of the ossifi - cation sites, speculating that an ossifi cation nest was

formed by endochondral ossifi cation [11]. Thus, ossifi cation mainly occurs by endochondral ossifi ca- tion, although intramembranous ossifi cation was also observed. In addition, because the ossifi ed lesion extends as far as the point at which the ligament is attached to the bone (enthesis) [10,11], events during enthesis are thought to play important roles in the pathogenesis of this ectopic ossifi cation. However, because many of the previous clinicopathological data were obtained either from autopsy or during surgery, they showed only a late stage or the end stage of the process. Virtually no documentation is available regard- ing the initial or developing stages of the process, and therefore an important key to elucidating the pathogen- esis has not been obtained.

BMPs and OPLL

Using immunohistochemical analyses, Kawaguchi et al.

documented that BMP-2 and TGF β were localized around ossifi ed areas of the longitudinal ligaments in surgical specimens from OPLL patients [5]. BMPs had been originally defi ned as substances inducing new bones when transplanted subcutaneously or intramus- cularly with some carriers. Thereafter, they were shown to have a variety of functions, including nonosteogenic development. TGF β and its homologous molecules comprise a large, diverse group of morphogens, the TFG β superfamily, to which BMPs also belong. TGFβ does not induce ectopic bone formation by itself but enhances bone formation when it is transplanted sub- periosteally and so can participate in bone formation, similar to BMPs. OP-1/BMP-7, another member of the BMP family, was also expressed in chondrocytes near the calcifi ed zone of ossifi ed ligament tissues in patients with OPLL [12] or OLF [13]. Kawaguchi et al. also observed the localization of BMP receptors, including types IA, IB, and II, in the cells of the areas around the ossifi ed tissues. Those fi ndings suggested that BMPs may be involved in promoting endochondral ossifi ca- tion at ectopic ossifi cation sites in OLF and OPLL.

Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 -8655, Japan

Present address:

1

Department of “Fuji Soft ABC” Cartilage & Bone Regeneration, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

93

Exogenous BMPs were shown to induce ossifi ca- tion of the ligamentum fl avum in mice and rabbits.

Miyamoto et al. surgically implanted BMP pellets (0.2 mg) that had been partially purifi ed from murine osteosarcoma (Dunn) in the epidural spaces of lumbar spines of ICR-strain mice [14]. At 4 weeks, the ligamen- tum fl avum became hypertrophied and contained fi brous, cartilaginous, and even bony tissues. Those tissues protruded into the spinal cord from the contigu- ous laminae in all the mice in the experimental group.

Eight weeks later, the ossifi cation was still more advanced, showing spinal canal stenosis at 46% by the canal narrowing ratio (CNR). Demyelination occurred in the posterior and lateral white columns, and neuro- nal loss or chromatolysis was observed in the gray matter. Mimatsu et al. also implanted crude BMP (1 mg), extracted from long bones of Japanese white rabbits, into the ligamentum fl avum of the rabbit lumbar spine [15]. Ectopic bones were found in approx- imately 40% of the BMP-treated rabbits, and the spinal cord was chronically compressed, becoming fl attened to 87% of its normal size. No pathological changes were detected in the intramedullary tissues in this model by light microscopic examination. Mimatsu et al. noted that the differences in the degree of the intramedullary changes between rabbits and mice may be due to species differences regarding ossifi cation. In any case, how liga- mentous tissues are altered under the effects of BMPs, especially during the initial or developing stages of the ossifi cation, had not been examined in detail because surgical manipulation of the host’s ligaments during implantation of the BMP pellets damaged the anatomi- cal structures and disturbed the fi ne observation of tissues.

During the late 1990s recombinant human BMP-2 (rhBMP-2) was provided by a pharmaceutical company, and highly purifi ed BMP-2 solution became available for experiments. This enabled us to treat a ligament with BMP-2 atraumatically using an injection method and to observe the time-course alterations of ligamen- tous cells at the cellular level. Employing the injection method, we established an experimental murine model in which the entire process, from the initial to the end stages, of ossifi cation in spinal ligaments could be examined in detail [16,17].

Time Course Changes in the Experimental Model

The authors used ligamenta fl ava in the lumbar spines of 12-week-old male ddY mice. The skin in the midline of the mouse back was incised following anesthesia with diethyl ether. After the tips of the spinous pro- cesses were recognized through the back fascia, 40 µg

of rhBMP-2 diluted in 100 µl of glutamate buffer (pH 4.5) (kindly provided by Astellas Pharma, Tokyo, Japan) was injected through a 27-gauge hypodermic needle vertically into the intraspinous space at a depth of 3 mm, which was the average length between the back fascia and the ligamentum fl avum; rhBMP-2 solu- tion was gradually infi ltrated (Fig. 1a) [16]. The BMP- 2 -treated group was compared with a group in which the solution lacked the protein (control group) or another group given no treatment (normal), both of which consequently showed similar fi ndings in each experiment.

Of 50 mice injected with BMP-2, there were 14 that showed signs of chondrogenesis or osteogenesis around the areas of the ligamenta fl ava. The remainder showed such reactions in areas other than the ligamenta fl ava or no change at all (Fig. 1b). The extent of spinal canal stenosis gradually increased until the third week. This worked out to approximately 10% the fi rst week follow- ing BMP-2 injection, 20% the second week, and 30%–

50 % the third week, after which it dropped to less than 10 % by the sixth week [16].

Under light microscopy, the ligamentum fl avum of the control group was seen to interconnect the cranial and caudal spinal laminae (Fig. 2, 0 weeks) (Fig. 2).

During the fi rst week following BMP-2 injection, carti- laginous tissue appeared at the enthesis site, and fi brous bundles decreased in number (Fig. 2, at 1 week). At the second week, ligamentous tissue was replaced by carti- laginous tissues (Fig. 2, at 2 weeks). In the cartilaginous tissues, the cells at the enthesis site appeared hypertro- phic. Moreover, vascular invasion was observed in the vicinity of the enthesis site (arrow in Fig. 2, at 2 weeks).

By the third week, compression of BMP-2-induced bone and cartilage against the spinal cord became evident (Fig. 2, at 3 weeks). At the sixth week, osseous tissues had completely replaced BMP-2-induced cartilage, leaving only slight traces of residual cartilage in the center of what was previously ligament (Fig. 2, at 6 weeks).

Under higher magnifi cation, fl attened fi broblasts normally existed in the central portion of the ligament (Fig. 3, at 0 weeks). At the fi rst week, chondrocytes were surrounded by metachromatic areas in toluidine blue staining interspersed with ligamentous fi bers (Fig. 3, at 1 week). By the second week, the chondrocytes became hypertrophic, with extracellular portions deeply stained by toluidine blue, which were regarded to be areas of calcifi cation (arrow in Fig. 3, at 2 weeks).

Electron microscopic images also indicated that chondrogenic alteration of ligamentous fi broblasts occurred chronologically. In the control group, fl at- tened fi broblasts existed alongside thick fi brils, re- garded as type I collagen in the central portion (Fig. 4a).

At the fi rst week, ligamentous cells became slightly

bloated (Fig. 4c). In this matrix the number of type I

BMP-2-induced Ossification of Spinal Ligaments 95

collagen fi brils decreased, while at the same time thin fi brils of type II collagen began to make their appear- ance among type I collagen fi brils (Fig. 4d). By the second week, chondrocytes were surrounded by a large number of type II collagen fi brils. These chondrocytes were located in the cartilage lacunae and resembled hypertrophic cells in the hyaline cartilage (Fig. 4e).

Areas of endochondral calcifi cation surrounded the hypertrophic cells. Matrix vesicles (30 to hundreds of nanometers in diameter) containing needle-like crys- tals or calcifi ed collagen were present in the calcifi ed areas (Fig. 4f ).

The localization of alkaline phosphatase (ALPase) activity, which is one of the chondrogenic markers, was examined. In the control group, no reaction was seen in the fl attened fi broblasts of the central portion (Fig. 5a). During the fi rst week, ALPase activity was observed in some fi broblasts in the central portion (Fig.

5 b), and by the second week chondrocytes had occu- pied the region where the ligamentous tissues originally existed and showed signifi cant ALPase activity (Fig. 5c).

The area containing such ALPase-positive chondro- cytes experienced substantial vascular invasion. In this area, tartrate-resistant acid phosphatase (TRAPase)- positive chondroclasts were also present (Fig. 5d).

Interpretation and Perspectives

The electron microscopic fi ndings suggested that the fl attened fi broblasts differentiated into chondrocytes through the action of exogenous BMP-2. An enzyme histochemical study for ALPase also showed that after injection of BMP-2 some fl attened fi broblasts initiated ALPase activity, which is known to appear in osteo- blasts or chondrocytes. The fl attened fi broblasts in the central portion of the spinal ligaments contained BMP receptor types IA and II [16], implying that fl attened fi broblasts are the target of BMP-2 and supporting the possibility that ligamentous fi broblasts differentiate into chondrocytes. The alteration of the extracellular matrix was accompanied by differentiation of fi bro- blasts into chondrocytes. Ligamentous fi broblasts embedded in type I collagen-based matrix differenti- ated, at fi rst, into chondrocytes in fi brous cartilage con- taining both types I and II collagen fi brils; and they later became those in hyaline cartilage consisting of type II collagen fi brils (Fig. 6).

Vascular invasion of hyaline cartilage induced by BMP-2 was followed by TRAPase-positive chondro- clasts (Fig. 6). This provides ample evidence of endo- chondral ossifi cation. Previous clinicopathological Fig. 1. a Injection method. b Data showing the

areas where ossifi cation occurred in each animal (n = 50)

a

b

fl ava with Azan staining. CA, cartilaginous tissue;

SC, spinal cord; SL, spinal lamina; arrows, vascular invasion. (Modifi ed from Hoshi et al. [16])

Fig. 3. Light micrographic observation with toluidine blue staining under a higher magnifi cation. Cc, chondrocytes; Fb, fi bro-

blasts; vertical arrows, ligamentous fi bers; horizontal arrows, calcifi cation areas. (Modifi ed from Hoshi et al. [17])

BMP-2-induced Ossification of Spinal Ligaments 97

Fig. 4. Electron micrographic alteration of ligamentous fi bro- blasts. a Normal. b–f Bone morphogenetic protein (BMP-2)- treated fi broblasts. b Some fi broblasts contain abundant lysosomes (Ly) and invagination of striated collagen fi brils (arrows), suggesting digestion of the type I collagen-based matrix. d Area in the square in c. Thin fi brils of type II collagen

(arrows) are visible among the thick fi brils of type I (Co).

e Endochondral calcifi cation occurs in the matrix of hypertro- phic chondrocyte. f Collagen calcifi cation (arrows) is observed.

Cal, calcifi cation areas; Cc, chondrocytes; CN, calcifi ed

nodules; MV, matrix vesicles; N, nucleus. (Modifi ed from

Hoshi et al. [17])

observations have indicated that the ossifi cation process is due, for the most part, to endochondral ossifi cation [10,11], a process similar to that in this experiment.

Matrix vesicle calcifi cation was recognized in BMP- 2 -induced cartilage followed by endochondral ossifi ca- tion. The matrix vesicles formed a scaffold during calcifi cation, of endochondral ossifi cation in the growth plate, or intramembranous ossifi cation in the calvaria.

The BMP-2-induced matrix vesicles observed in this model were larger than usual, exceeding hundreds of Fig. 5. Alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry. a Liga- mentum fl avum (Lig) is observed between large horizontal arrows. b, c Some fi broblasts (small horizontal arrows) or

chondrocytes in ectopic cartilage (CA) are ALPase-positive.

d Arrows indicate TRAPase-positive chondroclasts. (Modifi ed from Hoshi et al. [16])

Fig. 6. Summary of morphological changes in ligamentous fi broblasts and the surrounding matrices

nanometers in diameter. These larger matrix vesicles

may be derived from coarse cell budding or the rough

cell debris produced during rapid alteration of the liga-

mentous fi broblasts. The calcifi cation that provides the

hard property in a soft tissue is a major milestone

during ectopic bone formation. The mechanisms by

which such a specifi c matrix vesicle is produced or

crystallization occurs in the matrix vesicles should be

elucidated to provide a key to inhibiting the occurrence

or progression of this pathological ossifi cation.

BMP-2-induced Ossification of Spinal Ligaments 99 In this model, BMP-2-induced ossifi cation pro-

gressed from both enthesis sites of the ligamentum fl avum. Previous reports involving the spinal ligaments also indicated that bone formation progressed via enthesis of the ligament [10,11]. Interesting differences were noted between the characteristics of fl attened fi broblasts in the central portion of ligaments and those seen in the cells located at the enthesis site. First, cells having the BMP receptor were more abundant at the enthesis site than in the central portion [16]. Second, those cells also exhibited ALPase activity, whereas fl at- tened fi broblasts of the central portion did not [16].

Some previous papers noted that cells in the ligament or tendon joint to the bone, “enthesis,” had a fi ne struc- ture similar to that of chondrocytes in fi brous cartilage [18,19]. The cells at the enthesis site may easily differ- entiate into chondrocytes because of their similarities, and therefore ossifi cation at and around the enthesis site had progressed compared with that in the central portion.

Based on two fi ndings—fi rst, that BMP-2-induced bone formation occurred during the process of endo- chondral ossifi cation and, second, that the ossifi cation process was initiated at the enthesis site—the author believes the ossifi cation of spinal ligaments in this experimental model to be similar to that observed in clinics. Although stenosis resulting from protruding bone induced by BMP-2 increased up to the third week following BMP-2 injection, it was observed to decrease after that. Active resorption of the bony protrusion by osteoclasts located ventrally was observed at the second week. Compression of the spinal cord may be quickly eliminated by certain mechanisms. In contrast, patients suffering from ossifi cation of spinal ligaments contin- ued to experience spinal cord compression for several decades. Some unidentifi ed factors may be responsible for maintaining the compression of the spinal cord.

Acknowledgments. I express my sincere appreciation to Dr. Hidehiro Ozawa (Matsumoto Dental University), Dr. Norio Amizuka (Division of Oral Anatomy, Faculty of Dentistry, Niigata University), and Drs. Takahide Kurokawa and Kozo Nakamura (Department of Ortho- paedic Surgery, Faculty of Medicine, the University of Tokyo) for their valuable collaboration and kind instructions in this research.

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