Non-invasive estimation of vascular compliance and distensibility in the arm vessels: a novel ultrasound-based protocol.

Background: Performance and durability of arterio-venous grafts depend on their ability to mimic the mechanical behavior of the anastomized blood vessels. To select the most suitable synthetic graft, in vivo evaluation of the radial deformability of peripheral arteries and veins could be crucial; however, a standardized non-invasive strategy is still missing. Herein, we sought to define a novel and user-friendly clinical protocol for in vivo assessment of the arm vessel deformability. Methods: A dedicated protocol, applied on 30 volunteers, was specifically designed to estimate both compliance and distensibility of the brachial and radial arteries, and of the basilic and cephalic veins. Bi-dimensional ultrasound imaging was used to acquire cross-sectional areas (CSAs) of arteries in clinostatic configuration, and CSAs of veins combining clinostatic and orthostatic configurations. Arterial pulse pressure was measured with a digital sphygmomanometer, while venous hydrostatic pressure was derived from the arm length in orthostatic configuration. Results: For each participant, all CSAs were successfully extracted from ultrasound images. The basilic vein and the radial artery exhibited the largest (21.5±8.9 mm2) and the smallest (3.4±1.0 mm2) CSAs, respectively; CSA measurements were highly repeatable (Bland-Altman bias <10% and Pearson correlation ≥0.90, for both arteries and veins). In veins, compliance and distensibility were higher than in arteries; compliance was significantly higher (P<0.0001) in the brachial than in the radial artery (3.52×10-4 vs. 1.3×10-4 cm2/mmHg); it was three times larger in basilic veins than in cephalic veins (17.4×10-4 vs. 5.6×10-4 cm2/mmHg, P<0.0001). Conclusions: The proposed non-invasive protocol proved feasible, effective and adequate for daily clinical practice, allowing for the estimation of patient-specific compliance and distensibility of peripheral arteries and veins. If further extended, it may contribute to the fabrication of biohybrid arterio-venous grafts, paving the way towards patient-tailored solutions for vascular access.

A novel hybrid silk-fibroin/polyurethane three-layered vascular graft: towards in situ tissue-engineered vascular accesses for haemodialysis.

Clinically available alternatives of vascular access for long-term haemodialysis-currently limited to native arteriovenous fistulae and synthetic grafts-suffer from several drawbacks and are associated to high failure rates. Bioprosthetic grafts and tissue-engineered blood vessels are costly alternatives without clearly demonstrated increased performance. In situ tissue engineering could be the ideal approach to provide a vascular access that profits from the advantages of vascular grafts in the short-term (e.g. early cannulation) and of fistulae in the long-term (e.g. high success rates driven by biointegration). Hence, in this study a three-layered silk fibroin/polyurethane vascular graft was developed by electrospinning to be applied as long-term haemodialysis vascular access pursuing a 'hybrid' in situ engineering approach (i.e. based on a semi-degradable scaffold). This Silkothane® graft was characterized concerning morphology, mechanics, physical properties, blood contact and vascular cell adhesion/viability. The full three-layered graft structure, influenced by the polyurethane presence, ensured mechanical properties that are a determinant factor for the success of a vascular access (e.g. vein-graft compliance matching). The Silkothane® graft demonstrated early cannulation potential in line with self-sealing commercial synthetic arteriovenous grafts, and a degradability driven by enzymatic activity. Moreover, the fibroin-only layers and extracellular matrix-like morphology, presented by the graft, revealed to be crucial in providing a non-haemolytic character, long clotting time, and favourable adhesion of human umbilical vein endothelial cells with increasing viability after 3 and 7 d. Accordingly, the proposed approach may represent a step forward towards an in situ engineered hybrid vascular access with potentialities for vein-graft anastomosis stability, early cannulation, and biointegration.

Electrospun fibroin/polyurethane hybrid meshes: Manufacturing, characterization, and potentialities as substrates for haemodialysis arteriovenous grafts.

Several attempts made so far to combine silk fibroin and polyurethane, in order to prepare scaffolds encompassing the bioactivity of the former with the elasticity of the latter, suffer from critical drawbacks concerning industrial and clinical applicability (e.g., separation of phases upon processing, use of solvents unaddressed by the European Pharmacopoeia, and use of degradable polyurethanes). Overcoming these limitations, in this study, we report the successful blending of regenerated silk fibroin with a medical-grade, non-degradable polyurethane using formic acid and dichloromethane, and the manufacturing of hybrid, semi-degradable electrospun tubular meshes with different ratios of the two materials. Physicochemical analyses demonstrated the maintenance of the characteristic features of fibroin and polyurethane upon solubilization, blending, electrospinning, and postprocessing with ethanol or methanol. Envisioning their possible application as semidegradable substrates for haemodialysis arteriovenous grafts, tubular meshes were further characterized, showing submicrometric fibrous morphologies, tunable mechanical properties, permeability before and after puncture in the same order of magnitude as commercial grafts currently used in the clinics. Results demonstrate the potential of this material for the development of hybrid, new-generation vascular grafts with disruptive potential in the field of in situ tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 807-817, 2019.

Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges.

Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage.

Cyclic mechanical stimulation favors myosin heavy chain accumulation in engineered skeletal muscle constructs.

PURPOSE: Since stretching plays a key role in skeletal muscle tissue development in vivo, by making use of an innovative bioreactor and a biodegradable microfibrous scaffold (DegraPol(R)) previously developed by our group, we aimed to investigate the effect of mechanical conditioning on the development of skeletal muscle engineered constructs, obtained by seeding and culturing murine skeletal muscle cells on electrospun membranes. METHODS: Following 5 days of static culture, skeletal muscle constructs were transferred into the bioreactor and further cultured for 13 days, while experiencing a stretching pattern adapted from the literature to resemble mouse development and growth. Sample withdrawal occurred at the onset of cyclic stretching and after 7 and 10 days. Myosin heavy chain (MHC) accumulation in stretched constructs (D) was evaluated by Western blot analysis and immunofluorescence staining, using statically cultured samples (S) as controls. RESULTS: Western blot analysis of MHC on dynamically (D) and statically (S) cultured constructs at different time points showed that, at day 10, the applied stretching pattern led to an eight-fold increase in myosin accumulation in cyclically stretched constructs (D) with respect to the corresponding static controls (S). These results were confirmed by immunofluorescence staining of total sarcomeric MHC. CONCLUSIONS: Since previous attempts to reproduce skeletal myogenesis in vitro mainly suffered from the difficulty of driving myoblast development into an architecturally organized array of myosin expressing myotubes, the chance of inducing MHC accumulation via mechanical conditioning represents a significant step towards the generation of a functional muscle construct for skeletal muscle tissue engineering applications.

Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing.

Over the last decade, we have witnessed an increased recognition of the importance of 3D culture models to study various aspects of cell physiology and pathology, as well as to engineer implantable tissues. As compared to well-established 2D cell-culture systems, cell/tissue culture within 3D porous biomaterials has introduced new scientific and technical challenges associated with complex transport phenomena, physical forces, and cell-microenvironment interactions. While bioreactor-based 3D model systems have begun to play a crucial role in addressing fundamental scientific questions, numerous hurdles currently impede the most efficient utilization of these systems. We describe how computational modeling and innovative sensor technologies, in conjunction with well-defined and controlled bioreactor-based 3D culture systems, will be key to gain further insight into cell behavior and the complexity of tissue development. These model systems will lay a solid foundation to further develop, optimize, and effectively streamline the essential bioprocesses to safely and reproducibly produce appropriately scaled tissue grafts for clinical studies.

Electrospun degradable polyesterurethane membranes: potential scaffolds for skeletal muscle tissue engineering.

Skeletal muscle tissue engineering represents an attractive approach to overcome problems associated with autologous transfer of muscle tissue and provides a valid alternative in muscle regeneration enhancement. The aim of this study was to investigate the suitability, as scaffold for skeletal muscle tissue engineering, of a known biodegradable block copolymer (DegraPol) processed by electrospinning in the novel form of microfibrous membranes. Scaffolds were characterized with reference to their morphological, degradative and mechanical properties. Subsequently, cell viability, adhesion and differentiation on coated and uncoated DegraPol) slides were investigated using line cells (C2C12 and L6) and primary human satellite cells (HSCs). The membranes exhibited absence of toxic residuals and satisfactory mechanical properties (linear elastic behavior up to 10% deformation, E modulus in the order of magnitude of MPa). A promising cellular response was also found in preliminary experiments: both line cells and HSCs adhered, proliferated and fused on differently coated electrospun membranes. Positive staining for myosin heavy chain expression indicated that differentiation of C2C12 multinucleated cells occurred within the porous elastomeric substrate. Together the results of this study provide significant evidence of the suitability of electrospun DegraPol) membranes as scaffolds for skeletal muscle tissue engineering and that they represent a promising alternative to scaffolds currently used in this field.