The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen.

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R 2=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.

Egg related parameters affecting fertility and hatchability in the Italian bantam breed Mericanel della Brianza.

Local chicken breeds are a vital reservoir of gene resources and their conservation has a technical role related to the future development of the productive system, as well as a social-cultural role. The aim of this study was to evaluate the effects of egg weight, egg storage period and egg weight loss on hatchability of fertile eggs in the Italian bantam breed Mericanel della Brianza. Fourteen females and eight males were kept in floor pens and divided in 8 families (1M:1 or 2F) during the reproductive season (March-June). Birds received a photoperiod of 14L:10D and were fed ad libitum. Egg production and egg weight were recorded daily. Eggs were divided in 4 weight groups: EW1 =< 33 g, EW2 = 33-36 g, EW3 = 36-39 g and EW4 =≥ 39 g. Eggs were stored at 18 °C and classified in 3 egg storage groups: ES1 = 0-4, ES2 = 5-9 and ES3 = 10-15 days. Egg weight loss was recorded and distributed in 5 different classes: EWL1 =< 10%, EWL2 = 10-15%, EWL3 = 16-20%, EWL4 = 21-25%, EWL5 => 25%. Fertility, embryo mortality and hatchability were recorded. The mean values during the reproductive season were 82% fertility and 50% hatchability of fertile eggs. The best combination of fertility and hatchability values were recorded in EW2 and lower fertility was recorded in EW1 (P < 0.05). Hatchability decreased under 50% after 10 day storage period before incubation and the best hatchability was recorded in EWL1. The present results contribute to the knowledge on reproductive parameters necessary to improve the reproductive efficiency of this Italian breed within a conservation plan.

Effects of sterilization and storage on the properties of ALP-grafted biomaterials for prosthetic and bone tissue engineering applications.

Grafting of the biomaterial surfaces with biomolecules is nowadays a challenging research field for prosthetic and bone tissue engineering applications. On the other hand, very few research works investigate the effect of the sterilization processes on the properties of functionalized biomaterials. In this study, the effects of different sterilization techniques (e.g. gamma and electron beam irradiation, ethylene oxide) on the enzymatic activity of bioactive glasses and Ti6Al4V grafted with alkaline phosphatase (ALP) have been analyzed. Sterility maintenance and in vitro bioactivity of the sterilized surfaces have also been investigated. Finally the effect of packaging and storage conditions has been considered.

Surface modification of Ti-6Al-4 V alloy for biomineralization and specific biological response: part II, alkaline phosphatase grafting.

Titanium and its alloys are the most widespread materials for the realization of orthopaedic and dental implants due to their good mechanical properties and biocompatibility. Surface functionalization of biomaterials aimed to improve and quicken implant integration and tissue regeneration is an active research field. The opportunity to confer biological activity (ability to directly stimulate cells with proper biological signals) to the Ti6Al4 V alloy, previously modified to be bioactive from the inorganic point of view (apatite precipitation), was explored in this research work. The alkaline phosphatase (ALP) enzyme was grafted to metal surface via tresyl chloride activation, maintaining its activity. A synergistic effect between biological functionalization and inorganic bioactivity was observed.

Gene expression of markers of osteogenic differentiation of human mesenchymal cells on collagen I-modified microrough titanium surfaces.

Microrough, doubly acid etched titanium surfaces (Ti) were further modified by amination and covalent coupling of fibrillar collagen type I (ColTi). Human Mesenchymal Cells (HMC) adhesion and growth, and relevant osteogenic differentiation in nonosteogenic (basal) medium were evaluated by fluorescence microscopy, scanning electron microscopy, and RT-PCR for a three-week period. Results show strongly enhanced HMC adhesion and cell density at short experimental time on ColTi, together with complete spreading of the cell body over the microrough surface topography. RT-PCR analysis of several genes involved in osteogenesis indicate, since the first week of culturing, significant progression of HMC on ColTi along the osteogenic pathway. These results indicate that the adopted process of surface immobilization of collagen, mandatory to impart collagenase resistance in implant sites, does not impair biospecific interactions between HMC and collagen. Thus, it is possible to upgrade properties arising from the control of Ti surfaces topography by surface-chemistry driven enhanced recruitment of precursor osteogenic cells and pro-osteogenic stimula.

Multifunctional implant surfaces: surface characterization and bone response to acid-etched Ti implants surface-modified by fibrillar collagen I.

The goal of the study was the evaluation of the effect of biochemical surface modification by collagen on the bone response to acid-etched titanium surfaces. Fibrillar type I porcine collagen was adsorbed and covalently linked to acid-etched Ti disks and implants. Adhesion, growth, and specific alkaline phosphatase (ALP) activity of osteoblast-like SaOS2 cells were evaluated. Implants in the femur and tibia of rabbit were performed for 2 and 4 weeks and relevant bone to implant contact (BIC) was evaluated by histomorphometry. Results show that cell morphology and growth are controlled by the rough acid-etched implants topography. Specific metabolic activity (ALP) is significantly increased by the collagen overlayer. Importantly, surface modification by collagen increases the speed of periimplant bone formation, resulting in significantly higher BIC both in femur and tibia at 2 weeks. These results suggest that morphological (surface topography) and biochemical (surface linking of bioactive molecules) cues can cooperate and yield multifunctional implant surfaces. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

In vitro evaluation of the efficacy of a new laser surface implant: cellular adhesion and alkaline phosphatase production tests.

Bone tissue reacts in different ways to implant surfaces with different patterns. The aim of this study is to understand which laser generated surface pattern produces the best cell adhesion in vitro, evaluating both the activity of the alkaline phosphatase and the cells adhering to titanium samples. Tests were carried out on titanium samples with sandblasted surfaces with laser-produced holes with diameters of 5, 10, and 20 microm, and on sandblasted titanium cylinders without holes as controls. The samples were inserted into culture medium containing SaOS-2 cells for 3, 7 and 10 days. The results showed that at days 3 and 7 the laser surfaces stimulated a higher production of alkaline phosphatase (ALP) compared to the data from the control group. At day 10 there were no significant differences between the test group and the control group.

Differentiation of osteoblasts on pectin-coated titanium.

The gold standard for implant metals is titanium, and coatings such as collagen-I, RGD-peptide, chondroitin sulfate, and calcium phosphate have been used to modify its biocompatibility. We investigated how titanium coated with pectins, adaptable bioactive plant polysaccharides with anti-inflammatory effects, supports osteoblast differentiation. MC3T3-E1 cells, primary murine osteoblasts, and human mesenchymal cells (hMC) were cultured on titanium coated with rhamnogalacturonan-rich modified hairy regions (MHR-A and MHR-B) of apple pectin. Alkaline phosphatase (ALP) expression and activity, calcium deposition, and cell spreading were investigated. MHR-B, but not MHR-A, supports osteoblast differentiation. The MHR-A surface was not mineralized, but on MHR-B, the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The ALP activity of hMCs on MHR-A was 58.3% at day 7 and 9.3% from that of MHR-B at day 10. These data indicate that modified pectin nanocoatings may enhance the biocompatibility of bone and dental implants.

Biomaterials surface characterization and modification.

This paper presents several examples of recent work in the field of surface modification and characterization of biomaterials. Due to the explosion of techniques and approaches in this area, a complete review would be unmanageable in a single paper. Rather selected examples taken from such different areas as bone-contacting devices, drug eluting stents, and immobilization of novel biomolecules are presented. The aim is to place the existing and quickly developing background of analytical and synthetic biomaterial surface science into the current perspective of this rapidly evolving discipline.

Physical-chemical and biological characterization of silk fibroin-coated porous membranes for medical applications.

Membranes in artificial organs and scaffolds for tissue engineering are often coated with biomimetic molecules (e.g., collagen) to improve their biocompatibility and promote primary cell adhesion and differentiation. However, animal proteins are expensive and may be contaminated with prions. Silk fibroin (SF) made by Bombyx Mori silk worms, used as a scaffold or grafted to other polymers, reportedly promotes the adhesion and growth of many human cell types. This paper describes how commercial porous membranes were physically coated with SF, and their physical-chemical properties were characterized by SEM, AFM, tensile stress analysis and dynamic contact angle measurements. The effect of the SF coating on membrane biocompatibility and resistance to bacterial colonization is also examined. The proposed technique yields SF coats of different thickness that strengthen the membranes and make their surface remarkably more wettable. The SF coat is not cytotoxic, and promotes the adhesion and proliferation of an immortalized fibroblast cell line. Similarly to collagen, SF-coated membranes also exhibit a much better resistance to the adhesion of S. epidermidis bacteria than uncoated membranes. These preliminary results suggest that SF is a feasible alternative to collagen as a biomimetic coating for 3D scaffolds for tissue engineering or bioartificial (as well as artificial) prosthesis.

Effects of molecular weight and surface functionalization on surface composition and cell adhesion to Hyaluronan coated titanium.

This paper describes the effect of surface functionalization on surface composition and cell adhesion to titanium samples by high and low molecular weight Hyaluronan (HA). HA was covalently linked to aminated Ti surfaces obtained by two different surface functionalization techniques, that is polyethyleneimine (PEI) adsorption and deposition from allylamine plasma. The two approaches yield very different surface densities of available amino groups, affecting this way the number and frequency of surface-HA bonds and the configurational freedom of the latter. Results of cell adhesion test are dependent on the surface functionalization approach adopted, low molecular weight HA coupled to PEI functionalized Ti does not yield the same degree of resistance to cell adhesion found on other samples. These results indicate that the details of the surface functionalization step are crucial for surface engineering of implant devices by biological molecules.

Collagen I-coated titanium surfaces: mesenchymal cell adhesion and in vivo evaluation in trabecular bone implants.

The goal of the study was the evaluation of the effect of modification of titanium implants by acrylic acid surface grafting-collagen I coupling. Tests were performed on titanium samples treated by galvanostatic anodization to create a porous surface topography. Surface characterization by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) confirms the biochemical modification of the surface and shows a surface topography characterized by pores mostly below 1 mum diameter. In vitro evaluation involving human mesenchymal cells shows enhanced cell growth on collagen coated surfaces as compared to titanium ones. Four weeks in vivo evaluation of implants in rabbit femur trabecular bone shows improvements of bone-to-implant contact, while improvement of bone ingrowth is slightly not significant (p = 0.056), when compared to the control. Overall, these data indicate that integration in trabecular, or cancellous, bone can be enhanced by the surface collagen layer, confirming previous findings obtained by modification of machined surfaces by the same approach in cortical bone implants.

Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs.

Paving workers are exposed during road paving to several polycyclic aromatic hydrocarbons (PAHs) contained in asphalt fumes. In this study early genotoxic and oxidative effects of exposure to bitumen fumes were evaluated in 19 paving workers and 22 controls. Environmental and biological monitoring of exposure was carried out, measuring, on personal air samples from exposed workers collected during three working days, the concentration of 14 PAHs and urinary OH-pyrene at the end of each of the three working days. Genotoxic effect was evaluated analysing sister chromatid exchange (SCE) frequency and direct-oxidative DNA damage by formamido-pyrimidine-glycosylase (Fpg)-modified comet assay on lymphocytes. Tail moment values from Fpg-enzyme treated cells (TMenz) and from untreated cells (TM) were used as parameters of direct and oxidative DNA damage, respectively. For each subject, the TMenz/TM ratio >2.0 was used to indicate the presence of oxidative damage. DNA damage was also evaluated analysing comet percentage. Personal air samples showed low level of total PAHs (2.843 microg m(-3)) with prevalence of 2-3 ring PAHs (2.693 microg m(-3)). Urinary OH-pyrene after work-shift of the three working days was significantly higher than that found at the beginning of the working week. SCE analysis did not show any difference between two groups while an oxidative DNA damage was found in 37% of exposed with respect to the absence in controls. Comet percentage was significantly higher (P = 0.000 ANOVA) in the exposed than in controls. The results demonstrate the high sensitivity of comet assay to assess early oxidative effects induced by exposure to bitumen fumes at low doses and confirm the suitability of urinary OH-pyrene as a biomarker of PAH exposure. In conclusion the study suggests the use of Fpg-modified comet test as a biomarker of early genotoxic effects and that of urinary OH-pyrene as a biomarker of PAH exposure to furnish indications in terms of characterization, prevention and management of risk in occupational exposure to mixtures of potentially carcinogenic substances.

New polymers for drug delivery systems in orthopaedics: in vivo biocompatibility evaluation.

The use of biodegradable polymers for drug delivery systems excluded the need for a second operation to remove the carrier. However, the development of an avascular fibrous capsule, reducing drug release, has raised concern about these polymers in terms of tissue-implant reaction. Five novel polymers were evaluated in vivo after implantation in the rat dorsal subcutis and compared to the reference polycaprolactone (PCL). Poly(cyclohexyl-sebacate) (PCS), poly(L-lactide-b-1,5-dioxepan-2-one-b-L-lactide) (PLLA-PDXO-PLLA), two 3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (D400G and D600G), and a poly(organo)phosphazene (POS-PheOEt:Imidazole) specimens were histologically evaluated in terms of the inflammatory tissue thickness and vascular density at 4 and 12 weeks from surgery. The highest values of inflammatory tissue thickness were observed in D600G (P < 0.01), PCS (P < 0.001) and PLLA-PDXO-PLLA (P < 0.001) at 4 weeks, while POP-PheOEt:Imidazole showed the lowest value of inflammatory tissue thickness (P < 0.05) at 12 weeks. D400G, D600G, PLLA-PDXO-PPLA and POP-PheOEt:Imidazole showed higher (P < 0.001) values of vascular density near the implants in comparison to PCL at 4 weeks. Finally, D400G and D600G increased their vessel densities while POP-PheOEt:Imidazole and the synthetic polyester PLLA-PDXO-PLLA presented similar vessel density values during experimental times. These different behaviours to improve neoangiogenesis without severe inflammatory tissue-responses could be further investigated with drugs in order to obtain time-programmable drug delivery systems for musculoskeletal therapy.

Adsorption of cationic antibacterial on collagen-coated titanium implant devices.

Two different cationic antimicrobial molecules, chlorhexidine (CH) and poly(hexamethylenebiguanide) (PH), were adsorbed from aqueous solution to titanium implant devices surface-modified by the covalent coupling of collagen on a polyanionic acrylic acid overlayer. Results show that more antimicrobial was adsorbed on surface modified implants as compared to control titanium devices. Moreover, the kinetic of release was affected by the interaction between the polyanionic overlayer and the cationic antimicrobial, leading to slower kinetic of release in the case of CH and stable adsorption in the case of polycationic PH . These data indicate that biochemically modified collagen coated surfaces could be endowed also by antimicrobial properties, in the spirit of present researches on multifunctional implant surfaces.

Surface engineering of titanium by collagen immobilization. Surface characterization and in vitro and in vivo studies.

Collagen was covalently linked to the surface of Titanium (Ti) by a surface modification process involving deposition of a thin film from hydrocarbon plasma followed by acrylic acid grafting. The composition and properties of surface-modified Ti were investigated by a number of surface sensitive techniques: XPS, ATR-IR, atomic force microscopy and AFM force-separation curves. In vitro tests were performed to check samples cytotoxicity and the behavior of osteoblast-like SaOS-2 cells. In vivo experiments involved 12 weeks implants in rabbit muscle as general biocompatibility assessment and 1-month implants in rabbit bone to evaluate the effect of surface modification on osteointegration rate. Results of XPS measurements show how surface chemistry is affected throughout each step of the surface modification process, finally leading to a complete and homogeneous collagen overlayer on top of the Ti samples. AFM data clearly display the modification of the surface topography and of the surface area of the samples as a consequence of the grafting and coupling process. AFM force-distance curves show that the interfacial structure responds by shrinking or swelling to variations of ionic force of the surrounding aqueous environment, suggesting that the aqueous interface of the biochemically modified Ti samples has enhanced degrees of freedom as compared to the inorganic surface of plain Ti. As to biological evaluations, the biochemically modified Ti samples are safe in terms of cytotoxicity and in vivo biocompatibility assessment. SaOS-2 cells growth rate is lower on collagen modified surfaces, and no significant difference is detected in terms of alkaline phosphatase production as compared to control Ti. Importantly, implants in rabbit femur show a significant increase of bone growth and bone-to-implant contact in the case of the collagen modified samples, confirming that biochemical modifications of Ti surface can enhance the rate of bone healing as compared to plain Ti.

[Corrosion of titanium in presence of dental amalgams and fluorides]. / La corrosione del titanio in presenza di amalgami e fluoruri.

AIM: The aim of this study was to evaluate the behaviour of titanium (Ti) in precipitant condition, and more precisely the resistance against corrosion of Ti in presence of fluorides and the electrochemical interaction between Ti- amalgam couples in fluorinated solution. METHODS: The experimental test was made with the use of an electrochemical cell. The following materials were tested: commercially pure Ti and 2 types of amalgams, the Persistalloy (Prs) and the IQC. Palladium (IQC.P). The free corrosion potential of Ti and the amalgams, the polarization curves of both amalgams and the corrosion current of the Ti-amalgam couples in the measurements were performed in 3 different electrolytic solutions: Ringer solution, fluorinated neutral Ringer solution and acid fluorinated solution. The three corrosive media are described. RESULTS: The results showed that Ti could be damaged by the presence of fluorides with an acid pH: Ti potential becomes more negative in acid fluorinated solution. The corrosion currents between Ti and amalgam couples were considered: the amalgams underwent anodic oxidation in neutral Ringer, but a reversal phenomenon occurred in the fluorinated acid solution: Ti was damaged and the amalgams both Prs and IQC.P became the cathodic partner of the couple. In neutral fluorinated solution the IQC.P amalgam induced a significantly higher corrosion of Ti, when compared to the Prs one. CONCLUSION: Results clearly show the dependence of the Ti corrosion behaviour on the pH and composition of the solution and that the outcome of the damage is affected by the composition of other metals.

Force measurements on cell repellant and cell adhesive alginic acid coated surfaces.

The atomic force microscope (AFM) was used to acquire force versus distance curves between the cantilever tip and samples bearing a surface overlayer of covalently linked alginic acid. The alginic acid coating resists cell-adhesion in in vitro experiments involving a normal and a tumor cell line. However, the surface becomes cell adhesive when alginic acid coated samples are subjected to glow discharge treatment. Force curves show in both cases the typical features resulting from the interaction between the cantilever tip and a hydrophilic, compressible polymer overlayer, suggesting that in both cases a diffuse interface with water exists. Following some recent findings on oligoethyleneglycol-terminated self-assembled-monolayers, it is suggested that conformational and molecular aspects of hydrophilic surface layers, rather than steric repulsion effects, could play a significant role in the mechanism that controls resistance to bio-adhesion.

Evaluation of interfacial properties of hyaluronan coated poly(methylmethacrylate) intraocular lenses.

Polymethylmethacrylate intraocular lenses (IOLs) were surface-modified by the linking of a overlayer of hyaluronan. In vitro experiments show that the hydrophilic HA overlayer prevents fibroblasts adhesion and greatly reduces Staphyloccous epidermidis adhesion to the IOL surface. To gain insights into the interfacial properties of untreated and hyaluronan-coated PMMA IOLs, force-distance curves were obtained by atomic force microscopy, using standard and modified tips. These measurements allow clear appreciation of the marked difference between the mechanical and chemico-physical properties at the aqueous interface of the uncoated and hyaluronan-coated lenses.

Biological monitoring of occupational exposure to cyclohexane by urinary 1,2- and 1,4-cyclohexanediol determination.

OBJECTIVES: This article reports the results obtained with the biological and environmental monitoring of occupational exposure to cyclohexane using 1,2-cyclohexanediol (1,2-DIOL) and 1,4-DIOL in urine. The kinetic profile of 1,2-DIOL in urine suggested by a physiologically based pharmacokinetic (PBPK) model was compared with the results obtained in workers. METHODS: Individual exposure to cyclohexane was measured in 156 workers employed in shoe and leather factories. The biological monitoring of cyclohexane exposure was done by measurement of 1,2-DIOL and 1,4-DIOL in urine collected on different days of the working week. In all, 29 workers provided urine samples on Monday (before and after the work shift) and 47 workers provided biological samples on Thursday at the end of the shift and on Friday morning. Another 86 workers provided biological samples at the end of the work shift only on Monday or Thursday. RESULTS: Individual exposure to cyclohexane ranged from 7 to 617 mg/ m3 (geometric mean value 60 mg/m3). Urinary concentrations of 1,2-DIOL (geometric mean) were 3.1, 7.6, 13.2, and 6.3 mg/g creatinine on Monday (pre- and postshift), Thursday (postshift) and Friday (pre-shift), respectively. The corresponding values recorded for 1,4-DIOL were 2.8, 5.1, 7.8, and 3.7 mg/g creatinine. A fairly close, statistically significant correlation was found between environmental exposure to cyclohexane and postshift urinary 1,2-DIOL and 1,4-DIOL on Monday. Data collected on Thursday and Friday showed only a poor correlation to exposure with a wide scatter. Both metabolites have a urinary half-life of close to 18 h and accumulate during the working week. CONCLUSIONS: Comparison between data obtained from a PBPK model and those found in workers suggests that 1,2-DIOL and 1,4-DIOL are urinary metabolites suitable for the biological monitoring of industrial exposure to cyclohexane.