Simplified cell culture method for the diagnosis of atypical primary ciliary dyskinesia.

BACKGROUND: The diagnosis of primary ciliary dyskinesia (PCD) can be challenging, and it may be particularly difficult to distinguish primary ciliary disease from the secondary changes after infections. OBJECTIVES: The purpose of the study was to evaluate if nasal epithelial cells, obtained with nasal brushing instead of a biopsy, could be used in a culture system for the diagnosis of PCD in difficult cases. METHODS AND MAIN RESULTS: Ciliary motion analysis (CMA) and transmission electron microscopy (TEM) were performed on 59 subjects with persistent or recurrent pneumonia. These investigations allowed the diagnosis of PCD in 13 (22%) patients while the defect of the cilia was considered secondary to infections in 37 (63%) subjects. In the remaining nine (15%) patients the diagnostic evaluation with CMA and TEM remained inconclusive. Ciliogenesis in culture allowed the diagnosis of PCD in four of these patients, it was indicative of a secondary defect in two subjects, and it was not helpful in the remaining three patients. CONCLUSIONS: Culture of cells obtained with brushing of the nasal turbinate is not a perfect test, nevertheless it may offer diagnostic help in doubtful cases of PCD.

HEMET: mathematical model of biochemical pathways for simulation and prediction of HEpatocyte METabolism.

Many computer studies and models have been developed in order to simulate cell biochemical pathways. The difficulty of integrating all the biochemical reactions that occur in a cell in a single model is the main reason for the poor results in the prediction and simulation of cell behaviour under different chemical and physical stimuli. In this paper we have translated biochemical reactions into differential equations for the development of modular model of metabolism of a hepatocyte cultured in static and standard conditions (in a plastic multiwell placed in an incubator at 37 degrees C with 5% of CO(2)). Using biochemical equations and energetic considerations a set of non-linear differential equations has been derived and implemented in Simulink. This set of equations mimics some of the principal metabolic pathways of biomolecules present in the culture medium. The software platform developed is subdivided into separate modules, each one describing a different metabolic pathway; they constitute a library which can be used for developing new modules and models to project, predict and validate cell behaviour in vitro.

Microfabrication of fractal polymeric structures for capillary morphogenesis: applications in therapeutic angiogenesis and in the engineering of vascularized tissue.

Microfabrication techniques were combined with fractal algorithms to realize polymeric scaffolds resembling capillary networks. The scaffolds were seeded with human endothelial cells in monoculture as well as in coculture with human fibroblasts. To enhance the process of angiogenesis, endothelial cells were transfected with an adenoviral vector carrying the gene for human tissue kallikrien. The results demonstrate that both the presence of a structured scaffold as well as fibroblasts in coculture contribute synergically to the promotion of a metabolically active network. The fractal scaffolds have several possible applications for example in vascularized tissue engineering and therapeutic angiogenesis. A broader implication of these results is that cell-extra cellular matrix and cell-cell interactions cooperate dynamically both at a biochemical as well as microstructural level.

Pressure-activated microsyringe (PAM) fabrication of bioactive glass-poly(lactic-co-glycolic acid) composite scaffolds for bone tissue regeneration.

The aim of this work was the fabrication and characterization of bioactive glass-poly(lactic-co-glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA-CEL2 composite scaffolds were innovatively produced by a pressure-activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2-PLGA composite films (CEL2 is an experimental bioactive SiO2 -P2 O5 -CaO-MgO-Na2 O-K2 O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10-50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone-like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast-like cells and periosteal-derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone-like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT-PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd.

3D screening device for the evaluation of cell response to different electrospun microtopographies.

Micro- and nano-topographies of scaffold surfaces play a pivotal role in tissue engineering applications, influencing cell behavior such as adhesion, orientation, alignment, morphology and proliferation. In this study, a novel microfabrication method based on the combination of soft-lithography and electrospinning for the production of micro-patterned electrospun scaffolds was proposed. Subsequently, a 3D screening device for electrospun meshes with different micro-topographies was designed, fabricated and biologically validated. Results indicated that the use of defined patterns could induce specific morphological variations in human mesenchymal stem cell cytoskeletal organization, which could be related to differential activity of signaling pathways. STATEMENT OF SIGNIFICANCE: We introduce a novel and time saving method to fabricate 3D micropatterns with controlled micro-architectures on electrospun meshes using a custom made collector and a PDMS mold with the desired topography. A possible application of this fabrication technique is represented by a 3D screening system for patterned electrospun meshes that allows the screening of different scaffold/electrospun parameters on cell activity. In addition, what we have developed in this study could be modularly applied to existing platforms. Considering the different patterned geometries, the cell morphological data indicated a change in the cytoskeletal organization with a close correspondence to the patterns, as shown by phenoplot and boxplot analysis, and might hint at the differential activity of cell signaling. The 3D screening system proposed in this study could be used to evaluate topographies favoring cell alignment, proliferation and functional performance, and has the potential to be upscaled for high-throughput.

Material and structural tensile properties of the human medial patello-femoral ligament.

The medial patellofemoral ligament (MPFL) is considered the most important passive patellar stabilizer and acts 50-60% of the force of the medial soft-tissue which restrains the lateralization of the patella between 0° and 30°. In this work, 24 human knees have been tested to evaluate the material properties of MPFL and to determine the structural behavior of femur-MPFL-Patella complex (FMPC). Particular attention was given to maintain the anatomical orientation between the patella and MPFL and to the evaluation of the elongation during the mechanical tests. The ultimate stress of the isolated ligament was 16±11MPa, the ultimate strain was 24.3±6.8%, the Young׳s Modulus was 116±95MPa and the strain energy density was 2.97±1.69MPa. The ultimate load of the whole structure, FMPC, was 145±68N, the ultimate elongation was 9.5±2.9mm, the linear stiffness was 42.5±10.2N/mm and the absorbed energy was 818.8±440.7Nmm. The evaluation of material and structural properties of MPFL is fundamental to understand its contribution as stabilizer and for the selection of repair and reconstruction methods.

Reconstruction of medial patello-femoral ligament: Comparison of two surgical techniques.

The medial patello-femoral ligament is considered the most important passive patellar stabilizer and its proper functionality is essential for the patello-femoral joint stability. In this work, 18 human knees were randomly divided into two groups and reconstructed through two different surgical techniques: the "Through tunnel tendon" and the "Double converging tunnel" reconstructions. Subsequently, the samples were mechanically tested to evaluate the structural properties of reconstructed femur-MPFL-Patella complex (rFMPC). Particular attention was given to maintain the anatomical orientation between the patella and the graft. Both procedures showed lower stiffness and higher ultimate strain and absorbed energy compared to the native MPFL, but the advantages of the double converging tunnel technique are related to the restoration of the native MPFL sail-shape, to a better stress distribution on the patella, to the use of a single interference screw as fixation device and to the simplicity, rapidity and cost-effectivity of the surgical procedure. The evaluation of the structural properties of rMPFL is fundamental to evaluate the adequacy of the different techniques to restore the physiological structural properties of the native MPFL.

Design, fabrication and perivascular implantation of bioactive scaffolds engineered with human adventitial progenitor cells for stimulation of arteriogenesis in peripheral ischemia.

Cell therapy represents a promising option for revascularization of ischemic tissues. However, injection of dispersed cells is not optimal to ensure precise homing into the recipient's vasculature. Implantation of cell-engineered scaffolds around the occluded artery may obviate these limitations. Here, we employed the synthetic polymer polycaprolactone for fabrication of 3D woodpile- or channel-shaped scaffolds by a computer-assisted writing system (pressure assisted micro-syringe square), followed by deposition of gelatin (GL) nanofibers by electro-spinning. Scaffolds were then cross-linked with natural (genipin, GP) or synthetic (3-glycidyloxy-propyl-trimethoxy-silane, GPTMS) agents to improve mechanical properties and durability in vivo. The composite scaffolds were next fixed by crown inserts in each well of a multi-well plate and seeded with adventitial progenitor cells (APCs, 3 cell lines in duplicate), which were isolated/expanded from human saphenous vein surgical leftovers. Cell density, alignment, proliferation and viability were assessed 1 week later. Data from in vitro assays showed channel-shaped/GPTMS-crosslinked scaffolds confer APCs with best alignment and survival/growth characteristics. Based on these results, channel-shaped/GPTMS-crosslinked scaffolds with or without APCs were implanted around the femoral artery of mice with unilateral limb ischemia. Perivascular implantation of scaffolds accelerated limb blood flow recovery, as assessed by laser Doppler or fluorescent microspheres, and increased arterial collaterals around the femoral artery and in limb muscles compared with non-implanted controls. Blood flow recovery and perivascular arteriogenesis were additionally incremented by APC-engineered scaffolds. In conclusion, perivascular application of human APC-engineered scaffolds may represent a novel option for targeted delivery of therapeutic cells in patients with critical limb ischemia.

Triphasic scaffolds for the regeneration of the bone-ligament interface.

A triphasic scaffold (TPS) for the regeneration of the bone-ligament interface was fabricated combining a 3D fiber deposited polycaprolactone structure and a polylactic co-glycolic acid electrospun. The scaffold presented a gradient of physical and mechanical properties which elicited different biological responses from human mesenchymal stem cells. Biological test were performed on the whole TPS and on scaffolds comprised of each single part of the TPS, considered as the controls. The TPS showed an increase of the metabolic activity with culturing time that seemed to be an average of the controls at each time point. The importance of differentiation media for bone and ligament regeneration was further investigated. Metabolic activity analysis on the different areas of the TPS showed a similar trend after 7 days in both differentiation media. Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the TPS in mineralization medium compared to the controls. A different glycosaminoglycans amount between the TPS and its controls was detected, displaying a similar trend with respect to ALP activity. Results clearly indicated that the integration of electrospinning and additive manufacturing represents a promising approach for the fabrication of scaffolds for the regeneration of tissue interfaces, such as the bone-to-ligament one, because it allows mimicking the structural environment combining different biomaterials at different scales.

Quasi-linear viscoelastic properties of the human medial patello-femoral ligament.

The evaluation of viscoelastic properties of human medial patello-femoral ligament is fundamental to understand its physiological function and contribution as stabilizer for the selection of the methods of repair and reconstruction and for the development of scaffolds with adequate mechanical properties. In this work, 12 human specimens were tested to evaluate the time- and history-dependent non linear viscoelastic properties of human medial patello-femoral ligament using the quasi-linear viscoelastic (QLV) theory formulated by Fung et al. (1972) and modified by Abramowitch and Woo (2004). The five constant of the QLV theory, used to describe the instantaneous elastic response and the reduced relaxation function on stress relaxation experiments, were successfully evaluated. It was found that the constant A was 1.21±0.96MPa and the dimensionless constant B was 26.03±4.16. The magnitude of viscous response, the constant C, was 0.11±0.02 and the initial and late relaxation time constants τ1 and τ2 were 6.32±1.76s and 903.47±504.73s respectively. The total stress relaxation was 32.7±4.7%. To validate our results, the obtained constants were used to evaluate peak stresses from a cyclic stress relaxation test on three different specimens. The theoretically predicted values fit the experimental ones demonstrating that the QLV theory could be used to evaluate the viscoelastic properties of the human medial patello-femoral ligament.

Microsyringe-based deposition of two-dimensional and three-dimensional polymer scaffolds with a well-defined geometry for application to tissue engineering.

A technique for controlled deposition of biomaterials and cells in specific and complex architectures is described. It employs a highly accurate three-dimensional micropositioning system with a pressure-controlled syringe to deposit biopolymer structures with a lateral resolution of 5 microm. The pressure-activated microsyringe is equipped with a fine-bore exit needle and a wide variety of two- and three-dimensional patterns on which cells to be deposited can adhere. The system has been characterized in terms of deposition parameters such as applied pressure, motor speed, line width and height, and polymer viscosity, and a fluid dynamic model simulating the deposition process has been developed, allowing an accurate prediction of the topological characteristics of the polymer structures.

Microfabricated fractal branching networks.

In this article, we demonstrate how a combination of engineering and biological techniques could lead to the realization of branched microstructures that can be used for the repair of damaged vascularized tissue. Recursive "treelike" networks were first generated by using fractal algorithms based on Murray's equation for vascular branching as well as allometric scaling rules. Two- and three-dimensional branching patterns with different levels of complexity were then microfabricated from poly-lactide-co-glycolide (PLGA) by using the pressure-assisted microsyringe (PAM) system developed in our laboratory. Human endothelial cells isolated from umbilical cords were seeded on the microfabricated branched scaffolds to evaluate their effectiveness in supporting site-specific cell adhesion. The results show that cell densities on the networks increase with complexity up to the sixth level and are then constant independent of branching level. The implications of this finding are discussed in terms of contact inhibition of "capillaries."

Antepartum computerized CTG and neonatal acid-base status at birth.

OBJECTIVE: To assess the correlation between individual computerized cardiotocography (cCTG) variables and acid-base status at birth before the first breathing in uncomplicated term pregnancy delivered by cesarean section. STUDY DESIGN: A retrospective cohort study. SUBJECTS AND METHODS: Seventy singleton normal pregnancies delivered by elective cesarean section. The last antepartum cCTG performed within 4 h from delivery by the System 8002 (Oxford Sonicaid, UK) was correlated to umbilical blood gas analysis (UBGA) values (AVL compact 2 analyser). Considering blood gas analysis values as dependent and individual cCTG parameters as independent variables the best regression subsets followed by a backward stepwise linear regression was used. RESULTS: There was no significant correlation of cCTG parameters with any of the values of blood gas analysis. However, when neonatal conditions, as expressed by arterial pH > 7.20, Becf > - 4.0 mmol/l and Apgar score at 5 min > 7, were taken as an end-point, they could be predicted by Acc15 and FM/hour (sensitivity: 35%, positive predictive value: 92%, cut-off > 7 and 67%, 92%, > 21, respectively). CONCLUSION: In uncomplicated pregnancies, the prospect of a vigorous newborn seems particularly associated with the presence of Acc > 15 and FM/hour > 21.

Clinical and ultrasonographic implications of uterine leiomyomatosis in pregnancy.

OBJECTIVE: To study the complications related to leiomyomatosis in pregnancy by clinical and ultrasonographic assessment. DESIGN: A retrospective study. SUBJECTS: All pregnancies admitted to the 2nd Institute of Gynecology and Obstetrics, Policlinico Umberto I, in the period between January 1992 to December 1993 were surveyed. RESULTS: Gestational age at the time of ultrasonographic neoplasm diagnosis was 25.1 +/- 13.4 weeks, 'we found no correlation between maternal age or parity affecting pregnancy outcome, Leiomyomatosis complicated pregnancy rate was 1.68%. Myomatosis was diagnosed clinically in 25 of 67 cases (37.3%). Regarding the location of the neoplasm, 59% was located in the corpus-uteri, 21% was considered a diffuse neoplasm and the 14% was located in the fundus. Threatened abortion was the most frequent complication (20%), abortion was the second (16.4%). We observed an increased abortion threat rate (p < 0.001) in those cases where the leiomyoma was in relation with the placenta. We had a surgery rate of 76% in pregnancies complicated by myomatosis, and the indication for surgery was given either primarily or exclusively by the presence of myomatous formation in 19 cases (50%). CONCLUSIONS: Our study suggests that location of the leiomyoma in relation to the placenta is a higher risk factor than its size, and that there is a higher risk for threats of abortion and abortion rates in pregnancies complicated by leiomyomatosis. We recommend that every pregnant woman with a suspected myoma should be ultrasonographically scanned.

An obstetric and neonatal study on unplanned deliveries before arrival at hospital.

OBJECTIVE: To establish the prevalence of unplanned deliveries before arrival at a big metropolitan hospital and to determine the demographic characteristics of the group of women at risk of delivering before arrival. DESIGN: A random case control study. Each baby born before arrival and its mother were compared to the next baby born in the same Department. SUBJECTS: All babies born before arrival at the Department of Obstetrics and Gynecology, University "La Sapienza" in a 10 yr. period (Jan 1983-Dec 1993). RESULTS: Of 27,274 consecutive deliveries in the study period, 22 (0.8%) babies were born before arrival at hospital. Of the 22 women who delivered before arrival, 16 were Italian, 5 were considered nomad (no fixed address) and one was a Polish tourist. No statistical difference was found between groups regarding maternal age, parity, gestational age, birth weight and immediate delivery complications. No mortality cases were observed in the study or control group. Neonatal stay in the neonatal ward was longer in the study group (6.5 vs 3.5 days, P < 0.001). Hypothermia was the highest morbidity (P < 0.001) and neonatal complications were more prevalent in babies delivered before arrival than in-born babies (P < 0.001). CONCLUSIONS: Delivery before arrival to hospital does not seem to carry a higher neonatal mortality risk. However, the prevalence of complications was higher in such babies, with hypothermia being the highest morbidity.

A feasibility study of yarns and fibers with annexed electronic functions: the ARIANNE project.

In this paper several issues concerning the development of fibers endowed with electronic functions will be presented and discussed. In particular, issues concerning materials, structures, electronic models and the mechanical constraints due to textile technologies will be detailed. All these aspects have been studied in the framework of the project ARIANNE, funded by the European Community during the V Frame Programme.

Collagen-gelatin-genipin-hydroxyapatite composite scaffolds colonized by human primary osteoblasts are suitable for bone tissue engineering applications: in vitro evidences.

The application of porous hydroxyapatite (HAp)-collagen as a bone tissue engineering scaffold represents a new trend of mimicking the specific bone extracellular matrix (ECM). The use of HAp in reconstructive surgery has shown that it is slowly invaded by host tissue. Therefore, implant compatibility may be augmented by seeding cells before implantation. Human primary osteoblasts were seeded onto innovative collagen-gelatin-genipin (GP)-HAp scaffolds containing respectively 10%, 20%, and 30% HAp. Cellular adhesion, proliferation, alkaline phosphatase (ALP) activity, osteopontin (OPN), and osteocalcin (OC) expressions were evaluated after 3, 7, 15, and 21 days. The three types of scaffolds showed increased cellular proliferation over time in culture (maximum at 21 days) but the highest was recorded in 10% HAp scaffolds. ALP activity was the highest in 10% HAp scaffolds in all the times of evaluation. OC and OPN resulted in higher concentration in 10% HAp scaffolds compared to 20% and 30% HAp (maximum at 21 days). Finally, scanning electron microscopy analysis showed progressive scaffolds adhesion and colonization from the surface to the inside from day 3 to day 21. In vitro attachment, proliferation, and colonization of human primary osteoblasts on collagen-GP-HAp scaffolds with different percentages of HAp (10%, 20%, and 30%) all increased over time in culture, but comparing different percentages of HAp, they seem to increase with decreasing of HAp component. Therefore, the mechanical properties (such as the stiffness due to the HAp%) coupled with a good biomimetic component (collagen) are the parameters to set up in composite scaffolds design for bone tissue engineering.

Bone scaffolds with homogeneous and discrete gradient mechanical properties.

Bone TE uses a scaffold either to induce bone formation from surrounding tissue or to act as a carrier or template for implanted bone cells or other agents. We prepared different bone tissue constructs based on collagen, gelatin and hydroxyapatite using genipin as cross-linking agent. The fabricated construct did not present a release neither of collagen neither of genipin over its toxic level in the surrounding aqueous environment. Each scaffold has been mechanically characterized with compression, swelling and creep tests, and their respective viscoelastic mechanical models were derived. Mechanical characterization showed a practically elastic behavior of all samples and that compressive elastic modulus basically increases as content of HA increases, and it is strongly dependent on porosity and water content. Moreover, by considering that gradients in cellular and extracellular architecture as well as in mechanical properties are readily apparent in native tissues, we developed discrete functionally graded scaffolds (discrete FGSs) in order to mimic the graded structure of bone tissue. These new structures were mechanically characterized showing a marked anisotropy as the native bone tissue. Results obtained have shown FGSs could represent valid bone substitutes.

HEMETß: improvement of hepatocyte metabolism mathematical model.

This article describes hepatocyte metabolism mathematical model (HEMETß), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink®, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETß as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37° C with 5% of CO2) and with excess substrates concentration. The main improvements in HEMETß are the introductions of Michaelis-Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying 'peripheral' pathways or model inputs.