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Organ dysfunction is a major cause of morbidity and mortality. Transplantation is typically the only definitive cure, challenged by the lack of sufficient donor organs. Tissue engineering encompasses the development of biomaterial scaffolds to support cell attachment, proliferation, and differentiation, leading to tissue regeneration. For efficient clinical translation, the forming technology utilized must be suitable for mass production. Herein, uniaxial polyhydroxyalkanoate scaffolds manufactured by pressurized gyration, a hybrid scalable spinning technique, are successfully used in bone, nerve, and cardiovascular applications. Chorioallantoic membrane and in vivo studies provided evidence of vascularization, collagen deposition, and cellular invasion for bone tissue engineering. Highly efficient axonal outgrowth was observed in dorsal root ganglion-based 3D ex vivo models. Human induced pluripotent stem cell derived cardiomyocytes exhibited a mature cardiomyocyte phenotype with optimal calcium handling. This study confirms that engineered polyhydroxyalkanoate-based gyrospun fibers provide an exciting and unique toolbox for the development of scalable scaffolds for both hard and soft tissue regeneration.

Bioelectronic materials based on conjugated polymers are being developed in the hope to interface with electroresponsive tissues. We have recently demonstrated that a polyaniline chitosan patch can efficiently electro-couple with cardiac tissue modulating its electrophysiology. As a promising bioelectronic material that can be tailored to different types of devices, we investigate here the impact of varying the synthesis conditions and time of the in situ polymerization of aniline (An) on the sheet resistance of the bioelectronic patch. The sheet resistance increases significantly for samples that have either the lowest molar ratio of oxidant to monomer or the highest molar ratio of dopant to monomer, while the polymerization time does not have a significant effect on the electrical properties. Conductive atomic force microscopy reveals that the patch with the lowest sheet resistance has a connected network of the conductive phase. In contrast, patches with higher sheet resistances exhibit conductive areas of lower current signals or isolated conductive islands of high current signals. Having identified the formulation that results in patches with optimal electrical properties, we used it to fabricate patches that were implanted in rats for two weeks. It is shown that the patch retains an electroactive nature, and only mild inflammation is observed with fibrous tissue encapsulating the patch.

Cardiac tissue engineering (CTE) is currently a prime focus of research due to an enormous clinical need. In this work, a novel functional material, Poly(3-hydroxyoctanoate), P(3HO), a medium chain length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Our results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of the P(3HO) neat patches, assessed using Neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated to the patches. Also, no deleterious effect was observed on the adults cardiomyocytes’ contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO) based multifunctional cardiac patches are promising constructs for efficient CTE. This work will provide a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials.

In cardiac tissue engineering (TE), in vitro models are essential for the study of healthy and pathological heart tissues in order to understand the underpinning mechanisms. In this scenario, scaffolds are platforms that can realistically mimic the natural architecture of the heart, and they add biorealism to in vitro models. This paper reports a novel and robust technique to fabricate cardiovascular-mimetic scaffolds based on Parylene C and Polydimethylsiloxane (PDMS). Parylene C is employed as a mask material for inducing hybrid and non-hybrid micropatterns to the PDMS layer. Hybrid architectures present striped hydrophobic/hydrophilic surfaces, whereas non-hybrid scaffolds only corrugated topographies. Herein, we demonstrate that wavy features on PDMS can be obtained at the micro- and nanoscale and that PDMS can be integrated into the microfabrication process without changing its intrinsic physical properties. A study of the effects of these scaffolds on the growth of Neonatal Rat Ventricular Myocytes (NRVMs) cultures reveals that cell alignment occurs only for the case of hybrid architectures made of hydrophilic PDMS and hydrophobic Parylene C.

Hydrogels are promising materials for mimicking the extra-cellular environment. Here, we present a simple methodology for the formation of a free-standing viscoelastic hydrogel from the abundant and low cost protein serum albumin. We show that the mechanical properties of the hydrogel exhibit a complicated behaviour as a function of the weight fraction of the protein component. We further use X-ray scattering to shed light on the mechanism of gelation from the formation of a fibrillary network at low weight fractions to interconnected aggregates at higher weight fractions. Given the match between our hydrogel elasticity and that of the myocardium, we investigated its potential for supporting cardiac cells in vitro. Interestingly, these hydrogels support the formation of several layers of myocytes and significantly promote the maintenance of a native-like gene expression profile compared to those cultured on glass. When confronted with a multicellular ventricular cell preparation, the hydrogels can support macroscopically contracting cardiac-like tissues with a distinct cell arrangement, and form mm-long vascular-like structures. We envisage that our simple approach for the formation of an elastic substrate from an abundant protein makes the hydrogel a compelling biomedical material candidate for a wide range of cell types.

We recently demonstrated that patterned Parylene C films could be effectively used as a mask for directly copolymerizing proteins on polyacrylamide hydrogel (PAm). In this work, we have proved the applicability of this technique for studying the effect such platforms render on neonatal rat ventricular myocytes (NRVMs). Firstly, we have characterised topographically and mechanically the scaffolds in liquid at the nano-scale level. We thus establish that such platforms have physical properties that closely mimics the in vivo extracellular environment of cells. We have then studied the cell morphology and physiology by comparing cultures on flat uniformly-covered and collagen-patterned scaffolds. We show that micro-patterns promote the elongation of cells along the principal axis of the ridges coated with collagen. In several cases, cells also tend to create bridges across the grooves. We have finally studied cell contraction, monitoring Ca2+ cycling at a certain stimulation. Cells seeded on patterned scaffolds present significant responses in comparison to the isotropic ones.

The effect of O2 and Ar plasma etching on poly(chloro‐p‐xylylene) (Parylene C) is thoroughly studied by atomic force microscopy, X‐ray photoelectron spectroscopy, and static contact angle measurements. Results indicate that O2 plasma changes the topography more drastically than Ar plasma. Furthermore, despite the fact that Ar plasma is expected to be chemically inert, both plasmas introduce O2 to the surface of the Parylene C films, while Ar plasma additionally reduces the amount of Cl present in the polymer. The effect on the viability of cultured cardiomyocytes is also examined, indicating that cells attach and survive both on Ar and O2 treated films in contrast to untreated Parylene. These observations can provide useful insight into the field of material science and tissue engineering.

The application of tissue engineered patches made of conductive polymer scaffolds combined with cardiomyocytes (CMs) could provide a dual method of improving the damaged myocardium after an infarction: firstly by introducing functional CMs to the area; secondly the conductive polymer could modulate electrical transmission across the scar tissue. Polyaniline (PANI) scaffolds are one such example, however, the consequences of growing CMs on conductive PANI scaffolds with regards to CM electrophysiology are unknown. In this study we assess the hypothesis that conductive PANI scaffolds affect CM calcium transients and action potential morphology in culture. Neonatal rat ventricular myocytes (NRVMs) and neonatal rat fibroblasts were co-cultured on conductive and non-conductive (sodium hydroxide treated) PANI scaffolds and remained viable after four days of culture, covering the surface of the construct. Compared to those cultured on non-conductive PANI scaffolds, NRVM cultured on conductive PANI scaffolds show faster calcium transients, measured using Fluo-4AM and field stimulated at 1 Hz, with a decrease in the time to peak (t p non-conductive=105±6 ms, t p conductive= 85±5 ms, p50 non-conductive=212±12 ms, t 50 conductive= 116±7 ms, p90 non-conductive=404±24 ms, t 90 conductive= 266±15 ms, p

Tissue engineering provides a promising method of introducing functional cardiomyocytes (CMs) to damaged myocardium after myocardial infarction; however, finding a biocompatible construct with the chemical and mechanical properties capable of supporting CM function is challenging. Serum albumin hydrogels are novel autogenic scaffolds with elastic properties that can be tailored to mimic the stiffness of native adult myocardium. We assessed the hypothesis that culturing immature CMs on these serum albumin hydrogels would affect CM gene expression and calcium handling. Neonatal cardiomyocyte (NRVM) viability was maintained for at least 14 days on the hydrogels, with clear sarcomeric striations. Cardiac gene expression was quantified using RT-qPCR and demonstrated an up regulation in many genes of cells cultured on hydrogels compared to glass (e.g. relative expression (log 2-ΔΔCt) of ryanodine receptor 2: glass= -2.3±0.5, hydrogel= -0.3±0.1,pin vivo for cardiac repair.

Conjugated polymers have been proposed as promising materials for scaffolds in tissue engineering applications. However, the restricted processability and biodegradability of conjugated polymers limit their use for biomedical applications. Here we synthesized a block-co-polymer of aniline tetramer and PCL (AT–PCL), and processed it into fibrous non-woven scaffolds by electrospinning. We showed that fibronectin (Fn) adhesion was dependent on the AT–PCL oxidative state, with a reduced Fn unfolding length on doped membranes. Furthermore, we demonstrated the cytocompatibility and potential of these membranes to support the growth and osteogenic differentiation of MC3T3-E1 cells over 21 days.

Cell micropatterning has certainly proved to improve the morphological and physiological properties of cardiomyocytes in vitro; however, there is little knowledge on the single cell-scaffold interactions that influence the cells' development and differentiation in culture. In this study, we employ hydrophobic/hydrophilic micropatterned Parylene C thin films (2-10 μm) as cell microscaffolds that can control the morphology and microtubule density of neonatal rat ventricular myocytes (NRVM) by regulating their adhesion area on Parylene through a thickness-dependent hydrophobicity. Structured NRVM on thin films tend to bridge across the hydrophobic areas, demonstrating a more spread-out shape and sparser microtubule organization, while cells on thicker films adopt a cylindrical (in vivo-like) shape (contact angles at the level of the nucleus are 64.51° and 84.73°, respectively) and a significantly (p

Current platforms for in vitro drug development utilize confluent, unorganized monolayers of heart cells to study the effect on action potential propagation. However, standard cell cultures are of limited use in cardiac research, as they do not preserve important structural and functional properties of the myocardium. Here we present a method to integrate a scaffolding technology with multi-electrode arrays and deliver a compact, off-the-shelf monitoring platform for growing biomimetic cardiac tissue. Our approach produces anisotropic cultures with conduction velocity (CV) profiles that closer resemble native heart tissue; the fastest impulse propagation is along the long axis of the aligned cardiomyocytes (CVL) and the slowest propagation is perpendicular (CVT), in contrast to standard cultures where action potential propagates isotropically (CVL ≈ CVT). The corresponding anisotropy velocity ratios (CVL/CVT = 1.38 – 2.22) are comparable with values for healthy adult rat ventricles (1.98 – 3.63). The main advantages of this approach are that (i) it provides ultimate pattern control, (ii) it is compatible with automated manufacturing steps and (iii) it is utilized through standard cell culturing protocols. Our platform is compatible with existing read-out equipment and comprises a prompt method for more reliable CV studies.