A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1β exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-β1 pathway. Notably, TGF-β1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1β exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies

Tubular composite scaffolds produced via Diffusion Induced Phase Separation (DIPS) as a shaping strategy for anterior cruciate ligaments reconstruction

Injuries of tendons and ligaments are common, especially among the young population. Anterior cruciate ligament (ACL) injuries do not heal due to its limited vascularization and hence, surgical intervention is usually required. The ideal scaffold for ligament tissue engineering (TE) should be biocompatible and possess mechanical and functional characteristics comparable to the native ACL. The Diffusion Induced Phase Separation (DIPS) technique allows the preparation of homogenous porous tubular scaffold with micro-pores using a rather simple procedure. Composites based on biodegradable polymers and bioglass have attracted much attention in tissue reconstruction and repair because of their biological and physicochemical advantages. In this work a new approach in ACL TE will be proposed focussing on the development of a suitable technique for in vitro seeding of lapine ACL fibroblasts into tubular-shaped instructive Poly-lactic-acid (PLLA) scaffolds, supplemented or not with bioglass (BG) 1393, produced via DIPS. Tubular composite scaffold (diameters: 1.2 and 2 mm, +/- BG) were obtained through a dip coating around a cylindrical support followed by a DIPS. An 8%wt PLLA/dioxane solution was prepared with 5%wt of BG-1393 as filler. Preliminary in vitro cell culture trials were carried out by seeding lapine ACL fibroblasts inside the scaffolds (2 cm as length) employing different seeding strategies in order to find the best way that allows to obtain a homogeneous fibroblast distribution inside the tubes. (1) First trials consisted in the inoculating of the cell suspension inside the tubes and maintaining them in dynamical culture. (2) The second one was done by suspending the cells in a fibrin gel polymerized within the tubes by using of thrombin. (3) The third approach was carried out by using cell spheroids (three-dimensional self-assembled cell agglomerates). Cell attachment, viability and morphology were examined by live-death and Hematoxylin/Eosin stainings after 1, 7, 14 d and vimentin immunolabelings (7 d). Scanning electron microscopical analysis revealed that the internal surface of the tubes was homogeneously structured with micropores sized around 5 µm and a mean thickness of the wall of 60 µm. The results showed cell adhesion to the wall of the tubes with all seeding techniques applied even though with fibrin gel it was more homogenous. Furthermore, colonized areas expanded with culture time and the majority of cell survived irrespectively of seeding techniques. (1) In inoculation phase, many cells left the scaffold and attached on the plate. Even after the dynamic culture (rotating device) most cells covered only half the tube inner surface. (2) In the second trial, a fibrin gel was used to achieve a homogenous cell distribution during seeding. In the early stage (48 h) cells remained captured inside the fibrin, but after 7 d they become elongated and migrated from the fibrin to the inner tube surface forming a compact cell layer. So, the fibrin appears helpful to achieve an immediate high cell seeding efficiency and an almost homogeneous cell distribution inside the tubes. (3) Although using the spheroid technique the scaffold internal surface was not homogeneously colonized with cells, after 7 d cell migration to the inner scaffold surface from the attaching spheroids could be observed. In longitudinal sections cells were elongated like typical ligament fibroblasts parallel to the longitudinal tube axis. Therefore, it can be affirmed that employment of tubular scaffolds produced by DIPS could be a promising approach of ligament TE. In the future, it would be interesting to evaluate the effectiveness of seeding by combining the spheroids and the fibrin gel

Use of Modified 3D Scaffolds to Improve Cell Adhesion and Drive Desired Cell Responses.

In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement.In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement. Copyright © 2012, AIDIC Servizi S.r.l

Relationship between B-type natriuretic peptide levels and echocardiographic indices of left ventricular filling pressures in post-cardiac surgery patients

<p>Abstract</p> <p>Background</p> <p>B-type natriuretic peptide (BNP) is increased in post-cardiac surgery patients, however the mechanisms underlying BNP release are still unclear. In the current study, we aimed to assess the relationship between postoperative BNP levels and left ventricular filling pressures in post-cardiac surgery patients.</p> <p>Methods</p> <p>We prospectively enrolled 134 consecutive patients referred to our Center 8 ± 5 days after cardiac surgery. BNP was sampled at hospital admission and related to the following echocardiographic parameters: left ventricular (LV) diastolic volume (DV), LV systolic volume (SV), LV ejection fraction (EF), LV mass, relative wall thickness (RWT), indexed left atrial volume (_iLAV), mitral inflow E/A ratio, mitral E wave deceleration time (DT), ratio of the transmitral E wave to the Doppler tissue early mitral annulus velocity (E/E').</p> <p>Results</p> <p>A total of 124 patients had both BNP and echocardiographic data. The BNP values were significantly elevated (mean 353 ± 356 pg/ml), with normal value in only 17 patients (13.7%). Mean LVEF was 59 ± 10% (LVEF ≥50% in 108 pts). There was no relationship between BNP and LVEF (p = 0.11), LVDV (p = 0.88), LVSV (p = 0.50), E/A (p = 0.77), DT (p = 0.33) or RWT (p = 0.50). In contrast, BNP was directly related to E/E' (p < 0.001), LV mass (p = 0.006) and _iLAV (p = 0.026). At multivariable regression analysis, age and E/E' were the only independent predictors of BNP levels.</p> <p>Conclusion</p> <p>In post-cardiac surgery patients with overall preserved LV systolic function, the significant increase in BNP levels is related to E/E', an echocardiographic parameter of elevated LV filling pressures which indicates left atrial pressure as a major determinant in BNP release in this clinical setting.</p

Cultivation trials of coriander (Coriandrum sativum L.) in a semi-arid mediterranean environment

Coriander (Coriandrum sativum L. - family Apiaceae) fruits, commonly called "seeds," are used all over the world for flavouring many foods items. Unfortunately, most information about cropping techniques of coriander come from markedly different environments from the Mediterranean ones; it is worth, therefore, examining some of the most important aspects of this topic. In order to evaluate the possibility to grow coriander under semi-arid Mediterranean environments a field trial was performed in 1998/99 and 1999/2000. In both years, the experimental field was located in Sparacia (Cammarata- AG -Sicily). Coriander was sown, in rows 50 cm apart on December 4th, 1998 and December 7th, 1999. The harvest of seeds was performed in both years from the 10th to 20th of June, after reaching a thermic sum of 1586 and 1714 GDD (Growth Degree Days) respectively. Despite the very low rainfall (229 and 261 mm from sowing time to harvest in both years) the fruit yields were rather satisfactory, reaching 581.3 and 970.6 kg ha-1 (dry mass). In the case of the volatile oils the most important compounds detected in the fruits were linalool (24.8 to 31.7 %), α-pinene (27.5 to 26.1 %) and γ-terpinene (21.9 to 23.0 %)

Bio-agronomical evaluation of californian poppy (Eschscholtzia californica Cham.) in a semi-arid mediterranean environment

Californian poppy (Eschscholtzia californica Cham.) is an annual or perennial herb belonging to the Papaveraceae family. The aerial parts of the plant are claimed to be endowed with gentle and non addictive hypnotic and tranquillising effects, and for this reason they are traditionally used as a sedative hypnotic or analgesic both for adults and children. In order to evaluate the suitability of the species to field cultivation in the Mediterranean semi-arid areas, a two year long experiment was performed in Sicily in 1999 and 2000. The seed of the plant was sown in both years on the first days of December in rows 50 cm apart. During the cultivation the most important morphological and production characteristics were evaluated. It was proven that effective cultivation of the species is possible under the conditions of the experimental area. After the first year a considerable number of plants self-seeded and vigorous growth of these plants was observed on the same plots. Data proved significant effect of the year on the behaviour of the plants, especially on plant height and number of branches. Differences between sown and self-seeded plants were registered evaluating their height, fruit and seeds mass

Effect of the choice of different row arrangements on the bio-agronomical behaviour of Origanum heracleoticum

Origanum heracleoticum L. (oregano) is one of the most important aromatic crops growing spontaneously in the Mediterranean area. Besides being used since ancient times in many traditional recipes, oregano becomes increasingly appreciated even today. One of the reasons is the essential oil, containing a lot of principles possessing antioxidant and antimicrobial activities. Based on the demand a well established cultivation may economically supply the drug. This work summarises the results of a cultivation trial performed in Sparacia in a typical semi-arid Mediterranean environment. The maximum dry yields were obtained in the twin-row arrangement (1 m between and 0.5 m inside) in the second and third years of cultivation. Spacing effected the number of branches, age of the plants influenced almost all of the production characteristics. We found a significant interaction between the examined factors in case of the shoot mass, stem mass and number of flowers

Cultivation trials of some aromatic and medicinal plants in a semi-arid mediterranean environment

Aromatic and medicinal plants are widespread throughout the Mediterranean region, being dominant elements of the natural flora. Since the ancient time they have been utilised by local inhabitants for production of domestic preparations, beverages, confectionery and foods. Nowadays, their cultivation is increasing continuously due to the high demand for raw materials, especially products made with the help of "biological" techniques. In this work the results of cultivation trials performed in Sparacia (Cammarata - AG - Sicily) are summarised. In the experiments some aromatic and medicinal plants of major interest were tested under the representative semi-arid Mediterranean conditions in 1998, 1999 and 2000. Clary sage (Salvia sclarea L.), fennel (Foeniculum vulgare L.), dill (Anethum graveolens L.), marigold (Calendula officinalis L.) and milk thistle (Silybum marianum Gaertn.) were grown in experimental plots in order to evaluate their suitability to the field conditions. Based on the results the majority of the crops, grown in the trials grew well under semi-arid Mediterranean conditions and gave a satisfactory yield. The cultivation of these species provides an alternative for the farmers to increase their income, even in the areas unsuitable for conventional crops