A 12 month in vivo study on the response of bone to a hydroxyapatite-polymethylmethacrylate cranioplasty composite.

We investigated the osteoconductivity and biocompatibility in vivo of a new hydroxyapatite-polymethylmethacrylate (HA-PMMA) composite developed for use as an implant material for cranioplasty, which is expected to have the good osteoconductivity of HA together with the strength and ease of handling of PMMA. The HA-PMMA composites were implanted in eight full-grown beagles and then 6, 12, 24 weeks and 1 year after implantation, the animals were sacrificed and the implanted materials removed along with the surrounding tissues. Extirpated specimens were studied using an optical microscope and micro-computed tomography (micro-CT). Fibrous connective tissue was prominent in the interface of the composite at 6 weeks. New bone formation was seen around the implant, 12 and 24 weeks after operation. At 1 year, new bone filled in the interface of the HA-PMMA composite and adhered to the surrounding autogenous bone. Mixing HA and PMMA did not interfere with the osteoconductivity of the HA component. In micro-CT findings, the new bone growing on the HA-PMMA composite could be seen attaching preferentially to HA particles exposed at the composite surface, rather than the PMMA. This study demonstrated that this HA-PMMA composite is a good candidate for cranial bone implants due to its good osteoconductivity and biocompatibility.

Chromatographic separation of a small subunit (PsbW/PsaY) and its assignment to Photosystem I reaction center.

By using a hydroxyapatite column, the five major Photosystem I (PSI) subunits (PsaA,-B,-C,-D,-E) solubilized by sodium dodecyl sulfate (SDS) were fractionated from a spinach PSI reaction center preparation. Another small (5-6 kDa) polypeptide was also separated, and purified to homogeneity. Mass spectroscopy yielded its molecular weight to be 5942 +/- 10. This polypeptide had an N-terminal sequence homologous to those of previously reported 5-kDa subunits from spinach and wheat and a 6.1-kDa subunit of Chlamydomonas, which had all been assigned to Photosystem II (PSII) and designated as PsbW. However, we found similar 5-kDa polypeptides with highly conserved N-terminal sequences ubiquitously in PSI particles from other plants including Daikon (Raphanus sativus, Japanese radish), Chingensai (Brassica parachinensis, Chinese cabbage), parsley and Shungiku (Chrysanthemum coronarium, Garland chrysanthemum) as well. Preparations of spinach PSI particles prepared by using a mild detergent (digitonin) had this 5-kDa subunit, while PSII particles did not. Moreover, a bare-bone PSI reaction center preparation consisting of PsaA/B alone had a more than stoichiometric amount of this 5-kDa polypeptide. A mechanically (without detergent) fractionated stroma thylakoid preparation from Phytolacca americana, which lacked other PSII subunits, also contained this 5-kDa subunit. Thus, we propose that this 5-kDa polypeptide, previously designated as a PSII subunit (PsbW), is an integral subunit of PSI as well.

Adenosine receptor antagonists cancelled the ischemic tolerance phenomenon in gerbil.

Pretreatment of the brain with sublethal ischemia has been reported to induce neuronal resistance to otherwise lethal ischemia, a phenomenon designated as ischemic tolerance. The protective mechanisms of the phenomenon are not known yet, however, recent experimental data suggest the involvement of adenosine receptor activation in the acquisition of tolerance. In this study, the effect of theophylline, a non-selective adenosine receptor antagonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, were investigated to ascertain if these drugs could cancel the protective effect of ischemic tolerance in the gerbil. DPCPX or theophylline was administered at 3 h after a short preconditioning ischemia, and 21 h later animals were subjected to lethal ischemia of 5 min duration. DPCPX at a dose of 1.0 mg/kg (i.p) and theophylline at a dose of 20 mg/kg (i.p) significantly reduced the protective effect of preconditioning in the CA1 hippocampal neurons. These findings suggest the involvement of adenosine receptor activation for the development of ischemic tolerance phenomenon.

Expression of c-fos-like protein in the rat brain after injection of interleukin-1-beta into the gingiva.

Expression of the c-fos proto-oncogene in the rat brain was examined by immunostaining for fos, the nuclear protein product of the c-fos gene, after injection of interleukin-1-beta (IL-1 beta) into the gingiva of an incisor. The distribution pattern of labelled cells was compared with that induced by tooth pulp stimulation. Neurons that express fos-immunoreactivity (fos-IR) appeared in several regions in the neuraxis 1.5 h after IL-1 beta injection, peaked at 2 h, and then declined. Labelled cells were found bilaterally in regions that contribute to pain-relay and pain-inhibition. The distribution of labelled cells almost matched the pattern induced by noxious tooth pulp stimulation. In indomethacin-pretreated animals, no neurons expressing fos-IR were found in nuclei associated with relay of nociception nor in nuclei contributing to inhibition of nociception. The results suggest that a small amount of IL-1 beta at the site of periodontal disease can induce fos-IR in brain neurons through increased prostaglandin production.

Persistent primitive hypoglossal artery complicated by atrial septal defect and congenital intrahepatic shunts.

During early embryogenesis, anastomoses are formed between the carotid artery and the basilar or the vertebral artery, and subsequently, these anastomoses regress. In some cases, these anastomoses remain as persistent carotid-basilar or carotid-vertebral anastomoses. Atrial septal defect (ASD), a communication between the atria at the septal level, is a congenital heart anomaly. Intrahepatic venous shunts between the portal and hepatic veins are very rare and only some are considered congenital. We present the first case report of a patient with an ASD, a persistent primitive hypoglossal artery, and congenital portahepatic shunts.

Continuously applied compressive pressure induces bone resorption by a mechanism involving prostaglandin E2 synthesis.

In previous research, we devised a specific culture chamber to examine the effect of continuously applied compressive pressure (CCP) on bone formation and resorption. The chamber was infused with compressed mixed gases with different O2 and CO2 composition to maintain the pO2, pCO2, and pH in the culture medium under pressures of +0.5 atm (1.5 atm total) to +2.0 atm (3.0 atm total) at the same levels as those at the ordinary pressure (1 atm). Using the specific culture chamber, we demonstrated that CCP greatly suppressed the differentiation of mouse osteoblast-like MC3T3-E1 cells. The inhibition by CCP appeared to be mediated by prostaglandin E2 (PGE2). In the present study, we examined the effect of CCP on osteoclastic bone resorption. CCP treatment of mouse bone marrow culture markedly increased both the PGE2 production and the number of tartrate-resistant acid phosphatase (TRACP)-positive mononuclear cells (possibly precursors of multinucleated osteoclasts). An autoradiographic study using [125I]-salmon calcitonin showed clearly that those TRACP-positive cells had calcitonin receptors. The CCP effect was the greatest at +1.0 atm (2.0 atm total). Isobutylmethylxanthine potentiated the production of TRACP-positive cells induced by CCP. Adding indomethacin completely inhibited both the TRACP-positive cell formation and the PGE2 production induced by CCP. CCP also increased the release of 45Ca from prelabeled mouse calvaria during later stages (2-6 days) of the 6-day culture period. CCP markedly increased PGE2 but not interleukin 1 in the culture media of mouse calvaria. These results indicate that, besides inhibiting osteoblast differentiation, CCP stimulates bone resorption by generating new osteoclasts through a mechanism involving PGE2 production.

Cellular roles in relapse processes of experimentally-moved rat molars.

This histological study was designed to clarify the relapse processes of rat molars following orthodontic tooth movement by the Waldo method. An elastic band was inserted between the upper first and second molars, and removed either 7 or 21 days later. The relapse processes of first molars were examined at 1 or 4 days after completing the 7-day-insertion and at 5 or 10 days after the end of the 21-day-insertion. At the end of the 7-day-insertion period, the mean interdental space was 158 microns, but decreased rapidly, reaching 44 microns by 1 day after removal, and 16 microns by day 4. In the periodontal ligament (PDL) compressed by elastic band insertion, partial hyalinization and/or mineralization occurred. The stretched PDL consisted of irregularly-distributed, thin principal fibres, approximately 2.4 microns in diameter. On days 1 and 4 after removal, osteoclasts, macrophage-like cells and fibroblast-like cells were localized around hyalinized tissues, but thick principal fibres, approximately 3.8 microns in diameter, were regularly distributed throughout the PDL proper. The mean interdental space after the 21-day-insertion was 536 microns, but this had decreased to 108 microns by day 5, and 71 microns by day 10. On day 5 after removal, numerous osteoclasts were evenly distributed in the compressed PDL, while thick principal fibres, approximately 3.5 microns, were present in the stretched PDL. These results suggest that, during relapse of experimentally-moved rat molars, (i) rapid remodelling of the PDL and surrounding alveolar bones is the main cause of tooth relapse and (ii) hyalinized and/or mineralized tissues occurred by compression are rapidly resorbed by osteoclasts, macrophage-like cells and fibroblast-like cells.

Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro.

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.

Activation of cytosolic phospholipase A2 by platelet-derived growth factor is essential for cyclooxygenase-2-dependent prostaglandin E2 synthesis in mouse osteoblasts cultured with interleukin-1.

The synthesis of prostaglandins (PGs) is regulated by the arachidonic acid release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we examined the regulation of PG synthesis by interleukin (IL)-1alpha in primary mouse osteoblastic cells isolated from mouse calvaria. Although IL-1alpha greatly enhanced cox-2 mRNA expression and its protein levels, PGE2 was not produced until 24 h. When arachidonic acid was added to osteoblastic cells precultured with IL-1alpha for 24 h, PGE2 was produced within 10 min. Of several growth factors tested, platelet-derived growth factor (PDGF) specifically initiated the rapid synthesis of PGE2, which was markedly suppressed by a selective inhibitor of cox-2 (NS-398). In mouse osteoblastic cells, cytosolic PLA2 (cPLA2) mRNA and its protein were constitutively expressed and increased approximately 2-fold by IL-1alpha, but secretory PLA2 mRNA was not detected. PDGF rapidly stimulated PLA2 activity, which was blocked completely by a cPLA2 inhibitor (arachidonyltrifluoromethyl ketone). The PDGF-induced cPLA2 activation was accompanied by phosphorylation of its protein. These results indicate that cox-2 induction by IL-1alpha is not sufficient, but cPLA2 activation by PDGF is crucial for IL-1alpha-induced PGE2 synthesis in mouse osteoblasts.