Effects of processing on structural, mechanical and biological properties of collagen-based substrates for regenerative medicine.

The aim of this work was to investigate the structural features of type I collagen isoforms and collagen-based films at atomic and molecular scales, in order to evaluate whether and to what extent different protocols of slurry synthesis may change the protein structure and the final properties of the developed scaffolds. Wide Angle X-ray Scattering data on raw materials demonstrated the preferential orientation of collagen molecules in equine tendon-derived collagens, while randomly oriented molecules were found in bovine skin collagens, together with a lower crystalline degree, analyzed by the assessment of FWHM (Full Width at Half Maximum), and a certain degree of salt contamination. WAXS and FT-IR (Fourier Transform Infrared) analyses on bovine collagen-based films, showed that mechanical homogenization of slurry in acidic solution was the treatment ensuring a high content of super-organization of collagen into triple helices and a high crystalline domain into the material. In vitro tests on rat Schwannoma cells showed that Schwann cell differentiation into myelinating cells was dependent on the specific collagen film being used, and was found to be stimulated in case of homogenization-treated samples. Finally DHT/EDC crosslinking treatment was shown to affect mechanical stiffness of films depending on collagen source and processing conditions.

Interfibrillar packing of bovine cornea by table-top and synchrotron scanning SAXS microscopy.

Bovine cornea was studied with scanning small-angle X-ray scattering (SAXS) microscopy, by using both synchrotron radiation and a microfocus laboratory source. A combination of statistical (adaptive binning and canonical correlation analysis) and crystallographic (pair distribution function analysis) approaches allowed inspection of the collagen lateral packing of the supramolecular structure. Results reveal (i) a decrease of the interfibrillar distance and of the shell thickness around the fibrils from the periphery to the center of the cornea, (ii) a uniform fibril diameter across the explored area, and (iii) a distorted quasi-hexagonal arrangement of the collagen fibrils. The results are in agreement with existing literature. The overlap between laboratory and synchrotron-radiation data opens new perspectives for further studies on collagen-based/engineered tissues by the SAXS microscopy technique at laboratory-scale facilities.

An optimized table-top small-angle X-ray scattering set-up for the nanoscale structural analysis of soft matter.

The paper shows how a table top superbright microfocus laboratory X-ray source and an innovative restoring-data algorithm, used in combination, allow to analyze the super molecular structure of soft matter by means of Small Angle X-ray Scattering ex-situ experiments. The proposed theoretical approach is aimed to restore diffraction features from SAXS profiles collected from low scattering biomaterials or soft tissues, and therefore to deal with extremely noisy diffraction SAXS profiles/maps. As biological test cases we inspected: i) residues of exosomes' drops from healthy epithelial colon cell line and colorectal cancer cells; ii) collagen/human elastin artificial scaffolds developed for vascular tissue engineering applications; iii) apoferritin protein in solution. Our results show how this combination can provide morphological/structural nanoscale information to characterize new artificial biomaterials and/or to get insight into the transition between healthy and pathological tissues during the progression of a disease, or to morphologically characterize nanoscale proteins, based on SAXS data collected in a room-sized laboratory.

Three-dimensional coherent diffractive imaging on non-periodic specimens at the ESRF beamline ID10.

The progress of tomographic coherent diffractive imaging with hard X-rays at the ID10 beamline of the European Synchrotron Radiation Facility is presented. The performance of the instrument is demonstrated by imaging a cluster of Fe2P magnetic nanorods at 59 nm 3D resolution by phasing a diffraction volume measured at 8 keV photon energy. The result obtained shows progress in three-dimensional imaging of non-crystalline samples in air with hard X-rays.

Post-detection analysis for grating-based ultra-small angle X-ray scattering.

Until recently, the hard X-ray, phase-sensitive imaging technique called grating interferometry was thought to provide information only in real space. However, by utilizing an alternative approach to data analysis we demonstrated that the angular resolved ultra-small angle X-ray scattering distribution can be retrieved from experimental data. Thus, reciprocal space information is accessible by grating interferometry in addition to real space. Naturally, the quality of the retrieved data strongly depends on the performance of the employed analysis procedure, which involves deconvolution of periodic and noisy data in this context. The aim of this article is to compare several deconvolution algorithms to retrieve the ultra-small angle X-ray scattering distribution in grating interferometry. We quantitatively compare the performance of three deconvolution procedures (i.e., Wiener, iterative Wiener and Lucy-Richardson) in case of realistically modeled, noisy and periodic input data. The simulations showed that the algorithm of Lucy-Richardson is the more reliable and more efficient as a function of the characteristics of the signals in the given context. The availability of a reliable data analysis procedure is essential for future developments in grating interferometry.

Phasing diffuse scattering. Application of the SIR2002 algorithm to the non-crystallographic phase problem.

A new phasing algorithm has been used to determine the phases of diffuse elastic X-ray scattering from a non-periodic array of gold balls of 50 nm diameter. Two-dimensional real-space images, showing the charge-density distribution of the balls, have been reconstructed at 50 nm resolution from transmission diffraction patterns recorded at 550 eV energy. The reconstructed image fits well with a scanning-electron-microscope (SEM) image of the same sample. The algorithm, which uses only the density modification portion of the SIR2002 program, is compared with the results obtained via the Gerchberg-Saxton-Fienup HiO algorithm. The new algorithm requires no knowledge of the object's boundary and proceeds from low to high resolution. In this way, the relationship between density modification in crystallography and the HiO algorithm used in signal and image processing is elucidated.

Non-destructive determination of local strain with 100-nanometre spatial resolution

Structure sizes of approximately 180 nm are now standard in microelectronics, and state-of-the-art fabrication techniques can reduce these to just a few tens of nanometres. But at these length scales, the strain induced at interfaces can locally distort the crystal lattice, which may in turn affect device performance in an unpredictable way. A means of non-destructively characterizing such strain fields with high spatial resolution and sensitivity is therefore highly desirable. One approach is to use Raman spectroscopy, but this is limited by the intrinsic approximately 0.5-microm resolution limit of visible light probes. Techniques based on electron-beam diffraction can achieve the desired nanometre-scale resolution. But either they require complex sample preparation procedures (which may alter the original strain field) or they are sensitive to distortional (but not dilational) strain within only the top few tens of nanometres of the sample surface. X-rays, on the other hand, have a much greater penetration depth, but have not hitherto achieved strain analysis with sub-micrometre resolution. Here we describe a magnifying diffraction imaging procedure for X-rays which achieves a spatial resolution of 100nm in one dimension and a sensitivity of 10(-4) for relative lattice variations. We demonstrate the suitability of this procedure for strain analysis by measuring the strain depth profiles beneath oxidized lines on silicon crystals.

Long-term follow-up of patients enrolled in a randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small-cell lung cancer.

Our previously reported randomized study of patients with untreated, potentially resectable clinical stage IIIA non-small-cell lung cancer found that patients treated with perioperative chemotherapy and surgery had a significant increase in median survival compared to patients treated with surgery alone. We have now re-analyzed the results of the study with a median time from random allocation to analysis for all patients of 82 months. The increase in survival conferred by perioperative chemotherapy was maintained during the period of extended observation.

[Renal agenesis associated with homolateral ovarian dysplasia. A case report]. / Agenesia renale associata a displasia ovarica omolaterale. Descrizione di un caso.

The Authors describe a case of renal agenesis with ipsilateral ovarian dysplasia. The clinical features are abdominal mass and pain and then the treatment is laparoscopic ablation.

Concomitant cardiac and pulmonary operations for lung cancer.

From 1973 through 1990, 21 patients (17 men and 4 women) underwent concomitant cardiac operation and pulmonary resection for lung cancer. The mean age was 65.3 years (range, 50 to 80 years). Eighteen patients underwent coronary artery bypass; 1 underwent coronary bypass and mitral valve replacement; 1, aortic valve replacement; and 1, left ventricular aneurysmectomy. Pulmonary procedures included 16 lobectomies, 3 segmentectomies, and 2 wedge resections. Nine resections were performed during cardiopulmonary bypass, and 12 were performed either before or after bypass. On final pathologic diagnosis, 11 patients had adenocarcinoma, 7 had squamous cell carcinoma, and 3 had undifferentiated lesions. Twelve patients were in cancer stage 1 and 9 were in stage II. Placement of an intraaortic balloon pump was required in 3 patients. No patient sustained excessive blood loss requiring reoperation. Only 2 incidents (9.5%) of disseminated infection were reported. The overall 1-year survival rate was 90.5% and the 5-year survival rate was 52.4%. We found concomitant cardiac operation and pulmonary resection for lung cancer to be a safe and effective alternative to staged treatment in patients not requiring a pneumonectomy. Combined cardiac and pulmonary surgery spares the patient the risk and cost of a 2nd major surgical procedure without compromising long-term survival.

A randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small-cell lung cancer.

BACKGROUND: Patients with resectable stage IIIA non-small-cell lung cancer have a low survival rate following standard surgical treatment. Nonrandomized trials in which induction chemotherapy or a combination of chemotherapy and radiation prior to surgery were used to treat patients with regionally advanced primary cancers have suggested that survival is improved when compared with treatment by surgery alone. PURPOSE: We performed a prospective, randomized study of patients with previously untreated, potentially resectable clinical stage IIIA non-small-cell lung cancer to compare the results of perioperative chemotherapy and surgery with those of surgery alone. METHODS: This trial was designed to test the null hypothesis that the proportion of patients surviving 3 years is 12% for either treatment group against the alternate hypothesis that the 3-year survival rate would be 12% in the surgery alone group and 32% in the perioperative chemotherapy group. The estimated required sample size was 65 patients in each group. The trial was terminated at an early time according to the method of O'Brien and Fleming following a single unplanned interim analysis. The decision to terminate the trial was based on ethical considerations, the magnitude of the treatment effect, and the high degree of statistical significance attained. In total, 60 patients were randomly assigned between 1987 and 1993 to receive either six cycles of perioperative chemotherapy (cyclophosphamide, etoposide, and cisplatin) and surgery (28 patients) or surgery alone (32 patients). For patients in the former group, tumor measurements were made before each course of chemotherapy and the clinical tumor response was evaluated after three cycles of chemotherapy; they then underwent surgical resection. Patients who had documented tumor regression after preoperative chemotherapy received three additional cycles of chemotherapy after surgery. RESULTS: After three cycles of preoperative chemotherapy, the rate of clinical major response was 35%. Patients treated with perioperative chemotherapy and surgery had an estimated median survival of 64 months compared with 11 months for patients who had surgery alone (P < .008 by log-rank test; P < .018 by Wilcoxon test). The estimated 2- and 3-year survival rates were 60% and 56% for the perioperative chemotherapy patients and 25% and 15% for those who had surgery alone, respectively. CONCLUSIONS: In this trial, the treatment strategy using perioperative chemotherapy and surgery was more effective than surgery alone. IMPLICATIONS: This clinical trial strengthens the validity of using perioperative chemotherapy in the management of patients with resectable stage IIIA non-small-cell lung cancer. Further investigation of the perioperative chemotherapy strategy in earlier stage lung cancer is warranted.

[Correlation between availability of GM progenitors and endogenous CSA production in neutropenia]. / Correlazione tra disponibilità di progenitori-GM e produzione endogena di CSA nelle neutropenie.

We studied some cases with granulopoiesis disorders, owing to different causes, all showing peripheral neutropenia. Can these cases benefit by the growth factors G-CSF administration? To answer this question we assayed the bone marrow CFU-GM growth of the neutropenic patients with standard source of CSA and employing as CSA source the autologous peripheral blood mononucleated cells (APBMN). We proposed ourselves to investigate the granulopoiesis regulation referring to the relation between number of GM-progenitor cells and levels of CSA. The bone marrow CFU-GM cultures were performed by Pike and Robinson's double layer agar technique. For the evaluation of growth we applied the criterion of the dishes topographic lecture according to Ghizzi and De Caro. We plotted the experimental data both as absolute count of observed aggregates (tPP) and as dynamic patterns of in vitro aggregate proliferation, by differentiating the early proliferative events (AC-A and AC-B) from the later ones (AC-C). We observed cases showing growths higher under CSA standard source than under autologous source (CAA) and vice versa. In other cases there are not noteworthy differences. To estimate these growth patterns and to interpret the experimental data we correlated the autologous stimulus capacity to the CFU-GM absolute counts. Most cases of neutropenia showed inverse correlation between the two indexes. We consider a reduced stimulus capacity may be fit when the CFU-GM growth is high. We retain this capacity of modulating the CSA production as index of kept regulation of the granulopoiesis and, then, these cases can spontaneously evolve towards the neutropenia resolution.

[In vitro sensitivity of accessory hematopoietic cells from neutropenic subjects to the activity of human gamma-globulin]. / Sensibilità in vitro delle cellule accessorie emopoietiche di soggetti neutropenici all'azione delle gamma-globuline umane.

We studied eight patients all showing neutropenia: drug-induced isolated neutropenic failure (2 cases), immune cell-mediated neutropenia (2 case), severe bone marrow hypoplasia (2 cases), dysmyelopoietic syndrome (1 case), cyclic neutropenia (1 case). Aim of the study was to assay the effect on CSA production of the pretreatment of autologous peripheral blood mononucleated cells (APBMN) with immunoglobulins (Ig). The CFU-GM growth were tested in different experimental conditions: with standard source of CSA, with autologous source, with autologous source modified by means of the preincubation of the autologous cells with Ig. The bone marrow CFU-GM culture was performed by Pike and Robinson's double layer agar technique. The experimental data were pointed out with topographic lecture of the dishes repeated at two times so as to obtain besides the aggregates global growth their dynamic classes too. We observed that the APBMN cells pretreatment with Ig modulates CSA production: represses it in cases showing high production and increases it in those showing low production. The effect of Ig modulation appears clearer on the GM-progenitor proliferating late (AC-C) than on those proliferating early (AC-A). This effect turned out to be particularly evident in a case with immune cell-mediated neutropenia and in both cases with drug-neutropenia. We retain that the monomers Ig could block Fc receptors and modulate their expression on macrophagic cells, conditioning in this way the CSA production. The parenteral administration of intact Ig (in vivo treatment of the APBMN cells) appears therefore helpful both in cases with CSA defect and in cases with excess of CSA production.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of bisphosphonate on the in vitro proliferation dynamics of CFU-GM]. / Azione del bisfosfonato sulla dinamica proliferativa in vitro delle CFU-GM.

Bisphosphonates, potent inhibitors of osteoclast-mediated bone resorption, affect different ways of the intracellular metabolism of the osteoclasts. Likewise other cell lines are affected by bisphosphonate action, among these the macrophagic cells. So we assayed the dichloromethylene diphosphonate (clodronate, Cl2MBP) effect on the in vitro proliferation of granulocytic-macrophage progenitor cells (GM-progenitors). The bone marrow progenitor cells of four healthy volunteers were plated in double layer agar by Pike and Robinson's technique, with the addition of clodronate concentrations rising from 0.004 till 4.0 mMol. For each experimental point we evaluated: 1) the global growth of the proliferating GM-progenitors (tPP) deduced from the number of the counted cellular aggregates (CA); 2) the mean size (m.s.) of the CA; 3) the total proliferative capacity (TPC), deduced from the aggregate mean size per the number of the CA; 4) the contribution to the proliferative events by each subsets of proliferating GM-progenitors (early proliferating GM-progenitors, forming the CA present at the 7th day of the in vitro culture; later proliferating GM-progenitors, appearing only at the 12th day). The dose-effect curve showed decrease both of the tPP and TPC for increasing concentrations of clodronate up to a complete growth inhibition at the concentration 4.23 mMol of clodronate. Concerning the m.s. of the in culture growth aggregates, in three of the four cases it appears unvaried up to the Cl2MBP concentration 0.4 mMol. The trends of dose-effect curves have been analysed both for colonies (CA-P4) and for clusters (CA-P1, -P2, -P3 of our classification).(ABSTRACT TRUNCATED AT 250 WORDS)