Possible Roles of Bone Morphogenetic Proteins and Transforming Growth Factor- bs in the
Pathogenesis of OPLL and OLF
Hideki Yoshikawa
Introduction
Ossifi cation of the posterior longitudinal ligament (OPLL) and the ligamentum fl avum (OLF) is a patho- logical condition in the spinal ligament, with hetero- topic bone mainly through endochondral ossifi cation.
Bone morphogenetic proteins (BMPs) and transform- ing growth factor- βs, which belongs to the transform- ing growth factor- β superfamily (TGFβs), have been responsible for new bone and cartilage formation in vivo. The participating regulatory factors in the complex process (e.g., ligands of the TGF- β superfamily and responsive cell types that express their specifi c recep- tors) may resemble those that lead to pathological ectopic bone formation. Therefore, they might be caus- ative factors in the pathogenesis of OPLL and OLF. Pos- sible mechanisms are as follows: (1) systemic overexpression of BMPs/TGF βs, their receptors, or both in the patients, such as BMP-4 overexpression in fi brodysplasia ossifi cans progressiva [1]; (2) local over- expression of BMPs/TGF βs, their receptors, or both around or in the spinal ligaments; and (3) enhancement of responsiveness to BMPs/TGF βs in the mesenchymal cells around or in the ligaments. Systemic overexpres- sion of BMPs/TGF βs in OPLL or OLF patients has never been reported, but there have been several reports of local overexpression of BMPs/TGF βs, their receptors, or both and of enhancement of responsiveness to BMPs/TGF βs. These data suggest that BMPs/TGFβs may play a signifi cant role in the pathogenesis of OPLL and OLF. This chapter reviews the accumulated infor- mation on BMPs/TGF βs in OPLL and OLF and dis- cusses the biological and clinical signifi cance of BMPs/TGF βs.
Bone Morphogenetic Proteins and Transforming Growth Factor- bs
Bone morphogenetic proteins (BMPs) were originally identifi ed as proteins capable of inducing ectopic carti- lage and bone formation when implanted subcutane- ously or in muscle pouches. This ectopic cartilage/bone formation recapitulates the entire sequence of events that occurs during endochondral bone development in limb buds, where there is a sequential cascade of events:
chemotaxis of mesenchymal cells; mesenchymal cell condensation and proliferation and their differentia- tion into chondrocytes that produce cartilage matrix;
angiogenesis and vascular invasion; absorption of car- tilage with the appearance of osteoblasts that deposit bone matrix; and fi nally mineralization and remodel- ing. In 1988, Wozney et al. [2] cloned the BMP-1–4 genes, and subsequent studies have revealed that BMPs comprise a large subfamily of the TGFβ superfamily.
Abnormality of the gene coding for BMPs has never been reported in OPLL and OLF patients, but over- expression of BMP-4 in lymphocytes from patients with fi brodysplasia ossifi cans progressiva [1] suggests the possibility of some abnormality of BMP genes in patients with spinal ligament ossifi cation. BMPs bind to BMP receptors on the cell surface, and their signals are transduced intracellularly by Smad proteins [3]. Recent studies of tissue-specifi c activation and inactivation of BMP signals have revealed that BMP signals control proliferation and differentiation of chondrocytes, dif- ferentiation of osteoblasts, and bone quality [4]. BMPs are now used for regeneration of bone during fracture healing and spinal fusions as well as in dental tissue engineering. BMPs 2 and 7 are currently approved by the U.S. Food and Drug Administration (FDA) for clini- cal use.
On the other hand, TGF βs were identifi ed as a group of molecules that mediate many key events during normal development and growth of diverse tissues, including cartilage and bone [5]. Daily injections of TGF β into the periosteum of parietal bones or long bones of neonatal rats resulted in localized cartilage and bone formation [6]. The TGF βs seem to stimulate
Department of Orthopaedic Surgery, Osaka UniversityGraduate School of Medicine, 2-2 Yamadaoka, Suita 565-0871, Japan
59
new cartilage and bone formation by the periosteal chondroprogenitor and osteoprogenitor cells. Although they have no inductive activity of new cartilage or bone formation at extraskeletal sites, their biological effects suggest that TGF βs may contribute to the development, growth, and maintenance of ectopic bone formation, including OPLL and OLF. Several studies have been conducted to examine the relation between TGF β poly- morphism (T → C transition in the signal sequence) and OPLL. The T869 → C polymorphism of the TGFβ gene is a genetic determinant of a predisposition to OPLL, with the C allele representing a risk factor for genetic susceptibility to OPLL in Japanese subjects [7].
The polymorphism was not a factor associated with the occurrence of OPLL but, rather, a factor related to the area of the ossifi ed lesion. The “C” allele might be a risk factor for patients with OPLL in other areas as well as the cervical lesion [8].
Experimental and Clinical OLF Induced by BMP
Some reports have demonstrated that local implanta- tion of BMPs could induce OLF in animals [9–11].
Miyamoto et al. reported that OLF and secondary spinal cord compression were induced in mice by implanta- tion of partially purifi ed BMP [9]. A BMP–collagen composite was implanted into the posterolateral epidu- ral space. After 4 weeks, radiologic examination showed that the beak-like calcifi ed prominences arose from the laminae and protruded into the spinal canal, as seen on a lateral radiograph (Fig. 1). Histological examination of the ligamenta fl ava showed that the ligamentum fl avum became hypertrophied and the newly formed bony prominences protruded into the spinal canal at 4 weeks; the endochondral ossifi cation then became more advanced and matured at 8 weeks (Fig. 2). The spinal cords exhibited deformation secondary to compression by the protruded ossifi ed ligament. In those specimens with severe deformation of the spinal cord, both the white and gray matter had degenerated, and demyelin- ation in the posterior and lateral white columns was detected. The study indicates that OLF can be experi- mentally induced by BMP, and that mesenchymal cells able to respond to BMP and differentiate into chondro- cytes/osteoblasts do exist in or around the ligamentum fl avum of mice. Another important outcome was that the pathological fi ndings showed a close resemblance to those reported in clinicopathological studies: (1) ossifi cation is accompanied by degeneration and hyper- trophy of the ligament; (2) the bony prominences arise from the superfi cial surface of the laminae and extend along the superfi cial layer of the hypertrophied liga- ment; (3) the ossifi ed ligament develops through a
process of enchondral ossifi cation; and (4) the ossifi ed ligamentum fl avum increases in size with time and causes gradual compression and deformation of the spinal cord, resulting in the pathological changes.
Saito et al. studied chronic cord compression with OLF induced by a crude BMP fraction in rabbits [10].
The ossifi cation pattern was similar, and the histopath- ological changes in the spinal cord due to BMP-induced OLF seemed to be an early pathologic condition caused by chronic cord compression.
Fig. 1. Lateral radiologic picture of a lumber spine 8 weeks after bone morphogenetic protein (BMP) implantation. Beak- like bony prominences (arrow) are discernible
Fig. 2. Histological picture of a sagittal section of the ligamen- tum fl avum 4 weeks after BMP implantation. The ligament is hypertrophic and bony prominences (arrowheads) protrude into the spinal canal from the laminae (L) and compress the spinal cord (SC)
BMP/TGF-β in OPLL and OLF
61 Hoshi et al. injected an aqueous solution containing
recombinant human BMP-2 into murine ligamenta fl ava, and the ossifi cation process was analyzed mor- phologically [11]. During the week immediately follow- ing injection of BMP-2, ligamentous fi broblasts began to proliferate, differentiating into alkaline phosphatase- positive chondrocytes surrounded by an extracellular matrix composed of type I and II collagen. By the second week, differentiated chondrocytes at various stages were observed in type II collagen-rich matrix.
These chondrocytes showed an abundance of BMP receptors type IA and II. By the third week BMP-induced ossifi cation had compressed the spinal cord, and by the sixth week the ligamentous tissue had been almost completely replaced by bone. BMP receptors were up- regulated during chondrifi cation of ligamentous fi bro- blasts induced by exogenous BMP-2, suggesting that BMPs play an important role in the ossifi cation of spinal ligaments.
Clinically, there is an interesting phenomenon of OLF induced by BMP-producing osteoblastoma. Okuda et al. reported three cases of spinal osteoblastoma with OLF adjacent to the tumor [12]. Computed tomography (CT) demonstrated a typical radiolucent nidus in the spinal pedicle/lamina with a dense sclerotic rim. In addition, ectopic bone formation at the insertion point of the ligamentum fl avum adjacent to the tumor was clearly illustrated. Histological examination revealed endochondral OLF, which is quite unusual for normal young adults. Immunohistochemical assays demon- strated that BMP-2/4 was expressed in the osteoblastic tumor cells. The report raises the possibility that BMPs secreted from the tumor cells triggered ectopic ossifi ca- tion in the spinal ligament.
Expression of BMPs, TGF bs, and Their Receptors in OPLL and OLF
In 1992, Kawaguchi et al. reported expression and local- ization of BMP-2 and TGF βs at the ossifi cation site of the posterior longitudinal ligament of the cervical spine [13]. The immunohistochemical analysis showed that both BMP-2 and TGF β were present in the ossifi ed matrices and chondrocytes of adjacent cartilaginous areas of OPLL. Immunostaining for BMP-2 was also positive in mesenchymal cells with fi broblastic features in contact with the cartilaginous areas, but the staining for TGF β was negative in those cells. Expression of BMP-2 and TGF β was limited to the ossifi ed ligament, but no expression for BMP-2 or TGF β was detected in the nonossifi ed areas of the posterior longitudinal liga- ment from the same patients. These data suggested that BMP-2 and TGF β play important roles in the develop- ment of OPLL. BMP-2 may be responsible for initiating
the formation of OPLL by differentiating mesenchymal cells into chondrocytes or osteoblasts, and TGF β may stimulate bone formation at a later stage during ossifi cation.
In 1997, Yonemori et al. reported expression of OP- 1 /BMP-7 and their receptors (BMPR-IA, BMPR-IB, BMPR-II), as seen by immunohistochemistry, in ossi- fi ed ligament tissues of patients with OPLL and control ligament tissues from patients with cervical disc hernia- tion [14]. OP-1/BMP-7 was expressed in chondrocytes near the calcifi ed zone. Expression of BMPRs was ele- vated in OPLL patients compared with controls. BMPR- IA, BMPR-IB, and BMPR-II expression was observed not only in chondrocytes in fi brocartilage tissue around the calcifi ed zone but also in fi broblast-like spindle cells of the nonossifi ed ligament. OP1/BMP-7 was not detected in the control cases, and the BMPRs were only weakly expressed in fi brocartilage tissue at the site of ligament attachments to bone. Enhanced expression of BMPRs at the nonossifi ed ligament in OPLL patients suggests that these cells have a greater potential to dif- ferentiate into osteogenic cells than ligament cells from non-OPLL patients.
As for OLF, Hayashi et al. examined immunohisto- chemically the expression and localization of bone morphogenetic proteins (BMP-2/4 and OP1/BMP-7) and their receptors (BMPR-IA, BMPR-IB, BMPR-II) in the ligamentum fl avum of fi ve patients with OLF [15].
BMP-2/4 and OP-1/BMP-7 were co-localized in OLF patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcifi ed zone and in spindle-shaped cells and round cells in a part remote from ossifi ed foci in examined OLF tissue. In the control cases, expression of BMPs and BMPRs was reduced and was observed only around the calcifi ed zone at the insertion of the ligamentum fl avum to the bone. In summary, the expression profi le of BMPs and BMPRs in OPLL and OLF patients was entirely different from that of the control patients, suggesting that BMPs may be involved in promoting endochondral ossifi ca- tion at ectopic ossifi cation sites in patients with OPLL and OLF.
Analyses of Spinal Ligament Cells from OPLL and OYL Patients
Various cells derived from spinal ligaments and the
surrounding tissues have been cultured and character-
ized, and the effects of BMPs and TGF βs on those cells
have been examined in vitro [16,17]. The ligament cell
lines obtained from nonossifi ed sites in patients with
OPLL were found to have several phenotypic character-
istics for osteoblasts: high alkaline phosphatase (ALP)
activity, parathyroid hormone (PTH)- and prostaglan-
din E
2(PGE
2)-stimulated increases in cyclic AMP, and responses to both calcitonin (CT) and 1,25-dihydroxyvi- tamin D
3[1,25-(OH)
2D
3], suggesting that many cells with osteoblast-like characteristics are present [17].
ALP activity was high in the cultured cells of OPLL patients. Exogenous TGF β inhibited proliferation in the OPLL cells but promoted proliferation in control cells, suggesting that the spinal ligaments of OPLL patients have an osteogenic predisposition and that TFG β may play a role in the ossifi cation [18].
Kon et al. isolated spinal ligament cells from OPLL and non-OPLL patients and analyzed the effects of BMP- 2 on these cells [19]. BMP-2 caused a signifi cant increase in ALP activity in four OPLL cell lines, whereas the activ- ity did not change in the non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in fi ve cell lines, indicating that BMP-2 prefer- entially induces osteogenic differentiation in OPLL cells versus non-OPLL cells. OPLL cells exhibited a response to BMP-2 different from that of the non-OPLL cells, sug- gesting that the expression of BMP receptor(s) and the signal transduction initiated by BMP-2 in the spinal lig- ament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells.
More recently, Tanno et al. reported the effects of in vitro sinusoidal cyclic stretch on cultured spinal ligament cells derived from OPLL and non-OPLL patients [20]. The mRNA expressions of BMP-2, BMP- 4 , and BMP receptors as well as ALP activity in cell layers and production of BMPs in the conditioned medium were signifi cantly increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. Mechanical stress seemed to play a role in the progression of OPLL through the induction of osteogenic differentiation in spinal ligament cells and promotion of the autocrine/paracrine mechanism of BMPs.
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