Digital follow-up by means of dermatoscopy and reflectance confocal microscopy of actinic keratosis treated with Imiquimod 3.75% cream.

BACKGROUND: Imiquimod 3.75% cream (Zyclara® Meda, Stockholm, Sweden) is a new field-directed therapy for actinic keratosis (AK). OBJECTIVES: The aim is to evaluate efficacy and the morphologic dynamic changes induced by this treatment by means of dermatoscopy and reflectance confocal microscopy (RCM) of imiquimod 3.75% cream for the treatment of AKs of the face or scalp and to evaluate. METHODS: Thirty-two patients were treated with Imiquimod 3.75% cream. Demographic parameters, AK-FAS and AKASI scores and side-effects were collected. RCM and dermatoscopy on one target AKs were performed at each visit. We collected images at baseline (T0), after 1 week from the end of the first 2-week cycle (T1), after 1 week from the end of the entire treatment (T2) and 2 months after the end of treatment (T3). RESULTS: One target representative AK in the selected area of treatment of each patient was analysed. All dermoscopic and confocal parameters were reduced 2 months after the end of the therapy (T3) with a substantial reduction of AKASI and AK-FAS scores, and 17 cases (54.8%) were completely solved. Confocal microscopic analysis showed a reduction of keratinocytes disarray in 77.4% of cases; none showed crusts and parakeratosis. Inflammation was considerably decreased and was observed only in 12.9% of patients at the last visit. This improvement was not assessed on dermatoscopy because of inflammation and background erythema, which adversely influenced the assessments. LSRs were observed in almost all the patients during treatment being more severe after the first cycle of treatment (T1). CONCLUSIONS: Imiquimod 3.75% cream is effective in treating clinical and subclinical AKs with an easy management of side-effects. Dermatoscopy and mostly RCM allow non-invasive monitoring of treatment response in vivo.

In situ sol-gel synthesis of hyaluronan derivatives bio-nanocomposite hydrogels.

The main driving idea of the present study was the comparison between two different chemical modifications of hyaluronic acid (HA) followed by the development of nanocomposite hydrogels directly in situ by biomineralization of photocrosslinkable HA polymers through sol-gel synthesis. In this way, it has been possible to overcome some limitations due to classical approaches based on the physical blending of inorganic fillers into polymer matrix. To this aim, methacrylated and maleated HA, synthesized with similar degree of substitution (DS) were compared in terms of mechanical and physico-chemical properties. The success of in situ biomineralization was highlighted by reflect Fourier transform infrared spectroscopy and thermogravimetric analysis. Furthermore, mechanical characterization demonstrated the reinforcing effect of inorganic fillers evidencing a strong correlation with DS. The swelling behavior resulted to be correlated with filler concentration. Finally, the cytotoxicity tests revealed the absence of toxic components and an increase of cell proliferation over culture time was observed, highlighting these bio-nanocomposite hyaluronan derivatives as biocompatible hydrogel with tunable properties.

Diagnostic accuracy of ex vivo fluorescence confocal microscopy in Mohs surgery of basal cell carcinomas: a prospective study on 753 margins.

BACKGROUND: Frozen histological sections are used for intraoperative margin assessment during Mohs surgery. Fluorescence confocal microscopy (FCM) is a new tool that offers a promising and faster alternative to frozen histology. OBJECTIVES: To evaluate prospectively in a clinical setting the accuracy of FCM vs. frozen sections in margin assessment of basal cell carcinoma (BCC). METHODS: Patients with BCC scheduled for Mohs surgery were prospectively enrolled. Freshly excised surgical specimens were examined by FCM and then frozen sections were evaluated. Permanent sections were obtained, in order to validate the sample technique. A blind re-evaluation was also performed for discordant cases. Sensitivity and specificity levels, as well as positive and negative predictive values (PPV and NPV, respectively), were calculated and receiver-operating characteristic curves generated. RESULTS: We enrolled 127 BCCs in as many patients (40·2% females). Seven hundred and fifty-three sections were examined. All BCCs were located in the head and neck area. In evaluating the performance of FCM vs. frozen sections, sensitivity was 79·8%, specificity was 95·8%, PPV was 80·5% and NPV was 95·7% [area under the curve 0·88, 95% confidence interval 0·84-0·92 (P < 0·001)]. Forty-nine discordant cases were re-evaluated; 24 were false positive and 25 false negative. The performance of FCM and frozen sections was also evaluated according to the final histopathological assessment. CONCLUSIONS: We found high levels of accuracy for FCM vs. frozen section evaluation in intraoperative BCC margin assessment during Mohs surgery. Some technical issues prevent the wide use of this technique, but new devices promise to overcome these limitations.

Basal cell carcinoma: the utility of in vivo and ex vivo confocal microscopy.

The use of confocal microscopy is possible using two different modalities: first, at patient's bedside for a rapid in vivo diagnosis of basal cell carcinoma and second, in the operating room directly on freshly excised specimen for a fast ex vivo margin-controlled surgery. In the current review, we report the main application of confocal microscopy for basal cell carcinoma diagnosis and management in both modalities.

Tracking actinic keratosis of face and scalp treated with 0.015% ingenol mebutate to identify clinical and dermoscopic predictors of treatment response.

BACKGROUND: Ingenol mebutate (IngMeb) 0.015% gel is an approved field treatment option for non-hyperkeratotic non-hypertrophic actinic keratosis (AK) of face and scalp. Efficacy of IngMeb has been assessed only on a clinical ground, in the majority of studies. Dermoscopy is a pivotal tool for the diagnosis of AK, while its role in evaluating the response to non-surgical therapies for AK has not been fully defined. OBJECTIVES: Our study aims to determine whether some dermoscopic features of AK of the face and scalp areas may independently predict the response to IngMeb therapy. METHODS: Clinical and dermoscopic responses, 1 month after 0.015% IngMeb therapy, were retrospectively evaluated using a per-patient and per-lesion approach. Safety was evaluated through local skin reaction composite score calculation. Demographic, clinical and dermoscopic factors were then evaluated via univariate and multivariate logistic regression analysis to assess independent predictors of response. RESULTS: Fifty-five patients with 245 AKs were enrolled. Clinically, per-patient response evaluation identified 25 (45.4%) poor/partial and 30 (54.5%) complete responders, corresponding on a per-lesion approach to 66 (26.9%) and 179 (73.1%) AKs, respectively. Dermoscopy reclassified 14 patients in the per-patient and 48 AKs in the per-lesion analysis from complete to poor/partial responders. Multivariate logistic regression analysis showed that AKs dermoscopically characterized by red pseudonetwork and located on the face were independently associated with a complete dermoscopic response to 0.015% IngMeb therapy, while microerosions were negative predictors. CONCLUSION: Specific dermoscopic features of AK may predict the response to 0.015% IngMeb therapy, together with the location on the face.

Dermoscopic features predicting the presence of mitoses in thin melanoma.

BACKGROUND: The latest AJCC classification has included the number of mitoses as a factor for upstaging thin melanomas. Meanwhile, while dermoscopy has often been used to predict melanoma thickness, its value in predicting number of mitoses remains unknown. OBJECTIVE: Our aim is to evaluate the correlation between dermoscopic features and the presence of mitoses in a consecutive cohort of thin melanomas. METHODS: A case control study has been performed to identify specific dermoscopic parameters that could differentiate thin melanomas with 1 or more mitoses per mm2 from those without mitoses. RESULTS: Of 177 melanomas equal to or thinner than 1mm, 131 (74%) lesions had no mitoses and 46 (36%) lesions had at least 1 mitosis×mm2. Dermoscopic features associated with the presence of 1 or more mitoses were the following: peripheral streaks (OR 4.11; 95% CI 1.94-8.71) and black colour (OR 4.70; 95% CI; 2.28-9.68). In contrast, atypical pigment network (OR (0.30; 95% CI 0.15-0.61)) and brown colour (OR 0.36; 95% CI 0.18-0.75) were associated to melanomas without mitoses. The same variables were also associated to the increasing number of mitoses at linear regression. CONCLUSION: Black colour and peripheral streaks can predict the presence of mitoses in thin melanoma, while atypical pigment network and brown colour are associated to thin melanoma without mitoses.

Comparative facile methods for preparing graphene oxide-hydroxyapatite for bone tissue engineering.

Motivated by the success of using graphene oxide (GO) as a nanofiller of composites, there is a drive to search for this new kind of carbon material as a bioactive component in ceramic materials. In the present study, biomineralized GO was prepared by two different approaches, represented by in situ sol-gel synthesis and biomimetic treatment. It was found that in the biocomposites obtained by the sol-gel approach, the spindle-like hydroxyapatite nanoparticles, with a diameter of ca. 5 ± 0.37 nm and a length of ca. 70 ± 2.5 nm, were presented randomly and strongly on the surface. The oxygen-containing functional groups, such as hydroxyl and carbonyl, present on the basal plane and edges of the GO sheets, play an important role in anchoring calcium ions, as demonstrated by FT-IR and TEM investigations. A different result was obtained for biocomposites after biomimetic treatment: an amorphous calcium phosphate on GO sheet was observed after 5 days of treatment. These different approaches resulted in a diverse effect on the proliferation and differentiation of osteogenic mesenchymal stem cells. In fact, in biocomposites prepared by the sol-gel approach the expression of an early marker of osteogenic differentiation, ALP, increases with the amount of GO in the first days of cell culture. Meanwhile, biomimetic materials sustain cell viability and proliferation, even if the expression of alkaline phosphatase activity in a basal medium is delayed. These findings may provide new prospects for utilizing GO-based hydroxyapatite biocomposites in bone repair, bone augmentation and coating of biomedical implants and broaden the application of GO sheets in biological areas. Copyright © 2016 John Wiley & Sons, Ltd.

Both short-term and long-term dermoscopy monitoring is useful in detecting melanoma in patients with multiple atypical nevi.

BACKGROUND: Digital dermoscopy monitoring (DDM) is an effective strategy for melanoma detection. Two methods are currently employed. Short-term follow-up (STFU) for the evaluation of single, atypical lesions to detect subtle changes over a short period of time (3-6 months). Long-term follow-up (LTFU) is recommended for patients with multiple nevi. Although a study demonstrated that STFU improves the patients' compliance for DDM, little remains known about the impact and reliability of STFU in this setting. OBJECTIVES: The aim of this retrospective, observational study was to evaluate the impact and reliability of a schedule combining STFU and LTFU in patients with multiple atypical nevi. METHODS: We searched our database for all cases of patients with multiple atypical nevi occurring between 2006 and 2014. RESULTS: A total of 3823 lesions in 541 patients were dermoscopically monitored (mean number = 7 lesions per patient; median = 6 lesions; range, 2-51). In all, 264 (6.9%) lesions in 184 (34.4%) patients were excised (mean of 0.5 lesions per patient). In total, 197 (74.6%) lesions were excised at follow-up, with melanomas representing 30.5% of lesions excised after follow-up. A total of 30 (33.3%) melanomas were excised at baseline, 23 (25.6%) after STFU and 37 (41.1%) after LTFU. There was no difference in the number of in situ melanomas detected at baseline with those detected after follow-up. The mean Breslow thickness of melanomas detected at baseline was higher than those detected after STFU (P = 0.038) and LTFU (P = 0.055). CONCLUSIONS: Our study confirm that digital dermoscopy follow-up is a valid management strategy for patients with multiple atypical nevi, with short-term monitoring playing an effective role also in this setting of patients.

Properties of carbon nanotube-dispersed Sr-hydroxyapatite injectable material for bone defects.

This study concerns the synthesis of gel materials based on carbon nanotubes dispersed strontium-modified hydroxyapatite (Sr-HA) at different compositions obtained by sol-gel technology and their influence on human-bone-marrow-derived mesenchymal stem cells. Furthermore, an evaluation of the influence of nanotubes and Strontium on physico-chemical, morphological, rheological and biological properties of hydroxyapatite gel was also performed. Morphological analysis (scanning electron microscopy) shows a homogeneous distribution of modified nanotubes in the ceramic matrix improving the bioactive properties of materials. The biological investigations proved that Sr-HA/carbon nanotube gel containing 0-20 mol (%) of Sr showed no toxic effect and promote the expression of early and late markers of osteogenic differentiation in cell culture performed in basal medium without osteogenic factors. Finally, the SrHA/carbon nanotube gels could have a good potential application as filler in bone repair and regeneration and may be used in the osteoporotic disease treatment.

Reflectance confocal microscopy in the diagnosis of solitary pink skin tumours: review of diagnostic clues.

Reflectance confocal microscopy (RCM) is a noninvasive tool that can be helpful in the diagnosis of nonpigmented skin tumours. As RCM enables visualization of architectural and cytological structures at near-histological resolution, it can improve the diagnostic accuracy of dermoscopically equivocal solitary pink neoplasms. For management decisions, it is important to identify specific morphological clues that allow bedside classification of nonpigmented skin neoplasms into benign vs. malignant and melanocytic vs. nonmelanocytic. More specifically, the presence of a nested melanocytic proliferation at the dermoepidermal junction or dermis level permits the clinician to ascribe a given lesion as melanocytic; the identification of basaloid bright tumour islands is a key RCM feature for the diagnosis of basal cell carcinoma; and the presence of disarrayed epidermis along with small demarcated papillae is suggestive for the diagnosis of squamous cell carcinoma. The present review offers a comprehensive description of the main RCM diagnostic clues for solitary pink neoplasms that direct clinicians to the correct diagnosis and that may serve as groundwork for future prospective studies.

A novel CYLD germline mutation in Brooke-Spiegler syndrome.

BACKGROUND: Brooke-Spiegler syndrome (BSS) is a rare, inherited, autosomal dominant disorder characterized by the development of multiple adnexal neoplasms including spiradenomas, cylindromas, trichoepitheliomas and major and minor salivary glands neoplasms. This syndrome encompasses a wide variability of clinical phenotypes depending on the variable number of tumours present in the given patient. OBJECTIVE: Somatic mutations in adjunct to CYLD germline mutations may play a central role in the development of the tumour phenotype and in the genotype-phenotype correlations. METHODS: Blood sample and paraffin embedded tissue biopsied from three cylindromas, one trichoepithelioma and one spiradenomas were collected after obtaining informed consent from our patient and genomic DNA was isolated. RESULTS: We found out a novel germline mutation in the CYLD gene in exon 15 that resulted in the deletion of one nucleotide. This gives rise to a premature translational termination codon at amino acid position 693 prior to four Cys-X-X-Cys pairs and one of the two catalytic domains of ubiquitin carboxy-terminal hydrolases. In only one cylindroma we detected the same germline mutation (c.2070delT/p.F690FfsX3) in addition to two somatic events (I645V and R936X). The presence of this unique mutation could be linked to the peculiar phenotype of our patient who presented an attenuated form of BSS, an autosomal dominant inheritance with low penetrance and no additional visceral tumours. CONCLUSIONS: The overall phenotype of our patient may support the hypothesis that somatic mutations in adjunct to CYLD germline mutations may play a central role in the development of the tumour phenotype and in the genotype-phenotype correlations.

Effect of citric acid crosslinking cellulose-based hydrogels on osteogenic differentiation.

Understanding the relationships between material surface properties and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, cellulose hydrogels were crosslinked using a non-toxic and natural component namely citric acid. The chemical treatment induces COOH functional groups that improve the hydrophilicity, roughness, and materials rheological properties. The physiochemical, morphological, and mechanical analyses were performed to analyze the material surface before and after crosslinking. This approach would help determine if the effect of chemical treatment on cellulose hydrogel improves the hydrophilicity, roughness, and rheological properties of the scaffold. In this study, it was demonstrated that the biological responses of human mesenchymal stem cell with regard to cell adhesion, proliferation, and differentiation were influenced in vitro by changing the surface chemistry and roughness.

Heat treatment of Na2O-CaO-P2O5-SiO2 bioactive glasses: densification processes and postsintering bioactivity.

Because of their excellent bioactivity, bioactive glasses are increasingly diffused to produce biomedical devices for bone prostheses, to face the dysfunctions that may be caused by traumatic events, diseases, or even natural aging. However, several processing routes, such as the production of scaffolds or the deposition of coatings, include a thermal treatment to apply or sinter the glass. The exposure to high temperature may induce a devetrification phenomenon, altering the properties and, in particular, the bioactivity of the glass. The present contribution offers an overview of the thermal behavior and properties of two glasses belonging to the Na2O-CaO-P2O5-SiO2 system, to be compared to the standard 45S5 Bioglass(®). The basic goal is to understand the effect of both the original composition and the thermal treatment on the performance of the sintered glasses. The new glasses, the one (BG_Na) with a high content of Na2O, the other (BG_Ca) with a high content of CaO, were fully characterized and sintering tests were performed to define the most interesting firing cycles. The sintered samples, treated at 880°C and 800°C respectively, were investigated from a microstructural point of view and their mechanical properties were compared to those of the bulk (not sintered) glass counterparts. The effect of sintering was especially striking on the BG_Ca material, whose Vickers hardness increased from 598.9 ± 46.7 HV to 1053.4 ± 35.0 HV. The in vitro tests confirmed the ability of the glasses, both in bulk and sintered form, of generating a hydroxyapatite surface layer when immersed in a simulated body fluid. More accurate biological tests performed on the sintered glasses proved the high bioactivity of the CaO-rich composition even after a heat treatment.

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration.

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.

Sol-gel synthesis, characterization and bioactivity of poly(ether-imide)/TiO2 hybrid materials.

Novel organic-inorganic hybrid materials were synthesized by the sol-gel method from a multicomponent solution containing titanium butoxide, 6 weight % (wt%) or 12 wt% poly(ether-imide) (PEI), water and chloroform. The structure of the interpenetrating network is realized by hydrogen bonds between the Ti-OH group (H-donator) in the sol-gel intermediate species and the carboxylic group (H-acceptor) in the repeating units of the polymer. By Fourier transform infrared (FTIR) analysis the presence of hydrogen bonds between organic-inorganic components of the hybrid materials were proved. The morphology of the hybrid materials was studied by scanning electron microscopy (SEM). The structure of a molecular level dispersion was disclosed by an atomic force microscope (AFM), pore size distribution and surface measurements. The AFM and SEM analyzes confirmed that the PEI/TiO2 samples can be considered homogenous organic/inorganic hybrid materials because in both the compositions studied the average domains were less than 400 nm in size. The bioactivity of the synthesized hybrid materials was demonstrated by the formation of a layer of hydroxyapatite on the surface of the PEI/TiO2 samples soaked in a fluid simulating the composition of human blood plasma (SBF), demonstrated by SEM and energy dispersive spectroscopy (EDS) microscopy.

Characterization, bioactivity and ampicillin release kinetics of TiO2 and TiO24SiO2 synthesized by sol-gel processing.

Local drug delivery of antimicrobics by sustained release delivery system can be used to treat periodontal disease. Advantages of these systems may include maintaining high levels of antibiotic in the gingival crevicular fluid for a sustained period of time and ease of use with high patient acceptance. The materials used are TiO(2) and TiO(2)4SiO(2), mixed with sodium ampicillin, a broad-spectrum antibiotic, have been synthesized by sol-gel method. The amorphous nature of the gels was ascertained by X-ray diffraction analysis. Release kinetics in a simulated body fluid (SBF) have been subsequently investigated. The amount of sodium ampicillin released has been detected by UV-VIS spectroscopy and SEM. The release kinetics seems to occur in more than one stage. HPLC analysis has also been taken to ensure the integrity of ampicillin after the synthetic treatment. Finally, SEM micrographs and EDS analysis showed the formation of a hydroxyapatite layer on the surface of the samples soaked in SBF. Both the materials showed good release and could be used as drug delivery bioactive systems. High antimicrobial effects of samples against Escherichia coli and Streptococcus mutants were found.

Release kinetics of ampicillin, characterization and bioactivity of TiO2/PCL hybrid materials synthesized by sol-gel processing.

Poly(epsilon-caprolactone) (PCL 6, 12, and 24 wt %) and titanium (TiO2) organic-inorganic hybrid materials have been synthesized by the sol-gel method from a multicomponent solution containing titanium butoxide, poly(epsilon-caprolactone) (PCL), water, and chloroform (CHCl3). Sodium ampicillin was incorporated in the hybrid material to verify the effect as local controlled drug delivery system. The structure of a hybrid materials interpenetrating network is realized by hydrogen bonds between Ti-OH group (H-donator) in the sol-gel intermediate species and carboxylic group (H-acceptor) in the repeating units of the polymer. The presence of hydrogen bonds between organic/inorganic components of the hybrid materials was proved by FTIR analysis. The morphology of the hybrid materials was studied by scanning electron microscope (SEM). The structure of a molecular level dispersion has been disclosed by atomic force microscope (AFM), pore size distribution and surface measurements. The bioactivity of the synthesized hybrid materials has been showed by the formation of a layer of hydroxyapatite on the surface of TiO2/PCL samples soaked in a fluid simulating the composition of the human blood plasma. The amount of sodium ampicillin released has been detected by UV-vis spectroscopy and SEM. The release kinetics seems to occur in more than one stage. HPLC analysis has also been taken to ensure the integrity of ampicillin after the synthetic treatment.

Sol-gel processing of drug delivery materials and release kinetics.

Silica, calcium (5 mol%) silicate and silica/polycaprolactone hybrid inorganic/organic amorphous materials, all mixed with sodium ampicillin, a broad-spectrum antibiotic, have been synthesized by sol-gel method. The amorphous nature of the gels was ascertained by X-ray diffraction analysis. Release kinetics in a simulate body fluid (SBF) have been subsequently investigated. The amount of sodium ampicillin released has been detected by UV-Vis spectroscopy and SEM. The release kinetics seems to occur in more than one stage. Finally FTIR measurements and SEM micrograph showed the formation of a hydroxyapatite layer on the surface of the samples soaked in SBF. All data showed that these materials could be used as drug delivery bioactive systems.

Sol-gel synthesis, structure and bioactivity of polycaprolactone/CaO . SiO2 hybrid material.

A method has been developed to cast novel organic/inorganic polymer hybrids from multicomponent solutions containing tetramethyl orthosilicate, calcium nitrate tetrahydrate, polycaprolactone, water, and methylethyketone via sol-gel process. The existence of the hydrogen bonds between organic and inorganic components of the hybrid and hydroxyapatite formation on the surface was proved by Fourier transform infrared analysis. The morphology of the hybrid material was studied by scanning electron microscopy. The structure of a molecular level dispersion was disclosed by atomic force microscopy, pore size distribution, and surface measurements. The infrared spectra of the hybrid relative to sample soaked in a fluid simulating the composition of human blood plasma suggests that polycaprolactone/CaO * SiO(2) hybrid material synthesised via sol-gel process is bioactive as well as the CaO * SiO(2) gel glass.

Antibacterial and bioactive silver-containing Na2O x CaO x 2SiO2 glass prepared by sol-gel method.

The antibacterial effect of addition of silver oxide to Na2O x CaO x 2SiO2 glass have been studied. Silver containing and silver free Na2O x CaO x 2SiO2 glasses have been prepared by sol-gel synthesis using tetramethil orthosilicate, sodium ethoxide, calcium nitrate tetrahydrate and silver nitrate as starting materials and methyl ethyl ketone as solvent. The gel was examined by differential thermal analysis, thermo gravimetric analysis, FTIR spectroscopy and X-ray diffraction analysis. Antibacterial and bioactive tests on gel glass powders, obtained after a heat treatment of 2 h at 600 degrees C of the dried gel, were carried out. High antimicrobial effects of samples against Escherichia coli and Streptococcus mutans were found. FTIR measurements and SEM micrographs have ascertained the formation of a hydroxyapatite layer on the surface of samples soaked in a simulated body fluid for different times.