Influence of different drilling protocols and bone density on the insertion torque of dental implants.

BACKGROUND: The insertion torque of dental implants will depend on a combination of different factors such as bone density, the design of the implant and the drilling protocol used. However, it is not clear how the interaction of these factors affects the final insertion torque and which drilling protocol should be used in each clinical situation. The aim of this work is to analyse the influence of bone density, implant diameter and implant length on the insertion torque using different drilling protocols. MATERIAL AND METHODS: An experimental study was carried out in which the maximum insertion torque was measured, in standardised polyurethane blocks (Sawbones Europe AB) of four densities, for M12 Oxtein dental implants (Oxtein, Spain) with diameters of 3.5, 4.0, 4.5 and 5mm, and lengths of 8.5mm, 11.5mm and 14.5mm. All these measurements were carried out following four drilling protocols, a standard protocol, adding a bone tap, cortical drill or conical drill. In this way, a total of 576 samples were obtained. For the statistical analysis, the table of confidence intervals, mean, standard deviation and covariance was carried out, in total and grouped by the parameters used. RESULTS: The insertion torque for D1 bone obtained very high levels, reaching 77 6.95 N/cm, these values improved with the use of conical drills. In D2 bone, the mean torque obtained was 37.89± 13.70N/cm, with values within the standard. In D3 and D4 bone significantly low torques were obtained with values of 14.97± 4.40N/cm and 9.88± 4.16N/cm (p>0.001) respectively. CONCLUSIONS: In D1 bone, conical drills must be incorporated in drilling to avoid excessive torque, while in D3 and D4 bone, these would be contraindicated, as they drastically decrease the insertion torque, which may compromise the treatment.

Modification of physico-chemical surface properties and growth of Staphylococcus aureus under hyperglycemia and ketoacidosis conditions.

Diabetes is a widely spread disease affecting the quality of life of millions of people around the world and is associated to a higher risk of developing infections in different parts of the body. The reasons why diabetes enhances infection episodes are not entirely clear; in this study our aim was to explore the changes that one of the most frequently pathogenic bacteria undergoes when exposed to hyperglycemia and ketoacidosis conditions. Physical surface properties such as hydrophobicity and surface electrical charge are related to bacterial growth behavior and the ability of Staphylococcus aureus to form biofilms. The addition of glucose made bacteria more negatively charged and with moderate-intermediate hydrophobicity. Ketone bodies increased hydrophobicity to approximately 75% and pathological concentrations hindered some of the bacterial surface charge by decreasing the negative zeta potential of cells. When both components were present, the bacterial physical surface changes were more similar to those observed in ketone bodies, suggesting a preferential adsorption of ketone bodies over glucose because of the more favorable solubility of glucose in water. Glucose diabetic concentrations gave the highest number of bacteria in the stationary phase of growth and provoked an increase in the biofilm slime index of around 400% in relation to the control state. Also, this situation is related with an increase of bacterial coverage. The combination of a high concentration of glucose and ketone bodies, which corresponds to a poorly controlled diabetic situation, appears associated with an early infection phase; increased hydrophobic attractive force and reduced electrostatic repulsion between cells results in better packing of cells within the biofilm and more efficient retention to the host surface. Knowledge of bacterial response in high amount of glucose and ketoacidosis environments can serve as a basis for designing strategies to prevent bacterial adhesion, biofilm formation and, consequently, the development of infections.

Characterization of Magnesium-Polylactic Acid Films Casted on Different Substrates and Doped with Diverse Amounts of CTAB.

Polylactic acid (PLA) is a good candidate for the manufacture of polymeric biodegradable biomaterials. The inclusion of metallic particles and surfactants solves its mechanical limitations and improves its wettability, respectively. In this work, cetyltrimethylammonium bromide (CTAB) and magnesium particles have been incorporated into PLA films to evaluate the changes produced in the polymeric matrix cast on glass and silicone substrates. For this purpose, the surface of the films has been characterized by means of contact angle measurements and ToF-SIMS. Depth profiles and SEM images of the cross sections of the films have also been obtained to study their morphology. The results show that the CTAB in the polymer matrix with and without magnesium improves the wettability of the films, making them more suitable for cell adhesion. The higher the hydrophilicity, the higher the surfactant concentration. The depth profiles show, for the first time, that, depending on the surfactant concentration and the presence of Mg, there is a layer-like distribution near the surface where, in addition to the CTAB + PLA mixture, a surfactant exclusion zone can be seen. This new structure could be relevant in in vitro/in vivo situations when the degradation processes remove the film components in a sequential form.

3D-PLA-experimental set up to display the electrical background of the so-called geometric factor of electrokinetic cells.

The so-called geometric factor defined in electrokinetic cells, L/S (L being the length and S the cross-section of the channel), is relevant for providing the surface interaction electrical potential (zeta potential, ζ) of large surfaces, such as those used in the design of biomedical devices or water purification systems. Conversely, recent studies demonstrate that this factor is also employed to determine geometrical parameters, such as porosity in membrane-like systems. This factor, which has been attributed exclusively a geometrical character, can also be obtained from the electrical conductivity and resistance of the electrokinetic channel. In this work, we assess whether these two ways of obtaining the L/S factor are equivalent and how possible deviations can affect the value of the zeta potential. For this purpose, we work with channels of different geometries obtained by 3D printing using PLA (polylactic acid) as a polymer employed in biomedical applications. The discrepancies between the L/S factor obtained by electrical and purely geometrical measurements increase as the geometrical L/S factor becomes larger, reaching differences close to 80%. The results show that the so-called geometrical L/S factor also has an important electrical contribution and would be better denoted as electrogeometric factor. The differences found between the L/S factors are also propagated to the calculation of ζ but an optimum conductivity zone (from about 10 to 40 mS m-1) can be defined to obtain the zeta potential by selecting any of the L/S factors obtained from electrokinetic measurements. The results of this work should be taken into account in those investigations that use the L/S factor to obtain the geometry-porosity of permeable materials.

Structure, morphology, adhesion and in vitro biological evaluation of antibacterial multi-layer HA-Ag/SiO2/TiN/Ti coatings obtained by RF magnetron sputtering for biomedical applications.

Biocompatible and antibacterial multi-layer coatings of hydroxyapatite (HA)-Ag/SiO2/TiN/Ti were obtained on the Ti-6Al-4V alloy, by means of the magnetron sputtering technique. During characterization of the coatings, the chemical composition was evaluated by energy dispersive X-ray spectroscopy and the phase analysis was carried out by X-ray diffraction. The morphology of the coatings was observed by field emission scanning electron microscopy, while transmission electron microscopy was used to appreciate their structure. The adhesion of the coatings to the substrate was evaluated by micro scratch test. The in vitro biological response was evaluated in terms of cytotoxicity, adhesion and differentiation of mouse mesenchymal stem cells, as well as adhesion and bacterial viability of Staphylococcus aureus strain. Through the compositional study carried out, the deposition of the HA phase was verified, with a Ca/P ratio close to 1.67 and the characteristic diffraction peaks of this compound. The structural study of the coatings evidenced the obtention of multi-layer architectures. The use of an intermediate SiO2/TiN/Ti trilayer was found to improve adhesion between HA-Ag and the substrate by 84%. Finally, the in vitro biological tests carried out indicated a potentially non-toxic character in the coatings. Additionally, an antibacterial effect was registered at low concentrations of Ag (<0.25 mg/L).

The role of magnesium in biomaterials related infections.

Magnesium is currently increasing interest in the field of biomaterials. An extensive bibliography on this material in the last two decades arises from its potential for the development of biodegradable implants. In addition, many researches, motivated by this progress, have analyzed the performance of magnesium in both in vitro and in vivo assays with gram-positive and gram-negative bacteria in a very broad range of conditions. This review explores the extensive literature in recent years on magnesium in biomaterials-related infections, and discusses the mechanisms of the Mg action on bacteria, as well as the competition of Mg2+ and/or synergy with other divalent cations in this subject.

Impact of PLA/Mg films degradation on surface physical properties and biofilm survival.

New biocompatible and bioabsorbable materials are currently being developed for bone regeneration. These serve as scaffolding for controlled drug release and prevent bacterial infections. Films of polylactic acid (PLA) polymers that are Mg-reinforced have demonstrated they have suitable properties and bioactive behavior for promoting the osseointegration process. However little attention has been paid to studying whether the degradation process can alter the adhesive physical properties of the biodegradable film and whether this can modify the biofilm formation capacity of pathogens. Moreover, considering that the concentration of Mg and other corrosion products may not be constant during the degradation process, the question that arises is whether these changes can have negative consequences in terms of the bacterial colonization of surfaces. Bacteria are able to react differently to the same compound, depending on its concentration in the medium and can even become stronger when threatened. In this context, physical surface parameters such as hydrophobicity, surface tension and zeta potential of PLA films reinforced with 10% Mg have been determined before and after degradation, as well as the biofilm formation capacity of Staphylococcus epidermidis. The addition of Mg to the films makes them less hydrophobic and the degradation also reduces the hydrophobicity and increases the negative charge of the surface, especially over long periods of time. Early biofilm formation at 8 h is consistent with the physical properties of the films, where we can observe a reduction in the bacterial biofilm formation. However, after 24 h of incubation, the biofilm formation increases significantly on the PLA/Mg films with respect to PLA control. The explosive release of Mg ions and other corrosion products within the first hours were not enough to prevent a greater biofilm formation after this initial time. Consequently, the Mg addition to the polymer matrix had a bacteriostatic effect but not a bactericidal one. Future works should aim to optimize the design and biofunctionality of these promising bioabsorbable composites for a degradation period suitable for the intended application.

Relevance of Topographic Parameters on the Adhesion and Proliferation of Human Gingival Fibroblasts and Oral Bacterial Strains.

Dental implantology allows replacement of failing teeth providing the patient with a general improvement of health. Unfortunately not all reconstructions succeed, as a consequence of the development of infections of bacterial origin on the implant surface. Surface topography is known to modulate a differential response to bacterial and mammalian cells but topographical measurements are often limited to vertical parameters. In this work we have extended the topographical measurements also to lateral and hybrid parameters of the five most representative implant and prosthetic component surfaces and correlated the results with bacterial and mammalian cell adhesion and proliferation outcomes. Primary human oral gingival fibroblast (gum cells) and the bacterial strains: Streptococcus mutans, Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans, implicated in infectious processes in the oral/implant environment were employed in the presence or absence of human saliva. The results confirm that even though not all the measured surface is available for bacteria to adhere, the overall race for the surface between cells and bacteria is more favourable to the smoother surfaces (nitrided, as machined or lightly acid etched) than to the rougher ones (strong acid etched or sandblasted/acid etched).

Analysis of marginal bone loss and implant stability quotient by resonance frequency analysis in different osteointegrated implant systems. Randomized prospective clinical trial.

BACKGROUND: The aim of the present prospective clinical study is to compare the stability of the implant-bone interface by the ISQ quotient and marginal bone loss (MBL) rate during one year of follow-up in four system implants with the same surface and different design. MATERIAL AND METHODS: Prospective randomized clinical trial of 21 patients in which four implant systems with the same surface and different design were placed. Patients were treated by the same operator following a similar surgical protocol with submerged technique. The second surgery to perform the prosthesis was performed at 3 months. All patients went to their review at 6 months and a year. A periapical radiograph for crestal bone analysis and an Implant stability quotient by resonance frequency analysis (ISQ) analysis were taken at baseline and the reviews. RESULTS: No statistically significant differences were found in the Implant stability quotient by resonance frequency analysis and Marginal Bone Loss in the four types of implants. The ISQ increased from the moment of insertion of the implant until the revision to the year, showing an increase of the stability implant, being this increasing less between the 6 months and the year. CONCLUSIONS: Differences in the design of the four implants tested in this study did not show statistically significant differences in any of the variables studied, so the implant design does not influence implant stability and marginal bone loss in the first year after placement.

Kinetic of Adhesion of S. epidermidis with Different EPS Production on Ti6Al4V Surfaces.

Controlling initial bacterial adhesion is essential to prevent biofilm formation and implant-related infection. The search for surface coatings that prevent initial adhesion is a powerful strategy to obtain implants that are more resistant to infection. Tracking the progression of adhesion on surfaces from the beginning of the interaction between bacteria and the surface provides a deeper understanding of the initial adhesion behavior. To this purpose, we have studied the progression over time of bacterial adhesion from a laminar flow of a bacterial suspension, using a modified Robbins device (MRD). Comparing with other laminar flow devices, such as the parallel plate flow chamber, MRD allows the use of diverse substrata under the same controlled flow conditions simultaneously. Two different surfaces of Ti6Al4V and two strains of Staphylococcus epidermidis with different exopolymer production were tested. In addition, the modified Robbins device was examined for its convenience and suitability for the purpose of this study. Results were analyzed according to a pseudofirst order kinetic. The values of the parameters obtained from this model make it possible to discriminate the adhesive behavior of surfaces and bacteria. One of the fitting parameters depends on the bacterial strain and the other only on the surface properties of the substrate.

Botulinum toxin A for patients with orofacial dystonia: prospective, observational, single-centre study.

The objective of this study was to demonstrate the efficacy of intramuscular botulinum toxin type A (BTX-A) as a method of controlling the symptoms of focal facial dystonia. A prospective, longitudinal, observational, pre-post (case-series) single-centre study was conducted over a period of 3 months, involving 30 patients with focal dystonia. The patients were enrolled on a first-come, first-served basis. For all patients, the abnormal movements were evaluated using the Abnormal Involuntary Movement Scale (AIMS). The AIMS results were recorded immediately before BTX-A injection (primary predictor variable) and after 3 months (the toxin reaches its maximum effect 2 weeks after injection, and the effect is maintained for 3 months). An improvement in AIMS score was the primary outcome variable. Treatment efficacy was evaluated using the Pearson correlation index with a level of significance of P<0.05. The average age of the study subjects was 70.9±12.7years (20 female, 10 male). The mean dose of BTX-A used was 27.4±20.5U. The mean improvement in AIMS score after treatment was 5.2±4.2. A significant correlation was found between the dose applied and the reduction in AIMS score (P<0.05). BTX-A can be used in the treatment of focal dystonia and provides reproducible results.

Production of bone mineral material and BMP-2 in osteoblasts cultured on double acid-etched titanium.

BACKGROUND: The study of osteoblasts and their osteogenic functions is essential in order to understand them and their applications in implantology. In this sense, this study try to study BMP-2 production and bone matrix deposition, in addition to other biological variables, in osteoblasts cultured on a rough double acid-etched titanium surface (Osseotite®, Biomet 3i, Palm Beach Garden, Florida, USA) in comparison to a smooth titanium surface (machined) and a control Petri dish. MATERIALS AND METHODS: An in vitro prospective study. NHOst human osteoblasts from the femur were cultured on three different surfaces: Control group: 25-mm methacrylate dish (n = 6); Machined group: titanium discs with machined surface (n = 6) and Experimental group: titanium discs with a double acid-etched nitric and hydrofluoric Osseotite® acid surface (n = 6). A quantification of the mitochondrial membrane potential, and studies of apoptosis, mobility and adhesion, bone productivity (BMP-2) and cellular bone synthesis were carried out after culturing the three groups for forty-eight hours. RESULTS: A statistically significant difference was observed in the production of BMP-2 between the experimental group and the other two groups (22.33% ± 11.06 vs. 13.10% ± 5.51 in the machined group and 3.88% ± 3.43 in the control group). Differences in cellular bone synthesis were also observed between the groups (28.34% ± 14.4% in the experimental group vs. 20.03% ± 6.79 in the machined group and 19.34% ± 15.93% in the control group). CONCLUSIONS: In comparison with machined surfaces, Osseotite® surfaces favor BMP-2 production and bone synthesis as a result of the osteoblasts in contact with it.

Flavonoid and Antioxidant Capacity of Propolis Prediction Using Near Infrared Spectroscopy.

The use of propolis as a dietary supplement or as an ingredient in different food products is increasing, due to its antioxidant and bactericidal properties. These nutritional properties directly depend on its phenolic composition. For this reason, this study analysed the total contents of flavones and flavonols, flavanones and dihydroflavonols, and the antioxidant capacity by using the methods of ABTS and linoleic acid/ß-carotene in 99 samples of propolis from Spain and Chile. A rapid method was developed for quantifying these parameters in raw propolis using near infrared (NIR) spectroscopy with a remote reflectance fibre-optic probe applied directly to the ground-up sample. The models developed allow for the determination of the total flavones and flavonols (0-183 mg quercetin/g propolis and 0-72 mg rutin/g propolis), of the total flavanones and dihydroflavonols (9-109 mg pinocembrin/g propolis extract), and of its antioxidant capacity by the ABTS method based on the reduction of the 2.2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation(0-3212.6 nmol Trolox/mg of propolis) and of linoleic acid/ß-carotene (22-86% inhibition). The NIR spectroscopy models were applied in external validation to different samples of the calibration group, which led to the conclusion that the methods developed provide significantly identical data to the initial chemical data of reference.

Antibacterial effect of novel biodegradable and bioresorbable PLDA/Mg composites.

Polylactic acid/Mg composites have been recently proposed for biodegradable osteosynthesis devices because, with regards to the neat polymer, they combine an enhanced biocompatibility and bioactivity with better mechanical properties, particularly creep strength. A question still arises about their bacterial behavior. For this purpose, composites of poly-L-D-lactic acid (PLDA) loaded with 1 and 10 wt.% of Mg microparticles were evaluated using Staphylococcus epidermidis, with special emphasis on the study of bacterial adhesion and biofilm formation. During biofilm formation the bacteria viability of the composites decreased up to 65.3% with respect to PLDA. These antibacterial properties do not compromise the cytocompatibility of the material as the composites enhanced the viability of mesenchymal stem cells and their osteogenic commitment. These findings provide an important added value to the biodegradable and biocompatible PLDA/Mg composites for the manufacture of osteosynthesis devices.

Pesticide residues in propolis from Spain and Chile. An approach using near infrared spectroscopy.

This study analyses the presence of pesticides in 106 samples of raw propolis from Spain and Chile. The components detected and quantified using gas chromatography mass spectrometry were as follows: triamidefon present in 70.1% of the samples with concentrations between 0.35 and 42.17mg/kg, and dicofol detected in 7.5% of the samples with concentrations between 0.39 and 2.83mg/kg. Diclorofluanid, procymidone, folpet, propham and metazachlor were also detected but only in a small number of samples. Near infrared spectroscopy with a remote reflectance fibre-optic probe was used to detect samples contaminated with triadimefon via partial least squares discriminant, with a R2 value of 0.71. In addition, the presence of the pesticide triadimefon in propolis was quantified using the modified partial least squares (MPLS) regression method. The calibration results showed a coefficient of determination (R2) of 0.81, a root mean square error (RMSE) of 0.36, and a ratio of performance to deviation (RPD) of 2.5.

Fatty acids and fat-soluble vitamins in ewe's milk predicted by near infrared reflectance spectroscopy. Determination of seasonality.

The aim of the present work was to determine the fatty acid and fat-soluble vitamin composition and the season of ewe's milk production using NIR spectroscopy. 219 ewe's milk samples from different breeds and feeding regimes were taken each month over one year. Fatty acids were analyzed by gas chromatography, and retinol and α-, and γ-tocopherol by liquid chromatography. The results showed that the quantification was more accurate for the milk dried on paper, except for vitamins. Calibration statistical descriptors on milk dried on paper were good for capric, lauric, myristic, palmitoleic, stearic and oleic acids, and acceptable for caprilic, undecanoic, 9c, 11tCLA, ΣCLA, PUFA, ω3, ω6, retinol and α-tocopherol. The equations for the discrimination of seasonality was obtained using the partial least squares discriminant analysis (PLSDA) algorithm. 93% of winter samples and 89% of summer samples were correctly classified using the NIR spectra of milk dried on paper.

In vivo bactericidal efficacy of the Ti6Al4V surface after ultraviolet C treatment.

BACKGROUND: Biomaterial-associated infections are one of the most important complications in orthopedic surgery. The main goal of this study was to demonstrate the in vivo bactericidal effect of ultraviolet (UV) irradiation on Ti6Al4V surfaces. MATERIALS AND METHODS: An experimental model of device-related infections was developed by direct inoculation of Staphylococcus aureus into the canal of both femurs of 34 rats. A UV-irradiated Ti6Al4V pin was press-fit into the canal by retrograde insertion in one femur and the control pin was inserted into the contralateral femur. To assess the efficacy of UV radiation, the mean colony counts after inoculation in the experimental subjects and the control group were compared at different times of sacrifice and at different inoculum doses. RESULTS: At 72 h, the mean colony counts after inoculation in experimental femurs were significantly lower than those of the control group, with a reduction percentage of 76 % (p = 0.041). A similar difference between control and experimental pins was observed at 24 h using an inoculum dose <104 colony-forming units (CFU), for which the reduction percentage was 70.48 % (p = 0.017). CONCLUSION: The irradiated surface of Ti6Al4V is able to reduce early bacterial colonization of Ti6AlV pins located in the medullar channel and in the surrounding femur. The reductions depend on the initial inoculums used to cause infection in the animals and the greatest effects are detected for inoculums <104 CFU. LEVEL OF EVIDENCE: Not applicable.

CathepsinKCre mediated deletion of ßcatenin results in dramatic loss of bone mass by targeting both osteoclasts and osteoblastic cells.

It is well established that activation of Wnt/ßcatenin signaling in the osteoblast lineage leads to an increase in bone mass through a dual mechanism: increased osteoblastogenesis and decreased osteoclastogenesis. However, the effect of this pathway on the osteoclast lineage has been less explored. Here, we aimed to examine the effects of Wnt/ßcatenin signaling in mature osteoclasts by generating mice lacking ßcatenin in CathepsinK-expressing cells (Ctnnb1f/f;CtsKCre mice). These mice developed a severe low-bone-mass phenotype with onset in the second month and in correlation with an excessive number of osteoclasts, detected by TRAP staining and histomorphometric quantification. We found that WNT3A, through the canonical pathway, promoted osteoclast apoptosis and therefore attenuated the number of M-CSF and RANKL-derived osteoclasts in vitro. This reveals a cell-autonomous effect of Wnt/ßcatenin signaling in controlling the life span of mature osteoclasts. Furthermore, bone Opg expression in Ctnnb1f/f;CtsKCre mice was dramatically decreased pointing to an additional external activation of osteoclasts. Accordingly, expression of CathepsinK was detected in TRAP-negative cells of the inner periosteal layer also expressing Col1. Our results indicate that the bone phenotype of Ctnnb1f/f;CtsKCre animals combines a cell-autonomous effect in the mature osteoclast with indirect effects due to the additional targeting of osteoblastic cells.

In vivo bactericidal efficacy of farnesol on Ti6Al4V implants. / Eficacia bactericida in vivo del farnesol sobre implantes de Ti6Al4V.

OBJECTIVE: To evaluate the in vivo anti-staphylococcal bactericidal activity of farnesol on Ti6Al4V surfaces. MATERIAL AND METHODS: An experimental model of infection in biomaterials was developed by inoculation of Staphylococcus aureus ATCC 29213 into the canal of both femurs of 15 Wistar rats. A Ti6Al4V pin impregnated with 30mM of farnesol was inserted into study femur, and a Ti6Al4V control was inserted into the control femur. To evaluate the bactericidal efficacy, a comparison was made between the median of the colony forming units recovered after inoculation in the study group and the control group for different times of euthanasia and inoculum size. RESULTS: The median expressed as Log10 CFU counts obtained with farnesol titanium pin was 4.26, and in control group, it was 4.86, which was statistically significant (P=.001) on applying the Student t test for related samples. The median reduction obtained in farnesol pins relative to the control was 74%. CONCLUSIONS: Treatment with farnesol 30mM on Ti6Al4V pins appears to decrease the rate of colonisation by Staphylococcus aureus.

Antioxidant capacity of different cheeses: Affecting factors and prediction by near infrared spectroscopy.

In this study, we analyzed antioxidant capacity of 224 cheese samples prepared using 16 varied mixtures of milk from cows, ewes, and goats, in 2 manufacturing seasons (winter and summer), and over 6mo of ripening. Antioxidant capacity was evaluated using the spectrophotometric 2,2-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid) (ABTS) method. Total antioxidant capacity was significantly correlated with season of manufacturing and time of ripening but not with animal species providing the milk. Moreover, statistically significant correlations between the total antioxidant capacity and retinol (r=0.399), fat percentage (r=0.308), protein percentage (r=0.366), K (r=0.385), Mg (r=0.312), Na (r=0.432), and P (0.272) were observed. We evaluated the use of near infrared spectroscopy technology, together with the use of a remote reflectance fiber-optic probe, to predict the antioxidant capacity of cheese samples. The model generated allowed us to predict antioxidant capacity in unknown cheeses of different compositions and ripening times.