Takashi Sakou

Bone Morphogenetic Proteins: From Basic Studies to Clinical Approaches

There is growing evidence to suggest that BMPs and their receptors play an essential role in the formation of skeletal tissues, but the mechanism by which these molecules exert their functions in vivo remains to be elucidated. We have immunohistochemically studied the spatial and temporal localization of BMPs and receptors for BMPs during chondro-osteogenesis in various mammalian tissues, i.e., limb buds during embryonic development, fracture healing sites, articular cartilage, growth plate, and ectopic ossification sites of the spinal ligament. This review focuses on the functional roles of BMPs, their receptors and their signaling molecules in mammals during embryonic skeletal development, and chondro-osteogenesis in physiological and pathological conditions.

Strain on intervertebral discs after anterior cervical decompression and fusion.

STUDY DESIGN An analysis of the change in strain distribution of intervertebral discs present after anterior cervical decompression and fusion by an original method. The analytical results were compared to occurrence of herniation of the intervertebral disc on magnetic resonance imaging. OBJECTIVES To elucidate the influence of anterior cervical decompression and fusion on the unfused segments of the spine. SUMMARY OF BACKGROUND DATA There is no consensus regarding the exact significance of the biomechanical change in the unfused segment present after surgery. METHODS Ninety-six patients subjected to anterior cervical decompression and fusion for herniation of intervertebral discs were examined. Shear strain and longitudinal strain of intervertebral discs were analyzed on pre- and postoperative lateral dynamic routine radiography of the cervical spine. Thirty of the 96 patients were examined by magnetic resonance imaging before and after surgery, and the relation between alteration in strains and postsurgical occurrence of disc herniation was examined. RESULTS In the cases of double- or triple-level fusion, shear strain of adjacent segments had increased 20% on average 1 year after surgery. Thirteen intervertebral discs that had an abnormally high degree of strain showed an increase in longitudinal strain after surgery. Eleven (85%) of the 13 discs that showed an abnormal increase in longitudinal strain had herniation in the same intervertebral discs with compression of the spinal cord during the follow-up period. Relief of symptoms was significantly poor in the patients with recent herniation. CONCLUSIONS Close attention should be paid to long-term biomechanical changes in the unfused segment.

Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Localization of Smads, the TGF-β Family Intracellular Signaling Components During Endochondral Ossification

Members of the transforming growth factor‐β (TGF‐β) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF‐β signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF‐β superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF‐β1 and its receptors, and BMP‐2/4 and BMP receptors. The results show that TGF‐β1 and BMP‐2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF‐β/activin‐restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway‐restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF‐β superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.

Natural History of Degenerative Spondylolisthesis: Pathogenesis and Natural Course of the Slippage

To clarify the natural course of degenerative spondylolisthesis, the mechanism and progression of disk slippage were studied clinically and radiographically in 40 patients. Progressive slippage was observed in 12 patients (30%). No progression of slippage was noted in patients who showed narrowing of the interyertebral disk, spur formation, subcartilaginous sclerosis, or ossification of ligaments. These suggest that the mechanisms of spinal restabilization prevent progression of the disease. General joint laxity was observed in many patients (65%), and this was believed to be involved in the pathogenic mechanism of this disease. There was no correlation between the clinical symptoms and progression of slippage. These findings suggest that careful consideration of the natural mechanisms of spinal restabilization as well as the natural course of the disease is important.

Genetic mapping of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is recognized as a common disorder among Japanese and throughout Asia. Estimates of its prevalence are in the range of 1. 9%-4.3%. Although its etiology is thought to involve a multiplicity of factors, epidemiological and family studies strongly implicate genetic susceptibility in the pathogenesis of OPLL. In this study we report an identification of a predisposing locus for OPLL, on chromosome 6p, close to the HLA complex. The evidence for this localization is provided by a genetic-linkage study of 91 affected sib pairs from 53 Japanese families. In this sib-pair study, D6S276, a marker lying close to the HLA complex, gives evidence for strongly significant linkage (P = .000006) to the OPLL locus. A candidate gene in the region, that for collagen 11A2, was analyzed for the presence of molecular variants in affected probands. Of 19 distinct variants identified, 4 showed strong statistical associations with OPLL (highest P = .0004). These observations of linkage and association, taken together, show that a genetic locus for OPLL lies close to the HLA region, on chromosome 6p.

Preoperative and postoperative magnetic resonance image evaluations of the spinal cord in cervical myelopathy.

To evaluate the morphologic changes of the spinal cord in patients with cervical myelopathy due to cervical spondylosis and ossification of the posterior longitudinal ligament, the authors measured the thickness and signal intensity of the cervical cord with magnetic resonance imaging in healthy adults and patients with cervicla myelopathy, and compared these findings. In patients with cervical myelopathy, the preoperative and postoperative magnetic resonance imaging findings were compared with the severity of myelopathy and postoperative results. In healthy adults, the anteroposterior diameter of the cervical cord wad 7.8 mm at the C3 level and decreased at lower levels. In the patients with cervical myelopathy, the preoperative spinal anteroposterior diameter was significantly reduced at various levels corresponding to the stenosis site within the vertebral canal. In the group with ossification of the posterior longitudinal ligament, the minimal anteroposterior diameter of the cervical cord tended to decrease with increasing severity of myelopathy. However no relationship was observed between the two parameters in the cervical spondylotic myelopathy group. In the group with ossification of the posterior longitudinal ligament, surgical results were good when the postoperative anteroposterior diameter was increased, whereas in the cervical spondylotic myelopathy group there was no relationship between the parameters. In the patients with myelopathy, a high intensity area was observed in about 40% of all patients before operation and about 30% after operation. However, the presence or absence of a high intensity area did not correlate with the severity of myelopathy or with surgical results in the group with ossification of the posterior longitudinal ligament and the cervical spondylotic myelopathy groups.

Indication of fusion for lumbar spinal stenosis in elderly patients and its significance.

Study Design Selection of surgical therapy for lumbar canal stenosis in elderly patients is discussed. Decompression alone and decompression with fusion were evaluated. Objectives To determine the indication of decompression with fusion for lumbar spinal stenosis in elderly patients. Summary of Background Data Although there is no objection to posterior decompression, which is regarded as the first choice of surgical therapy for lumbar spinal stenosis in the elderly, it is debatable whether or not fusion should be used with decompression. Methods The presence or absence of instability was defined by Posners method from preoperative plain radiographic lateral findings. Thirty-four elderly patients with lumbar canal stenosis were studied. Seventeen of the 34 patients were found to have instability. Ten of the 17 patients with spinal instability underwent decompression and instrumented fusion. The seven remaining patients with spinal instability underwent decompression alone. The 17 patients without spinal instability were treated by decompression alone. Preoperative symptoms, postoperative results, and changes in radiographic findings were compared among the three groups. Results The group treated by decompression and fusion showed the best results. The group treated by decompression in the presence of instability showed the worst results by the Japanese Orthopaedic Association back scores. Good results can be obtained by decompression alone only if the patients do not have instability as defined by Posner. Conclusions The definition of instability by Posners method proved useful for selecting elderly patients with instability for fusion treatment. Fusion with instrumentation should be performed on elderly patients with instability after decompression.

Traumatic injury-induced BMP7 expression in the adult rat spinal cord

It has been reported that bone morphogenetic proteins (BMPs) are involved in the generation of the central nervous system during development. However, the roles of BMPs in mature spinal cord have not been clarified. We examined the expression of BMP7 mRNA before and after traumatic injury of the adult rat spinal cord. BMP7 mRNA was already detectable at a relatively low level in uninjured spinal cord, but was dramatically increased after injury. Semiquantitative RT-PCR study further confirmed upregulation of BMP7 mRNA in injured spinal cord. In situ hybridization indicated that expression of BMP7 mRNA was present only in glial cells in uninjured spinal cord. After injury, the number of BMP7-expressing glial cells was increased, BMP7 expression also became apparent in motor neurons. It has been suggested that BMPs promote survival of subventricular zone cells in adult rats. Thus, our results suggest that increase in the expression of BMP7 promotes survival of neurons and glial cells after acute traumatic injury. In contrast, there is increasing evidence that BMPs inhibit neurogenesis and alternatively promote gliogenesis of neural progenitors, which are also present in adult spinal cord, suggesting that injury-upregulated BMP7 may regulate differentiation of glial cells from neural progenitors and may induce gliosis after central nervous system injury.

Genetic study of ossification of the posterior longitudinal ligament in the cervical spine with human leukocyte antigen haplotype.

To evaluate the genetic background of ossification of the posterior longitudinal ligament, the relationship between the presence or absence of ossification and human leukocyte antigen haplotypes was studied in 33 families of patients with ossification of the posterior longitudinal ligament. The study revealed that human leukocyte antigen haplotypes formed certain types of clusters, and that some human leukocyte antigen haplotypes were very rare in the Japanese population, suggesting the involvement of human leukocyte antigen-linked factors in the pathogenesis of ossification of the posterior longitudinal ligament of the cervical spine. In the families of these patients, ossification of the posterior longitudinal ligament was demonstrated by radiography in 56% (10/18) of the siblings. Each of these siblings shared both human leukocyte antigen haplotypes with the patient. None of those who shared only one human leukocyte antigen haplotype with the patient had developed ossification of the posterior longitudinal ligament. From these findings, the presence of both pathogenic human leukocyte antigen haplotypes is considered to be necessary for the development of ossification of the posterior longitudinal ligament, and this genetic predisposition may be activated by multiple factors, including regressive degeneration due to aging and the environment.