Gene therapy improves dental manifestations in hypophosphatasia model mice.

BACKGROUND AND OBJECTIVE: Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. MATERIAL AND METHODS: Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. RESULTS: Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. CONCLUSIONS: Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.

Association of periodontal status with occlusal force and food acceptability in 70-year-old adults: from SONIC Study.

This cross-sectional study aimed to investigate the association of periodontal status with occlusal force and food acceptability. We hypothesised that mastication deteriorated with reduced periodontal support, even when posterior occlusal contacts with natural teeth were maintained and the patients remained clinically asymptomatic. Participants were 482 independently living 69-71-year-olds, classified as Eichner's group A, having no mobile teeth and no periodontal symptoms. The periodontal probing depth (PPD) and restoration status of each tooth were examined. Occlusal force in the intercuspal position was measured with pressure-sensitive films. Food acceptability was evaluated from the difficulty experienced in chewing apples, grilled beef, and hard rice crackers. Multivariate regression analysis was performed to investigate the association of periodontal status with occlusal force and food acceptability. A P-value of <0.05 was considered statistically significant. Multiple linear regression analysis showed that occlusal force had significant negative associations with maximal PPD (standardised partial regression coefficient (ß) = -0.121) after controlling for gender, handgrip strength, number of teeth, and percentage of restored teeth. Approximately 15% of participants were included in the compromised food acceptability group. Logistic regression analyses showed that compromised food acceptability was significantly associated with PPD, after controlling for gender, number of teeth, and percentage of restored teeth. Periodontal probing depth (PPD) was significantly correlated with occlusal force and self-rated food acceptability after controlling for the possible confounding factors in septuagenarians, even those with complete posterior occlusal contacts and no tooth mobility.

Influence of hypertension on pH of saliva in older adults.

BACKGROUND: Hypertension and/or antihypertensive medication reportedly induce dry mouth; however, an immediate relationship between salivary flow and hypertension has not been elucidated. OBJECTIVES: The purpose of the present study was to examine the association of hypertension and antihypertensive medication with salivary flow rate and pH in older adults. SUBJECTS: Subjects were independently living volunteers aged 60-78 years taking no medication (n = 138) or only antihypertensive medication (n = 27). Unstimulated saliva and stimulated saliva by the mastication method were collected, and salivary pH was measured. Hypertension was defined as systolic blood pressure of ≥ 140 mmHg and/or diastolic blood pressure of ≥ 90 mmHg. RESULTS: There was no significant correlation between either hypertension or intake of antihypertensive medication and unstimulated or stimulated salivary flow rate. However, multiple regression analysis showed that hypertension was significantly associated with pH of unstimulated saliva after controlling for other variables (ß = -0.270, P = 0.003). CONCLUSION: Hypertension regardless of antihypertensive medication was related to a lower pH of unstimulated saliva. Blood pressure might be a required consideration for maintenance of oral health in older individuals.

A biodegradable polymer as a cytokine delivery system for inducing bone formation.

Bone morphogenetic proteins (BMPs) that have the potential to elicit new bone in vivo have been used in a tissue-engineering approach for the repair of bone injuries and bone defects. Although it is now possible to generate large amounts of recombinant human (rh) BMPs for medical use, the major challenge remains in the development of optimal local delivery systems for these proteins. Here we describe the development of a synthetic biodegradable polymer, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG). This polymer exhibits promising degradation characteristics for BMP delivery systems and good biocompatibility under test conditions. PLA-DX-PEG/rhBMP-2 composite implants induced ectopic new bone formation effectively when tested in vivo, and can repair large bone defects orthotopically. This polymeric delivery system represents an advance in the technology for the enhancement of bone repair.

Symmetric Peripheral Running Sutures are Superior to Asymmetric Peripheral Running Sutures for Increasing the Tendon Strength in Flexor Tendon Repair.

BACKGROUND: The fatigue strength of three peripheral suture techniques for flexor tendon repair was compared by cyclic loading of the repairs in the porcine flexor digitorum tendon. METHODS: Thirty-six tendons were sutured using only peripheral sutures with 6-0 Nylon. An initial cyclic load of 10 N for 500 cycles was applied and increased by 10 N for an additional 500 cycles at each new load until rupture. RESULTS: The fatigue strength of the symmetric running peripheral suture was 85.0% and 144.8% greater than that of the two kinds of the asymmetric running peripheral sutures. CONCLUSIONS: Symmetric running sutures can enhance the suture strength and appears to be a useful technique for increasing the strength of the peripheral suture.

Asymmetric six-strand core sutures enhance tendon fatigue strength and the optimal asymmetry.

Under cyclic loading, we recorded the fatigue strength of a six-strand tendon repair with different symmetry in the lengths of suture purchase in two stumps of 120 dental rolls and in 30 porcine tendons. First, the strengths of the repairs with 1, 2, 3, 4 and 5 mm asymmetry were screened using the dental rolls. The asymmetric core suture repairs were then made with a Kessler repair of equal suture purchase (10 mm) in two tendon stumps, and shifting two other Kessler repairs by 1, 3 or 5 mm, respectively, along the longitudinal axis of the tendon in relation to the first (symmetric) Kessler repair. The core repairs with 3 mm or more asymmetry in suture purchases in two tendon ends showed significantly greater fatigue strength and significantly smaller gaps compared with 1 mm asymmetry in core suture repair. Our results support that asymmetric placement of core sutures in two tendon ends favour resisting gapping at the repair site and 3 mm or more asymmetry is needed to produce such beneficial effects.

Synthetic biodegradable polymers as drug delivery systems for bone morphogenetic proteins.

Bone morphogenetic proteins (BMP) induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to deliver BMP for clinical use need to be developed. We synthesized a new synthetic biodegradable polymer, poly-D,L-lactic acid-para-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG), to serve as a biocompatible, biodegradable polymer for recombinant human (rh) BMP-2 delivery systems. In animal experiments, new bone was efficiently formed and a large bone defect was repaired using PLA-DX-PEG/rhBMP-2 composites. In addition, this new polymer could be used as an injectable delivery system for rhBMP-2. The rhBMP-2/PLA-DX-PEG composites also could be combined with other materials such as hydroxyapatite or titanium. This new synthetic polymer might be used for rhBMP-2 delivery in various clinical situations involving repair of bone, leading to great changes in orthopedic treatment.

Local bone formation by injection of recombinant human bone morphogenetic protein-2 contained in polymer carriers.

The regenerating potential of human bone is limited. The repair of large bone defects often associated with bone tumor resections is not observed, and nonunion or delayed union of bone is a serious problem for fracture treatment. In these cases, autogeneic or allogeneic bone grafting has been routinely indicated, but these approaches require invasive surgical procedures. An alternative approach described in this paper involves the injection of bone morphogenetic proteins (BMPs) in a polymeric delivery system. We demonstrate that synthetic biodegradable polymers, poly-D,L-lactic acid-polyethylene glycol (PLA-PEG) block copolymers, which exhibit an exquisite temperature-dependent liquid-semisolid transition, work well as an injectable delivery system for recombinant human (rh) BMP-2. The thermosensitive property of the PLA-PEG/rhBMP-2 composite is permissive to percutaneous injection when heated. The fluidity of this composite decreases as it cools down to body temperature and the resultant semisolid form provides a scaffold for bone formation through the gradual local release of the rhBMP-2. This new type of injectable osteoinductive material will enable a less invasive approach to surgeries involving the restoration or repair of bone tissues.

Common atrium associated with anomalous high insertion of the inferior vena cava.

The case is reported of a 38-year-old housewife with a common atrium associated with anomalous high insertion of the inferior vena cava, absence of the coronary sinus, and drainage of a left hepatic vein into the left side of common atrium. Surgical repair was performed successfully. The procedure was closure of a nearly total atrial septal defect with a Dacron patch. Embroyologically, the development failure of the sinu-atrial fold appears to be the basic causal factor.

Growth hormone-releasing hormone (GHRH)-GH-somatic growth and luteinizing hormone (LH)RH-LH-ovarian axes in adult female transgenic mice expressing human GH gene.

We have examined alterations in the hypothalamo-pituitary GH-somatic growth axis and the hypothalamo-pituitary LH-ovarian axis in a line of transgenic ICR mice expressing human GH (hGH) under the influence of the whey acid protein promoter. Transgenic female mice weighed twice as much as control females and were infertile. The size of the anterior pituitary (AP) was 1/3 that of the controls. In transgenic mice, acinar cells in the mammary and mandibular glands displayed hGH-immunoreactivity, and plasma hGH was detected by radioimmunoassay. In the medial basal hypothalamus (MBH) of transgenic females, the immunoreactive-GHRH level was decreased (P<0.01). There was a corresponding reduction in the number of GHRH-immunoreactive neurons in the arcuate nucleus (ARC) and in the immunostaining of GHRH nerve terminals in the median eminence. The level of somatostatin (SRIH) in the MBH was increased (P<0.05), and SRIH-immunoreactive neurons in the periventricular nucleus (PeV) were increased in size and number in transgenic mice. The MBH level of LHRH in transgenic animals was greater (P<0.01) than in controls, although there was no apparent difference in the number of LHRH-immunoreactive neurons or in LHRH level in the preoptic area. There are fewer SRIH- and LHRH-immunoreactive neurons in the ARC in transgenic mice. Cells in the AP for GH, PRL, and LH were fewer in transgenic mice. The ovary suffered disturbance of follicular development and of corpora lutea formation. These results demonstrate that chronic overproduction of hGH may profoundly affect the organization of the GHRH/SRIH-GH-somatic growth axis and the LHRH-LH-ovarian axis due to reduction of GHRH-, SRIH- and LHRH-neurons in the ARC and increase of SRIH-neurons in the PeV.

Surface morphology and friction coefficient of various types of Foley catheter.

Urinary catheters are commonly used in chronic care facilities and geriatric homes. Problems associated with the catheters include encrustation, infection, physical trauma and inflammation. Many of these problems can be correlated to the catheter surface. Previous studies have dealt with problems associated with surface morphology and catheter composition and numerous catheter coatings have been developed to alter the surface including siliconized, Teflon and polyurethane hydrogel coatings. The hydrogel coating appears best, as it imparts a smooth, soft surface layer with lubricating properties. The paper describes a new poly(vinyl alcohol) hydrogel coating and compares its surface morphology and lubricity with other catheter coatings. Each catheter coating-type was examined by scanning electron microscopy for surface morphology. The new hydrogel coating was found to be smooth with no evidence of cracking. The apparent friction coefficient was used as an indication of surface lubricity. Friction measurement tests of the new hydrogen coating were run underwater and the friction coefficient was determined between the hydrated catheter surface and the hydrogel surface. The new hydrogel coating was found to have a high degree of lubricity in its hydrated state.

Influence of glycosaminoglycans on the collagen sponge component of a bilayer artificial skin.

A bilayer artificial skin composed of a silicone membrane and a collagen sponge layer containing glycosaminoglycans (GAGs) was first developed by Yannas and Burke. They reported that GAGs contained in the collagen sponge layer contributed to the function of the artificial skin. In an attempt to assess the effect of GAGs in the collagen sponge layer, the electron microscopic structure, mechanical strength of collagen sponges, and cell proliferation were examined in vitro, using four kinds of collagen sponges containing: no GAG, chondroitin 6-sulphate (C6S), dermatan sulphate (DER), and hyaluronic acid (HYA). The results indicated that: (1) addition of GAGs scarcely affected the mechanical structure of collagen sponges; (2) addition of C6S and DER reinforced mechanical strength, while addition of HYA did not; (3) addition of C6S and DER significantly decreased cell proliferation.

Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies.

We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyltransferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans. The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.

Biodegradable poly-D,L-lactic acid-polyethylene glycol block copolymers as a BMP delivery system for inducing bone.

BACKGROUND: Bone morphogenetic proteins (BMPs) are biologically active molecules capable of eliciting new bone formation. In combination with biomaterials, these proteins can be used in a clinical setting as bone-graft substitutes to promote bone repair. Collagen from animal sources has previously been the preferred carrier material in animal experiments. More recently, synthetic biodegradable polymers have been tested as a delivery vehicle for osteoinductive agents. In earlier studies performed in our laboratory, it was found that the polylactic acid homopolymers (PLA650) and poly-D,L-lactic acid-polyethylene glycol block copolymers (PLA650-PEG200) are viscous liquids that can be used as BMP delivery systems. METHODS: To obtain new PLA-PEG polymers that exhibit greater plasticity, the molecular sizes of PLA and PEG segments in the copolymer chains were increased. Plastic PLA-PEG polymers with various molecular sizes and PLA/PEG ratios were synthesized, mixed with recombinant human (rh) BMP-2, and implanted into the dorsal muscles of mice for 3 weeks to evaluate their capacity to elicit new bone formation. To compare the plastic PLA-PEG polymer with the liquid PLA650-PEG200 polymer, these two polymers were combined with rhBMP-2, implanted, and harvested after 3 weeks. Bone mineral content (BMC), bone area, and bone mineral density (BMD) of the ectopic new bone were measured by means of single energy X-ray absorptiometry (SXA). RESULTS: All of the PLA6,500-PEG3,000 implants with 10 or 20 microg of rhBMP-2 showed new bone formation. In contrast, little or no bone formation was seen in other plastic PLA-PEG implants with rhBMP-2. Control implants that lacked rhBMP-2 did not show new bone formation. Radiographic and histologic examinations showed that the PLA6,500-PEG3,000 implants with rhBMP-2 harvested 3 weeks after implantation had normal bone characteristics with hematopoietic marrow and osseous trabeculae. SXA analysis showed that the values for bone mineral content (BMC), bone area, and bone mineral density (BMD) of new bone resulting from the use of plastic PLA6,500-PEG3,000 polymers with rhBMP-2 were significantly higher than those obtained with the liquid PLA650-PEG200 polymers (p < 0.001 for each of the three values). CONCLUSIONS: These results indicate that the PLA6,500-PEG3000 block copolymer with plastic properties works effectively as a BMP delivery system. These data suggest that the total molecular size and ratio of PLA size to PEG size is an essential factor in determining the efficacy of a BMP delivery system. After implantation, it is possible that the PLA6,500-PEG3,000 pellets might have absorbed tissue fluids and become swollen, resulting in bone formation that exceeded the size of the original implants. This expansion characteristic is a potentially beneficial property, given the intended practical application of the polymer in the repair of bone defects. CLINICAL RELEVANCE: New synthetic biodegradable delivery systems will play an important role in the clinical applications of rhBMPs in which local formation of bone via an osteoinductive graft material is needed. Further pre-clinical and clinical work is necessary to establish the safety of these implants before they are adopted for widespread clinical use.

Synthesis of a novel lipopeptide with alpha-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability.

Introduction of liposomes into target cells is important for drug delivery systems. For this purpose, the surface of the liposome is equipped with ligand peptides, which may bind to specific receptors on the cell membrane. An artificial novel lipopeptide (MSH-C4A2) containing the alpha-melanocyte-stimulating hormone (alpha-MSH) sequence and two long alkyl chains was designed and synthesized, and the liposome, composed of egg phosphatidylcholine (EPC) and MSH-C4A2, was prepared. The stability of the liposome was estimated by measuring calcein leakage from the liposome inner phase. The stability of the liposome decreased upon addition of MSH-A4C2, which seemed to be attributable to the amphiphilic property of the peptide moiety (alpha-MSH) of MSH-A2C4. The stability was, however, recovered fairly well upon addition of cholesterol (Ch) or phosphatidylglycerol (PG). It was concluded therefore that the ternary system, MSH-C4A2/Ch/EPC or MSH-C4A2/PG/EPC, is suitable for preparing the functional liposome.

Preparation of functional liposomes with peptide ligands and their binding to cell membranes.

Two novel lipopeptides, which have the peptide ligands [alpha-melanocyte stimulating hormone (alpha-MSH)] sequence and repeated [Gly-Arg-Gly-Asp-Se (GRGDS) sequence], are designed, synthesized by the solid-phase method, and introduced into liposome membranes by the freeze-thaw method. These liposomes bearing the peptide ligands on their surface are expected to bind to cell membranes. We have confirmed that the lipopeptides are introduced into liposome membranes almost quantitatively, while such a high degree of incorporation has not been accomplished in conventional methods. In this respect, the present method is superior to prepare surface-modified liposomes that are applicable to drug carriers and so on. We have also confirmed by using immunoelectron microscopy that the peptide ligands are actually located in an aqueous phase. It has been shown by flow cytometry that the liposome bearing alpha-MSH peptide ligand binds to B16 cells and the liposome bearing the repeated GRGDS sequence binds to NIH3T3 cells.

Effect of silicone oil on ocular tissues.

The effect of silicone oil on ocular tissues was investigated histopathologically in rabbit eyes by injecting silicone oil into the anterior chamber or the vitreous cavity after vitrectomy. The eyes were enucleated 3, 6, 9, 12 or 18 months after the injection and various tissues were observed by light and electron microscopy. Silicone oil was first observed in the retrocorneal membrane at 18 months after the injection. In the trabecular meshwork silicone oil was seen for the first time at 12 months after injection. Migrating cells engulfing silicone oil attached to the internal limiting membrane of the retina 3 months after the injection. Twelve and 18 months after the injection, silicone oil passed through the internal limiting membrane and was engulfed by Müller cells. These results indicate that attention should be paid to the effects of silicone oil on ocular tissues when it remains in the eye for a long time since, after a certain period, cells of the trabecular meshwork and retina may engulf silicone oil.

Case report: facial asymmetry and early condylar fracture.

This report describes the surgical-orthodontic treatment of two adult females who exhibited dentofacial asymmetry. Their medical histories included early condylar fractures. Growth was disturbed on the fractured side, resulting in mandibular deviation and maxillary canting. Following the completion of growth, the asymmetries were corrected by combined orthodontic-surgical treatment. The treatment planning procedures and the results are presented.

[The effect of silicone oil on the ocular tissues].

It has been pointed out that the silicone oil tamponade can result in complications such as corneal damage, elevation of intraocular pressure and retinal toxicity against. The effect of silicone oil on the ocular tissues was investigated histopathologically by injecting silicone oil into the anterior chamber of the eyes of rabbits. In addition, in order to study its effect on the retina, silicone oil was injected into the vitreous cavity after vitrectomy. The eyes were extracted 3, 6, 9, 12 and 18 months after injection and various tissues were observed by light and electron microscopy. Silicone oil particles were first observed in the retro-corneal membrane 18 months after injection. In the trabecular meshwork silicone oil particles were seen for the first time 12 months after injection. Migrating cells engulfing silicone oil particles were attached to the internal limiting membrane of the retina three months after injection. Twelve and 18 months after injection, silicone oil particles passed through the internal limiting membrane and were engulfed by Mueller cells.

[A double-blind comparison between cefaclor and cephalexin in the treatment of dental infections].

A comparative clinical study of cefaclor (CCL) with cephalexin (CEX) was carried out by randomized double-blind techniques in order to contemplate the clinical efficacy, side effects and usefulness in treatment of 243 patients with dental infections. In the CEX group, CEX was orally administered 4 times a day at a daily dosage of 1,000 mg for 3 to 5 days. In the CCL group, CCL was orally administered 3 times a day at a daily dosage of 750 mg for 3 to 5 days and 1 capsule of placebo was also given every evening in order to keep the blindness of the administration. Evaluable cases for efficacy of athe drugs were 213 consisting of 108 for CCL and 105 for CEX. There were no significant differences in background of the patients and severity of the disease between 2 treatment groups. Clinical effectiveness on the 3rd day was 89.7% in CCL group and 78.6% in CEX group, showing significant difference between 2 treatment groups. Clinical effectiveness on the final day of administration was 94.4% in CCL group and 92.4% in CEX group, showing no significant difference between 2 treatment groups. Side effects were found in 10.5% of 114 patients receiving CCL and in 4.5% of 112 patients with CEX, and there was no significant difference between 2 treatment groups. The side effects were mostly gastrointestinal origin. According to the judgement by physicians in charge, no significant difference was seen in clinical usefulness between the 2 drugs.