Hierarchical electrospun tendon-ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions.

The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high-resolution X-ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L-lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads. LAY DESCRIPTION: To enhance the regeneration of injured tendons and ligaments cells need to growth on dedicated structures (scaffolds) with mechanical properties and a fibrous morphology similar to the natural tissue. In particular, the morphological organisation of scaffolds is fundamental in leading cells to colonise them, regenerating the collagen extracellular matrix. Electrospinning is a promising technique to produce fibres with a similar to the human collagen fibres, suitable to design complex scaffolds able to guide cells in the reconstruction of the natural tissue. Moreover, it is well established that the cyclic stretching of these scaffolds inside dedicated systems called bioreactors, can speed up cells growth and their shape modification. The aim of the present study was to investigate how hierarchically structured electrospun scaffolds, made of resorbable material such as poly(L-lactic acid) and collagen, could induce morphological changes in human fibroblasts, while cultured during static and dynamic conditions. These scaffolds were composed by an external electrospun membrane that grouped inside it a ring-shaped bundle, made of axially aligned nanofibres, resembling the morphological arrangement of tendon and ligament tissue. After 7 days of parallel cultures, the scaffolds were investigated using the following imaging techniques: (i) high-resolution X-ray tomography (XCT); (ii) scanning electron microscopy (SEM); (iii) fluorescence microscopy and (iv) histology. The results showed that fibroblasts were able to grow on the external nanofibrous sheath of the static scaffolds, by elongating themselves along their circumference. The dynamic cultures revealed instead a preferential axial orientation of fibroblasts grown on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds allowed an axial distribution of the fibroblasts along the nanofibres direction. This study has demonstrated that the electrospun hierarchically structured scaffolds investigated can modify the fibroblasts morphology both in static and dynamic conditions, in relation with the direction of the applied loads.

High-resolution x-ray tomographic morphological characterisation of electrospun nanofibrous bundles for tendon and ligament regeneration and replacement.

Repair of ligaments and tendons requires scaffolds mimicking the spatial organisation of collagen in the natural tissue. Electrospinning is a promising technique to produce nanofibres of both resorbable and biostable polymers with desired structural and morphological features. The aim of this study was to perform high-resolution x-ray tomography (XCT) scans of bundles of Nylon6.6, pure PLLA and PLLA-Collagen blends, where the nanofibres were meant to have a predominant direction. Characterisation was carried out via a dedicated methodology to firmly hold the specimen during the scan and a workflow to quantify the directionality of the nanofibres in the bundle. XCT scans with 0.4 and 1.0 µm voxel size were successfully collected for all bundle compositions. Better image quality was achieved for those bundles formed by thicker nanofibres (i.e. 0.59 µm for pure PLLA), whereas partial volume effect was more pronounced for thinner nanofibres (i.e. 0.26 µm for Nylon6.6). As expected, the nanofibres had a predominant orientation along the axis of the bundles (more than 20% of the nanofibres within 3° and more than 60% within 18° from the bundle axis), with a Gaussian-like dispersion in the other directions. The directionality assessment was validated by comparison against a similar analysis performed on SEM images: the XCT analysis overestimated the amount of nanofibres very close to the bundle axis, especially for the materials with thinnest nanofibres, but adequately identified the amount of nanofibres within 12°. LAY DESCRIPTION: Repair of ligaments and tendons requires dedicated materials (scaffolds) mimicking the spatial organisation of the collagen (the main material composing such natural tissue). Electrospinning is a promising technique that allows production of fibres with nanometric dimension using high voltage to stretch very tiny drops of polymeric solutions. Electrospinning allows processing both polymers that can be resorbed by the host tissue, and nonresorbable ones, to obtain the desired structural and morphological features by arranging the nanofibres in bundles. The aim of this study was to perform high-resolution x-ray computed tomography (XCT) scans of bundles, where the nanofibres were meant to have a predominant direction. The investigation included bundles of different compositions: a biostable polymer (Nylon) and bioresorbable ones (pure Poly-L-lactic acid (PLLA) and PLLA-Collagen blends). The electrospun bundles were produced using a validated method (Sensini et al 2017: https://doi.org/10.1088/1758-5090/aa6204). To this end, we developed a dedicated methodology to scan such small specimens, and a workflow to quantify the directionality of the nanofibres in the bundle. For all the compositions, XCT scans with extremely high resolution (i.e. down to 0.4 µm) were successfully collected. As expected, better images were obtained for those bundles where the nanofibres were thicker than the scanning resolution (i.e. 0.59 µm for pure PLLA). The images of the thinnest nanofibres (i.e. 0.26 µm for Nylon) were poorer because the fibre diameter was smaller than the resolution (partial volume effect). The nanofibres had a predominant orientation along the axis of the bundles (more than 60% of the nanofibres were within 18° from the bundle axis). The nanofibres had a Gaussian-like dispersion in the other directions. As this is the first time that XCT is used to quantify the directionality of this kind of bundles, the directionality assessment was further validated by comparison against a similar analysis performed on SEM images. Overall, this study has demonstrated the usefulness and reliability of using high-resolution x-ray computed tomography (XCT) scans to investigate the morphology of polymeric scaffolds made of electrospun nanofibres.

Toxoplasma gondii infection in pregnancy: opportunities and pitfalls of serological diagnosis.

Because of its life cycle, the recovery of Toxoplasma gondii from biological samples is often impracticable. Consequently, a serological diagnosis represents the first and the most widely used approach to defining the stage of infection. The detection of IgG, IgM, IgA, IgE and IgG avidity by different methods offers this opportunity. However, the results may be affected by difficulties in interpretation, as the same antibody pattern may have a different valency, contingent upon subjects and clinical settings, e.g., pregnant women vs. neonates, and treated vs. untreated patients. This review describes the various factors that should be taken into account when performing serological tests for T. gondii, as well as the pitfalls that may be encountered during the interpretative process.

[Organotypic culture of normal and neoplastic laryngeal mucosa]. / Cultura organotipica di mucosa laringea normale e neoplastica.

Mucous membrane's phragments from human normal and neoplastic larynx have been maintained in a organ culture system. A set of cultures has been prepared for optical microscope studies and a parallel set for incorporation studies using 3H Thymidine and 3H Leucine. The results show that there is a good incorporation of 3H Thymidine and a greater incorporation of 3H Leucine at the 7th day due to a differentiation in vitro with a new synthesis of cheratin. These data agree with the morphologic evidence of considerable horny structures in the pathological mucous membrane after 11 days at culture.

[Hexokinase activity of erythrocytes in cancer patients]. / Attività esocinasica negli eritrociti di soggetti neoplastici.

In the present work we have considered the following erythrocyte enzymatic activities; hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphate-dehydrogenase (G6PD), 6-phosphogluconate-dehydrogenase (6PGD), glicero phosphate-isomerase (PHI) and glutathione-reductase (GR) in the red blood cells of 130 subjects of which 55 were healthy or with non cancerous pathology and 75 with malignant tumors. Evident modification has been shown only for the HK which together with fructose-6-phosphate-kinase (PFK) and PK are limiting steps of glycolysis. The increased HK activity value resulted particularly significant in red blood cells of patients affected by well differentiated adenocarcinoma.

Modification of the hexokinase activity in the red blood cells of subjects with differentiated adenocarcinoma.

In the present work we found a significant increase of erythrocyte HK activity in subjects suffering from differentiated adenocarcinoma. Some erythrocyte enzymatic activities (such as HK, PK, G-6-PD, 6-PGD, PHI and GR) in the red blood cells of 96 subjects, of which 55 were healthy or with noncancerous pathology and 41 had malignant tumors, are considered. Evident modification was shown only for the HK, which together with PFK and PK are limiting steps of glycolysis.

[Our criteria for surgical treatment of acute obstructions of the large gut of neoplastic origin (author's transl)]. / Nostri indirizzi nella terapia chirurgica delle occlusioni acute neoplastiche del grosso intestino.

The authors review a total of 132 cases of acute neoplastic obstruction of the large gut seen in their clinical material over the last twenty years. In the light of the results obtained they discuss the indications and limitations of the various surgical strategies adopted (radical surgery, derivative surgery, and palliative resection); they conclude that radical procedures are best for the majority of cases.

[Reflections on ten years' experience in the treatment of hemorrhagic colorectitis and Crohn's disease (author's transl)]. / La nostra esperienza nel trattamento della rettocolite emorragica e del m. di Crohn.

The authors offer a critical review of eight cases of hemorrhagic colorectitis and Crohn's disease (regional ileitis) managed with medical treatments, and eleven like cases treated surgically, during the last ten years. After a discussion of the resources and limitations of medical therapy, the authors describe the various surgical strategies adopted in their cases; they define the possibility of obtaining final cures in Crohn's disease with extended ileocolectomy; and concerning ulcerative forms, they point out the greater efficacy of surgical procedures involving sacrifice of the rectum compared to those in which the terminal segment of the gut is used immediately or later for restoring continuity of the intestinal canal.

[Personal experience with anterior resection of the rectum]. / La nostra esperienza nelle resezioni anteriori del retto.

Authors experience in the Dixon's operation regarding the surgical treatment of cancer of the rectum and recto-sigmoid junction is reviewed. It consists in 224 cases of malignant lesions which were treated by Miles operation (106 cases--47%), Dixon's operation (49 cases--21%), palliation anterior resection (16 cases--7%), other operations (53 cases--24%). After a brief discussion on anatomo-pathological conditions and tecniques, the Authors consider their results: operatory mortality 4%; complications: total anastomosis dehiscence 1 case, partial anastomosis dehiscence 14%; local repetitions 10%; hepatic metastasis within 5 ys 40%; survival at 5 ys 46%. Functional results were always very good. The Authors conclude that, even considering the large rate (56%) of wide-spread cancers treated, Dixon's operation, at least in their experience, doesn't seem to satisfy those needs of radically requested by a correct surgical therapy, which only Miles' operation guarantees.