Synthesis of osteotropic hydroxybisphosphonate derivatives of fluoroquinolone antibacterials.

1-Hydroxybisphosphonate derivatives of ciprofloxacin, gatifloxacin and moxifloxacin have been synthesized using Cu(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction. The 1,2,3-triazol linked hydroxybisphosphonate derivative of ciprofloxacin exhibited antibacterial activity comparable to the parent antibiotic and all fluoroquinolone-bisphosphonates displayed osteotropic properties in a bone model.

The effects of enamel matrix derivative and cyclic mechanical strain on human gingival fibroblasts in an in vitro defect healing model.

Gingival fibroblasts (GFs) play a considerable role in the maintenance of the gingival apparatus as well as in connective tissue repair. Mobility of a periodontal wound or soft tissue graft can impair connective tissue healing from the GFs. Enamel matrix derivative (EMD) is an enamel matrix protein used clinically for periodontal regeneration of intrabony defects and furcations, as well as treatment of gingival margin recessions. The goal of this project was to compare the effects of varying concentrations of EMD, with and without cyclic mechanical strain, on cellular wound fill of human GFs using an in vitro defect healing model. GFs were seeded and cultured in six-well flexible-bottomed plates. A 3-mm wound was created in the central portion of each confluent well. Three wells were treated with each EMD concentration of 0 Μg/mL (control), 30 Μg/mL, 60 Μg/mL, or 120 Μg/mL. The plates were placed in an incubator containing a strain unit to subject test plates to cyclic strain. An identical set of control plates were not flexed. Cells were examined on days 4, 8, 12, and 16. Microphotographs were taken and wound fill measurements made using image analysis software. The percent wound fill was calculated. All nonflexed plates, regardless of EMD concentration, reached > 90% defect fill at similar rates by day 16. However, in the flexed plates, EMD had a significant negative effect on defect fill. The defect fill was 55.7% for 0 Μg/mL EMD, 48.2% for 30 Μg/mL EMD, 36.7% for 60 Μg/mL EMD, and 34.1% for 120 Μg/mL EMD on day 16 for the flexed GFs. EMD, in concentrations as high as 120 Μg/mL, did not significantly affect the amount of defect fill with nonflexed GFs. However, when the GFs were flexed, the addition of EMD had a significant negative effect on defect fill in a dose-dependent manner.

An acute osteomyelitis model in traumatized rat tibiae involving sand as a foreign body, thermal injury, and bimicrobial contamination.

The multfactorial nature of bone injuries in modern warfare and emergency trauma patients warrants enhancement of existing models. To develop a more appropriate model, rat tibiae (n = 195) were mechanically injured, divided into 2 groups (with or without thermal injury), and contaminated with a range of Staphylococcus aureus (Cowan 1) inocula. In some experiments, S. aureus inocula also contained Escherichia coli or foreign bodies (sand or soil). The primary outcome measure was the amount of S. aureus remaining in the tibia (tibial bacterial load) 24 h after contamination, reported as log10 cfu/g bone. S. aureus showed ID50 and ID95 values of 72 and 977 cfu, respectively. Values were lower than seen previously by using S. aureus strain SMH. S. aureus tibial bacterial loads were higher in tibiae with mechanical and thermal injury (log10 4.15 +/- 0.27 cfu/g) versus mechanical injury alone (log10 3.1 +/- 0.47 cfu/g, P = 0.028). The addition of E. coli to the S. aureus inoculum had no effect on tibial bacterial loads (S. aureus only, log10 4.24 +/- 0.92 cfu/g; S. aureus + E. coli, log10 4.1 +/- 1.0 cfu/g, P = 0.74). Sand, added as a foreign body, increased tibial bacterial load. Combined mechanical and thermal trauma of the tibia is associated with increased S. aureus tibial bacterial loads, increasing the risk of acute osteomyelitis. Understanding the interplay of mechanical and thermal injuries, bimicrobial contamination, and foreign bodies may improve our understanding of traumatic bone injuries and the pathogenesis of osteomyelitis.

Influence of matrix-suspended demineralized bone on osseous repair using a critical-sized defect in the rat (Rattus norvegicus) calvarium.

Demineralized freeze-dried bone (DFDB) in matrix form must be rehydrated with a carrier medium which allows for easy manipulation during periodontal surgery. The purpose of this study was to evaluate how human DFDB suspended in a polyol matrix affects new bone formation in the rat calvarium critical-sized defect (CSD) model. Fifty-five adult male Harlan Sprague-Dawley rats were assigned to 1 of 5 treatment groups: polyol, 100% DFDB, 47% DFDB/polyol, 47% DFDB, or an unfilled control. They were then placed into 8-m calvarial CSDs. The bone donor source company for the DFDB and DFDB/polyol groups was the same. Calvaria were harvested 10 weeks after surgery and evaluated histomorphometrically. The diameter of bone particles from the 3 groups containing DFDB was measured by scanning electron microscopy. There was no statistically significant difference in the percentage of bone fill between any of the groups, although the 100% DFDB group exhibited the most bone fill. The 47% DFDB/polyol and 47% DFDB groups had similar amounts of bone formation. The average size of the demineralized bone particles from the 100% DFDB group was significantly smaller than that of the other 2 groups containing DFDB. Adding a polyol to DFDB produced similar osseous regeneration in the rat calvarium defect model vs DFDB alone. Yet from a clinical standpoint, the polyol enhanced graft handling and stability. Graft particle size may have an effect on bone fill.

Effect of a hydroxyapatite tricalcium phosphate alloplast on osseous repair in the rat calvarium.

BACKGROUND: Bony defects caused by periodontitis are often treated by regenerative therapy using autografts and/or allografts. Alloplasts, such as hydroxyapatite or ceramics, are also used as osteoconductive materials that serve as a scaffold for new bony ingrowth. The purpose of this study was to determine the effect of hydroxyapatite tricalcium phosphate (HA-TCP) on osseous repair in the rat calvarium. METHODS: Forty-four adult male Harlan Sprague-Dawley rats were assigned to one of four treatment groups: HA-TCP macroporous disk, HA-TCP microporous disk, HA-TCP granules, and demineralized freeze-dried bone (DFDB). The materials were placed into 8-mm calvarial critical-size defects (CSD). Calvariae were harvested at 10 weeks post-surgery and evaluated histomorphometrically. RESULTS: The DFDB group had significantly (P <0.05) more new bone formation (47%) than any other group. The HA-TCP macroporous disk group had significantly (P <0.05) more new bone formation (19.7%) than the HA-TCP microporous disk (8.5%) or HA-TCP granule (6.9%) groups. CONCLUSIONS: The HA-TCP macroporous disk may elicit significant new bone formation due to its rigid space-maintaining scaffold and pore size for vascular ingrowth. It is well tolerated by host tissues and may be a suitable carrier for growth factors.

An in vitro evaluation of the antibacterial efficacy of chlorine dioxide on E. faecalis in bovine incisors.

This study investigated the ability of chlorine dioxide to eliminate Enterococcus faecalis from dentinal tubules of bovine incisors. Thirty-seven extracted bovine incisor roots were sectioned into seventy-four 5 mm disks. Standardized lumens were filled with either sterile Brain Heart Infusion Broth (contamination controls, n = 10) or BHI containing E. faecalis (1.0 x 10 cfu/ml). Disks were incubated in 5% CO2 at 37 degrees C for 72 h. To simulate endodontic instrumentation the lumens were again enlarged. Sixty disks were randomly divided into four experimental groups and filled with one of the following irrigants: 10% Clidox-S (chlorine dioxide), 13.8% BioClenz (chlorine dioxide), 5.25% Clorox, or saline. The disks were incubated for 30 min and were then frozen, pulverized, serially diluted in phosphate buffered saline, and plated on BHI plates in triplicate. Total colony forming units were counted macroscopically. Statistical analysis of the data was performed with a Kruskal-Wallis one-way ANOVA on ranks (p < 0.05, n = 60). Bacterial counts, expressed in log10 cfu/disk were as follows (">" denotes significant differences): Saline > Clidox-S = BioClenz > Clorox. All negative controls were sterile. Chlorine dioxide and NaOCL were both effective in eliminating E. faecalis from the dentinal disks within 30 min.

Low-dose infectivity of Staphylococcus aureus (SMH strain) in traumatized rat tibiae provides a model for studying early events in contaminated bone injuries.

Animal models of post-traumatic acute osteomyelitis (OM) that closely mimic human scenarios, including infection prophylactic procedures such as debridement and lavage, may provide a better understanding of OM. We contaminated mechanically traumatized rat tibiae (n = 69) with various doses of a Staphylococcus aureus strain (SMH) known to cause human OM and then performed curettage and lavage. Tibiae were harvested 24 h after lavage for assessment of bacterial load and determination of minimal infective doses for 50% (ID50) and 95% (ID95) of rats. Some experiments varied tibial harvest time after lavage (n = 10); for progressive infection, tibiae were evaluated at 7 and 15 days after contamination (n = 17 for each time point). At 24 h after contamination, the ID50 was 1.8 x 10(3) CFU, and the ID95 was 9.2 x 10(3) CFU. Tibial bacterial loads did not increase with inocula greater than the ID95. Lavage removed many bacteria from bone, but it did not prevent subsequent infection or disease. At 15 days after contamination, most tibiae (14 of 17) were infected, with macroscopic and radiological signs of established OM. This newly described rat OM model, with a low ID95 despite prophylactic curettage and lavage, closely mimics events in contaminated human bone injuries. This situation will allow study of early factors in contaminated bone injuries, including clinical interventions that may reduce infection and prevent disease.

Apical diffusion of calcium hydroxide in an in vitro model.

An in vitro agar model was developed to study the effect of intracanal medicaments on periapical tissues and was used to study the diffusion of three calcium hydroxide (Ca(OH)2) medicaments of varying viscosity through simulated root canals with various sizes of apical foramina. Experimental medicaments were added to pipette tips used to represent tooth roots, which were fixed in syringes containing brain heart infusion agar and calcium-reactive dye. OH and Ca concentrations were measured in the agar at 30 minutes and 24 hours. Ca concentration and pH increased with larger aperture sizes, and higher pH and Ca diffusion was produced by a 10% Ca(OH)2 solution than was produced by Pulpdent or a Ca(OH)2 paste. The results suggest that the properties of the Ca(OH)2-containing vehicle could affect the action of the medicament in the periapical tissues.

Antibacterial efficacy of calcium hydroxide, iodine potassium iodide, betadine, and betadine scrub with and without surfactant against E faecalis in vitro.

OBJECTIVE: This study investigated the ability of endodontic irrigants and medicaments to eliminate Enterococcus faecalis from infected dentinal tubules, and whether their antimicrobial action was enhanced by surfactant. STUDY DESIGN: For the study, 5-mm dentin disks were sectioned from bovine incisor roots and infected with E faecalis. Lumens were instrumented, and 1 of 7 medicaments (10% Ca(OH) 2, Betadine, or IKI, each with or without surfactant, or Betadine Scrub) was used to flush and fill each lumen. Positive controls received saline. Specimens were incubated for 15 minutes or 24 hours. Quantitative microbiology of the remaining bacteria was performed and groups were compared using a 1-way ANOVA. RESULTS: The addition of surfactant did not enhance the antibacterial action of any medicament. When used as a 24-hour medicament, Ca(OH) 2 consistently failed to eliminate E faecalis, whereas both Betadine Scrub and IKI rendered 90% of samples sterile. IKI was the only agent shown to consistently eliminate E faecalis in a 15-minute time frame. CONCLUSION: Under the in vitro conditions of this study, IKI was able to eliminate E faecalis from bovine root dentin when used with a 15-minute contact time.

Antibacterial efficacy of calcium hydroxide and chlorhexidine gluconate irrigants at 37 degrees C and 46 degrees C.

This study investigated the ability of two endodontic irrigants to eliminate Enterococcus faecalis from dentinal tubules, and whether their antimicrobial action was enhanced by heat. The lumens of disks prepared from extracted bovine roots were infected with E. faecalis and incubated for 72 h. Specimens were then filled with saline, 10% calcium hydroxide (Ca(OH)2), or 0.12% chlorhexidine gluconate (CHX) at 24 degrees C or 46 degrees C and incubated at 37 degrees C or 46 degrees C. The samples were then pulverized and plated to quantify residual bacteria. No statistical difference (p > 0.05) in bacterial growth was seen between the two saline groups, or between the two medication groups at a given temperature. CHX and Ca(OH)2 at either temperature produced significantly less growth than either saline group, and CHX or Ca(OH)2 at 46 degrees C produced significantly less growth than either group at 37 degrees C. Heat enhanced the antibacterial action of both experimental irrigants against E. faecalis, but heating saline produced no increase in bactericidal effect.

In vitro antimicrobial activity of various medication preparations on E. faecalis in root canal dentin.

The purpose of this study was to evaluate the antimicrobial activity of several medication preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens, 5 mm in height. The smear layer was removed and the samples were autoclaved and then incubated at 37 degrees C/5% CO2 for 24 h in brain-heart infusion (BHI) broth containing 7.0 x 10(4) colony forming units per ml of E. faecalis. The samples were washed in phosphate buffered saline and mounted to individual culture wells with sticky wax. Test medications were applied to fill the canal lumina; medication groups were: (a) sterile H2O (positive control); (b) a 10% mixture of 1.0 g Ca(OH)2 USP in 10 ml sterile H2O; (c) 10% Ca(OH)2 in 0.12% chlorhexidine gluconate (Peridex); (d) Peridex; and (e) uninoculated BHI (negative control). The samples were incubated at 37 degrees C/5% CO2 for 24 h. Dentin samples for quantitative microbiology were then obtained with consecutive sterile burs (ISO 029, 035, 042). All three experimental groups demonstrated significantly greater antimicrobial activity than the positive control (p < 0.001). Group 2 demonstrated significantly greater antimicrobial activity than Group 3 or Group 4 at all dentin depths (p < 0.05). These results suggest that 10% Ca(OH)2 may be more effective than Peridex or 10% Ca(OH)2 in Peridex for the elimination of E. faecalis from dentin tubules.

Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials.

Bisphosphonates conjugated to fluoroquinolone antibacterials through an intermediate carbon had better activity than conjugates lacking the carbon. Virtually all molar-based activity of these esterified bisphosphonate derivatives was identical to that of its parent. De-esterified free-acid forms retained good activity against most Gram-negative bacteria, but not against Gram-positives. A free-acid derivative remained bound to washed bone and completely inhibited Staphylococcus aureus growth. The more potent parent, ciprofloxacin, failed to bind significantly, and bacterial growth occurred.