Heparin-Modified Collagen Gels for Controlled Release of Pleiotrophin: Potential for Vascular Applications

A fast re-endothelialization, along with the inhibition of neointima hyperplasia, are crucial to reduce the failure of vascular bypass grafts. Implants modifications with molecules capable of speeding up the re-endothelialization process have been proposed over the last years. However, clinical trials of angiogenic factor delivery have been mostly disappointing, underscoring the need to investigate a wider array of angiogenic factors. In this work, a drug release system based on a type I collagen hydrogel has been proposed for the controlled release of Pleiotrophin (PTN), a cytokine known for its pro-angiogenetic effects. Heparin, in virtue of its ability to sequester, protect and release growth factors, has been used to better control the release of PTN. Performances of the PTN drug delivery system on endothelial (ECs) and smooth muscle cells (SMCs) have been investigated. Structural characterization (mechanical tests and immunofluorescent analyses of the collagen fibers) was performed on the gels to assess if heparin caused changes in their mechanical behavior. The release of PTN from the different gel formulations has been analyzed using a PTN-specific ELISA assay. Cell viability was evaluated with the Alamar Blue Cell Viability Assay on cells directly seeded on the gels (direct test) and on cells incubated with supernatant, containing the released PTN, obtained from the gels (indirect test). The effects of the different gels on the migration of both ECs and SMCs have been evaluated using a Transwell migration assay. Hemocompatibility of the gel has been assessed with a clotting/hemolysis test. Structural analyses showed that heparin did not change the structural behavior of the collagen gels. ELISA quantification demonstrated that heparin induced a constant release of PTN over time compared to other conditions. Both direct and indirect viability assays showed an increase in ECs viability while no effects were noted on SMCs. Cell migration results evidenced that the heparin/PTN-modified gels significantly increased ECs migration and decreased the SMCs one. Finally, heparin significantly increased the hemocompatibility of the collagen gels. In conclusion, the PTN-heparin-modified collagen here proposed can represent an added value for vascular medicine, able to ameliorate the biological performance, and integration of vascular grafts

Role of mechanical stretching in the modulation of myocytes phenotype: implications for tissue engineering

The phenotype of myocytes is regulated by various stimuli, including mechanical environment (Davis-Dusembery et al., 2011; Lu et al., 2011). Several studies examined the role of mechanical strain on myogenesis in skeletal muscle cells, but the mechanisms that dictate the effects of cyclic strain on myocytes phenotype are still not understood (Simmons et al., 2004). Cellular responses to mechanical stress depend on to the substrate deformation, frequency and duration of the applied mechanical stress (Kook et al., 2008). Physiologic mechanical stimuli may affect the properties of the tissues, leading also to the development of several pathologies. In this work, we studied the effects of different cyclic strains on C2C12 myoblasts phenotype. Cellular mechanisms involved in the mechanical stress-mediated modulation of myogenesis or osteogenesis were considered. In particular, low (2%) and high (15%) substrate deformations were applied and cell proliferation and differentiation markers (Myf5, Myogenin, Osteopontine, ALP) were observed by RT-PCR and western blot analyses. Results showed that cell phenotype switches from myogenic to osteogenic, depending on the dynamic conditions applied. In particular, the myogenic differentiation was inhibited through the down-regulation of muscle specific markers, and the up-regulation of the osteogenetic phenotype markers

Collagen-based tissue engineering strategies for vascular medicine

Cardiovascular diseases (CVDs) account for the 31% of total death per year, making them the first cause of death in the world. Atherosclerosis is at the root of the most life-threatening CVDs. Vascular bypass/replacement surgery is the primary therapy for patients with atherosclerosis. The use of polymeric grafts for this application is still burdened by high-rate failure, mostly caused by thrombosis and neointima hyperplasia at the implantation site. As a solution for these problems, the fast re-establishment of a functional endothelial cell (EC) layer has been proposed, representing a strategy of crucial importance to reduce these adverse outcomes. Implant modifications using molecules and growth factors with the aim of speeding up the re-endothelialization process has been proposed over the last years. Collagen, by virtue of several favorable properties, has been widely studied for its application in vascular graft enrichment, mainly as a coating for vascular graft luminal surface and as a drug delivery system for the release of pro-endothelialization factors. Collagen coatings provide receptor-ligand binding sites for ECs on the graft surface and, at the same time, act as biological sealants, effectively reducing graft porosity. The development of collagen-based drug delivery systems, in which small-molecule and protein-based drugs are immobilized within a collagen scaffold in order to control their release for biomedical applications, has been widely explored. These systems help in protecting the biological activity of the loaded molecules while slowing their diffusion from collagen scaffolds, providing optimal effects on the targeted vascular cells. Moreover, collagen-based vascular tissue engineering substitutes, despite not showing yet optimal mechanical properties for their use in the therapy, have shown a high potential as physiologically relevant models for the study of cardiovascular therapeutic drugs and diseases. In this review, the current state of the art about the use of collagen-based strategies, mainly as a coating material for the functionalization of vascular graft luminal surface, as a drug delivery system for the release of pro-endothelialization factors, and as physiologically relevant in vitro vascular models, and the future trend in this field of research will be presented and discussed

Short-term effects of microstructured surfaces: role in cell differentiation toward a contractile phenotype

Cell adhesion plays a key role in cell behavior, in terms of migration, proliferation, differentiation and apoptosis. All of these events concur with tissue regeneration and remodeling mechanisms, integrating a complex network of intracellular signaling modules. Morphogenetic responses, which involve changes in cell shape, proliferation and differentiation, are thought to be controlled by both biochemical and biophysical cues. Indeed, the extracellular matrix not only displays adhesive ligands necessary for cell adhesion but also plays an essential biomechanical role - responsible, for instance, for the acquisition of the contractile phenotype. The substrate topography around the forming tissues and the associated mechanical stresses that are generated regulate cellular morphology, proliferation and differentiation. Thus, the ability to tailor topographical features around cells can be a crucial design parameter in tissue engineering applications, inducing cells to exhibit the required performances.In this work, we designed micropillared substrates using highly spaced arrays (interspacing equal to 25 µm) to evaluate the effects of topography on C2C12 myoblasts' adhesion and differentiation. Optical and fluorescence microscopy images were used to observe cell adhesion, together with Western blot analysis on vinculin and focal adhesion kinase (FAK) expression, a protein highly involved in adhesive processes. Differentiation marker (Myf5, myogenin and myosin heavy chain [MHC]) expression was also studied, in relation to the effect of different substrate topographies on the enhancement of a contractile phenotype. Our results demonstrated that microstructured surfaces may play a key role in the regeneration of functional tissues

A Gut-Ex-Vivo System to Study Gut Inflammation Associated to Inflammatory Bowel Disease (IBD)

Inflammatory bowel disease (IBD) is a complex, chronic, and dysregulated inflammatory condition which etiology is still largely unknown. Its prognosis and disease progression are highly variable and unpredictable. IBD comprises several heterogeneous inflammatory conditions ranging from Ulcerative Colitis (UC) to Crohn's Disease (CD). Importantly, a definite, well-established, and effective clinical treatment for these pathologies is still lacking. The urgent need for treatment is further supported by the notion that patients affected by UC or CD are also at risk of developing cancer. Therefore, a deeper understanding of the molecular mechanisms at the basis of IBD development and progression is strictly required to design new and efficient therapeutic regimens. Although the development of animal models has undoubtedly facilitated the study of IBD, such in vivo approaches are often expensive and time-consuming. Here we propose an organ ex vivo culture (Gut-Ex-Vivo system, GEVS) based on colon from Balb/c mice cultivated in a dynamic condition, able to model the biochemical and morphological features of the mouse models exposed to DNBS (5-12 days), in 5 h. Indeed, upon DNBS exposure, we observed a dose-dependent: (i) up-regulation of the stress-related protein transglutaminase 2 (TG2); (ii) increased intestinal permeability associated with deregulated tight junction protein expression; (iii) increased expression of pro-inflammatory cytokines, such as TNFα, IFNγ, IL1β, IL6, IL17A, and IL15; (iv) down-regulation of the anti-inflammatory IL10; and (v) induction of Endoplasmic Reticulum stress (ER stress), all markers of IBD. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of IBD, in a time- and cost-effective manner

Mechanisms involved in the cross-talk between humoral and mechanical cues underlying muscle wasting in cachexia

Introduction. Exercise training improves quality of life and survival of cancer patients. In an animal model of cancer cachexia we demonstrated that wheel running counteracts cachexia by releasing the autophagic flux. Exercise pleitropic effects include the alteration of circulating factors in favour of an anti-inflammatory environment and the activation of mechanotransduction pathways in muscle cells. Our goal is to assess whether mechanostransduciton per se is sufficient to elicit exercise effects in the presence of pro-cachectic factors of tumor origin. Serum response factor (SRF) is a transcription factor of pivotal importance for muscle homeostasis, which is activated with its co-factor MRTF by mechanostranduction in a way dependent on actin polymerisation. Methods. We use C26 tumor-bearing mice, in the absence or presence of wheel running, and mixed cultures of C2C12 myotubes and myoblasts treated with C26 conditioned medium (CM) in the absence or presence of cyclic stretch to mimic the mechanical stimulation occurring upon exercise. Results. In vivo both SRF expression and activity are differentially modulated by the C26 tumor, i.e. by humoral factors, and by exercise. In vitro we showed that CM had a negative effect on muscle cell cultures, both in terms of myotube atrophy and of myoblast recruitment and fusion, and that these effects were counteracted by cyclic stretch. We showed that CM repressed SRF-MRTF transcriptional activity, while mechanical stretch rescued their transcriptional activity; in addition, loss of function experiments demonstrated that SRF was necessary to mediate the beneficial effects of mechanical stimulation on muscle cells. At least part of the observed effects were mediated by the balance of pro- and anti-myogenic factor of the TGFbeta superfamily. Conclusions. We propose that the positive effects of exercise on cancer patients and mice may be specifically due to a mechanical response of muscle fibers affecting the secretion of myokines

Global summer schools: developing multicultural competencies staying at home

International summer schools are a well know approach to improve technical skills as well as to develop multicultural competencies (Lerke, 2020). University students benefit particularly from these as they can also learn specialist knowledge which might inspire them to pursue a specific research topic or to consider options abroad for further education or employment after graduation. The global disruption of COVID-19 prevented international summer schools in 2020 and 2021, and continues to restrict international travel, with some higher education institutions holding a stricter policy to protect students and staff, limiting travel until further notice. A virtual alternative global summer school was coordinated by an international team of universities: Unidad Profesional Interdisciplinaria de Biotecnologia (UPIBI-IPN, Mexico), Escuela Nacional de Educacion Superior-Juriquilla (ENES-Juriquilla, Mexico), Universita de Piemonte Orientale-Novara (UPO-Novara, Italy) and Manchester Metropolitan University (MMU, UK) to enable students in biomed/biotech/bioeng areas, to experience a virtual summer school, supporting their employability while fostering international academic collaborations between departments. The aim was to deliver an online summer school that would provide an insight into the latest research projects, as much as, allow students to learn about other cultures. The programme was planned for 66% for scientific talks, and 33% cultural talks, the latter including virtual tours of cities, live food sessions, recorded videos of participants performing folkloric dances and introduction to languages. The programme ran over four days, each hosted by a different institution. Prior institutional experience on blended teaching and virtual collaborations allowed the team to run the online school with appropriate technologies to coordinate questions and polls to monitor students’ experience and assess their learning. The official language of the programme was English; speakers, chairs and coordinators connected via zoom, with sessions being watched by 200long-term viewers in Facebook live and Youtube live. Sessions were interactive with quizzes and polls using Kahoot and Vevox, the latter, allowed real time data analysis to compare the perception of students about their multicultural and scientific experience. Finally, students were assessed on the scientific content with a final VLE MCQ, which also allowed one institution (UPO) to award credits for a module, and another institution (MMU) to provide extracurricular points for their RISE Award. Student engagement was constant and feedback showed their satisfaction for attending a summer school that not only exposed them to high-quality international research, but also to other cultures; with data showing 66% of students enjoyed cultural sessions as much as scientific talks (n=35±10). In addition, 95% of students confirmed that they were more likely to travel to one of the hosting countries, and 93% more likely to learn one of the hosting languages after the global summer school, indicating a strong correlation between a virtual international activity, and an inclusive attitude and new-found multicultural proficiency. Despite the success, our current structure should evolve to include virtual spaces to facilitate group work and students’ interactions (de Hei, 2020) to assist intercultural learning and complement the high-quality scientific engagement and the rich cultural exchange