C. De Maria

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.

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.

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.

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–P2O5–CaO–MgO–Na2O–K2O 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

HEMETβ: improvement of hepatocyte metabolism mathematical model

This article describes hepatocyte metabolism mathematical model (HEMETβ), 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. HEMETβ 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 HEMETβ 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.

A new library of HEMET model: Insulin effects on hepatic metabolism

Prediction and simulation of cell culture behaviour, under different chemical and physical stimuli by a mathematical model, represent an innovative way to create a virtual cell laboratory, where it is possible to perform and optimize experimental protocol, saving time and money. In silico experiments permit to reproduce pathological and physiological situations and make toxicological tests. In this paper we introduce a new library of HEMET (HEpatocyte METabolism) software that allows the insulin effects on hepatic metabolism to be simulated. This new set of nonlinear differential equations, derived from biochemical reactions which involve this pancreatic hormone, allows the catabolites concentration in hepatic cell culture after insulin infusion to be predicted. The validation procedures were carried out using data obtained from specifically designed cell experiments and from literature. A user friendly interface allows to easily change model parameters, rate constants and inputs simulating a wide range of physiological and pathological scenarios.

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 recipients 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.

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.

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.

DataBrain: a web-accessible database for three-dimensional reconstructions and quantitative morphometrics of neurons

Thanks to the new advanced tools and the innovative methods to image deep in the brain at cell resolution, neuroanatomy is quickly redefining its protocols for quantitatively studying neurons in their own three-dimensional arrangement. The huge amount of data generated has to be managed and shared among labs: this need has led us to develop DataBrain, an on-line archive of three-dimensional single neuron reconstructions and their associated morphometrics. DataBrain interface allows users to upload and download data, to easily search neuron using filters and to on-line view both three-dimensional reconstructions and morphological parameters. Here we describe DataBrain’s main features and show an example of how it can be used to store morphological quantitative datasets of Purkinje cells from murine clarified cerebellum slices acquired using a confocal microscope.