Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro.

PURPOSE: It is widely thought that inflammation and osteoclastogenesis result in hydroxyapatite (HA) resorption and sequestrum formation during osseous infections, and microbial biofilm pathogens induce the inflammatory destruction of HA. We hypothesized that biofilms associated with infectious bone disease can directly resorb HA in the absence of host inflammation or osteoclastogenesis. Therefore we developed an in vitro model to test this hypothesis. MATERIALS AND METHODS: Customized HA discs were manufactured as a substrate for growing clinically relevant biofilm pathogens. Single-species biofilms of Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans and mixed-species biofilms of C albicans plus S mutans were incubated on HA discs for 72 hours to grow mature biofilms. Three different non-biofilm control groups also were established for testing. HA discs were then evaluated by means of scanning electron microscopy, micro-computed tomography metrotomography, x-ray spectroscopy, and confocal microscopy with planimetric analysis. In addition, quantitative cultures and pH assessment were performed. Analysis of variance was used to test for significance between treatment and control groups. RESULTS: All investigated biofilms were able to cause significant (P < .05) and morphologically characteristic alterations in HA structure as compared with controls. The highest number of alterations observed was caused by mixed biofilms of C albicans plus S mutans. S mutans biofilm incubated in medium with additional sucrose content was the most detrimental to HA surfaces among single-species biofilms. CONCLUSIONS: Our findings suggest that direct microbial resorption of bone is possible in addition to immune-mediated destruction, which has important translational implications for the pathogenesis of chronic bone infections and for targeted antimicrobial therapeutics.

Pamidronate enhances bacterial adhesion to bone hydroxyapatite. Another puzzle in the pathology of bisphosphonate-related osteonecrosis of the jaw?

PURPOSE: Bacterial colonization of the denuded bone in bisphosphonate-related osteonecrosis of the jaw suggests that bisphosphonates increase bacterial adhesion and biofilm formation. This study evaluated the adhesion of gram-positive and gram-negative bacteria on hydroxyapatite coated with pamidronate, one of the most potent bisphosphonates. MATERIALS AND METHODS: Twenty-five Staphylococcus aureus and 25 Pseudomonas aeruginosa strains were cultured on pamidronate-coated and uncoated hydroxyapatite discs. After incubation, nonadherent bacteria were removed by rinsing and centrifugation. Formation of a biofilm was confirmed by confocal laser 3-dimensional and scanning electron microscopy. The number of bacterial colonies was counted using quantitative cultures and mean numbers were compared using the Mann-Whitney rank sum test (statistical significance defined as P ≤ .05). The Hartree-Fock method was used for the calculation of electron interactions between hydroxyapatite ions and pamidronate. RESULTS: Fold increases in the number of colonies formed by S aureus and P aeruginosa in the presence of pamidronate compared with controls were 7.19 ± 4.127 and 2.87 ± 0.622, respectively. Hartree-Fock analysis showed that the reactive NH3(+) group of pamidronate may act as a steric factor, facilitating anchoring of bacteria to the hydroxyapatite surface. Alternatively, the NH3(+) group may attract bacteria by direct electrostatic interaction. CONCLUSIONS: Increased bacterial adhesion in the presence of bisphosphonates can promote osteomyelitis in patients with bisphosphonate-related osteonecrosis of the jaw. There may be increased infection rates when bisphosphonates are used for stabilization of prostheses in joint arthroplasty and in osteotomies and open fractures in patients treated with bisphosphonates.

Carbon dioxide directly suppresses spontaneous migration, chemotaxis, and free radical production of human neutrophils.

BACKGROUND: Carbon dioxide (CO(2)) insufflation during laparoscopy has been shown to dampen the systemic stress response to surgery. This is related to a suppression of peritoneal macrophage functions. In vivo data suggest that CO(2) can also affect neutrophils (polymorphonuclear cells, PMNs), the most abundant cell type in the inflamed peritoneal cavity. Nonetheless, the direct effects of CO(2) on PMNs have not yet been investigated. METHOD: PMNs were isolated from peripheral blood of healthy volunteers and incubated with (1) CO(2) (100% CO(2), pH 6.2), (2) hypoxic control (95% helium/5% CO(2), pH 7.4), and (3) control (95% air/5% CO(2), pH 7.4). Spontaneous and IL-8-induced migrations (chemokinesis and chemotaxis) during 2 h of exposure to different gases were measured with a transwell chamber system. The release of reactive oxygen species (ROS, luminometry) was determined after 15-min and 2-h exposures. In other sets of experiments, PMNs were exposed for 2 h or 4 h and kept under normal conditions for 18 h with lipopolysaccharide (LPS) stimulation thereafter. Final viability and apoptosis were assessed with fluorometry. RESULTS: Exposure to 100% CO(2) completely blocked spontaneous and IL-8 induced migration of PMNs (p < 0.001 vs. controls). Neutrophil migration was slightly diminished in the hypoxic control group. PMA-stimulated ROS production was reduced even after short exposure to 100% CO(2)(p < 0.05). We observed a slight increase of caspase-3/7 activity after exposure to 100% CO(2) and/or hypoxia; however, total viability was not affected. CONCLUSIONS: CO(2) incubation directly and temporarily suppresses the proinflammatory functions of PMNs; this is caused only partially by the concomitant hypoxia. This effect will contribute to the dampened inflammatory response to laparoscopic surgery. Further studies are needed to investigate whether the temporary suppression of neutrophil functions could affect the clearance of bacterial contaminations.

Impact of tumour characteristic and treatment modality on the local recurrence and the survival in patients with oral squamous cell carcinoma.

PURPOSE OF THE STUDY: To evaluate the impact of tumour location, local and regional advancement, histological differentiation, status of the surgical margins and radiotherapy on the disease-free time and overall survival rates in patients with oral squamous cell carcinoma. MATERIAL AND METHODS: A retrospective analysis of 67 patients treated with surgery (61 pts.), radiotherapy (6 pts.) and their combination (28 pts.). Follow time on average 40 months. The probabilities of survival were assessed using the Kaplan-Meier estimates, the differences were calculated with the log-rank test. An analysis of the influence of the neck recurrences on the prognosis was additionally performed. Relationship between independent categorical variables as: primary local advancement, location of the tumour, histological grading and lymph node metastases was evaluated with Fisher's Exact Test. RESULTS: Disease-free time rate amounted to 40.1%. There was no independent prognostic importance of primary location, T-staging and N-staging, histological grading of the tumour or radiation on disease-free time, just opposite to the status of the resection margins. However, the number of neck metastases was directly proportional to the tumour dimension and poor histological differentiation. Overall survival rate amounted to 87,5%. Posterior location in the oral cavity, involvement of cervical lymph nodes, surgical margins with the presence of tumour cells, poor histological differentiation and necessity of irradiation negatively correlated with the survival. CONCLUSIONS: A complete resection of the tumour was the most important independent prognostic parameter for the disease-free and overall survivals in oral squamous cell carcinomas in this study. An adjuvant radiation therapy could improve the results of treatment of oral squamous cell carcinoma also in cases were so far considered only for surgical management.

Advantages of a new technique of neck dissection using an ultrasonic scalpel.

UNLABELLED: The aim of the study was to see whether a new technique of a neck dissection applying an ultrasonic activated (harmonic) scalpel would improve patient recovery. MATERIAL AND METHODS: A prospective, non-randomized study was undertaken on 40 supraomohyoid neck dissections performed using a harmonic scalpel (20 pts) or electrocoagulation (20 pts). The evaluation included: operation time, intraoperative blood loss, postoperative seroma formation and pattern of wound healing. RESULTS: The operative time (mean+/-SD) using a harmonic scalpel was significantly shorter (52+/-10 min vs. 86+/-22 min; p

[Results of treatment of orbital fractures with polydioxanone sheet]. / Ocena wyników leczenia zlaman oczodolów z uzyciem folii polidioksanowej.

UNLABELLED: Orbital fractures are one of the most frequent consequences following midfacial trauma. If not treated they can lead to serious optical complications as: double vision (diplopia), restriction of ocular motility, eyeball sinking (enophthalmos). Autogenic bone grafts although still wide and effectively used for reconstruction of the fractured orbital walls present some disadvantages. This is morbidity of the donor site, unforeseen resorption, time-consuming procedure and longer postoperative care. Because of that introduction of new materials for orbital reconstruction seems to be desirable. The aim of the work was to evaluate usefulness of the polydioxanone sheets (PDS) as a reconstructive material for orbital floor fractures. MATERIAL AND METHODS: From 1. 09. 2004 to 1. 02. 2006 111 patients with orbital fractures were treated in the Department of Maxillofacial Surgery Klinikum Minden. Age of the patients was between 15- 89 years (mean 43 y.); m:w ratio 78:33. There were 54 isolated orbital floor fractures (in 3 cases with additional medial wall fracture) and 57 zygomatico-orbito-maxillary fractures. The diagnosis was based on the clinical picture and coronal CT scans. The patients were operated through a transconjunctival (72%) or an infraorbital-Mustarde (28%) access and the orbital floor (medial orbital wall) was reconstructed with PDS sheet. Control examination was performed immediately, 1 moth and 6 months after the operative treatment. RESULTS: There were 47% true "blow-out", 34% "trap-door" and 19% "en-clapet" fractures. The most important preoperative symptom was double vision (23%), restriction of ocular motility (18%), enophthalmos (3,6%), impairment of function of the infraorbital nerve (41%). Size of the defect was 3+/-1,13 cm(2) in "blow out" fracture, 1,8 +/-0,9 cm(2) in "trap-door" and 2+/-0,5 cm(2) in ,"en-clapet" ones. The primary reconstruction was successful in 97,3% of the cases. Any inflammation or reaction against implant was noted. Persistent double vision was present in 2,7%, restriction of ocular motility in 1,8% and enophthalmos in 0,9% after the primary procedure. These patients were submitted to reoperation. In 1 case badly positioned PDS sheet causing diplopia was removed. In another one, eyeball movement restriction was due to adhesions between the sheet and periorbital tissue. Visual status of this patient has improved after adhesiolysis. In 1 patient with a large bony destruction reconstruction only with PDS sheet was an inadequate treatment. In 7,2% of patients disturbances in the field of innervation of the infraorbital nerve were present 6 months after the surgery. CONCLUSIONS: Alloplastic, resorbable PDS sheets in most cases were a valuable material for the reconstruction of the orbital floor (medial orbital wall). Mechanical properties of PDS seem to be not sufficient for the reconstruction of extremely large bony defects. In these cases use of autogenic bone grafts or a titanium mesh should be rather considered.

Incidence and risk predictors for osteonecrosis of the jaw in cancer patients treated with intravenous bisphosphonates.

INTRODUCTION: The aim of this study was to establish the incidence of bisphosphonate-related osteonecrosis of the jaws (BRONJ) in oncologic patients and to determine risk predictors with respect to this condition. MATERIAL AND METHODS: This retrospective review included 197 oncologic patients treated from January 2005 to December 2010 with administration of bisphosphonates (BPs) as part of management. Sex, age, type of cancer diagnosed, period of substantial disease, oral surgery, type of bisphosphonate, number of doses, and cases of BRONJ diagnosis were recorded. The cumulative incidence and incidence rate of BRONJ were calculated. The factors that influenced BRONJ were assessed with multivariate logistic regression and with estimations of 95% confidence intervals and odd ratios. Values of p ≤ 0.05 were considered significant. RESULTS: The BRONJ appeared in 9.64% of patients. The BRONJ incidence rate was 1 in 28 patients per year of BP treatment. Logistic regression showed that the odds of osteonecrosis increased 1.0172-fold with each given dose of BP. The BRONJ risk with zoledronate was 5-fold higher than that with pamidronate or ibandronate. The risk of BRONJ increased by 40-fold after dental surgery. CONCLUSIONS: Period of BP administration and type of BP used are important risk predictors for the development of BRONJ in oncologic patients treated with intravenous administration of these drugs. Patient-related factors are dental or periodontal events connected with need for oral surgery.

Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite.

BACKGROUND: Because of the suspicion that bisphosphonates enhance bacterial colonization, this study evaluated adhesion and biofilm formation by Streptococcus mutans 25175, Staphylococcus aureus 6538, and Pseudomonas aeruginosa 14454 reference strains on hydroxyapatite coated with clodronate, pamidronate, or zoledronate. MATERIAL AND METHODS: Bacterial strains were cultured on bisphosphonate-coated and noncoated hydroxyapatite discs. After incubation, nonadhered bacteria were removed by centrifugation. Biofilm formation was confirmed by scanning electron microscopy. Bacterial colonization was estimated using quantitative cultures compared by means with Kruskal-Wallis and post-hoc Student-Newman-Keuls tests. Modeling of the interactions between bisphosphonates and hydroxyapatite was performed using the Density Functional Theory method. RESULTS: Bacterial colonization of the hydroxyapatite discs was significantly higher for all tested strains in the presence of bisphosphonates vs. CONTROLS: Adherence in the presence of pamidronate was higher than with other bisphosphonates. Density Functional Theory analysis showed that the protonated amine group of pamidronate, which are not present in clodronate or zoledronate, forms two additional hydrogen bonds with hydroxyapatite. Moreover, the reactive cationic amino group of pamidronate may attract bacteria by direct electrostatic interaction. CONCLUSION: Increased bacterial adhesion and biofilm formation can promote osteomyelitis, cause failure of dental implants or bisphosphonate-coated joint prostheses, and complicate bone surgery in patients on bisphosphonates.

Treatment of monostotic fibrous dysplasia with pamidronate.

INTRODUCTION: Fibrous dysplasia is a condition where fibrous tissue and bone with an altered structure replace normal bone. The craniofacial involvement is difficult to treat due to location, uncontrolled proliferation and compression of nerves. These result in facial asymmetry, pain, cranial nerve deficiencies, loss of vision or hearing, alterations in breathing, etc. The use of pamidronate, which inhibits the resorptive activity of osteoclasts may be an alternative to surgical intervention and may improve the results of treatment. AIM OF STUDY: To evaluate the treatment of monostotic craniofacial fibrous dysplasia with pamidronate. MATERIAL: A prospective pilot study was undertaken in 6 children with progressive fibrous dysplasia located in the mandible (3 pts), maxilla (2 pts) or cranial base (1 pt). Surgery consisted only of a biopsy and implantation of gentamycin sponge into the biopsy bed in 3 cases in which an infection was initially suspected. All patients received pamidronate infusions (1mg/kg i.v. for 3 days, every 4-6 months). The changes in bone density and extension of the disease were evaluated by serial orthopantomograms and CT scans. Follow up time was 9-24 months. RESULTS: Pain relief was achieved in all cases, decrease in swelling in 3, and stabilization in 3 cases. Radiological investigation revealed any progression but reduction in size and calcification of osteolytic lesions. The local bone density increased and there was no further spread of the disease in any of the patients. Inflammatory symptoms resolved in all cases. The only side effect was an increase in body temperature up to 38-40 degrees C. CONCLUSION: Pamidronate appears to be an effective and well-tolerated therapeutic option for patients with fibrous dysplasia. In the future, the combination of the metabolic blockage of the dysplasia by pamidronate and limited non-mutilating surgical interventions are possible.

Tissue reaction after implantation of Spongostan, as the carrier of xenogenic bone morphogenetic protein. Experimental study.

Reconstruction or filling of bone defects, especially in the maxillofacial region, often requires use of biomaterials. An implant should fasten healing of the bone gap or it should replace autogenic bone grafts. The combination of bone morphogenetic proteins with suitable carrier may fulfill these requirements. Proteins causing differentiation of mesenchymal cells in chondroblasts and osteoblasts were called Bone Morphogenetic Proteins--BMPs. The authors extracted BMP from bovine bones and placed it into collagen carrier formed from generally accessible hemostatic sponge--Spongostan. The implants were grafted into rat femoral muscle pouches in order to trace the tissue response. Pathologic examinations were performed 3, 6 and 8 weeks after implantation. On the basis on macroscopic and microscopic examinations it was stated that collagen sponge speckled with BMP caused minimal tissue response and evolved characteristic thin connective tissue capsule formation around the implant. The connective tissue penetrated spongious structure of the implant, filling the spaces, which became growing due to sponge resorption. Characteristic hyalinization and sparse chondroblasts were visible 8 weeks after implantation.

Association of dental and periodontal status with bisphosphonate-related osteonecrosis of the jaws. A retrospective case controlled study.

INTRODUCTION: To assess the association of oral hygiene, dental caries, and periodontal status with bisphosphonate-related osteonecrosis of the jaws. MATERIAL AND METHODS: A retrospective case-control study on 81 patients treated for neoplasms with bone metastases. Twenty-nine patients with bone necrosis and 52 controls treated with bisphosphonates were compared using the Oral Hygiene Index, Decay, Missing, Filled Teeth, Community Periodontal Index of Treatment Needs, and Residual Periodontal Bone. The null hypothesis stated that there was no difference in parameters of oral health between patients with and without bone necrosis. Differences of means of above-mentioned variables were compared between the groups with Student's t-test or Mann-Whitney rank sum test and χ(2) test. Value of p ≤ 0.05 was considered significant. RESULTS: Poorer oral hygiene (OHIs 1.94 vs. 1.32; p = 0.065), more advanced dental caries (DMFT 26.85 vs. 22.87; p = 0.05), and more advanced periodontal disease (CPITN: = 0: 21.05% vs. 42.51%; = 1 13.16% vs. 7.29%; = 2: 0% vs. 15.38%; = 3: 65.79% vs. 28.34%; = 4: 0% vs. 6.48%, Residual periodontal bone 73.1% vs. 80.51%; p = 0,001) were characteristic of patients with bisphosphonate related jaw necrosis when compared with control group. An advanced dental caries or periodontal disease required surgical intervention which directly contributed to the development of the bone necrosis. CONCLUSIONS: Dental and periodontal disease can lead to bisphosphonate-related osteonecrosis of the jaw. Oncologic patients treated with bisphosphonates should be offered preventive care to reduce dental plaque, calculus, dental caries, and periodontal disease.

Bisphosphonate-related osteonecrosis of the jaws: a review of 34 cases and evaluation of risk.

INTRODUCTION: The purpose of this study was to identify factors that influence bisphosphonate-related osteonecrosis of the jaws (BRONJ). PATIENTS AND METHODS: Patients undergoing treatment for BRONJ (n=34) were evaluated. Sex, age, underlying diagnosis, type of bisphosphonate (BP), duration and route of administration, location of osteonecrosis, clinical symptoms, Actinomyces colonisation, treatment and outcome were recorded. Symptom onset was analysed with respect to BP potency and cumulative dose. RESULTS: Underlying diagnoses indicating BP-treatment included multiple myeloma, breast carcinoma, prostate carcinoma and osteoporosis. In 31 patients, BRONJ was preceded by tooth extraction, root apicotomy, ill-fitting dentures, cystenucleation, implant insertion or trauma; in 3 patients, the precipitating event was not identified. Actinomyces colonisation was observed in 18 patients (53%). The occurrence of BRONJ was not directly related to BP dose or potency. More women with multiple myeloma had BRONJ than did males. BRONJ was observed in osteoporotic patients treated with both corticosteroids and BPs. CONCLUSIONS: BRONJ was not primarily associated with BP potency or dose. Factors that increased the risk of osteonecrosis were female sex, oral surgery and corticosteroids plus intravenous or oral BP administration. BP deposition in the jaw bones might enhance BRONJ by promoting bacterial colonisation; however, this hypothesis requires more study.

Carbon dioxide suppresses macrophage superoxide anion production independent of extracellular pH and mitochondrial activity.

BACKGROUND: Superoxide anions released by activated macrophages during surgery are considered to be responsible for local cellular damage. Application of CO2 pneumoperitoneum during laparoscopy affects superoxide anion release, but the underlying mechanism remains unclear and the data reported are conflicting. We investigated the direct and pH-mediated impact of CO2 and air on macrophage superoxide anion production. METHODS: Cells of the NR 8383 rat macrophage cell line were incubated for 2 hours in 5% CO2, 100% CO2, and room air or pH 7.4, pH 6.5, and pH 5.5. The extracellular pH was monitored during incubation. At 0, 2, and 6 hours after incubation, the release of superoxide anions was determined fluorometrically. The mitochondrial activity was determined via the conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] during and after incubation. RESULTS: Extracellular pH decreased to 6.4 during incubation in a CO2 atmosphere. The release of superoxide anions was significantly reduced immediately after CO2 incubation. It was restored at all other time-points. Decreasing the extracellular pH to 6.5 had no effect on superoxide anion release, whereas acidification of the extracellular milieu to pH 5.5 significantly suppressed subsequent superoxide release. Mitochondrial activity was significantly decreased by CO2 up to 2 hours and by acidic milieu up to 6 hours. Incubation in room air had no effect. CONCLUSIONS: Incubation in CO2 can directly suppress macrophage superoxide anion production. This effect is of short duration, fully reversible, and not correlated to changes in extracellular pH or mitochondrial activity. Air contamination does not affect macrophage superoxide anion release. We speculate that CO2 pneumoperitoneum could attenuate the intraoperative free radical production by directly inhibiting superoxide anion release of macrophages without long-lasting suppression of macrophages and their capacity to release superoxide anions postoperatively.