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

Biocompatible electrospun polycaprolactone-polyaniline scaffold treated with atmospheric plasma to improve hydrophilicity

Conductive polymers (CPs) have recently been applied in the development of scaffolds for tissue engineering applications in attempt to induce additional cues able to enhance tissue growth. Polyaniline (PANI) is one of the most widely studied CPs, but it requires to be blended with other polymers in order to be processed through conventional technologies. Here, we propose the fabrication of nanofibers based on a polycaprolactone (PCL)-PANI blend obtained using electro-spinning technology. An extracellular matrix-like fibrous substrate was obtained showing a good stability in the physiological environment (37 °C in PBS solution up 7 days). However, since the high hydrophobicity of the PCL-PANI mats (133.5 ± 2.2°) could negatively affect the biological re-sponse, a treatment with atmospheric plasma was applied on the nanofibrous mats, obtaining a hydrophilic surface (67.1 ± 2°). In vitro tests were performed to confirm the viability and the physi-ological-like morphology of human foreskin fibroblast (HFF-1) cells cultured on the plasma treated PCL-PANI nanofibrous scaffolds

Wound dressing products: A translational investigation from the bench to the market

Chronic skin wounds affect more than 40 million patients globally and represent a severe growing burden for the healthcare systems, with annual costs expected to exceed $15 billions by 2022. To satisfy the huge demand for effective wound care products, different types of wound dressings have been introduced on the market during the last decades. Based on “the moist wound healing theory” postulated by Prof Winter in 1962, bandages were initially designed to recreate the optimal wound environment to favor the healing process. Then, thanks to the advancements achieved in biomaterial design and processing, biotechnology, imaging and electronic fields, great effort has been devoted to the development of formulations able to actively participate to tissue healing. Indeed, both the literature and the market report the design of medicated wound dressings, i.e., wound care products releasing anti-microbial agents, anti-inflammatory drugs, or bioactive molecules. In this scenario, this review aims at critically describing the currently available wound care products, highlighting their proved effectiveness in wound management. Moreover, an overview of the main strategies exploited to design personalized wound dressings has been reported. Lastly, concerns on regulatory affairs and practical issues limiting the clinical translation of advanced research platforms have also been discussed

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

In Vitro Models of Bacterial Biofilms: Innovative Tools to Improve Understanding and Treatment of Infections

Bacterial infections are a growing concern to the health care systems. Bacteria in the human body are often found embedded in a dense 3D structure, the biofilm, which makes their eradication even more challenging. Indeed, bacteria in biofilm are protected from external hazards and are more prone to develop antibiotic resistance. Moreover, biofilms are highly heterogeneous, with properties dependent on the bacteria species, the anatomic localization, and the nutrient/flow conditions. Therefore, antibiotic screening and testing would strongly benefit from reliable in vitro models of bacterial biofilms. This review article summarizes the main features of biofilms, with particular focus on parameters affecting biofilm composition and mechanical properties. Moreover, a thorough overview of the in vitro biofilm models recently developed is presented, focusing on both traditional and advanced approaches. Static, dynamic, and microcosm models are described, and their main features, advantages, and disadvantages are compared and discussed

Towards 3d multi-layer scaffolds for periodontal tissue engineering applications: Addressing manufacturing and architectural challenges

Reduced periodontal support, deriving from chronic inflammatory conditions, such as periodontitis, is one of the main causes of tooth loss. The use of dental implants for the replacement of missing teeth has attracted growing interest as a standard procedure in clinical practice. However, adequate bone volume and soft tissue augmentation at the site of the implant are important prerequisites for successful implant positioning as well as proper functional and aesthetic reconstruction of patients. Three-dimensional (3D) scaffolds have greatly contributed to solve most of the challenges that traditional solutions (i.e., autografts, allografts and xenografts) posed. Nevertheless, mimicking the complex architecture and functionality of the periodontal tissue represents still a great challenge. In this study, a porous poly(ε-caprolactone) (PCL) and Sr-doped nano hydroxyapatite (Sr-nHA) with a multi-layer structure was produced via a single-step additive manufacturing (AM) process, as a potential strategy for hard periodontal tissue regeneration. Physicochemical characterization was conducted in order to evaluate the overall scaffold architecture, topography, as well as porosity with respect to the original CAD model. Furthermore, compressive tests were performed to assess the mechanical properties of the resulting multi-layer structure. Finally, in vitro biological performance, in terms of biocompatibility and osteogenic potential, was evaluated by using human osteosarcoma cells. The manufacturing route used in this work revealed a highly versatile method to fabricate 3D multi-layer scaffolds with porosity levels as well as mechanical properties within the range of dentoalveolar bone tissue. Moreover, the single step process allowed the achievement of an excellent integrity among the different layers of the scaffold. In vitro tests suggested the promising role of the ceramic phase within the polymeric matrix towards bone mineralization processes. Overall, the results of this study demonstrate that the approach undertaken may serve as a platform for future advances in 3D multi-layer and patient-specific strategies that may better address complex periodontal tissue defects

Listeria monocytogenes contamination of Tenebrio molitor larvae rearing substrate: Preliminary evaluations

Today, edible insects represent a hot topic as an emerging and eco-friendly source of protein. The mealworm (Tenebrio molitor L.) is among the most employed insects for human consumption and feed purposes. So far Listeria monocytogenes, have never been detected either in products sold on the market or during the rearing process. In this study, the substrate employed for mealworm rearing was deliberately contaminated with L. monocytogenes and the bacterium was enumerated during the rearing period and after technological treatments of the larvae. L. monocytogenes persisted during the rearing period. Washing the larvae did not produce any significant effect, while fasting the larvae for 24 or 48 h reduced the L. monocytogenes load (P &lt; 0.001). Oven cooking eliminated L. monocytogenes cells from the product, reducing the risk associated to this foodborne pathogen to zero