Phenotypic spectrum of hereditary neuralgic amyotrophy caused by the SEPT9 R88W mutation.

BACKGROUND: Hereditary neuralgic amyotrophy (HNA), also known as hereditary brachial plexus neuropathy, has phenotypic and genetic heterogeneity. Mutations in the septin 9 (SEPT9) gene were recently identified in some HNA patients. The phenotypic spectrum of HNA caused by SEPT9 mutations is not well known. OBJECTIVE: To characterise the phenotype of a large family of HNA patients with the SEPT9 R88W mutation. METHODS: We report clinical, electrophysiological, neuroimaging and genetic findings of six HNA patients from a Japanese family. RESULTS: All 17 neuropathic episodes identified were selectively and asymmetrically distributed in the upper-limb nerves. Severe pain was an initial symptom in 16 episodes (94%). Motor weakness occurred in 15 (88%) and sensory signs in 10 (59%). A minor dysmorphism, hypotelorism, was seen in all. Nerve conduction studies revealed focal demyelination as well as prominent axonal degeneration changes. Needle electromyography revealed chronic neurogenic patterns only in the upper limbs. An MRI study showed a gadolinium-enhanced brachial plexus. The missense mutation c.262C>T; p.R88W was found in exon 2 of SEPT9 in all patients. CONCLUSIONS: The SEPT9 R88W mutation in this family causes selective involvement of the brachial plexus and upper-limb nerves. Wider and more universal recognition of clinical hallmarks and genetic counselling are of diagnostic importance for HNA caused by the SEPT9 mutation.

Reduced angiotensin II levels in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

BACKGROUND: Recent studies suggest that angiotensin II, a major substrate in the renin-angiotensin system, protects neurons through stimulation of its type 2 receptors. However, quite a few clinical studies of angiotensin II levels have shown their relation to disease severity in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). AIMS OF THE STUDY: To clarify the significance of angiotensin II in ALS. METHODS: We assayed angiotensin II concentrations in cerebrospinal fluid (CSF) samples from 23 patients with ALS, nine patients with spinocerebellar degeneration (SCD) and 24 control individuals. We evaluated the disability levels of patients with ALS using the Revised ALS Functional Rating Scale (ALSFRS-R) and calculated the disease progression rate (DPR). RESULTS: CSF angiotensin II levels were significantly lower in the ALS group compared with that in the control group (P = 0.00864), and showed a significant positive correlation with scores on the ALSFRS-R, and a significant negative correlation with the DPR. CONCLUSIONS: In the present study, we reveal for the first time that angiotensin II levels in the CSF from patients with ALS are significantly reduced and significantly associated with disease severity and progression rate. These findings suggest that reduced levels of intrathecal angiotensin II may play a role in ALS.

Pathological gambling associated with cabergoline in a case of recurrent depression.

Pathological gambling has been described frequently in patients with Parkinson disease or other movement disorders who were treated with dopamine agonists. Here, we report a patient with recurrent depression who developed pathological gambling after administration of the dopamine agonist cabergoline. A 36-year-old male Japanese patient presented with his third episode of depression. His depressive symptoms responded minimally to fluvoxamine. Cabergoline was then added to augment the antidepressant's efficacy. Although this regimen resulted in dramatic improvement, he started to spend considerable money and time every day in pachinko parlors and go to the horse racing track every weekend. He spent more than twenty thousand US dollars in total. He tried to stop gambling many times but failed to control his urge. His gambling behavior did not stop even though he was experiencing a marital crisis. He had not displayed any manic symptoms during this entire period. This complication fulfilled the criteria for pathological gambling according to the Diagnostic and Statistical Manual of MentalDisorders, Fourth Edition, Text Revision edition. The patient's perplexing behavior did not end until cabergoline was discontinued. Thus far, pathological gambling associated with cabergoline has rarely been reported while gambling associated with pramipexole and ropinirole, dopamine agonists, has frequently been documented. In addition, this is the first case of depression in which the patient developed pathological gambling during treatment with a dopamine agonist. In conclusion, clinicians should be aware of the potential for pathological gambling when prescribing cabergoline to patients with depression.

Distinct CSF cytokine/chemokine profiles in atopic myelitis and other causes of myelitis.

BACKGROUND: We reported the emergence of a distinct myelitis in patients with atopic diathesis (atopic myelitis [AM]) by a nationwide survey throughout Japan. Similar cases have recently been reported in Caucasians. Pathologic studies of biopsied spinal cord specimens revealed chronic active inflammation with eosinophilic infiltration. OBJECTIVE: To clarify the cytokine/chemokine alterations in CSF from patients with AM in comparison to other causes of myelitis. METHODS: We measured 27 cytokines, chemokines, and growth factors simultaneously in CSF from 22 patients with AM, 20 with opticospinal multiple sclerosis (OSMS), 11 with HTLV-1-associated myelopathy (HAM), 9 with Sjögren syndrome-related myelitis (SM), and 20 with other noninflammatory neurologic diseases (OND), using a fluorescent bead-based immunoassay. RESULTS: In patients with AM, CCL11 and interleukin (IL)-9 were significantly increased as compared with patients with OND and other myelitis while in patients with OSMS interferon-gamma and granulocyte-colony stimulating factor levels were significantly higher than in patients with OND and other causes of myelitis. Significant increase of IL-17 in comparison to patients with OND was found only in patients with OSMS, irrespective of presence or absence of anti-aquaporin-4 (AQP4) antibody. In patients with HAM, CXCL10 and CCL5 were higher than in patients with OND and other myelitis. In patients with SM, CCL3 and CCL4 were higher than in patients with OND. In patients with AM, CCL11, IL-9, and IL-1 receptor antagonist (IL-1ra) showed positive correlations with the final Kurtzke Expanded Disability Status Scale scores while IL-1ra and IL-12(p70) had positive correlations with disease duration. CONCLUSION: Intrathecal upregulation of CCL11 and Th2 cytokines is characteristic of atopic myelitis, which is distinct from interleukin-17/interferon-gamma-related autoimmune condition of opticospinal multiple sclerosis.

The effect of CYP2C19 genotypes on the pharmacokinetics of warfarin enantiomers.

The aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of warfarin enantiomers in relation to cytochrome P450 2C19 (CYP2C19) genotypes. Fourteen subjects, of whom seven were homozygous extensive metabolizers (hmEMs) and seven were poor metabolizers (PMs) for CYP2C19, were enrolled. After a single oral 10 mg dose of racemic warfarin, the plasma concentrations of the warfarin enantiomers and prothrombin time expressed as international normalized ratio (PT-INR) were measured over the course of 120 h. The mean plasma concentrations and elimination half-life of (R)-warfarin of all the subjects were about 2-fold greater than those of (S)-warfarin. Additionally, the area under the plasma concentration-time curve from zero to infinity (AUC(0-infinity)) and the elimination half-life of (R)-warfarin in PMs were significantly greater than those in hmEMs (P = 0.0005 and P = 0.0101 respectively). The S/R ratios of AUC of warfarin enantiomers were 0.51 in hmEMs and 0.37 in PMs (P = 0.0052). Whereas no difference was found in all pharmacokinetic parameters of (S)-warfarin in hmEMs compared with PMs. No significant difference in PT-INR, used as a measure of anticoagulant effect, was found between the hmEMs and PMs. These results show that CYP2C19 activity is important in the pharmacokinetics of (R)-warfarin. However, when warfarin is administered as a racemate, this difference is not translated into any significant effect in the pharmacodynamics of warfarin.

Intestinal CYP3A4 is not involved in the enantioselective disposition of lansoprazole.

The contribution of (S)-lansoprazole to CYP3A4-catalysed sulfoxidation is greater than that of (R)-lansoprazole. The aim was to investigate the effect of grapefruit juice on the enantioselective disposition of lansoprazole among three CYP2C19 genotype groups. Eighteen healthy subjects, consisting of six each of homozygous extensive metabolizers (homEMs), heterozygous extensive metabolizers (hetEMs) and poor metabolizers (PMs), ingested a single oral dose of 60 mg racemic lansoprazole after taking either 200 ml grapefruit juice or water. There was no effect of grapefruit juice on the mean maximum plasma concentrations (C(max)) or the elimination half-life for each lansoprazole enantiomer in all three CYP2C19 genotype groups. Similarly, the pharmacokinetic parameters of lansoprazole sulfone remained unaltered by grapefruit juice in all three groups. The CYP3A4-mediated first-pass sulfoxidation of (R)- and (S)-lansoprazole were not influenced by grapefruit juice. In addition, stereoselectivity of the intestinal CYP3A4-catalysed sulfoxidation of (R)- and (S)-lansoprazole was not observed.

[Rapidly enlarging giant left ventricular pseudo-false aneurysm after myocardial infarction; report of a case].

A 71-year-old man was admitted to our hospital with acute myocardial infarction and cardiac tamponade. After pericardial drainage, his hemodynamics was improved. Because more than 3 days had been passed after the onset of myocardial infarction and he had severe renal dysfunction, emergent coronary angiography (CAG) was not performed. After improvement of his general status, coronary angiography and percutaneous catheter intervention was carried out, and his course was uneventful. But transthoracic echocardiography before discharge revealed a giant posterior psudoaneurysm. Patch closure and coronary artery bypass grafting was carried out under cardiopulmonary bypass, and postoperative course was uneventful. Postoperative left ventriculogram revealed disappearance of pseudoaneurysm, but relatively large akinetic area of posterior-inferior wall was left around a patch. Pseudo-false aneurysm was diagnosed by histological examination.

Pharmacokinetics and pharmacodynamics of etizolam are influenced by polymorphic CYP2C19 activity.

OBJECTIVE: To examine the effect of cytochrome P450 (CYP) 2C19 activity on the single-dose pharmacokinetics and pharmacodynamics of etizolam. METHODS: The subjects were 21 healthy Japanese volunteers. The two mutated alleles (CYP2C19*2 and CYP2C19*3) causing absent CYP2C19 activity were identified by a polymerase chain reaction method. Twelve subjects were extensive metabolizers (EMs) with no or one mutated allele, and nine subjects were poor metabolizers (PMs) with two mutated alleles. The subjects received a single oral 1-mg dose of etizolam, and blood samplings and evaluation of psychomotor function were conducted up to 24 h after dosing. RESULTS: The PMs had significantly larger total area under the plasma concentration-time curve (287+/-74 vs 178+/-122 ng.h/ml, p<0.05) and longer elimination half-life (14.8+/-4.2 vs 10.5+/-3.9 h, p<0.05) of etizolam than the EMs. The area under the score-time curve from 0 to 8 h of the Stanford Sleepiness Scale was significantly larger in the PMs than in EMs (28.9+/-5.2 vs 22.9+/-6.9 score.h, p<0.05). CONCLUSION: The present study suggests that the single-dose pharmacokinetics and pharmacodynamics of etizolam are influenced by polymorphic CYP2C19 activity.

Induction of the metabolism of etizolam by carbamazepine in humans.

OBJECTIVE: To examine the effect of carbamazepine on the single oral dose pharmacokinetics of etizolam. METHODS: Eleven healthy male volunteers received carbamazepine 200 mg/day or placebo for 6 days in a double-blind, randomized, crossover manner, and on the sixth day they received a single oral 1-mg dose of etizolam. Blood samplings and evaluation of psychomotor function by the Digit Symbol Substitution Test and Stanford Sleepiness Scale were conducted up to 24 h after etizolam dosing. Plasma concentration of etizolam was measured using high-performance liquid chromatography. RESULTS: Carbamazepine treatment significantly decreased the peak plasma concentration (17.5+/-4.1 ng/ml versus 13.9+/-4.1 ng/ml; P<0.05), total area under the plasma concentration-time curve (194.8+/-88.9 ng h/ml versus 105.9+/-33.0 ng h/ml; P<0.001), and elimination half-life (11.1+/-4.6 h versus 6.8+/-2.8 h; P<0.01) of etizolam. No significant change was induced by carbamazepine in the two pharmacodynamic parameters. CONCLUSIONS: The present study suggests that carbamazepine induces the metabolism of etizolam.

Pharmacokinetic differences between the enantiomers of lansoprazole and its metabolite, 5-hydroxylansoprazole, in relation to CYP2C19 genotypes.

OBJECTIVE: The purpose of this study was to elucidate the pharmacokinetics of each enantiomer of lansoprazole and 5-hydroxylansoprazole in three different CYP2C19 genotype groups of Japanese subjects. METHODS: Healthy subjects ( n=18), of whom 6 were homozygous extensive metabolizers (homEMs), 6 were heterozygous extensive metabolizers (hetEMs) and 6 were poor metabolizers (PMs), participated in the study. After a single oral dose of 60 mg of racemic lansoprazole, the plasma concentrations of the lansoprazole enantiomers, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone were measured for 24 h post-dose. RESULTS: The plasma concentrations of ( R)-lansoprazole were remarkably higher in all three CYP2C19 genotype groups than those of the corresponding ( S)-enantiomer. The mean maximum plasma concentration ( C(max)) of ( S)-lansoprazole differed significantly among the three groups, whereas there was no difference for the ( R)-enantiomer. The relative area under the plasma concentration (AUC) ratios of ( R)- and ( S)-lansoprazole in the homEMs, hetEMs, and PMs were 1:1.5:4.0 and 1:1.8:7.4, respectively. Yet, the relative AUC ratios of 5-hydroxylansoprazole to lansoprazole for the ( R)- and ( S)-enantiomers in the homEMs, hetEMs, and PMs were almost the same (1:0.73:0.12 and 1:0.77:0.13, respectively). However, the AUC ratios of the ( S)-enantiomer were 13-fold greater for the three CYP2C19 genotypes than those of the corresponding ( R)-enantiomer. CONCLUSIONS: The magnitude of the contribution of CYP2C19 to the 5-hydroxylation of ( S)-lansoprazole was greater than that of the ( R)-enantiomer. The R/S ratios for the AUC of lansoprazole for the homEMs, hetEMs and PMs were 12.7, 8.5 and 5.8, respectively, suggesting a significant effect of CYP2C19 polymorphisms on the stereoselective disposition of lansoprazole.

Inhibition of the metabolism of etizolam by itraconazole in humans: evidence for the involvement of CYP3A4 in etizolam metabolism.

OBJECTIVE: To clarify the involvement of cytochrome P450 (CYP) 3A4 in the metabolism of etizolam. METHODS: The effects of itraconazole, a potent and specific inhibitor of CYP3A4, on the single oral dose pharmacokinetics and pharmacodynamics of etizolam were examined. Twelve healthy male volunteers received itraconazole (200 mg/day) or placebo for 7 days in a double-blind randomized crossover manner, and on the 6th day they received a single oral 1-mg dose of etizolam. Blood samplings and evaluation of psychomotor function using the Digit Symbol Substitution Test and Stanford Sleepiness Scale were conducted up to 24 h after etizolam dosing. Plasma concentration of etizolam was measured by means of high-performance liquid chromatography. RESULTS: Itraconazole treatment significantly increased the total area under the plasma concentration-time curve (AUC; 213+/-106 ng rectangle h/ml versus 326+/-166 ng rectangle h/ml, P<0.001) and the elimination half-life (12.0+/-5.4 h versus 17.3+/-7.4 h, P<0.01) of etizolam. The 90% confidence interval of the itraconazole/placebo ratio of the total AUC was 1.38-1.68, indicating a significant effect of itraconazole. No significant change was induced by itraconazole in the two pharmacodynamic parameters. CONCLUSION: The present study suggests that itraconazole inhibits the metabolism of etizolam, providing evidence that CYP3A4 is at least partly involved in etizolam metabolism.

Reproducibility of nifedipine absorption from GITS tablets: comparison of single-dose pharmacokinetics using 10, 20, 40 and 60 mg nifedipine.

OBJECTIVE: To examine the reproducibility of nifedipine absorption from gastrointestinal therapeutic system (GITS) tablets by comparing the single-dose pharmacokinetic profiles of 4 different dosages administered orally. METHODS: Twelve healthy male volunteers, aged between 22 and 29 years were enrolled in the open, 4-way, dose escalation study with single oral doses of 10, 20, 40 and 60 mg (two 30 mg) nifedipine GITS tablets. Each administration was separated by a 1-week washout period. Coefficients of variation (CV) of dose-corrected area under the concentration-time curve (AUC) and peak plasma drug concentrations (Cmax) were calculated from the pharmacokinetic profiles. RESULTS: Mean AUC and mean Cmax were dose-proportional from 10 to 60 mg. Although the CV of 4 mean dose-corrected AUC and Cmax were 5.5% and 17.5%, respectively, CV of dose-corrected AUC and Cmax in each subject varied from 5.1 to 37.4% (mean 11.0%) and from 14.1% to 46.4% (mean 25.8%), respectively. CONCLUSIONS: Whereas mean plasma nifedipine concentration remained markedly stable over a 16- to 24-hour interval and mean dose-corrected AUC showed good reproducibility with nifedipine GITS, the CV of dose-corrected AUC of the nifedipine GITS tablets in each subject showed large variability.

Development of cone and plate-type rheometer for quantitative analysis of endothelial cell detachment by shear stress.

We developed an apparatus, which has a structure based on a cone and plate-type rheometer, to facilitate quantitative analysis of the detachment process of endothelial cells (EC) from diverse materials including 3-dimensional scaffolds such as plate, membrane and porous-shaped materials. As an artificial vascular model, a material inoculated with EC to a polycarbonate membrane coated with laminin was prepared. In consequence, reduced cell number, medium volume, and material size was enough to evaluate cell detachment from various materials by shear stress in our system. When shear stress was loaded to a material with EC, the ratio retained of initially inoculated cells decreased with time. Increase of magnitude of shear stress also decreased the ratio. We conclude that our apparatus could analyze quantitatively detachment of EC with ease for screenings to find materials that enhance adhesive force of EC. To produce artificial blood vessels with small diameter, our apparatus could become a useful instrument.

Long-term implantation of zinc-releasing calcium phosphate ceramics in rabbit femora.

Zinc is an essential trace element that has stimulatory effects on bone formation. Recently, we developed zinc-releasing calcium phosphate ceramics in order to add the pharmacologic effect of zinc to calcium phosphate ceramics. In our previous study, we showed that the optimum zinc content for promoting bone formation was 0.316 wt %. Therefore a zinc composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite, with a zinc content of 0.316 wt %, was chosen for long-term implantation. Cylindrical rods of the zinc composite ceramic were implanted in rabbit femora for 2 to 60 weeks. Using computer-aided image analysis, a histomorphometric study was carried out to investigate bone formation and resorption around the implants. The control was a composite ceramic of beta-tricalcium phosphate and hydroxyapatite without zinc. The addition of zinc to the implant demonstrated both favorable and unfavorable effects on bone remodeling. The favorable effect was enhanced bone apposition to the implant surface, demonstrated by a significant increase in intramedullary bone apposition rate at 6 weeks and in cortical bone apposition rate at 24 and 60 weeks (p < 0.05). The unfavorable effect was increased bone resorption, demonstrated by a significant increase in medullary cavity area at 60 weeks (p < 0.05). In order to utilize the favorable effect and avoid the unfavorable effect of zinc, either a reduction in zinc content in the zinc composite ceramic or the selection of implantation sites that do not have excessive exposure to bone marrow are required.

Tyrosine hydroxylase antisense gene therapy causes hypotensive effects in the spontaneously hypertensive rats.

We investigated the effect of antisense oligodeoxynucleotides (AS ODN) against tyrosine hydroxylase (TH) on hypertension and sympathetic nervous system activity in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) in SHR treated with TH AS ODN (50, 200 microg/rat, i.v.) was significantly lower than that of control SHR. Epinephrine and norepinephrine levels, TH activity, and TH protein levels in the adrenal medulla of SHR were reduced concomitant with TH AS ODN treatment-induced changes in SBP. In contrast, TH AS ODN (200 microg/rat) had no effect on SBP in Wistar-Kyoto rats (WKY), despite significantly decreased catecholamine levels, TH activity, and TH protein levels. These findings suggest that peripheral systemic injection of TH AS ODN may be effective as hypotensive therapy in SHR.

Formation of human fibroblast aggregates (spheroids) by rotational culture.

In the current study, we attempted to form aggregates of fibroblasts by rotationally shaking, declining fibroblast-material interactions, and augmenting cell-cell interactions. In addition, to promote cell-cell interactions, the medium was supplemented with insulin, dexamethasone, and basic fibroblast growth. Under such improved culture conditions, normal neonatal human dermal fibroblasts formed spheroidal aggregates within 1 day of rotation on a rotational shaker. The aggregates that formed had irregular shapes and were composed from only several cells after 12 h. However, they became nearly spheroidal after 24 h of shaking. The aggregates were approximately 240 microm in diameter. After 36 h of shaking, their shape became more rounded and their surfaces became smoother. No evidence of necrosis in the center of the aggregates was observed, although a small number of dead cells was scattered throughout the aggregates. After 24-36 h, aggregates of normal human fibroblasts were collected and reinoculated onto a scaffold composed of polyglycolic acid. which is used commercially as a scaffold for artificial skin, coated with collagen. The aggregates were successfully trapped to the mesh of polyglycolic acid and became attached within 24 h. Therefore, the aggregates could provide an alternative method for seeding fibroblasts to scaffold for an artificial skin, such as a mesh of polyglycolic acid.

In vitro organization of biohybrid rat liver tissue incorporating growth factor- and hormone-releasing biodegradable polymer microcapsules.

To enhance the liver-specific functions of rat hepatocyte aggregates without the addition of exogenous growth factors, polylactic acid-polyglycolic acid (PLGA)/gelatin microcapsules that release insulin, dexamethasone, epidermal growth factors, and glucagon were prepared and incorporated into the hepatocyte aggregates in suspension culture. Precoating the capsules with fibronectin enhanced the incorporation of the microcapsules into the hepatocyte aggregates. In a growth factor- and hormone-free culture medium, these microcapsule-containing aggregates showed a sustained cell number and an ammonium detoxification capacity compared with two types of control culture. One was the culture of microcapsule-free aggregates with albumin-containing control capsules and the other was the culture of capsule-free aggregates that were supplied with the same factors and hormones from the culture medium at concentration levels expected from the release kinetics of the microcapsules. Our new methodology demonstrates that the performance and duration of bioartificial liver systems can be enhanced due to a more efficient maintenance of cell number by using such growth factor- and hormone-releasing microcapsules.

Preparation of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams by use of ice microparticulates.

Biodegradable foams of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) for tissue engineering were fabricated by a porogen-leaching technique using ice microparticulates as the porogen material. PLLA or PLGA solution in chloroform was mixed with ice microparticulates. The mixtures were frozen by being placed in molds in liquid nitrogen and freeze-dried to form the foams. Scanning electron microscopic observation of the PLLA and PLGA foams showed that evenly distributed and interconnected pore structures were formed in these foams. The porosity and surface area of the foams increased with an increase in the weight fraction of the ice microparticulates, while the median pore size remained unchanged. The pore structures of the foams could be manipulated by controlling processing variables such as the size and weight fraction of the ice microparticulates and polymer concentration.

In vivo evaluation of a novel porous hydroxyapatite to sustain osteogenesis of transplanted bone marrow-derived osteoblastic cells.

Biosynthetic bone grafts are considered to contain one or more of three critical components: osteoprogenitor cells, an osteoconductive matrix, and osteoinductive growth factors. The basic requirements of the scaffold material are biocompatibility, mechanical integrity, and osteoconductivity. A major design problem is satisfying these requirements with a single composite. In this study, we hypothesize that one composite that combines bone marrow-derived osteoblasts and a novel mechanical reinforced porous hydroxyapatite with good biocompatibility and osteoconductivity (HA/BMO) can reach these requirements. A novel sintered porous hydroxyapatite (HA) was prepared by the following procedures. The HA slurry was foamed by adding polyoxyethylenelaurylether (PEI) and mixing. The pores were fixed by crosslinking PEI with diepoxy compounds and the HA porous body was sintered at 1200 degrees C for 3 h. The HA sintered porous body had a high porosity (77%), and was completely interconnected. Average pore diameter was 500 microm and the interconnecting path 200 microm in diameter. The compressive (17 MPa) and three-point bending (7 MPa) strengths were high. For in vivo testing, the 2-week subcultured HA/BMO (+) composites were implanted into subcutaneous sites of syngeneic rats until 8 weeks after implantation. These implants were harvested at different time points and prepared for the biochemical analysis of alkaline phosphatase activity (ALP) and bone osteocalcin content (OCN), and histological analysis. ALP and OCN in the HA/BMO group were much higher than those in the HA without BMOs control group 1 week after implantation (p < 0.001). Light microscopy revealed mature bone formation in the HA/BMO composite 4 weeks after implantation. In the SEM study, mineralized collagenous extracellular matrix was noted in HA/BMO composite 2 weeks after implantation with numbers of active osteoblasts. We conclude that the composite of the novel HA and cultured BMOs has osteogenic ability in vivo. These results provide a basis for further studies on the use of this composite as an implant in orthopaedic surgery.

Poly(DL-lactic-co-glycolic acid) sponge hybridized with collagen microsponges and deposited apatite particulates.

A novel three-dimensional porous scaffold has been developed for bone tissue engineering by hybridizing synthetic poly(DL-lactic-co-glycolic acid) (PLGA), naturally derived collagen, and inorganic apatite. First, a porous PLGA sponge was prepared. Then, collagen microsponges were formed in the pores of the PLGA sponge. Finally, apatite particulates were deposited on the surfaces of the collagen microsponges in the pores of PLGA sponge. The PLGA-collagen sponge served as a template for apatite deposition, and the deposition was accomplished by alternate immersion of PLGA-collagen sponge in CaCl(2) and Na(2)HPO(4) aqueous solutions and centrifugation. The deposited particulates were small and scarce after one cycle of alternate immersion. Their number and size increased with the number of alternate immersion cycles. The surfaces of collagen microsponges were completely covered with apatite after three cycles of alternate immersion. The porosity of the hybrid sponge decreased gradually as the number of alternate immersion increased. Energy-dispersive spectroscopy analysis and X-ray diffraction spectra showed that the calcium-to-phosphorus molar ratio of the deposited particulates and the level of crystallinity increased with the number of alternate immersion cycles, and became almost the same as that of hydroxyapatite after four cycles of alternate immersion. The deposition process was controllable. Use of the PLGA sponge as a mechanical skeleton facilitated formation of the PLGA-collagen-apatite hybrid sponge into desired shapes and collagen microsponges facilitated the uniform deposition of apatite particulates throughout the sponge. The PLGA-collagen-apatite hybrid sponge would serve as a useful three-dimensional porous scaffold for bone tissue engineering.