Expression of CXCL10 in cultured cortical neurons.

Chemokines expressed in neurons are important mediators in neuron-neuron and neuron-glia signaling. One of these chemokines is CCL21 that activates microglia via the chemokine receptor CXCR3. As neurons also express CXCL10, a main ligand for CXCR3, we have thus investigated in detail the expression pattern of CXCL10 in neurons. We show that CXCL10 is constitutively expressed by neurons, is stored in large dense-core vesicles and is not regulated by neuronal injury or stress. Neuronal CXCL10 release occurred constitutively at low level. In vivo CXCL10 expression was found in the developing brain at various embryonic stages and its peak expression correlates with the presence of CD11b- and GFAP-positive cells expressing CXCR3. These results suggest a possible role of neuronal CXCL10 in recruitment and homing of glial cells during embryogenesis.

Comparison of closure of gastric perforation ulcers with biodegradable lactide-glycolide-caprolactone or omental patches.

BACKGROUND: The current treatment of perforated peptic ulcers is primary closure, supported by the application of an omental patch. It is difficult and time consuming to perform this procedure by laparoscopic surgery, largely because of the required suturing. It was our aim to develop and test a new method of closure for gastric perforation that is similar in efficacy and safety to a traditional repair. This technique could have utility in laparoscopic repair, as it does not require sutures or mobilization of the omentum. METHOD: The new method, called the "stamp" method consists of closure of the perforation by gluing a biodegradable patch made of lactide-glycolide-caprolacton (LGC, Polyganics, B.V. Groningen, The Netherlands) on the outside of the stomach. It was compared with the omental patch procedure. Perforations were made in the stomach of 20 rats and closed by either method (10 rats in each group). The rats were followed for 10 weeks. RESULTS: No complications were seen in any of the rats. In both groups, histological degradation of the patch by giant cells started at week 2. No signs of inflammation existed in either group. Signs of closure of the mucosa were seen after 2 weeks, and the muscular layer started to regenerate after 8 weeks in both groups. CONCLUSION: Results of both methods were similar, which means that treatment of a gastric perforation through the application of a biodegradable patch to the outside of the stomach is a feasible option and might even be an interesting technique for closure of other perforations in the digestive tract.

Age-related changes of periodontal ligament surface areas during force application.

OBJECTIVE: To investigate the age-dependent morphology of the periodontal ligament (PDL) tissue and changes in its surface area (SA) during force application provided with a standardized orthodontic setup for a period of 12 weeks in young and adult rats. METHODS: Two groups of 30 rats, age 6 weeks and 9 to 12 months, were used. Orthodontic appliances were placed to move the maxillary molars mesially with the contralateral sides used as controls. At 1, 2, 4, 8, and 12 weeks, groups of animals were killed. The PDL SA and the PDL SA ratio between pressure and tension regions were determined. RESULTS: An age-related decrease in the PDL SA was noted at control sides. Significant changes during the experimental period occurred only at experimental sides: The PDL SA was smaller at pressure than at tension regions only at week 1 in young rats; in adult rats, the difference between the two regions was significant at week 8. These changes were confirmed by the morphologic disorganization of the PDL and alterations in the PDL SA ratio. CONCLUSIONS: During force application, the PDL at the pressure regions became disorganized and subsequently was reorganized, as is shown by the histologic changes and SA of the PDL over time. This process occurred earlier and was more prominent in young rats; it occurred later and was more prolonged in adult animals.

Effect of duration of force application on blood vessels in young and adult rats.

INTRODUCTION: Age effects on orthodontically induced periodontal vascular reactions have not been studied. The aim of the present study was to test the hypothesis that prolonged tooth movement induces age-related increases in periodontal vascularity. METHODS: A standardized orthodontic appliance was placed in 2 groups of 30 rats aged 6 weeks and 9 to 12 months. At 1, 2, 4, 8, and 12 weeks, animals were killed. Blood vessels (BV) were identified based on their morphology and by immunohistochemical staining for alpha-smooth muscle actin. At each study region, surface areas (SA) of the periodontal ligament space and each BV were measured; BV mean SA, BV relative SA (the summed BV SA as a percentage of the periodontal ligament SA), and BV numbers were calculated. RESULTS: Pressure and tension regions showed similar vascular changes. Young rats had lower BV relative SA and BV mean SA in the early phase of force application (< 4 weeks); this increased in the late phase, reaching the same level as adult rats. In the late phase (4-12 weeks), young rats had increases of both small- and large-sized BV that did not affect the BV mean SA; adult rats had an increase of small-sized BV only; this resulted in decreased BV mean SA. CONCLUSIONS: The hypothesis was confirmed that prolonged tooth movement increases periodontal vascularity, which is age related. These results suggest that clinicians should consider age-related difference in tissue reactions during orthodontic tooth movement.

Age-dependent external root resorption during tooth movement in rats.

OBJECTIVE: To investigate the effect of age on root resorption and distribution along different parts of the root during prolonged light force application. MATERIAL AND METHODS: Orthodontic appliances were placed in two groups of 30 rats (one group 6 weeks old, the other 9-12 months old), with contralateral sides as controls. Groups of animals were killed at 1, 2, 4, 8, and 12 weeks. At the study site, incidence was counted as either 1 (with resorption) or 0 (without); severity was measured as the summed length of all resorption lacunae as a percentage of study total root length. RESULTS: Young and adult rats had the same incidence of root resorption in the early phase (<4 weeks), and both increased in the late phase (4-12 weeks) to the same level. Severity of resorption increased with prolonged tooth movement only in adult rats. However, there was no age-related difference in either the early or the late phase. In both groups, the middle part of the root had the highest incidence of resorption; the most severe resorption occurred exclusively at this part. CONCLUSION: Orthodontic intervention even with light forces increased both the incidence and severity of root resorption, the more so in the middle part of the root. Adult rats had increased incidence and severity with prolonged tooth movement.

Resorbable screws for fixation of autologous bone grafts.

The aim of this study was to evaluate the suitability of resorbable screws made of poly (D.L-lactide) acid (PDLLA) for fixation of autologous bone grafts related to graft regeneration and osseointegration of dental implants. In eight edentulous patients suffering from insufficient retention of their upper denture related to a severely resorbed maxilla, the floor of both maxillary sinus and width of the alveolar crest were augmented with an autologous bone graft from the iliac crest. Randomly, the bone graft used to augment the alveolar crest was fixed with two titanium screws on one side and two resorbable screws on the other side (split-mouth design). Three months after the reconstruction, a bone biopsy was taken with a trephine including one resorbable screw (N = 8). Subsequently, six implants were placed in the left and right posterior maxilla. Six months later, at the abutment connection, a bone biopsy was taken including the other resorbable screw (N = 8). The biopsies were processed for light microscopic examination. In addition, clinical parameters were scored. Wound healing was uneventful. Clinically no difference in wound healing was observed between sides treated with either a resorbable or titanium screw. No implants were lost. Six months after implantation, implant retained overdentures could be fabricated in all patients. All patients functioned well with their overdentures (follow-up 22.2 +/- 4.3 months). Three as well as 9 months after insertion (remnants of), the resorbable screws were still visible after reflecting the mucoperiosteum. Histological examination confirmed that a considerable quantity of remnants of the resorbable screws was still present, although areas with some fragmentation of the PDLLA were also observed. The screws were separated by a fibrous tissue layer containing many giant cells from the bone. Particles of PDLLA were observed within these giant cells. This study revealed that resorbable screws made of PDLLA can be used for fixation of bone grafts. The bulk of the PDLLA material is still present after 9 months.

Vesicle-mediated transport and release of CCL21 in endangered neurons: a possible explanation for microglia activation remote from a primary lesion.

Whenever neurons in the CNS are injured, microglia become activated. In addition to local activation, microglia remote from the primary lesion site are stimulated. Because this so-called secondary activation of microglia is instrumental for long-term changes after neuronal injury, it is important to understand how microglia activity is controlled. The remote activation of microglia implies that the activating signals are transported along neuronal projections. However, the identity of these signals has not yet been identified. It is shown here that glutamate-treated neurons rapidly express and release the chemokine CCL21. We also provide evidence that neuronal CCL21 is packed in vesicles and transported throughout neuronal processes to reach presynaptic structures. Chemotaxis assays show that functional CCL21 is released from endangered neurons and activate microglia via the chemokine receptor CXCR3. Based on these findings, we suggest that neuronal CCL21 is important in directed neuron-microglia signaling and that this communication could account for the remote activation of microglia, far distant from a primary lesion.

Does platelet-rich plasma promote remodeling of autologous bone grafts used for augmentation of the maxillary sinus floor?

The aim of this study was to evaluate the effect of platelet-rich plasma (PRP) on remodeling of autologous bone grafts used for augmentation of the floor of the maxillary sinus. In five edentulous patients suffering from insufficient retention of their upper denture related to a severely resorbed maxilla, the floor of both maxillary sinus was augmented with an autologous bone graft from the iliac crest. Randomly, PRP was added to the bone graft used to augment the floor of the left or right sinus (split-mouth design). Three months after the reconstruction, bone biopsies were taken with a trephine from the planned implant sites (N=30). Subsequently, three implants were placed in the left and right posterior maxilla. Microradiograms were made of all biopsies (N=30), whereafter the biopsies were processed for light microscopic examination. In addition, clinical parameters were scored. Wound healing was uneventful, clinically no difference was observed between the side treated with PRP or not. Also microradiographical and histomorphological examination of the biopsies revealed no statistical difference between the PRP- and non-PRP side. One implant placed in the PRP side of the graft was lost during the healing phase. Implant-retained overdentures were fabricated 6 months after implantation. All patients functioned well (follow-up 20.2+/-4.3 months). In this study, no beneficial effect of PRP on wound healing and bone remodeling was observed. It is posed that PRP has no additional value in promoting healing of grafted non-critical size defects.

Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor.

Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.

Effects of maxillary sinus floor elevation surgery on maxillary sinus physiology.

In a prospective study, the effects of elevation surgery of the maxillary sinus floor on maxillary sinus physiology were assessed. Seventeen consecutive patients without preoperative anamnestic, clinical and radiological signs of maxillary sinusitis underwent sinus floor elevation surgery with iliac crest bone grafts. All patients were subjected to unilateral endoscopic examination of the maxillary sinus, taking of a biopsy specimen from the sinus floor mucosa, and collection of a sinus lavage-fluid aspirate. This triad of evaluations was performed immediately preceding the elevation procedure, and 3 months (at implant insertion) and 9 months (at uncovering of implants) postoperatively. All procedures were performed under general anesthesia. Preoperatively, three out of 17 patients showed pre-existing mucosal pathology endoscopically, while the 3- and 9-month results revealed the presence of mucosal pathology in four and two patients, respectively. The 3-month microbiological evaluation showed a significant increase in cultures with bacterial growth, while the 9-month culture results were comparable to the preoperative status of the maxillary sinus. Morphologically, neither fibrosis nor an altered inflammatory response or thickening of the epithelium and lamina propria was observed postoperatively. The number of goblet cells in the epithelial layer was increased. From this study it is concluded that the effect of maxillary sinus floor elevation surgery with autogenous bone grafts does not appear to have clinical consequences in patients without signs of pre-existing maxillary sinusitis.

Crosslinking of fibrinogen and fibronectin by free radicals: a possible initial step in adhesion formation in osteoarthritis of the temporomandibular joint.

PURPOSE: Adhesion formation in osteoarthritis (OA) of the temporomandibular joint (TMJ) typically results in a sustained limitation of joint movement. We propose the hypothesis that free-radical-mediated crosslinking of proteins underlies this adhesion formation in affected joints. Free radicals may cause oxidative modification of proteins, creating an opportunity for the formation of intramolecular and intermolecular crosslinks via covalent bonds. This may stabilize protein aggregates, rendering them more resistant to degradation. In this study, the free-radical-mediated crosslinking of model proteins (fibrinogen and fibronectin) was investigated to test our hypothesis that free radicals contribute to adhesion formation via this mechanism in OA of the TMJ. MATERIALS AND METHODS: Physiological clot formation of fibrinogen by thrombin and free-radical-induced crosslinking of fibrinogen and of fibronectin were analyzed using spectrophotometric turbidity measurements, light-scattering techniques, polyacrylamide gel electrophoresis (PAGE), and rotary shadowing. RESULTS: Fibrinogen was shown to aggregate after free radical treatment, as detected using turbidity measurements and light-scattering techniques. Using PAGE, fibrinogen as well as fibronectin was shown to degrade under low oxidative stress. Under high oxidative stress, however, fragments from both proteins were found to be covalently crosslinked, resulting in high-molecular-weight protein aggregates. The aggregation was shown to be at random with rotary shadowing. CONCLUSION: The study shows that high oxidative stress contributes to the formation of crosslinked proteins that may serve as an initial scaffolding for the development of adhesions frequently seen in OA of the TMJ.

Vertical distraction of the severely resorbed edentulous mandible: a clinical, histological and electron microscopic study of 10 treated cases.

The aim of this study was to evaluate the clinical and histomorphological results of distraction of the severely resorbed edentulous mandible. In a group of 10 edentulous patients suffering from insufficient retention of their mandibular denture related to a severely resorbed mandible, the anterior segment was augmented as a preimplant surgical procedure using the nonvoluminous Groningen Distraction Device (GDD). Two months after the last day of distraction, a bone biopsy was taken with a trephine, both distraction screws were replaced by endosseous implants and the guide screw was removed. The biopsies were examined by means of light microscopy (LM) and transmission electron microscopy (TEM). Radiographical and histomorphological examination of the biopsies revealed the presence of two cortical zones, one at each end of the biopsies, a poorly mineralized, fibrous interzone in the middle of the distraction gap, and two zones of mineralization between the central fibrous and the peripheral cortical zones. Formation of lamellar bone parallel to the distraction vector was clearly visible in the mineralization zone as well as signs of remodelling at the borderline between the native cortical bone and the generate. Clinical examination showed in all patients that the anterior segment distracted from the mandible body was sufficiently enlarged to enable insertion of endosseous implants with a length of at least 12 mm. One implant was lost during the healing phase, but was successfully replaced thereafter. Implant retained overdentures were fabricated 3 months after implantation. All patients have good function 11.2 +/- 4.3 months after the end of treatment. From this study it is concluded that the GDD has proven to be a reliable tool for augmentation of the anterior segment of a severely resorbed edentulous mandible enabling osseointegration of endosseous load-bearing implants.