Mutsumi Matsushita

Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin (PG)E2 promotes both bone resorption and bone formation. By infusing PGE2 to mice lacking each of four PGE receptor (EP) subtypes, we have identified EP4 as the receptor that mediates bone formation in response to this agent. Consistently, bone formation was induced in wild-type mice by infusion of an EP4-selective agonist and not agonists specific for other EP subtypes. In culture of bone marrow cells from wild-type mice, PGE2 induced expression of core-binding factor α1 (Runx2/Cbfa1) and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggest that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

Association of the human NPPS gene with ossification of the posterior longitudinal ligament of the spine (OPLL).

Abstract OPLL (ossification of the posterior longitudinal ligament of the spine) is a common form of human myelopathy with a prevalence of as much as 4% in a variety of ethnic groups. To clarify the genetic factors that predispose to OPLL, we have studied ttw (tiptoe walking), a mouse model that presents ectopic ossification of the spinal ligaments similar to OPLL and have found that the ttw phenotype is caused by the nonsense mutation of the gene encoding nucleotide pyrophosphatase (NPPS), a membrane-bound glycoprotein thought to produce inorganic pyrophosphate, a major inhibitor of calcification and mineralization. To investigate a possible role of NPPS in the etiology of OPLL, we have examined its genetic variations in OPLL patients. A total of 323 OPLL patients was screened by means of polymerase chain reaction/single-strand conformation polymorphism analysis covering all the exons and their surrounding introns, plus about 1.5-kb of the promoter region. We identified ten nucleotide variations in the NPPS gene; five of the alterations caused amino-acid substitutions, and two of them were found specifically in OPLL patients. Subsequently, we performed an association study using these variations and found a significant association of an allele, viz., a deletion of T at a position 11 nucleotides upstream from the splice acceptor site of intron 20 (IVS20–11delT), with OPLL; the proportion of the individuals having this deletion was significantly higher (P = 0.0029) in OPLL patients than in controls, indicating that those who have this variation may be more susceptible to the abnormal ossification of the spinal ligaments. Thus, our study suggests that NPPS plays an important role in the etiology of human OPLL.

Identification of peak bone mass QTL in a spontaneously osteoporotic mouse strain

Abstract. The whole genome scan for quantitative trait loci (QTLs) specifying peak bone mass was performed with the F2 intercrosses of SAMP6, an established murine model of senile osteoporosis, exhibiting a significantly lower peak bone mass, and SAMP2, exhibiting a higher peak bone mass. Cortical thickness index (CTI), a parameter of bone mass of femurs, was measured in 488 F2 progeny at 4 months of age, when the animals attained peak bone mass by microphotodensitometry. Genetic markers were typed at 90 loci spanning all chromosomes except the Y. By interval mapping of 246 male F2 mice, two loci were identified with significant linkage to peak bone mass, one on Chromosome (Chr) 11 and another on Chr 13, with a maximum lod score of 10.8 (22.2% of the total variance) and 5.8 (10.0%), respectively. Another locus on the X Chr was suggestive of a QTL associated oppositely with a low peak bone mass to the SAMP2 allele. This association was consistent with the distribution of peak bone mass in the F1 and F2. These findings should be useful to elucidate the genetics of osteoporosis.

Atlantoaxial transarticular screw fixation for a high-riding vertebral artery.

Study Design. The feasibility of inserting a screw for the narrow isthmus with a high-riding vertebral artery was evaluated in patients subjected to posterior atlantoaxial transarticular screw fixation. Objective. To demonstrate the feasibility of inserting bilateral screws and obtaining a stable atlantoaxial complex for patients with a high-riding vertebral artery. Summary of Background Data. Posterior atlantoaxial transarticular screw fixation entails the potential risk of vertebral artery injury, which may be lethal. The risk is much higher for the narrow isthmus caused by a high-riding vertebral artery, and many authors recommend that the procedure should be abandoned if the isthmus is too narrow. On the other hand, bilateral screw fixation is stronger than unilateral screw fixation. Methods. For this study 27 consecutive patients who submitted to atlantoaxial transarticular screw fixation were evaluated before surgery for the position of the vertebral artery grooves using computed tomography (CT) reconstruction. Seven of the patients were defined as having a unilateral high-riding vertebral artery. For these patients, bilateral screw insertion through the most posterior and medial part of the isthmus was performed. Results. No massive bleeding or major complications were encountered in any patients with a high-riding vertebral artery. Postoperative computed tomography reconstruction demonstrated that five of the screws cleared the vertebral artery groove successfully, and two slightly breached it. No screws penetrated into the vertebral artery groove. Conclusions. It is possible to insert a screw safely, even into the narrow isthmus with a high-riding vertebral artery, if the surgeon realizes where the screw should be inserted and has the requisite insertion technique. Bilateral screw fixation should provide a high fusion rate.

Periodic acid-Schiff (PAS)-positive, granular structures increase in the brain of senescence accelerated mouse (SAM)

SummaryAbnormal granular structures, which stained positively with periodic acid-Schiff (PAS-positive granular structures; PGS), were observed in the brain of senescence accelerated mouse (SAM). They were small, round to ovoid, homogenous structures measuring up to 5 μm in diameter and usually grouped in clusters. PGS were localized in the hippocampus, piriform cortices, olfactory tubercle, nucleus of the trapezoid body, and cerebellar cortices. Quantitative analysis revealed that PGS remarkably increased in the hippocampus of SAM-P/8, a substrain of SAM, with advancing age, although a few PGS also appeared in the aged control mice, SAM-R/1 and DDD. Their histochemical nature, morphological features and distribution pattern were different from those of corpora amylacea and other similar bodies. A close anatomical relationship between PGS and glial fibrillary acidic protein-positive astrocytes was inferred from immunohistochemical studies. PGS is considered to be one of the morphological manifestations of senescence in mice brains, and are found to occur more numerously in the brains of learning or memory deficit mice, SAM-P/8.

Lumbar posterolateral fusion with biphasic calcium phosphate ceramic.

The authors conducted a retrospective observational study of patients who needed lumbar posterolateral fusion (PLF) using a biphasic calcium phosphate ceramic implant as a substitute for bone graft. The findings of clinical, radiographic, and histologic examinations were reviewed. Thirty-two patients underwent single-level PLF with instrumentation. In all cases, to decrease the occurrence of donor-site complications and morbidity, locally harvested morselized bone from the decompressive site was mixed with hydroxyapatite and beta-tricalcium phosphate (HAP-TCP) granules and sticks and used for fusion at the posterolateral aspect of the lumbar spine. The histologic findings of three biopsy specimens obtained during second operations for metallic implant removal were reviewed. The minimum follow-up period was 26 months. There was no evidence of instrument loosening or breakage. However, bone-graft incorporation was difficult to evaluate radiographically, because image quality was inferior to that with conventional autogenous iliac bone graft. Partial graft bone resorption was revealed on radiographs in 75% of cases. The results showed clinical improvement in all but one case. Solid fusion was observed during the second operation in all three cases. Histologic analysis showed excellent bone incorporation around the HAP-TCP granules. These findings suggest that, although the bulk of the fusion mass with HAP-TCP was smaller than that with autogenous bone, this technique combined with rigid instrumentation is a safe and effective procedure.

Decreased endosteal formation during cortical bone modelling in SAM-P/6 mice with a low peak bone mass.

Inter-strain differences in bone mass and density during growth were followed in three strains of mice: SAM-P/2, SAM-R/1 and SAM-P/6 (a murine model of senile osteoporosis, Matsushita et al., Am J Pathol 1986;125:276-283). Photometrically, the inter-strain disparities first appeared in mice at about age 28 days and increased until age 60 days. During this period, tetracycline labelling revealed significant strain differences regarding rate of the appositional formation at the endosteal surface but not at the periosteal surface. The order coincided with results of the photometrical assay, that is, highest in SAM-P/2, followed by SAM-R/1 and SAM-P/6, respectively. Therefore, strain differences, especially the osteopenic state of SAM-P/6, occur, at least in part, by disparities in endosteal formation rates during cortical bone modelling.

Chromosome 13 locus, Pbd2, regulates bone density in mice.

Bone density is inherited as a complex polygenic trait. Previously, we identified two quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected by bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains: SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. In this study, we report the effects of the Chr 13 QTL on peak bone density (Pbd2). First, we constructed a congenic strain P6.P2‐Pbd2b, which carried a single genomic interval from the Chr 13 of SAMP2 on an SAMP6‐derived osteoporotic background, to dissect this polygenic trait into single gene factors. This congenic strain had a higher bone density than the background strain using three measurement methods with different principles for bone density. Next, we measured the peak relative bone mass of the AKR/J strain and the 13 senescence‐accelerated mouse (SAM) strains, which are considered to be a series of recombinant‐like inbred (RI) strains derived from the AKR/J strain and other unspecified strains. We then determined the microsatellite marker haplotypes of these strains around the Pbd2 locus, in which three strains with a high relative bone mass shared the same haplotype over the 26‐centimorgan (cM) region. In the Pbd2 locus, a high relative bone mass was associated with alleles of the unknown strain, whereas a low relative bone mass was associated with the alleles from the AKR/J strain. These results confirmed the existence of a Pbd2 locus regulating bone density in the SAM strains.

Consistency of low bone density across bone sites in SAMP6 laboratory mice

The development of bone densitometry has made it clear that there are discrepancies in bone density at various measurement sites in a given individual. This study examined the consistency of bone density measurements across various sites in a strain of laboratory mouse (senescence-accelerated mouse; SAM). A systemic evaluation of the bone density was performed by dual-energy X-ray absorptiometry (DXA) on SAMP6 (P6) mice, a strain with low peak bone density, as measured by microphotodensitometry of the femoral bones, whereas the SAMP2 (P2) and SAMR1 (R1) strains have high peak bone density. We modified Jilka’s method to more comprehensively measure the whole body and additional regions of interest (ROIs; head, right foreleg, left foreleg, right hindleg, left hindleg, spine, and tail). The age-related changes in the total (whole-body) BMD showed a common pattern among the strains studied, and the peak value was seen at 4 months old. P6 showed the lowest peak BMD. A detailed comparison of the bone density between P6 and P2 at the age of 4 months revealed significantly lower regional BMD values for P6 in all seven ROIs. The strain difference in BMD could not be attributed to a difference in size. In conclusion, P6 mice showed low bone density not only in their femurs but also in the subregions and over their entire body. This strain can be potentially useful in the investigation of the genetic basis of senile osteoporosis.

Chylous leakage after circumferential thoracolumbar fusion for correction of kyphosis resulting from fracture. Report of three cases.

Study Design. A description of the clinical picture of chylous leakage after spinal surgery. Objectives. To present the clinical course of three cases of chylous leakage after spinal surgery and to discuss the pathogenesis of the disease. Summary of Background Data. Chylous leakage is a rare complication after spinal surgery. It has been attributed to direct injury of a lymphatic trunk or one of its major tributaries by surgical maneuver. Methods. Three cases of chylous leakage after circumferential thoracolumbar fusion for correction of kyphosis resulting from fracture were reported. Results. All of the three cases were managed successfully; two cases of chyloretroperitoneum detected within 4 days after surgery were healed conservatively, but one case of chylothorax, of which the onset was noticed 5 weeks after spinal surgery, required surgical ligation of the thoracic duct and pleurodesis. Conclusion. Early detection of this disease is important for a good prognosis. Retroperitoneal drainage is necessary for the detection and management of chyloretroperitoneum. The pathogenesis and management of the chylous leakage are discussed in this report.