Garrit Koller

Effects of bioactive glass and beta-TCP containing three-dimensional laser sintered polyetheretherketone composites on osteoblasts in vitro

Because of their excellent physical properties nonresorbable thermoplastic polymers have become more important for the field of reconstructive surgery. Aim of the present study was to investigate the effects of laser sintered polyetheretherketone (PEEK) with incorporated osteoconductive and bioactive bone substitution materials on osteoblasts in vitro. Human osteoblasts (hFOB 1.19) were seeded onto laser sintered PEEK samples containing nano-sized carbon black, beta-tricalciumphosphate (beta-TCP), and bioactive glass 45S5. Osteoblasts were investigated for cell viability, cell proliferation and cell morphology. A constant proliferation of osteoblasts could be observed on all samples with the highest values for bioactive glass containing samples at day 7 (OD 1.76 +/- 0.22) and day 14 (OD 3.75 +/- 0.31) and lowest values for beta-TCP containing probes throughout the study compared with the PEEK pure control group. Highest cell viability was observed for Bioglass containing probes (95.5 +/- 3.32)% whereas osteoblasts seeded on beta-TCP containing probes showed reduced viability (84.4 +/- 4.32)%. Laser sintered PEEK implants seem to be attractive candidates for use as bone substitutes for reconstructive surgery because of their biocompatibility, individual shape, and the possibility of compounding bioinert polymer powder with osteoconductive and bioactive materials which might benefit bone formation in vivo.

Neutrophil elastase-mediated increase in airway temperature during inflammation

BACKGROUNDnHow elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection.nnnMETHODSnWe determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq.nnnRESULTSnHere we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes.nnnCONCLUSIONnNeutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF.

Combined Systems Approaches Reveal Highly Plastic Responses to Antimicrobial Peptide Challenge in Escherichia coli

Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies.

An MMP-inhibitor modified adhesive primer enhances bond durability to carious dentin

OBJECTIVESnTo evaluate the effect of adding a matrix metalloproteinase (MMP) inhibitor (BB94, Batimastat) to the primer of a three-step etch and rinse adhesive system on caries-affected dentin (CaD) MMP activity, and to assess the effect of such an inclusion on the chemical content of the CaD-adhesive interface.nnnMETHODSnCaries-infected dentin (CiD) was excavated selectively from freshly extracted human carious teeth using a chemo-mechanical agent. Each tooth was sectioned into three slabs through the CaD retained cavity. These were treated with either Optibond FL OB (Kerr, Orange, USA) without MMP inhibitor, or with 500 μM BB94 prior to the application of OB primer and bond, or with OB primer that contained 5 μM BB94. In situ zymography and Raman micro-spectroscopy were used to investigate MMP activity and the changes in the chemical content at the CaD/adhesive interface, respectively.nnnRESULTSnData showed the use of OB adhesive with BB94 resulted in immediate interfacial MMP inhibition, by direct application (93.3%) and by means of a drug delivery system (80%), as demonstrated by in situ zymography. Raman imaging revealed 33% higher resin infiltration into MMP-inhibited adhesive interfaces (SE 3.88).nnnSIGNIFICANCEnThrough competitive inhibition by batimastat (BB94), a proportion of the MMPs found in CaD were inhibited immediately and irreversibly. Such a competitive mechanism brings the adhesive primer close to the collagen matrix and enhances the dental adhesive wettability, which is a proposed mechanism to explain the presence of more resin within the hybrid layer.

A technique for placement of apical MTA plugs using modified Thermafil carriers for the filling of canals with wide apices

AIMnTo describe a technique for the placement of apical Mineral trioxide aggregate (MTA) plugs in canals with wide apices.nnnSUMMARYnA novel technique to fill root canals with an apical diameter larger than 0.4xa0mm is presented. The technique includes three main stages; three Thermafil carriers of increasing size, previously de-sheathed by removing the Gutta-percha coating, are selected to engage 1, 2 and 3xa0mm short of the apex. Their use allows the negotiation of acute curvatures and ledged canals. Subsequently, an MTA plug matching the apical gauge is pre-formed with a pellet block, placed and condensed using the modified carriers in sequence. The presented protocol for the management of teeth with apices of a diameter greater than 0.4xa0mm allows a favourable apical control of the MTA. Clinical cases completed using this methodology are presented.nnnKEY LEARNING POINTSnMTA placement in teeth with wide apices was facilitated by using de-sheathed Thermafil carriers, to create an appropriate seal and stable platform for Gutta-percha backfilling or subsequent fibre post placement. The use of de-sheathed Thermafil carriers of different sizes allows predictable placement of pre-formed MTA plugs. Gauging of Thermafil carriers enhances control of the condensation phase to limit the extrusion of MTA.

Antimicrobial potential of bioactive bone cements

Poly(methyl methacrylate) (PMMA) based bone cement is widely used to anchor artificial joints. In recent years, antibiotics have been incorporated in bone cements and administered systemically to either prevent or reduce the severity of infections. Certain antibiotics, such as tobramycin or vancomycin antibiotics have been incorporated at high concentrations into PMMA bone cements when dealing with infected hip joints, however, the inclusion of some antibiotics adversely affect the mechanical properties of the cement. Furthermore, studies have indicated that the incorporation of gentamicin in PMMA cements does not have a statistically significant effect on the biofilm formation of species such as Staphylococcus aureus (S. aureus). Bioactive glasses have been shown to have numerous applications in the biomedical field because of their properties in bonding to both hard and soft tissues. Bioglass(R) undergoes surface dissolution in a physiological environment forming a hydroxycarbonate apatite layer and, in addition, exhibits the anti-inflammatory properties. Recent studies have shown that two paste bioactive bone cement systems that use Bioglass(R) as filler exhibit physical and mechanical properties comparable to PMMA cements with low polymerization exotherm and better mechanical properties with improved adhesion to bone and implant surfaces. In this study, Bioglass(R) containing two paste bone cements were investigated for their potential bacteriostatic properties and compared with PMMA cements with and without antibiotics. The results of this study indicated that the PMMA cement containing the antibiotic, gentamycin and the Bioglass(R) containing cements both pre- and post-immersion in simulated body fluid (SBF) for brief periods showed the inhibition zones were not statistically significantly different in their average size for any of the three bacterial species, namely Pseudomonas aeruginosa, S. aureus and Staphylococcus epidermidis. However, the PMMA cement without any antibiotic, namely gentamycin (CMW1) did not show any inhibition zones around the specimens for any of the three bacterial species. It was also noted that both Bioglass(R) filled bioactive cements immersed in SBF showed statistically significant increases in inhibition zones for all three specimens compared to specimens that were not immersed (p<0.001).

The metalloprotease-disintegrin ADAM8 contributes to temozolomide chemoresistance and enhanced invasiveness of human glioblastoma cells.

BACKGROUNDnDespite multimodal treatment, glioblastoma (GBM) therapy with temozolomide (TMZ) remains inefficient due to chemoresistance. Matrix metalloproteinase (MMP) and a disintegrin and metalloprotease (ADAM), increased in GBM, could contribute to chemoresistance and TMZ-induced recurrence of glioblastoma.nnnMETHODSnTMZ inducibility of metalloproteases was determined in GBM cell lines, primary GBM cells, and tissues from GBM and recurrent GBM. TMZ sensitivity and invasiveness of GBM cells were assessed in the presence of the metalloprotease inhibitors batimastat (BB-94) and marimastat (BB-2516). Metalloprotease-dependent effects of TMZ on mitochondria and pAkt/phosphatidylinositol-3 kinase (PI3K) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) pathways were analyzed by fluorescence activated cell sorting, morphometry, and immunoblotting. Invasiveness of GBM cells was determined by Matrigel invasion assays. Potential metalloprotease substrates were identified by proteomics and tested for invasion using blocking antibodies.nnnRESULTSnTMZ induces expression of MMP-1, -9, -14, and ADAM8 in GBM cells and in recurrent GBM tissues. BB-94, but not BB-2516 (ADAM8-sparing) increased TMZ sensitivity of TMZ-resistant and -nonresistant GBM cells with different O(6)-methylguanine-DNA methyltransferase states, suggesting that ADAM8 mediates chemoresistance, which was confirmed by ADAM8 knockdown, ADAM8 overexpression, or pharmacological inhibition of ADAM8. Levels of pAkt and pERK1/2 were increased in GBM cells and correlated with ADAM8 expression, cell survival, and invasiveness. Soluble hepatocyte growth factor (HGF) R/c-met and CD44 were identified as metalloprotease substrates in TMZ-treated GBM cells. Blocking of HGF R/c-met prevented TMZ-induced invasiveness.nnnCONCLUSIONSnADAM8 causes TMZ resistance in GBM cells by enhancing pAkt/PI3K, pERK1/2, and cleavage of CD44 and HGF R/c-met. Specific ADAM8 inhibition can optimize TMZ chemotherapy of GBM in order to prevent formation of recurrent GBM in patients.

Acrylic bone cement: genesis and evolution

Abstract: This chapter describes the story of modern bone cements and their evolution over the last five decades. The 1960s saw the application of poly(methylmethacrylate) (PMMA)-based cement for the fixation of both the femur and acetabulum in hip replacement surgery ( Charnley, 1960 ). Since then PMMA acrylic bone cement has gained a distinctive place in the domain of synthetic biomaterials although the composition of the cements remains essentially unaltered, newer mixing and dispensing techniques are increasingly being used to improve the performance of the cement. Furthermore, the addition of additives such as antibiotics, fluoride salts and bioactive glass fillers has been researched to enhance the clinical function of the PMMA cement.

Self-Limiting versus Conventional Caries Removal: A Randomized Clinical Trial

A single-blind randomized controlled clinical trial in patients with deep caries and symptoms of reversible pulpitis compared outcomes from a self-limiting excavation protocol using chemomechanical Carisolv gel/operating microscope (self-limiting) versus selective removal to leathery dentin using rotary burs (control). This was followed by pulp protection with mineral trioxide aggregate (MTA) and restoration with glass ionomer cement and resin composite, all in a single visit. The pulp sensibility and periapical health of teeth were assessed after 12 mo, in addition to the differences in bacterial tissue concentration postexcavation. Apical radiolucencies were assessed using cone beam computed tomography/periapical radiographs (CBCT/PAs) taken at baseline 0 mo (M0) and 12 mo (M12). In total, 101 restorations in 86 patients were placed and paired subsurface, and deep (postexcavation) dentin samples were obtained. DNA was extracted and bacteria-specific 16S ribosomal RNA gene quantitative polymerase chain reaction was performed. No significant difference was found in bacterial copy numbers normalized to mass of dentin (“bacterial tissue concentration”) between the self-limiting (96.3% reduction) and control protocols (97.1%, P = 0.33). The probability of 12-mo success was 4 times (odds ratio [OR] = 4.33; confidence interval [CI], 1.2–15.6; P = 0.025) higher in the self-limiting protocol compared to the control (conventional excavation technique), with pulp survival rates of 73.3% and 90%, respectively (P = 0.049). Molars had a 4 times higher probability of success compared to premolars (OR, 4.17; CI, 1.17–14.9; P = 0.028), and symptom severity did not statistically predict outcome (OR, 0.41; CI, 0.12–13.9, P = 0.153). CBCT detected significantly more periapical (PA) lesions than PA radiographs at the baseline visit (P < 0.001). In conclusion, the self-limiting caries excavation protocol under magnification increased pulp survival rate compared to rotary bur excavation (ClinicalTrials.gov NCT03071588).

Bacterial Contamination of Endodontic Materials before and after Clinical Storage

Introduction The aim of this study was to evaluate the bacterial contamination in endodontic consumables (gutta‐percha points, rubber dams, paper mixing pads, caulking agents, and endodontic instrument sponges [EISs]) before and after clinical use and storage. Methods Materials were randomly sampled in triplicates at 3 time points (t0, at package opening; t1, at 7 days; and t2, at 14 days) during their clinical usage. The gutta‐percha points and caulking agent (25 mg) were added to 1 mL phosphate‐buffered saline (PBS). The rubber dam, paper mixing pad, and EIS were added to 25 mL PBS. After vortexing, centrifuging, and removing the supernatant, the pellet was resuspended in 1 mL PBS, plated on fastidious anaerobic agar, and incubated aerobically and anaerobically. The grown colonies were identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The total bacterial load was calculated in the remaining volume (800 &mgr;L) from each sample by quantitative polymerase chain reaction after DNA extraction. Results All tested materials showed a varied number of contaminated samples at the 3 time points (except EIS at t0) using MALDI‐TOF MS. The most isolated genera were Propionibacterium (42%) and Staphylococcus (32%). By using non–culture‐based approaches, all tested materials at the 3 time points (except gutta‐percha at t0 and the caulking agent at t0, t1, and t2) carried bacterial DNA. Conclusions The majority of the tested materials harbored bacteria in their samples before and after clinical storage. Nosocomial infection derived from commonly used consumables could have an impact on the outcome of endodontic treatment. HighlightsConsumables commonly used in endodontics are often in nonsterile packaging.Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectroscopy is a valid technique to identify the contaminants of endodontic materials.Clinical storage can induce environmental contamination.The presence of bacteria in endodontic materials may induce nosocomial infection.Nosocomial infections may induce failure in de novo root canal treatments