Impact of polyplex micelles installed with cyclic RGD peptide as ligand on gene delivery to vascular lesions.

Gene therapy is expected to open a new strategy for the treatment of refractory vascular diseases, so the development of appropriate gene vectors for vascular lesions is needed. To realize this requirement with a non-viral approach, cyclo(RGDfK) peptide (cRGD) was introduced to block copolymer, poly(ethylene glycol)-block-polycation carrying ethylenediamine units (PEG-PAsp(DET)). cRGD recognizes α(v)ß(3) and α(v)ß(5) integrins, which are abundantly expressed in vascular lesions. cRGD-conjugated PEG-PAsp(DET) (cRGD-PEG-PAsp(DET)) formed polyplex micelles through complexation with plasmid DNA (pDNA) and the cRGD-PEG-PAsp(DET) micelles achieved significantly more efficient gene expression and cellular uptake as compared with PEG-PAsp(DET) micelles in endothelial cells and vascular smooth muscle cells. Intracellular tracking of pDNA showed that cRGD-PEG-PAsp(DET) micelles were internalized via caveolae-mediated endocytosis, which is associated with a pathway avoiding lysosomal degradation and that, PEG-PAsp(DET) micelles were transported to acidic endosomes and lysosomes via clathrin-mediated endocytosis. Further, in vivo evaluation in rat carotid artery with a neointimal lesion revealed that cRGD-PEG-PAsp(DET) micelles realized sustained gene expression, whereas PEG-PAsp(DET) micelles facilitated rapid, but transient gene expression. These findings suggest that introduction of cRGD to polyplex micelles might create novel and useful functions for gene transfer and contribute to the establishment of efficient gene therapy for vascular diseases.

Selective and sustained delivery of basic fibroblast growth factor (bFGF) for treatment of peripheral arterial disease: results of a phase I trial.

OBJECTIVES: The aim of this study was to evaluate the safety of selective and sustained delivery of basic fibroblast growth factor (bFGF) using acidic gelatine hydrogel microspheres (AGHMs) for the treatment of peripheral arterial disease (PAD). MATERIALS AND METHODS: We conducted a non-randomised and uncontrolled trial involving prospective observation of eight patients (eight limbs) with PAD - five limbs with arteriosclerosis obliterans and three limbs with thromboangiitis obliterans, five limbs (three arms and two legs) with critical limb ischaemia (CLI) and three limbs with intermittent claudication (IC) - who were followed up for 6 months or more. AGHM suspension containing 100 microg bFGF was infused into the artery of the affected limb. Besides evaluation of safety and changes in symptoms, resting ankle-brachial pressure index measurement and transcutaneous PO(2) (tcPO(2)), angiography were conducted at baseline and then at various time points. Skin perfusion pressure as an index of CLI and claudication distance as an index of IC were also used to assess clinical improvement and limb perfusion. RESULTS: No serious adverse events were observed. All cases showed improvement in symptoms, although this was temporary in some patients. CONCLUSION: Selective delivery of bFGF using AGHMs was suggested to be safe and well-tolerated in patients with PAD.

A case of tumoural calcinosis in the temporomandibular joint associated with systemic sclerosis.

Systemic sclerosis (SSc) is a relatively rare condition characterized by the excessive production and deposition of collagen within tissue. This condition is thought to be immunologically mediated and, in addition to its notorious cutaneous manifestations, often involves multiple organs. A case is presented of systemic sclerosis associated with extensive tumoural calcinosis in the temporomandibular joint. There has been no evidence of recurrence or complications during approximately 2 years of follow up, but long-term follow up is essential.

Changes in maxillofacial morphology and velopharyngeal function with two-stage maxillary distraction-mandibular setback surgery in patients with cleft lip and palate.

Maxillary distraction is increasingly used for the correction of severe maxillary retrusion in patients with cleft lip and palate. However, control of the maxillary movement is difficult, and the need to wear visible distractors for a long period of time causes psychosocial problems. A two-stage surgical approach consisting of maxillary distraction and mandibular setback was developed to overcome these problems. In this study, changes in maxillofacial morphology and velopharyngeal function were examined in 22 patients with cleft lip and palate who underwent this two-stage approach. Lateral cephalograms taken just before the first surgery, immediately after the second surgery, and at completion of the active post-surgical orthodontic treatment were used to examine maxillofacial morphology. Velopharyngeal function was evaluated by speech therapists using a 4-point scale for hypernasality. The average forward movement of the maxilla with surgery at point A was 7.5mm, and the average mandibular setback at pogonion was 8.6mm. The average relapse rate during post-surgical orthodontic treatment was 25.2% for the maxilla and 11.2% for the mandible. After treatment, all patients had positive overjet, and skeletal relapse was covered by tooth movement during postoperative orthodontics. Velopharyngeal function was not changed by surgery. This method can shorten the period during which the distractors have to be worn and reduce the patient burden.

Cyclooxygenase-2 activity is essential for the osseointegration of dental implants.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone response after the placement of implants in the femurs of mice. titanium implants 1.0mm in diameter were placed into the middle of the femurs of 9-week-old male COX-2 wild-type (COX-2(+/+)) and knockout (COX-2(-/-)) mice. For RNA analysis, the mice were killed 0, 1, 2, 4, 7 and 56 days after implantation. RNA was extracted from the bone surrounding the implants. For histological analysis, the mice were killed 4 and 8 weeks after treatment, and undecalcified sections were prepared. Contact microradiography was performed, and the sections were stained with 1% toluidine blue for histological examination. Histomorphometric measurements were obtained with a computer-based image analyser to quantify bone newly formed around the implant and the rate of implant-bone contact. Expression of COX-2 and osteocalcin mRNA was induced in bone surrounding implants in COX-2(+/+) mice, but not in COX-2(-/-) mice. In cortical bone, the implant surface was in direct contact with newly formed bone lamellae in COX-2(+/+) mice; new bone formation was minimal in COX-2(-/-) mice. These results suggest that COX-2 plays an essential role in osseointegration and provide evidence that COX-2-selective non-steroidal anti-inflammatory drugs may interfere with osseointegration clinically.

A 2-stage procedure combining maxillary advancement by distraction technique with mandibular setback surgery in patients with cleft lip and palate.

A 2-stage procedure combining maxillary advancement by distraction technique with mandibular setback surgery was used to correct jaw deformities in 5 patients with severe maxillary retrusion secondary to cleft lip and palate. First, a Le Fort I maxillary osteotomy was performed. Immediately after maxillary distraction, the distraction device was removed. The advanced maxilla was fixed with miniplates after adjusting the length and direction of advancement, and mandibular setback surgery was performed simultaneously to obtain a normal occlusal relationship. This 2-stage procedure resulted in stable occlusion and a markedly improved facial profile.

Complications after intraoral vertical ramus osteotomy: relationship to the shape of the osteotomy line.

Intraoral vertical ramus osteotomy (IVRO) is used widely to correct mandibular prognathism. However, several disadvantages of this procedure have been reported, such as condylar luxation and bony interference at the osteotomy site. The aim of this study was to survey the incidence of complications (condylar luxation and bony interference) based on the shape of the osteotomy line. One hundred and eighty-five rami in 118 patients with jaw deformities, which were treated with IVRO, were examined retrospectively. The shape of the osteotomy line and the postoperative complications were examined on panoramic radiographs. Osteotomy lines were classified into three types: vertical, C-shaped, and oblique. Of the 185 osteotomy sites, 98 were vertical, 37 C-shaped, and 50 oblique. Condylar luxation was found in six rami (3.2%); four had undergone vertical osteotomy and two had undergone C-shaped osteotomy. Bony interference occurred in seven rami (3.8%), all with vertical type osteotomy lines. Most complications occurred in the vertical type cases and no complications were found in oblique type cases. Condylar luxation was found mainly in unilateral IVRO cases and bony interference was found in bilateral IVRO cases. These results suggest that the oblique type of osteotomy line has the advantage of avoiding complications.

Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

A novel synthetic triazolotriazepine derivative JTT-606 inhibits bone resorption by down-regulation of action and production of bone resorptive factors.

In the search for a new class of bone-sparing agents, we have conducted random screening of the domestic chemical library using 45Ca release assay from prelabeled cultured neonatal mouse calvariae and identified a novel synthetic triazolotriazepine JTT-606 as a candidate for a potent inhibitor of bone resorption. JTT-606 inhibited 45Ca release dose dependently not only in the control calvarial culture but also in the stimulated cultures by interleukin-1alpha (IL-1alpha), fibroblast growth factor 2 (FGF-2), and parathyroid hormone (PTH). JTT-606 also inhibited both basal and stimulated osteoclast-like (OCL) cell formation in the coculture of mouse osteoblastic cells and bone marrow cells dose dependently, indicating its inhibitory effect on osteoclast differentiation. Ex vivo OCL cell formation by cultured bone marrow cells collected from ovariectomized (OVX) mice also was decreased dose dependently by in vivo application of JTT-606 to a level similar to that from sham-operated mice. Furthermore, JTT-606 inhibited resorbed pit formation by isolated mature osteoclasts as well as by unfractionated bone cells derived from rabbit long bones in the control and FGF-2-stimulated cultures dose dependently, indicating both the direct and the indirect actions of JTT-606 on mature osteoclast function. In addition, JTT-606 reduced production of IL-1alpha, tumor necrosis factor alpha (TNF-alpha), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the human peripheral blood mononuclear cell culture. In vivo analyses of mature OVX rats revealed that the application of JTT-606 for 12 weeks increased the BMD of the lumbar spine and decreased the levels of serum osteocalcin and urine deoxypyridinoline to levels similar to those of 17beta-estradiol-treated OVX rats. We propose that JTT-606 may inhibit both osteoclast differentiation and function by down-regulating both the action and the production of bone resorptive factors. It is speculated that JTT-606 could be a potent agent for the treatment of osteopenic disorders with elevated osteoclastic bone resorption.

Regulation of osteoclast differentiation by fibroblast growth factor 2: stimulation of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor expression in osteoblasts and inhibition of macrophage colony-stimulating factor function in osteoclast precursors.

This study investigated the mechanism of direct and indirect actions of fibroblast growth factor 2 (FGF-2) on osteoclast differentiation using two mouse cell culture systems. In the coculture system of osteoblasts and bone marrow cells, FGF-2 stimulated osteoclast formation. This effect was decreased markedly by osteoprotegerin (OPG) or NS-398, a selective cyclo-oxygenase 2 (COX-2) inhibitor. FGF-2 (> or = 10(-9) M) stimulated receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) messenger RNA (mRNA) expression from 2 h to 7 days in cultured osteoblasts. NS-398 did not affect the early induction but decreased the later one, indicating that the later effect is mediated by COX-2 induction in osteoblasts. To study the direct action of FGF-2 on osteoclast precursors, we used mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of soluble RANKL/ODF (sRANKL/ODF) and macrophage colony-stimulating factor (M-CSF). Although osteoblasts expressed all FGF receptors (FGFR-1 to -4), only FGFR-1 was detected in C7 cells at various differentiation stages. FGF-2 alone or in combination with sRANKL/ODF did not induce osteoclastogenesis from C7 cells; however, FGF-2 from lower concentrations (> or = 10(-11) M) significantly decreased osteoclast formation induced by M-CSF in the presence of sRANKL/ODF. FGF-2 did not alter mRNA levels of M-CSF receptor (Fms) or RANK in C7 cells. Immunoprecipitation/ immunoblotting analyses revealed that tyrosine phosphorylation of several cellular proteins including Fms in C7 cells induced by M-CSF was inhibited by FGF-2 in the presence of sRANKL/ODF. We conclude that FGF-2 regulates osteoclast differentiation through two different mechanisms: (1) an indirect stimulatory action via osteoblasts to induce RANKL/ODF partly through COX-2 induction and prostaglandin production and (2) a direct inhibitory action on osteoclast precursors by counteracting M-CSF signaling.

Direct action of nitric oxide on osteoblastic differentiation.

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin-1beta and tumor necrosis factor-alpha expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by L-NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by L-NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose-dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine-induced inhibition of AIPase activity is mediated via mechanism other than NO.

Oral and pharyngeal reconstruction using the free forearm flap.

Recent progress in myocutaneous and free flaps has facilitated the primary closure of various complex defects resulting from ablative surgery of head and neck cancers. The musculocutaneous flap (such as the pectoralis major flap or the latissimus dorsi flap) has in particular proved its greater versatility. The forearm free flap for intraoral defects can provide thin and pliable skin and has great potential in the reconstruction of oropharyngeal defects. Between November 1982 and August 1985, we achieved 74 free forearm flap transfers for patients with head and neck cancers. Herein we present several clinical applications of the free forearm flap and discuss its advantages and disadvantages, especially hand morbidity after flap elevation.

Clinical application of the erbium:YAG laser for apicoectomy.

Recently, an Er:YAG laser has attracted attention because of the possibility of cutting hard tissues with extremely small thermal effects. In this article, we report 8 cases (13 teeth) of apicoectomy using Er:YAG laser. All procedures were performed without using an air turbine or an electric drill. Although the cutting speed of this laser was slightly slower than ordinary methods, its clinical application for apicoectomy has many advantages including absence of discomfort and vibration, less chance for contamination of the surgical site, and reduced risk of trauma to adjacent tissue.

Flexible rhombic flap.

A flexible rhombic flap which can be designed in accordance with the degree of tension of skin bordering a part of the defect has been described. This procedure provides sufficient coverage of a skin defect with very little dog-ear.

The histologic analysis of distraction osteogenesis of the mandible in rabbits.

The process of bone formation in mandibular lengthening by distraction was studied in 30 rabbits. The mandible was subjected to a corticotomy, held in a neutral position for 2 weeks, and then lengthened at 0.18 mm per 12 hours for 24 days using a unilateral external fixation device (Orthofix M-100). On completion of the distraction, x-ray analysis showed that the distracted gap was filled with callus organized into three zones, namely, two sclerotic zones with a central radiolucent zone. These zones became indistinguishable from the adjacent preexisting mandible at 10 weeks after distraction. Histologically, new bone, which was formed by both intramembranous and endochondral ossification, underwent remodeling and resulted in cortical bone by 8 to 10 weeks after completion of distraction.

Primary osteoma cutis of the back.

A case of primary osteoma cutis on the back is reported. This case showed an unusual form of calcification on soft x-ray. Histologic examination disclosed mature bone in the subcutis. There was no history or evidence of any skin disease prior to the development of the osteoma. The literature is reviewed and discussed.

Vascularized periosteal grafts: an experimental study using two different forms of tibial periosteum in rabbits.

Three groups of rabbits were studied to investigate the osteogenic capacity of vascularized periosteum. It was found that such tissue, especially in the form of musculoperiosteal flaps, produced a significant amount of bone. It is concluded that vascularized periosteal flaps have an excellent osteogenic capacity even though they are not subjected to weight or stress.

The outcome of long-term follow-up after palatoplasty.

The speech outcome was studied retrospectively in 140 cleft-palate patients who underwent push-back palatoplasty. Velopharyngeal function and articulation disorders were evaluated serially at 4, 7, 10, and more than 10 years of age. On comparison of velopharyngeal function between 4 years of age and the most recent review (>10 years), it was unchanged in 90 patients (64.3 percent), whereas it showed deterioration in 14 patients and showed improvement in 8 patients. The other 28 patients underwent pharyngeal flap surgery; this group also included patients with functional deterioration. Changes of velopharyngeal function often occurred between 4 and 7 years of age but sometimes occurred after 10 years of age. Articulation disorders were observed in 49 subjects (35.0 percent) at 4 years of age. Many of the patients with glottal stop showed improvement from 4 to 7 years of age. Palatalized articulation showed less improvement than glottal stop (p < 0.01). The number of patients with articulation disorders decreased significantly between 4 years of age and the most recent review (p < 0.001). These findings suggest that speech does not become stable before 10 years of age and that patients with cleft palate should be carefully followed until they are beyond this age.

Hamartoma of the tongue: two case reports.

Hamartoma designates an excessive focal overgrowth of mature normal cells and tissues in an organ or tissue composed of identical cellular elements. Two cases of hamartoma at the back of the tongue are reported. Histologically they comprised fibrous tissue, fat, salivary gland lobules, and smooth muscle. The literature is reviewed and discussed.

Pharyngoesophageal reconstruction using a fabricated forearm free flap.

A new microsurgical alternative in reconstruction of the pharynx and cervical esophagus is reported. A trapezoidal forearm flap is fabricated into an inverted skin tube and placed in the pharyngoesophageal defect. Although microvascular anastomoses are required to revascularize the transferred forearm flap, the long and large nutrient vessels of the flap make anastomoses easy and reliable. None of our 12 patients demonstrated any necrosis of the transferred flap. This one-stage, less invasive operation for pharyngoesophageal reconstruction greatly benefits older persons, who are the more likely to be involved with pharyngoesophageal carcinomas.