TENOGENIC DIFFERENTIATION PROTOCOL IN XENOGENIC-FREE MEDIA ENHANCES TENDON-RELATED MARKER EXPRESSION IN ADIPOSE-DERIVED STEM CELLS

Tendon injuries are common and current therapies often are unsuccessful. Cell-based therapy using mesenchymal stem cells (MSCs) seems to be the most promising approach to heal tendon. Moreover, providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices (GMP). Adipose-derived stem cells (n=4) were cultured in 6-well plates coated with type-I collagen in a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL). At passage 4, ASCs were induced to tendon lineage for 14 days using 100ng/ml CTGF, 10ng/ml TGFβ3, 50ng/ml BMP12 and 50µg/ml ascorbic acid in the SF (SF-TENO) or in the hPL (hPL-TENO) medium. Cells cultured without any supplements are used as control. Morphological appearance, cell viability and FACS were performed in undifferentiated cells to evaluate the xenogenic-free culture conditions; the gene and protein expression were performed by RT-PCR and immunofluorescence to evaluate to expression of stem cell- and tendon-related markers upon cell differentiation. SF-CTRL and hPL-CTRL showed similar viability and MSC's surface proteins and expressed the stemness markers NANOG, OCT4 and Ki67. Moreover, both SF-TENO and hPL-TENO expressed significant higher levels of SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 genes already at 3d (p<0.05) respect to CTRLs. Scleraxis and collagen were also detected in both SF-TENO and hPL-TENO at protein level in higher amount than CTRLs. In conclusion, ASCs exposed to CTGF, BMP12, TGFb3 and AA in both serum and xenogenic-free media possess similar tenogenic differentiation ability moving forward the GMP-compliant approaches for the clinical use of ASCs

Adipose-derived mesenchymal stem cells cultured in tenogenic serum-free medium express tendon-specific markers

Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long-term functional impairment. Poor tendon healing responses are mainly attributed to insufficient or failed tenogenesis. For optimal treatment, enhanced understanding of tendon physiology is necessary. Among others, growth factors (GFs) and cytokines modulate the differentiation of tendons during embryogenesis and the healing process of injured tendons. Cell-based therapy using mesenchymal stem cells (MSCs) in combination with GFs and biomaterials seems to be the most promising approach to heal tendon injuries. Adipose- derived MSCs (ASCs) are multipotent and immunoprivileged, making them ideal candidates for therapeutic purposes. Moreover, providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices (GMP), and GMP guidelines should be adhered to throughout the process of isolating, expanding and differentiating MSCs. For these reasons, the aims of this study were: i) to investigate the effect of several GFs already known to be involved in tendon development/healing process on human ASCs proliferation and expression of tendon-related markers; ii) to develop a tenogenic GMP-compliant serum free medium. Subcutaneous fat was obtained from 5 healthy donors by lipoaspiration, after written consent. Primary cultures of the stromal vascular fraction were established and characterized by flow cytometry analysis to evaluate cell viability (7AAD(-) and SYTO 40(+) expression), and ASC surface marker expression (CD45(-), CD146(-) and CD34(+)) and then cryopreserved. After thawing, ASCs were expanded until P3 culturing in a commercial human platelet lysate- supplemented culture medium (hPL) or in a well-defined serum free medium (SF) developed in our laboratories. At P4, tenogenic induction was performed: ASCs were cultured in 6-well plates coated with the tendon matrix protein type-I collagen and in tenogenic medium (TENO) consisting in hPL or SF medium supplemented with 100ng/ml CTGF, 10ng/ml TGFβ3, 50ng/ml BMP12 and 50μg/ml Ascorbic acid (AA) for 1, 3, 7 and 14 days. Cells cultured without any supplementations at the same time points were used as control (CTRL). Morphological appearance (optical microscopy), cell proliferation (lactate assay), gene (RT-PCR) and protein (immunofluorescence, SIRIUS-RED staining) expression were performed in all groups at all time points. Both SF-TENO and hPL-TENO cells appeared more rounded and with more cytoplasmic content and proliferated faster than respective CTRL. Tendon-marker genes (SCX, COL1A1, COL3A1, TNC, MMP3, MMP13) were significantly upregulated already after 3 to 14 days of differentiation in respect to CTRL without any significant differences between hPL and SF groups. In the meantime, stem cell gene (KLF4, NANOG, OCT4) expression decreased in TENO cells vs CTRL. SCX protein expression and the increase of collagen-matrix deposition were also observed in all TENO cells vs CTRL. These results demonstrate that ASCs possess tenogenic differentiation ability when exposed to CTGF, BMP12, TGFb3 and AA in both hPL and SF medium providing insights of the earliest events of tendon development and move forward the GMP-compliant approaches needed for cell-therapy strategies

3D bioprinting for orthopaedic applications: Current advances, challenges and regulatory considerations

In the era of personalised medicine, novel therapeutic approaches raise increasing hopes to address currently unmet medical needs by developing patient-customised treatments. Three-dimensional (3D) bioprinting is rapidly evolving and has the potential to obtain personalised tissue constructs and overcome some limitations of standard tissue engineering approaches. Bioprinting could support a wide range of biomedical applications, such as drug testing, tissue repair or organ transplantation. There is a growing interest for 3D bioprinting in the orthopaedic field, with remarkable scientific and technical advances. However, the full exploitation of 3D bioprinting in medical applications still requires efforts to anticipate the upcoming challenges in translating bioprinted products from bench to bedside. In this review we summarised current trends, advances and challenges in the application of 3D bioprinting for bone and cartilage tissue engineering. Moreover, we provided a detailed analysis of the applicable regulations through the 3D bioprinting process and an overview of available standards covering bioprinting and additive manufacturing

Soluble Guanylate Cyclase Generation of cGMP Regulates Migration of MGE Neurons

Here we have provided evidence that nitric oxide-cyclic GMP (NO-cGMP) signaling regulates neurite length and migration of immature neurons derived from the medial ganglionic eminence (MGE). Dlx1/2(-/-) and Lhx6(-/-) mouse mutants, which exhibit MGE interneuron migration defects, have reduced expression of the gene encoding the alpha subunit of a soluble guanylate cyclase (Gucy1A3). Furthermore, Dlx1/2(-/-) mouse mutants have reduced expression of NO synthase 1 (NOS1). Gucy1A3(-/-) mice have a transient reduction in cortical interneuron number. Pharmacological inhibition of soluble guanylate cyclase and NOS activity rapidly induces neurite retraction of MGE cells in vitro and in slice culture and robustly inhibits cell migration from the MGE and caudal ganglionic eminence. We provide evidence that these cellular phenotypes are mediated by activation of the Rho signaling pathway and inhibition of myosin light chain phosphatase activity

Hand Held 3D Scanning for Cultural Heritage: Experimenting Low Cost Structure Sensor Scan.

In the last years 3D scanning has become an important resource in many fields, in particular it has played a key role in study and preservation of Cultural Heritage. Moreover today, thanks to the miniaturization of electronic components, it has been possible produce a new category of 3D scanners, also known as handheld scanners. Handheld scanners combine a relatively low cost with the advantage of the portability. The aim of this chapter is two-fold: first, a survey about the most recent 3D handheld scanners is presented. As second, a study about the possibility to employ the handheld scanners in the field of Cultural Heritage is conducted. In this investigation, a doorway of the Benedictine Monastery of Catania, has been used as study case for a comparison between stationary Time of Flight scanner, photogrammetry-based 3D reconstruction and handheld scanning. The study is completed by an evaluation of the meshes quality obtained with the three different kinds of technology and a 3D modeling reproduction of the case-study doorway

‘Coming back Home’ . Il Modello Virtuale della Statua Romana di Asclepio del Museo di Siracusa (Italia)

The colossal torso of the god Asclepius, kept into the Castello Maniace during the Spanish domination of Sicily, is now one of the most significant examples of roman statuary in the Syracuse Museum. The recent restoration of the Castello Maniace has been celebrated in 2008 with an exhibition of archaeological finds obtained in the various excavations of the castle. The statue of Asclepius, which had for centuries been a part of the architecture there, should have been the most significant piece on display. However, its large size and frailty made such a temporary move inadvisable, and it was decided that a plaster cast should be made and exhibited instead. A team of archaeologists and information technicians from the Archeomatica Project of Catania University were able to contribute to this task by creating a 3D model of the statue through the use of laser scanning techniques

The OPERA magnetic spectrometer

The OPERA neutrino oscillation experiment foresees the construction of two magnetized iron spectrometers located after the lead-nuclear emulsion targets. The magnet is made up of two vertical walls of rectangular cross section connected by return yokes. The particle trajectories are measured by high precision drift tubes located before and after the arms of the magnet. Moreover, the magnet steel is instrumented with Resistive Plate Chambers that ease pattern recognition and allow a calorimetric measurement of the hadronic showers. In this paper we review the construction of the spectrometers. In particular, we describe the results obtained from the magnet and RPC prototypes and the installation of the final apparatus at the Gran Sasso laboratories. We discuss the mechanical and magnetic properties of the steel and the techniques employed to calibrate the field in the bulk of the magnet. Moreover, results of the tests and issues concerning the mass production of the Resistive Plate Chambers are reported. Finally, the expected physics performance of the detector is described; estimates rely on numerical simulations and the outcome of the tests described above.Comment: 6 pages, 10 figures, presented at the 2003 IEEE-NSS conference, Portland, OR, USA, October 20-24, 200

Hypoxia promotes the inflammatory response and stemness features in visceral fat stem cells from obese subjects

Low-grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of 2 subpopulations of adipose-derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of 2 subpopulations of ASC from the same isolation sites of non-obese individuals. In particular, the behavior of ASC of obese vs non-obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV-ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation-related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue-specific NF-kB activation and an NF-kB-mediated increase in inflammatory and fibrogenic responses. Moreover, obV-ASC, which showed a less stem-like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV-ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF-kB inhibitors. These novel findings suggest that the deregulated hyper-responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low-grade chronic inflammation, which has been implicated in obesity-related morbidity

Integrated three-dimensional models for noninvasive monitoring and valorization of the Morgantina silver treasure (Sicily)

The Morgantina silver treasure belonging to the Archaeological Museum of Aidone (Sicily) was involved in a three-dimensional (3-D) survey and diagnostics campaign for monitoring the collection over time in anticipation of their temporary transfer to the Metropolitan Museum of Art in New York for a period of 4 years. Using a multidisciplinary approach, a scientific and methodological protocol based on noninvasive techniques to achieve a complete and integrated knowledge of the precious items and their conservation state, as well as to increase their valorization, has been developed. All acquired data, i.e., 3-D models, ultraviolet fluorescence, x-ray images, and chemical information, will be made available, in an integrated way, within a web-oriented platform, which will present an in-progress tool to deepen existing archaeological knowledge and production technologies and to obtain referenced information of the conservation state before and after moving of the collection from its exposure site