Cappelletti S, Caimi A, Caldiroli A, Baroni I, Votta E, Riboldi SA, Marrocco-Trischitta MM, Redaelli A, Sturla F. Non-invasive estimation of vascular compliance and distensibility in the arm vessels: a novel ultrasound-based protocol. Quant Imaging Med Surg. 2022 Jul;12(7):3515-3527. doi: 10.21037/qims-21-987. PMID: 35782271; PMCID: PMC9246759. Abstract 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.

Infrared scattering-type scanning near-field optical microscopy (IR s-SNOM) and imaging is here exploited together with attenuated total reflection (ATR) IR imaging and scanning electron microscopy (SEM) to depict the chemical composition of fibers in hybrid electrospun meshes. The focus is on a recently developed bio-hybrid material for vascular tissue engineering applications, named Silkothane®, obtained in the form of nanofibrous matrices from the processing of a silk fibroin-polyurethane (SFPU) blend via electrospinning. Morphology and chemistry of single fibers, at both surface and subsurface level, have been successfully characterized with nanoscale resolution, taking advantage of the IR s-SNOM capability to portray the nanoscale depth profile of this modern material working at diverse harmonics of the signal. The applied methodology allowed to describe the superficial characteristics of the mesh up to a depth of about 100 nm, showing that SF and PU do not tend to co-aggregate to form hybrid fibers, at least at the length scale of hundreds of nanometers, and that subdomains other than the fibrillar ones can be present. More generally, in the present contribution, the depth profiling capabilities of IR s-SNOM, so far theoretically predicted and experimentally proven only on model systems, have been corroborated on a real material in its natural conditions with respect to production, opening the room for the exploitation of IR s-SNOM as valuable technique to support the production and the engineering of nanostructured materials by the precise understanding of their chemistry at the interface with the environment.

BackgroundPerformance 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.MethodsA 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.ResultsFor 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).ConclusionsThe 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.

To address the need of alternatives to autologous vessels for small-calibre vascular applications (e.g. cardiac surgery), a bio-hybrid semi-degradable material composed of silk fibroin (SF) and polyurethane (Silkothane®) was herein used to fabricate very small-calibre grafts (Ø in = 1.5 mm) via electrospinning. Bio-hybrid grafts were in vitro characterized in terms of morphology and mechanical behaviour, and compared to similar grafts of pure SF. Similarly, two native vessels from a rodent model (abdominal aorta and vena cava) were harvested and characterized. Preliminary implants were performed on Lewis rats to confirm the suitability of Silkothane® grafts for small-calibre applications, specifically as aortic insertion and femoral shunt. The manufacturing process generated pliable grafts consisting of a randomized fibrous mesh and exhibiting similar geometrical features to rat aortas. Both Silkothane® and pure SF grafts showed radial compliances in the range from 1.37 ± 0.86 to 1.88 ± 1.01% 10 −2 mmHg −1 , lower than that of native vessels. The Silkothane® small-calibre devices were also implanted in rats demonstrating to be adequate for vascular applications; all the treated rats survived the surgery for three months after implantation, and 16 rats out of 17 (94%) still showed blood flow inside the graft at sacrifice. The obtained results lay the basis for a deeper investigation of the interaction between the Silkothane® graft and the implant site, which may deal with further analysis on the potentialities in terms of degradability and tissue formation, on longer time-points.

BACKGROUNDIn an attempt at answering the need for a valuable and durable hemodialysis vascular access, a novel semi‐degradable hybrid vascular graft, manufactured by electrospinning using silk fibroin and polyurethane (Silkothane®), has been developed and characterized in vitro according to standards.OBJECTIVESIn this proof‐of‐principle animal study, we aimed at evaluating the performances of Silkothane® grafts in a sheep model of arteriovenous shunt, with respect to patency and short‐term graft remodeling.MATERIALS AND METHODSNine Silkothane® grafts (6mm inner diameter, ≈7cm long, ≈400μm thick) were surgically placed into the left cervical fossa of nine female sheep, between the common carotid artery and the external jugular vein. During the housing period the animals were treated with an inhibitor of platelets aggregation. Grafts were examined by palpation three times per week, and by duplex ultrasound every two weeks to exclude significant stenosis, dilation, presence of thromboses, neointimal proliferation or other complicating features. The sheep were euthanized at 30, 60 and 90 days (N=3 for each group). At sacrifice, grafts were harvested, fixed, and submitted for histologic, immunohistochemical and Scanning Electron Microscopy examination.RESULTSNo cases of graft‐related complications (e.g. seroma formation, transgraft bleeding, aneurismal dilation, calcification, structural yielding) were recorded in this study. One graft failed due to a thrombotic event likely related to a technical error (11%). One sheep had post‐operative wound infection, surgically resolved (11%). Four out of nine sheep (44%) showed post‐operative edema, usually disappearing in 7 days. Eight of nine sheep (89%) showed 100% primary unassisted patency at the respective time of sacrifice, as confirmed by palpation and ultrasound evaluation (average flow rate 1.76 ± 0.61 l/min). Histological, immunoistochemical and SEM analysis evidenced signs of inflammation and formation of neointimal hyperplasia at the venous anastomosis, as it typically happens with arteriovenous fistulae and synthetic grafts.CONCLUSIONSIn our ovine model of arteriovenous shunt, Silkothane® grafts provided evidences of safety and efficacy in granting 100% patency up to 90 days. Further studied need to be conducted in order to directly compare the novel grafts with commercially available synthetic grafts.

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.

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.

The cover image, by G. Talò et al., is based on the Research Article Industrialization of a perfusion bioreactor: Prime example of a non-straightforward process, DOI: 10.1002/term.2480. This work was partially funded by POR FESR 2007–2013 resources (grant ID: ATP 2009, No. 13396272”.

Bioreactors are essential enabling technologies for the translation of advanced therapies medicinal products from the research field towards a successful clinical application. In order to speed up the translation and the spread of novel tissue engineering products into the clinical routine, tissue engineering bioreactors should evolve from laboratory prototypes towards industrialized products. In this work, we thus challenged the industrialization process of a novel technological platform, based on an established research prototype of perfusion bioreactor, following a GMP-driven approach. We describe how the combination of scientific background, intellectual property, start-up factory environment, wise industrial advice in the biomedical field, design, and regulatory consultancy allowed us to turn a previously validated prototype technology into an industrial product suitable for serial production with improved replicability and user-friendliness. The solutions implemented enhanced aesthetics, ergonomics, handling, and safety of the bioreactor, and they allowed compliance with the fundamental requirements in terms of traceability, reproducibility, efficiency, and safety of the manufacturing process of advanced therapies medicinal products. The result is an automated incubator-compatible device, housing 12 disposable independent perfusion chambers for seeding and culture of any perfusable tissue. We validated the cell seeding process of the industrialized bioreactor by means of the Design of Experiment approach, whilst the effectiveness of perfusion culture was evaluated in the context of bone tissue engineering.

In the last years, the interest in bioreactors is constantly increasing in several fields of bioengineering and for various applications, thanks to the progresses in technology, engineering, stem cell biology, and material science. At different levels and for various applications bioreactors can be used in research as actuator systems (i.e., to apply complex stimuli to cells in dynamic culture), as model systems to study the development of biological tissues, or as systems to monitor the developmental parameters. Exploiting the potentiality of bioreactors, the biomedical industry invested in developing reliable and automated devices for 2D or 3D cell culture, having specific features such as modularity, scalability, and process traceability, to make bioreactors essential tools for the development of safe and reproducible biological constructs and for the study of new therapeutic drugs. Thanks to the efforts of researchers and industry, bioreactors can potentially be applied in the clinics for the development of products for Advanced Therapies, acting as automatic platforms for the economically and clinically sustainable fabrication of bioprocesses and cell products. This chapter is focused on the analysis of the translation of bioreactors from research to clinical application, providing information on the fundamental features and issues, as well as practical examples of devices design at each stage of development.

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.

Purpose: We previously reported that a cocktail of bone morphogenetic protein (BMP)-2, insulin and triiodothyronine (BIT) could trigger redifferentiation of human chondrocytes after their amplification on plastic, with cartilage-characteristic matrix reconstruction when the chondrocytes were seeded in collagen sponges (Claus et al., 2011). However, this matrix was not homogenously distributed in the scaffolds, most likely because the collagen sponges were cultivated in static conditions. With the aim of enhancing cellular access to nutrients and the soluble factors, bi-directional flow was tested to perfuse the scaffolds during cartilage reconstruction. Methods: After 3 weeks of amplification on plastic, HAC were seeded then cultivated for 21 days in collagen sponges under bi-directional flow, by using a prototype of OPB (Oscillating Perfusion Bioreactor). We established a program of perfusion including phases of high and low perfusion speeds to alternate sequences of cell stimulation and matrix deposition. For comparison, cultures of HAC in collagen sponges were performed in static conditions. The status of the chondrocyte phenotype and the nature of the matrix synthesized in collagen sponges were evaluated by real time PCR, Western Blotting and immunohistochemistry analyses. The viability and cell proliferation were also monitored. Results: The results clearly indicate that perfusion improves cartilage matrix deposition within the sponges, in comparison with static conditions. More precisely, in the sponges cultured in the bioreactor, redifferentiated and metabolically active HAC produced a cartilaginous matrix rich in type II and type IX collagens and in glycosaminoglycans, with no sign of hypertrophy. Interestingly, a much lower amount of type I collagen was produced in the sponges cultivated in dynamic conditions, indicating therefore that bi-directional perfusion limits the risk of fibrocartilage formation. Conclusions: The combination of HAC, collagen sponges, and the BIT cocktail with the bi-directional perfusion bioreactor favors the reconstruction of hyaline cartilage. Importantly, bi-directional perfusion abolishes the spatial concentration gradients routinely observed in scaffolds in static culture. This study also demonstrates the value of a multi-factorial approach for the design of cell-based grafts for cartilage repair.

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell-seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactonetricalcium phosphate (PCLTCP) scaffolds increases human bone marrow stromal cells (hBMSCs) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCLTCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSCs directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSCs under perfusion for 3weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non-crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture. Copyright (c) 2011 John Wiley & Sons, Ltd.

In this chapter, the functions and potential applicability of bioreactors from a technical, scientific and clinical perspective will be reviewed in the context of tissue engineering and regenerative medicine. In particular, examples will be given to illustrate the role of bioreactors in (a) establishing and maintaining 3D cell cultures, (b) standardizing physicochemical culture parameters, (c) physically conditioning engineered grafts, (d) predicting mechanical functionality of constructs to be implanted, (e) automating conventional tissue culture processes, (f) streamlining tissue manufacturing strategies. The critical role of bioreactors to make tissue engineered products clinically accessible, safe and commercially competitive will finally be discussed.