Katleen Vandamme

Repurposing as a means to increase the activity of amphotericin B and caspofungin against Candida albicans biofilms

OBJECTIVES Biofilms of Candida species, often formed on medical devices, are generally resistant to currently available antifungal drugs. The aim of this study was to identify compounds that increase the activity of amphotericin B and caspofungin, commonly used antifungal agents, against Candida biofilms. METHODS A library containing off-patent drugs was screened for compounds, termed enhancers, that increase the in vitro activity of amphotericin B against Candida albicans biofilms. Biofilms were grown in 96-well plates and growth was determined by the cell titre blue assay. Synergy between identified enhancers and antifungal agents was further characterized in vitro using fractional inhibitory concentration index (FICI) values and in vivo using a worm biofilm infection model. In light of the application of these enhancers onto implants, their possible effect on the growth potential of MG63 osteoblast-like cells was assessed. RESULTS Pre-incubation of C. albicans biofilms with subinhibitory concentrations of the enhancers drospirenone, perhexiline maleate or toremifene citrate significantly increased the activity of amphotericin B or caspofungin (FICI  < 0.5) against C. albicans and Candida glabrata biofilms. Moreover, these enhancers did not affect the growth potential of osteoblasts. Interestingly, toremifene citrate also enhanced the in vitro activity of caspofungin in a mixed biofilm consisting of C. albicans and Staphylococcus epidermidis. Furthermore, we demonstrate synergy between toremifene citrate and caspofungin in an in vivo worm C. albicans biofilm infection model. CONCLUSIONS Our data demonstrate an in vitro and in vivo enhancement of the antibiofilm activity of caspofungin by toremifene citrate. Furthermore, our results pave the way for implant-related applications of the identified enhancers.

Histological, histomorphometrical, and radiological evaluation of an experimental implant design with a high insertion torque

OBJECTIVES The aim of this study was to compare bone behaviour around an experimental implant design with a high insertion torque with the Astra-Tech implant (control). MATERIALS AND METHODS In ten 18-month-old male minipigs, the last premolars and first molars were extracted to provide space for two implants in each quadrant. A first set of 40 implants were placed 3 months after the extraction and 40 additional implants were installed another 2 months later. The animals were sacrificed 3 months after the first implant installation so that half of the implants had healed for 1 month and the other half for 3 months. Radiological evaluation was performed at baseline, 1 month, 2 months, and 3 months after implant installation. Bone defect depth and area, bone level changes, bone-to-implant contact density, and peri-implant bone fraction were measured histomorphometrically. RESULTS Radiological analyses revealed a significantly higher bone loss around the experimental implants. Histomorphometric analyses confirmed significantly more bone loss, larger marginal bone defects, and a lower overall peri-implant bone fraction around the experimental implants. CONCLUSION The experimental implant design caused significantly more peri-implant bone loss compared with the control implant. As strain gauge measurements indicate excessive marginal strains around the experimental implants, osseocompression might have played a role in the observed marginal bone loss.

The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs.

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation

OBJECTIVES Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Overnight storage of removable dentures in alkaline peroxide-based tablets affects biofilm mass and composition☆

BACKGROUND Clinical guidelines for denture care are available, but evidence for optimal nocturnal storage is scarce. The aim of the study was to compare the role of the overnight storage state on plaque growth and composition on acrylic removable dentures. METHODS In a parallel-group randomized controlled trial of 51 institutionalized participants, 3 denture overnight preservation methods were considered: (i) in water, (ii) dry or (iii) in water with added alkaline peroxide-based cleansing tablet. Biofilm samples were taken on day 7 (developing biofilm - dBF) and day 14 (maturing biofilm - mBF) from a mechanically uncleaned, standardized region, situated distally to the second lower premolars. Total and individual levels of selected perio-pathogenic and commensal species (n=20), and of Candida albicans were calculated by PCR. Differences between storage conditions (water/dry/tablet) and between the samples (dBF/mBF) were assessed by means of unpaired and paired t-tests respectively, with α=5%. RESULTS Overnight denture storage with cleansing tablet significantly decreased the total bacterial level of dBF and mBF up to 13.8%. Fn, Ec, Cs, Sc, Ao and Vp counts were particularly affected by tablet care. Significant lower amounts of Candida albicans for tablet storage compared to water preservation were recorded in dBF and mBF (-69.3 ± 3.8% and -75.9 ± 3.2% respectively). The mass and pathogenicity of dBF and mBF was equal, irrespective of the overnight storage intervention. CONCLUSIONS The use of cleansing tablets for acrylic removable denture overnight storage reduces denture biofilm mass and pathogenicity compared to dry and water preservation, and may contribute to the overall systemic health. CLINICAL SIGNIFICANCE Evidence-based clinical guidelines for overnight storage of removable acrylic dentures are lacking. The findings of this study indicate that alkaline peroxide-based cleansing tablets decrease bacterial and Candida levels in denture biofilms in case of poor oral hygiene. This provides evidence for a clinical guideline to minimize microbial load of dentures, thereby reducing associated systemic health risks.

Effect of insertion torque on titanium implant osseointegration: an animal experimental study

OBJECTIVE To evaluate the effect of implant insertion torque on the peri-implant bone healing and implant osseointegration. MATERIAL AND METHODS Bilaterally in the tibia of five adult New Zealand white rabbits, 20 implants were installed, subdivided into four groups, corresponding to two insertion torque conditions (low, < 10 Ncm vs. high > 50 Ncm) and 2 experimental periods (2 weeks vs. 4 weeks of healing). The implant insertion torque was determined by the surgical drill diameter relative to the implant diameter. Implant osseointegration was evaluated by quantitative histology (bone-to-implant contact with host bone [BIC-host], with neoformed bone [BIC-de novo], with both bone types [BIC-total], and peri-implant bone [BA/TA]). Every response was modelled over time using GEE (general estimation equation) with an unstructured variance-covariance matrix to correct for dependency between the measurements from one animal. The statistical significance level of α = 0.05 was applied. RESULTS Significantly, more BIC-host and BIC-total were recorded for H implants compared with L implants after 2 week of healing (P = 0.010 and P = 0.0001, respectively). However, this result was no longer found for the extended healing period. Furthermore, BIC-total significantly increased over time for L implants (P < 0.00001). In contrast, the significant increase in BA/TA over time was found for H implants (P < 0.01). Finally, H insertion torque led to an increased BA/TA after 4 week of healing (P < 0.02) compared with the L insertion protocol. CONCLUSION L insertion torque implants installed in the rabbit tibial bone osseointegrate with considerable de novo bone formation. This bone neoformation enables L implants to catch up, already during the early osseointegration stage, the initial inferior amount BIC contact compared with that of H implants. A negative impact of the created strain environment accompanying H insertion torque implant installation on the biological process of osseointegration could not be observed, at least not at tissue level.

In vivo assessment of the effect of controlled high- and low-frequency mechanical loading on peri-implant bone healing

The aim of this study was to investigate the effect of controlled high- (HF) and low-frequency (LF) mechanical loading on peri-implant bone healing. Custom-made titanium implants were inserted in both tibiae of 69 adult Wistar rats. For every animal, one implant was loaded by compression through the axis of tibia (test), whereas the other one was unloaded (control). The test implants were randomly distributed among four groups receiving different loading regimes, which were determined by ex vivo calibration. Within the HF (40 Hz) or LF (2 Hz) loading category, the magnitudes were chosen as low- (LM) and high-magnitude (HM), respectively, leading to constant strain rate amplitudes for the two frequency groups. This resulted in the four loading regimes: (i) HF-LM (40 Hz–0.5 N); (ii) HF-HM (40 Hz–1 N); (iii) LF-LM (2 Hz–10 N); and (iv) LF-HM (2 Hz–20 N) loading. Loading was performed five times per week and lasted for one or four weeks. Tissue samples were processed for histology and histomorphometry (bone-to-implant contact, BIC; and peri-implant bone fraction, BF) at the cortical and medullar level. Data were analysed statistically with ANOVA and paired t-tests with the significance level set at 0.05. For the one-week experiments, an increased BF adjacent to the implant surface at the cortical level was exclusively induced by the LF-HM loading regime (2 Hz–20 N). Four weeks of loading resulted in a significant effect on BIC (and not on BF) in case of HF-LM loading (40 Hz–0.5 N) and LF-HM loading (2 Hz–20 N): BIC at the cortical level significantly increased under both loading regimes, whereas BIC at the medullar level was positively influenced only in case of HF-LM loading. Mechanical loading at both HF and LF affects osseointegration and peri-implant BF. Higher loading magnitudes (and accompanying elevated tissue strains) are required under LF loading to provoke a positive peri-implant bone response, compared with HF loading. A sustained period of loading at HF is needed to result in an overall enhanced osseointegration.

Enhancement of implant osseointegration by high-frequency low-magnitude loading.

Background Mechanical loading is known to play an important role in bone remodelling. This study aimed to evaluate the effect of high- and low-frequency axial loading, applied directly to the implant, on peri-implant bone healing and implant osseointegration. Methodology Titanium implants were bilaterally installed in rat tibiae. For every animal, one implant was loaded (test) while the other one was not (control). The test implants were randomly divided into 8 groups according to 4 loading regimes and 2 experimental periods (1 and 4 weeks). The loaded implants were subject to an axial displacement. Within the high- (HF, 40 Hz) or low-frequency (LF, 8 Hz) loading category, the displacements varied 2-fold and were ranked as low- or high-magnitude (LM, HM), respectively. The strain rate amplitudes were kept constant between the two frequency groups. This resulted in the following 4 loading regimes: 1) HF-LM, 40 Hz-8 µm; 2) HF-HM, 40 Hz-16 µm; 3) LF-LM, 8 Hz-41 µm; 4) LF-HM, 8 Hz-82 µm. The tissue samples were processed for resin embedding and subjected to histological and histomorphometrical analyses. Data were analyzed statistically with the significance set at p<0.05. Principal Findings After loading for 4 weeks, HF-LM loading (40 Hz-8 µm) induced more bone-to-implant contact (BIC) at the level of the cortex compared to its unloaded control. No significant effect of the four loading regimes on the peri-implant bone fraction (BF) was found in the 2 experimental periods. Conclusions The stimulatory effect of immediate implant loading on bone-to-implant contact was only observed in case of high-frequency (40 Hz) low-magnitude (8 µm) loading. The applied load regimes failed to influence the peri-implant bone mass.

Novel anti-infective implant substrates: Controlled release of antibiofilm compounds from mesoporous silica-containing macroporous titanium

Bone implants with open porosity enable fast osseointegration, but also present an increased risk of biofilm-associated infections. We design a novel implant material consisting of a mesoporous SiO2 diffusion barrier (pore diameter: 6.4 nm) with controlled drug release functionality integrated in a macroporous Ti load-bearing structure (fully interconnected open porosity: 30%; pore window size: 0.5-2.0 μm). Using an in vitro tool consisting of Ti/SiO2 disks in an insert set-up, through which molecules can diffuse from feed side to release side, a continuous release without initial burst effect of the antibiofilm compound toremifene is sustained for at least 9 days, while release concentrations (up to 17 μM daily) increase with feed concentrations (up to 4mM). Toremifene diffusivity through the SiO2 phase into H2O is estimated around 10(-13)m(2)/s, suggesting configurational diffusion through mesopores. Candida albicans biofilm growth on the toremifene-release side is significantly inhibited, establishing a proof-of-concept for the drug delivery functionality of mesoporous SiO2 incorporated into a high-strength macroporous Ti carrier. Next-generation implants made of this composite material and equipped with an internal reservoir (feed side) can yield long-term controlled release of antibiofilm compounds, effectively treating infections on the implant surface (release side) over a prolonged time.

The impact of autologous platelet concentrates on endodontic healing: a systematic review

Abstract The current literature was reviewed to determine the impact of autologous platelet concentrates (APCs) on endodontic healing. All types of clinical study designs concerning any kind of endodontic treatment involving the application of APCs were included. Two independent reviewers searched three databases (PubMed, Embase, and Cochrane Library) for studies, complemented by hand search, until 16/1/2016. From the 423 identified records, 48 articles met the inclusion criteria. Selected randomized controlled clinical trials (RCTs) underwent Cochrane Collaboration’s risk-of-bias assessment and data extraction. Only two RCTs showed low risk of bias. There was considerable heterogeneity between the RCTs with regard to the type of therapy, type of APCs, assessment method, and study quality, and therefore the data could not be analyzed quantitatively. The included case reports/series and non-randomized comparative studies underwent qualitative analysis with the revised Methodological Index for Non-Randomized Studies (MINORS) and data extraction. The two comparative non-randomized studies scored qualitatively high, though the MINORS-scores of the case series and reports were dispersed. APCs were involved in five endodontic treatment modalities, namely apexification, regenerative endodontic procedures, pulpotomy, apical surgery, and treatment of endo-perio/perio-endo lesions. APCs seem to accelerate postoperative bone healing, augment the patients’ postoperative quality of life, aid further root development, and support maintenance/regaining of pulp vitality. No adverse events were reported. APCs in endodontic treatments seem to contribute to the healing of soft and hard tissues, though there is a lack of long-term high quality clinical trials and standardized treatment protocols.