Periodontal Inflammation Primes the Systemic Innate Immune Response.

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

Oral and Blood Neutrophil Activation States during Experimental Gingivitis.

Polymorphonuclear neutrophils (PMNs) are the primary leukocytes present in the healthy and inflamed oral cavity. While unique PMN activation states have been shown to differentiate health and periodontitis, little is known about the changes in PMN activation states that occur during the transition from periodontal health to gingivitis. The objective of this study was to characterize oral and circulatory PMNs during induction and resolution of experimental gingivitis. Healthy volunteers were recruited to undergo experimental gingivitis. Clinical assessment of pocket depths, bleeding on probing, gingival index, and plaque index, as well as flow cytometric analysis of CD (cluster of differentiation) activation markers on blood and oral PMNs, was performed weekly. All clinical parameters increased significantly during the induction period and returned to baseline levels during the resolution phase. During the induction phase, while oral PMN counts increased, oral PMN activation state based on surface expression of CD63, CD11b, CD16, and CD14 was diminished compared to those seen in health and during the resolution phase. PMNs in circulation during onset showed increased activation based on CD55, CD63, CD11b, and CD66a. Using clinical parameters and oral PMN counts assessed at day 21, we noted 2 unique disease patterns where one-third of subjects displayed an exaggerated influx of oral PMNs with severe inflammation compared to the majority of the population who experienced a moderate level of inflammation and PMN influx. This supports the notion that PMN influx and severe inflammatory changes during gingivitis could identify subjects at risk for the development of severe gingival inflammation and progression toward destructive periodontitis. This study demonstrates that oral PMN activation states are reduced in gingivitis and suggest that only in periodontitis do PMNs become hyperactivated and tissue damaging. Knowledge Transfer Statement: Our article creates a paradigm for future studies of the evolution of essential oral and circulatory biomarkers to identify individuals at risk to develop periodontitis at an early stage of periodontal disease, which is reversible upon proper oral hygiene practices and dental treatments.

Resolving Macrophages Counter Osteolysis by Anabolic Actions on Bone Cells.

Progression of inflammatory osteolytic diseases, including rheumatoid arthritis and periodontitis, is characterized by increased production of proinflammatory mediators and matrix-degrading enzymes by macrophages and increased osteoclastic activity. Phenotypic changes in macrophages are central to the healing process in virtually all tissues. Using a murine model of periodontitis, we assessed the timing of macrophage phenotypic changes and the impact of proresolving activation during inflammatory osteolysis and healing. Proinflammatory macrophage activation and TNF-α overproduction within 3 wk after induction of periodontitis was associated with progressing bone loss. Proresolving activation within 1 wk of stimulus removal and markers of resolving macrophages (IL-10, TGF-ß, and CD206) correlated strongly with bone levels. In vivo macrophage depletion with clodronate liposomes prevented bone resorption but impaired regeneration. Induction of resolving macrophages with rosiglitazone, a PPAR-γ agonist, led to reduced bone resorption during inflammatory stimulation and increased bone formation during healing. In vitro assessment of primary bone marrow-derived macrophages activated with either IFN-γ and LPS (proinflammatory activation) or IL-4 (proresolving activation) showed that IL-4-activated cells have enhanced resolving functions (production of anti-inflammatory cytokines; migration and phagocytosis of aged neutrophils) and exert direct anabolic actions on bone cells. Cystatin C secreted by resolving but not inflammatory macrophages explained, in part, the macrophage actions on osteoblasts and osteoclasts. This study supports the concept that therapeutic induction of proresolving functions in macrophages can recouple bone resorption and formation in inflammatory osteolytic diseases.

The learning curve with a new cephalomedullary femoral nail.

INTRODUCTION: The Cephalomedullary Nail (CMN) (Zimmer, Warsaw) was introduced in 2010 as part of a multicenter trial to evaluate its performance. At one year the CMN had results in keeping with other intramedullary devices with good union rates and low complication rates. In the second and third years of use an increased rate of implant failure was observed, towards the higher end of the 1-5% nail breakage rate seen in other studies. This study aims to evaluate if there any common features in this cohort of patients. MATERIALS AND METHODS: This is a retrospective cohort study looking at patients who underwent femoral fracture fixation using the cephalomedullary nail between January 2011 and June 2014. The primary outcome measure was implant failure; secondary outcomes were; fracture reduction and bisphosphonate use. RESULTS: 201 patients were included (135 female, 66 male) with an average age of 81 years. Ten (5%) nail breakages occurred in the study period at an average of 39 weeks (24-92); 9 were 125° nails 1 was a 130° nail and all fractured at the lag screw junction. CONCLUSIONS: Implant failure is a recognised complication of intramedullary nailing in cases of non-union. The increased rate of implant failure in our department required a change to a 130° CMN implant and a 3.2mm diameter guide wire for placement of the lag screw. We continue to monitor this difficult group of patients very closely.

Effect of a calcium-enriched diet on the colonic epithelial hyperproliferation induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats on a low calcium and fat diet.

We examined whether hyperproliferation of colonic crypt epithelium during cancer induction by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), in rats on a low fat and calcium diet could be reduced by added calcium p.o. From the age of 4 weeks, 104 male Sprague-Dawley rats received a low fat (3.5%), low calcium (0.05% calcium ion), and low vitamin D (0.4 IU/g) diet. Sixty-four also had calcium salts, derived from either calcium lactate or solubilized calcium carbonate, added to their drinking water; therefore their total calcium intake was about 1% of daily diet. At age 12 weeks the rats were divided into 4 treatment groups: 8 rats, not receiving added calcium, had rectal saline instillations weekly (saline control group) and were sacrificed after a further 28 weeks; 3 groups of 32 rats each received intrarectal MNNG (1.5 mg) weekly. One group, not receiving added calcium, was the MNNG control group; while the second group also received added calcium lactate, and the third group received calcium carbonate. Groups of 24 were sacrificed periodically until 28 weeks of treatment. Rats were sacrificed and epithelial proliferation was estimated, 1 week after the last intrarectal instillation, by in vivo labeling with tritiated thymidine and measuring the ratio of labeled to total colonic crypt epithelial cells. The mean labeling index of the MNNG treated and added calcium groups were significantly higher (8.7-9.5%) than that of the saline controls (2.8%) only at week 28; however, it was then still significantly less than that of the MNNG controls not having added calcium (17.9%). Hyperproliferation, during induction of colonic cancer by MNNG in rats on a low calcium diet, can be reduced by a calcium enriched diet even in the presence of a low fat intake.

Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state.

Autoradiographic localization of 24R,25-dihydroxyvitamin D3 in epiphyseal cartilage.

There is emerging evidence for specific binding sites and biologic action for 24,25(OH)2D3 in the epiphyseal cartilage. The present study was undertaken to identify the target cells of 24,25(OH)2D3 in the epiphyses of rat bone using an autoradiographic technique. Pieces of epiphyseal cartilage obtained from 4-day-old vitamin D-deficient rats were incubated for 15 or 60 min with [3H]-24,25(OH)2D3 in the presence or absence of 100-fold excess of 25(OH)D3, 1,25(OH)2D3, or 24R,25(OH)2D3. The pieces were prepared for autoradiographic study by a new modified fixation method. Cytoplasmic and nuclear concentration of radioactivity was observed in all cell layers of the epiphyseal cartilage except for the hypertrophic cartilage zone. The highest concentration of radioactivity was seen in the proliferating chondroblasts of the columnar zone. After 15 min of incubation the radioactivity was seen mainly in the cell membrane and cytoplasm, whereas at 60 min radioactivity was also prominent in the nuclei. The competition with excess of cold metabolites revealed that only 24R,25(OH)2D3 caused a significant decrease in cytoplasmic and nuclear radioactivity. These data support the biochemical studies showing that the epiphyseal cartilage is a target tissue for 24,25(OH)2D3.

Influence of demographic parameters on rectal epithelial proliferation.

Measurement of rectal epithelial proliferation is now being used as a biomarker for assessing risk for colorectal cancer and response within dietary intervention studies. We examined the possible confounding effects of demographic parameters on the proliferation of 52 healthy middle-aged volunteers without known risk factors for colorectal cancer. No significant effects on proliferation of age, sex or ethnic grouping were found other than marked urban-rural differences amongst men. We hypothesise that these could be explained by differences in dietary habits and their deleterious effects in the older male population. Careful matching of controls are probably needed in order to demonstrate the minor changes in mucosal proliferation that could reflect risk for neoplasia. Further human studies are needed to examine the effects of diet and extremes of age on proliferation.

Reproducibility studies and effects of bowel preparations on measurements of rectal epithelial proliferation.

Measurements of rectal epithelial proliferation (REP), using tritiated labelled thymidine, correlate with colonic epithelial proliferation, risk for cancer and response to therapies. There have been criticisms regarding its reproducibility and the possible deleterious effects of bowel preparations on this biomarker. We studied paired observations on 7 patients repeated without bowel preparation, 11 repeated after tap-water enema, and 8 repeated after PEG-electrolyte solution or extract of senna purgative and found no significant differences between paired observations. In addition, in a high-risk group for colorectal cancer, 31 persons received PEG or senna preparation and their REP was not significantly different from that of 23 examined without these preparations. Thus, REP is a reproducible biomarker and not affected by several commonly used bowel preparations.

Rectal epithelial proliferation characteristics of first degree relatives of sporadic colon cancer patients.

Hyperproliferation of rectal epithelium is characteristic of families at high genetic risk for large bowel neoplasia, but has not been well-documented in families of sporadic colorectal cancer patients. This was studied in 119 such first degree relatives and 44 comparison subjects without this family history. All screened negative for large bowel neoplasia. Within the family group proliferation was significantly higher in the men and those aged less than 45 years, also higher (insignificantly) in non-Europeans and those having greater than 1 first degree colorectal cancer relative. In comparison to the nonfamily group the labelling index (LI) of the relatives showed a significant negative correlation with age (R = -0.20, P = 0.03). Within this family group the probability of having an elevated LI (greater than 6.0%) was greatest in the young (less than 50 years old) men (odds ratio = 2.0). Measurements of rectal epithelial proliferation (REP) in these first degree relatives, at a young age, might help delineate a high risk subgroup for prospective primary and secondary intervention.

Rectal epithelial proliferation in women recovered from breast cancer.

Women recovered from breast cancer are at increased risk for colorectal neoplasia. The reasons may be genetic, dietary or endogenous hormonal risk factors. Measurements of rectal epithelial proliferation are a useful biomarker of risk for large bowel cancer. This was studied in 12 women after (mean 7.8 years) cured breast cancer, who had a mean % labelling index of 7.5 +/- 3.5 (S.D.) as compared to 5.8 +/- 1.8 (S.D.) in a disease-free comparison group of 25 women. In addition, analysis of labelled crypt compartments demonstrated a significantly higher proportion in the study group with thymidine uptake, mainly in the mid crypt zone, and an extension of crypt cell DNA synthesis towards the surface epithelium. Using proliferative activity as a biomarker of risk in a larger study group, we may learn more about common etiological factors for both malignancies and also identify a higher-risk subgroup for long-term follow-up and possible therapeutic intervention.

Rectal epithelial proliferation in persons post sporadic colorectal neoplasia.

Some studies have shown diffuse large bowel epithelial hyperproliferation in persons having colorectal neoplasia. Thus, measurements of rectal epithelial proliferation (REP) could be useful as a screening biomarker of risk for sporadic neoplasia. We examined REP, by autoradiography with tritiated thymidine, in 84 persons: 32 healthy volunteers, 37 who had had sporadic adenomas and 15 post cured sporadic colorectal cancer. Measurements of the labelling index (ratio of labelled to total number of crypt cells) showed a statistically insignificant but increasing gradient of hyperproliferation related to degree and invasiveness of neoplasia. However, this became significant when examining the proportion of labelled crypt compartments in each group and by comparing combined compartments 3 and 4 of cancer patients to non-cancer patients. Gender and age were found to be parameters that influenced the results. Using standard methods of analysis of REP, the lack of clear separation between risk groups limits the usefulness of REP measurements as a screening biomarker of risk for sporadic large bowel neoplasia.

Protracted ambulatory venous infusion of 5-fluorouracil.

5-Fluorouracil (5-Fu) was administered by a constant venous infusion schedule at a dose of 300 mg/m2/d for 30-180 days. The dose schedule was associated with minimal toxicity in 32 patients with gastrointestinal cancer treated by employing a portable infusion pump for ambulatory drug delivery. Cumulative dose of 5-Fu was extended to three to four times that achieved by intermittent bolus therapy or short-term 5-day infusion therapy. Objective tumor regression was observed in six of 22 patients with measurable disease; 10 patients had stable disease, five of whom had a decrease in CEA levels. The responses according to tumor type were as follows: 1/1 gastric cancer; 1/2 hepatoma; and 4/18 colon cancer. The superiority of this new treatment schedule for 5-Fu will need to be established by prospective randomized clinical trials.

Mitomycin C: phase I study of a constant infusion ambulatory treatment schedule.

Thirty patients received mitomycin C by constant infusion for 5 consecutive days (16 patients ) or for extended period by an ambulatory infusion pump (Cor-Med model) for 9 to 30 days (14 patients). The short-term 5-day infusions were delivered at dose rates of 2, 3, 4, 5 and 6 mg/m2/d with a cumulative dose of 15-50 mg. The protracted infusions were delivered at dose rates of 0.75-3 mg/m2/d with a cumulative dose of 21.6-65.2 mg. Marrow suppression was dose-limiting and occurred in 5/6 evaluable patients receiving more than 30 mg in the short-term infusion schedule and 8/10 evaluable patients receiving more than 20 mg in the protracted infusion schedule. The characteristics of the marrow suppression are that: a) thrombocytopenia precedes or is observed without concomitant leukopenia and b) the nadir day is delayed (WBC day 42, platelet day 36). Mitomycin C delivered by constant infusion leads to dose-limiting marrow toxicity at 20 to 30 mg cumulative dose depending upon the dose rate and duration of treatment. For short-term 5-day therapy, 3 mg/m2/d and for protracted therapy (up to 30 d) 0.75 mg/m2/d are the recommended dose rates for the constant infusion schedule.

Regional accelerated phenomenon in the mandible following mucoperiosteal flap surgery.

Striking remodeling activity occurs adjacent to the site of injury in orthopedic surgery. This reaction has been described as regional accelerated phenomenon (RAP), as it speeds up the healing stage. The phenomenon is a transient burst of localized remodeling process following surgical wounding of cortical bone. We explored whether RAP occurs following mucoperiosteal flap surgery in the jaw bone. Mucoperiosteal flaps were performed on 60 Wistar rats, either only on the buccal aspect or both on buccal and lingual aspects of the mandible. The surgical procedure lasted an average of 30 seconds and the flap was readapted without sutures. The rats were sacrificed at 3, 7, 10, 14, 21, and 120 days. High resolution x-ray microradiography of 1 to 1.5 mm thick ground sections between premolar and molar regions of the mandible were analyzed and revealed large areas of radiolucency which correlated to massive resorption of the alveolar bone, as well as areas in the bone proper. The RAP was observed as early as 10 days in the treated side group. Striking resorption of the cortical bone, both on the surface and the bone proper, occurred on the periodontal aspect of the crestal bone leading to widening of the periodontal ligament space, where a mucoperiosteal flap was performed on the buccal aspect. The resorption was more prominent when a mucoperiosteal flap was performed both on the lingual and buccal aspect. The alveolar bone recovered almost to control levels 120 days after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of bisphosphonate on alveolar bone resorption following mucoperiosteal flap surgery in the mandible of rats.

Following elevation of a full thickness flap a transient burst of regional remodeling occurs. This phenomenon is termed in orthopedic surgery as regional accelerated phenomenon (RAP), beginning with accelerated resorption activity followed by a slow process of bone regeneration. Recently we have demonstrated that a mucoperiosteal surgical flap of rat mandible is producing a typical RAP process. Bisphosphonates are synthetic compounds that are taken up preferentially by the skeleton and suppress osteoclast-mediated bone resorption by a mechanism that is not yet fully understood. Amino bisphosphonate has been shown to inhibit active bone resorption without interfering with bone formation. In this study we evaluated the effect of amino bisphosphonate on bone resorption associated with a mucoperiosteal flap used as a resorptive model. We compared the effect of amino bisphosphonate in rats using IV administration with topical application at 3 dose levels. The results show that topical application of all 3 doses (0.15, 0.75, and 1.5 mg/ml) had no inhibiting effect on bone resorption after surgery, while IV administration at 0.5 mg/kg body weight significantly reduced the bone resorption. Interestingly, in the non-operated side, amino bisphosphonate increased mineral density.

Mechanical and hormonal stimulation of cell cultures derived from young rat mandible condyle.

Collagenase digestion of young rat condyles released cells which were grown in culture during two weeks. Morphologically, two populations of cells were distinguished, one of which reacted to alkaline phosphatase and resembled chondroblast or osteoblast-like cells. Parathyroid hormone stimulated a 2-fold increase in cellular cyclic AMP, whereas calcitonin had no effect. Physical forces activated cellular cyclic AMP to a 2.5-fold of control levels and increased the incorporation of radioactive thymidine into DNA by 50 per cent. In contrast to cultured bone cells, the response to physical forces was not inhibited by indomethacin in cultured condyle cells. It seems, therefore, that condyle cells are specific in their response to bone-seeking hormones and physical forces.

Uptake of 45Ca from a topically-applied monofluorophosphate sodium fluoride calcium hydroxide dentifrice by rat enamel.

Relative 45Ca-uptake in vivo was determined by etchings after dentifrice application between 18 and 22 days of age; uptake increased and the Ca and P that dissolved decreased with application time. The mechanism of 45Ca-uptake from the dentifrice may be due to either isotopic exchange or to the precipitation or crystal growth of calcium and phosphate.

Percutaneous drainage of subcapsular splenic hematoma: an experimental model in dogs.

Preservation of the spleen in patients with posttraumatic subcapsular hematoma is still controversial. To determine the best therapeutic approach for this type of splenic injury, we designed an experimental model of subcapsular hematoma of the spleen in dogs. A total of 23 subcapsular hematomas were caused in 19 dogs, which were followed both clinically and ultrasonographically for 12 weeks. Fifty-seven per cent of the hematomas resolved spontaneously; those that persisted (43%) were aspirated percutaneously with ultrasound guidance, after which they were all resolved. Our results support a conservative approach. When the hematoma does not resolve spontaneously, percutaneous ultrasound-guided aspiration may be considered.

Prefabricated and prelaminated flaps for head and neck reconstruction.

Flap prefabrication and prelamination are evolving, new techniques that are useful in reconstructing complex defects of the head and neck. Flap prefabrication involves the introduction of a new blood supply by means of a vascular pedicle transfer into a volume of tissue. After a period of neovascularization, this volume of tissue may be transferred, based only on its implanted vascular pedicle. The transfer may be local transposition or by microsurgical transfer. Flap prelamination refers to a technique in which additional tissue is added to an existing flap (without manipulation of its axial blood supply) to make a multilayered flap that may be used for complex, three-dimensional multilayered reconstructions. This technique may be used locally or at a distance, requiring microvascular transfer. Examples of each are described in this article.