Conjugated polymers mediate intracellular Ca2+ signals in circulating endothelial colony forming cells through the reactive oxygen species-dependent activation of Transient Receptor Potential Vanilloid 1 (TRPV1).

Endothelial colony forming cells (ECFCs) represent the most suitable cellular substrate to induce revascularization of ischemic tissues. Recently, optical excitation of the light-sensitive conjugated polymer, regioregular Poly (3-hexyl-thiophene), rr-P3HT, was found to stimulate ECFC proliferation and tube formation by activating the non-selective cation channel, Transient Receptor Potential Vanilloid 1 (TRPV1). Herein, we adopted a multidisciplinary approach, ranging from intracellular Ca2+ imaging to pharmacological manipulation and genetic suppression of TRPV1 expression, to investigate the effects of photoexcitation on intracellular Ca2+ concentration ([Ca2+]i) in circulating ECFCs plated on rr-P3HT thin films. Polymer-mediated optical excitation induced a long-lasting increase in [Ca2+]i that could display an oscillatory pattern at shorter light stimuli. Pharmacological and genetic manipulation revealed that the Ca2+ response to light was triggered by extracellular Ca2+ entry through TRPV1, whose activation required the production of reactive oxygen species at the interface between rr-P3HT and the cell membrane. Light-induced TRPV1-mediated Ca2+ entry was able to evoke intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors, followed by store-operated Ca2+ entry on the plasma membrane. These data show that TRPV1 may serve as a decoder at the interface between rr-P3HT thin films and ECFCs to translate optical excitation in pro-angiogenic Ca2+ signals.

A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs.

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.

Whole rat stomach decellularisation using a detergent-enzymatic protocol.

BACKGROUND: Conditions leading to reduced gastric volume are difficult to manage and are associated to poor quality-of-life. Stomach augmentation using a tissue-engineered stomach is a potential solution to restore adequate physiology and food reservoir. Aim of this study was to evaluate the decellularisation of whole rat stomach using a detergent-enzymatic protocol. METHODS: Stomachs harvested from rats were decellularised through luminal and vascular cannulation using 24-h detergent-enzymatic treatment and completely characterized by appropriate staining, DNA and Extracellular matrix -component quantifications. RESULTS: The detergent-enzymatic protocol allows a complete decellularisation of the gastric tissue, with a complete removal of the DNA with two cycles as confirmed by both quantifications and histological analysis. Extracellular matrix components, collagen, fibronectin, laminin and elastin, were optimally preserved by the treatment, while glycosaminoglycans were reduced. CONCLUSION: Gastric tissue can be efficiently decellularised. Scaffolds retained original structure and important components that could enhance integration with other tissues for in vivo transplant. The use of naturally derived material could be potentially considered for the treatment of both congenital and acquired conditions.

Whole Organ Tissue Vascularization: Engineering the Tree to Develop the Fruits.

Tissue engineering aims to regenerate and recapitulate a tissue or organ that has lost its function. So far successful clinical translation has been limited to hollow organs in which rudimental vascularization can be achieved by inserting the graft into flaps of the omentum or muscle fascia. This technique used to stimulate vascularization of the graft takes advantage of angiogenesis from existing vascular networks. Vascularization of the engineered graft is a fundamental requirement in the process of engineering more complex organs, as it is crucial for the efficient delivery of nutrients and oxygen following in-vivo implantation. To achieve vascularization of the organ many different techniques have been investigated and exploited. The most promising results have been obtained by seeding endothelial cells directly into decellularized scaffolds, taking advantage of the channels remaining from the pre-existing vascular network. Currently, the main hurdle we need to overcome is achieving a fully functional vascular endothelium, stable over a long time period of time, which is engineered using a cell source that is clinically suitable and can generate, in vitro, a yield of cells suitable for the engineering of human sized organs. This review will give an overview of the approaches that have recently been investigated to address the issue of vascularization in the field of tissue engineering of whole organs, and will highlight the current caveats and hurdles that should be addressed in the future.

Terminal sterilization of equine-derived decellularized tendons for clinical use.

In the last few years, the demand for tissue substitutes has increased and decellularized matrices has been widely proposed in the medical field to restore severe damages thanks to high biocompatibility and biomechanical properties similar to the native tissues. However, biological grafts represent a potential source of contamination and disease transmission; thus, there is the need to achieve acceptable levels of sterility. Several sterilization methods have been investigated with no consensus on the outcomes in terms of minimizing structural damages and preserving functional features of the decellularized matrix for transplantation in humans. With the aim of making decellularized tendons safe for clinical use, we evaluated the cytocompatibility, and biochemical, structural and biomechanical variations of decellularized equine tendons sterilized with peracetic acid or ß-irradiation and differently wet- or dry- stored at 4°C or -80°C, respectively. Considering that both sterilization and long-term storage are crucial steps that could not be avoided, our results pointed at ionizing ß-rays as terminal sterilization method for decellularized grafts followed by frozen dry storage. Indeed, this approach can maintain the integrity of collagen-based structures and can avoid biomechanical changes, thus making xenogeneic decellularized tendons a promising candidate for clinical use.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

BACKGROUND: In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. METHODS: A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. RESULTS: The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. CONCLUSIONS: The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

Arterial Decellularized Scaffolds Produced Using an Innovative Automatic System.

There is still an unmet clinical need for small-caliber artery substitution. Decellularized scaffolds in tissue engineering represent a promising solution. We have developed an innovative system for the automatic decellularization of blood vessels, used to process pig arteries. The system is able to automatically drive a decellularization process in a safe and reliable environment, with complex time patterns, using up to three different decellularization solutions, and providing at the same time a physical stress to improve the decellularization. The decellularization of pig arteries was evaluated by means of histology, DNA quantification and mechanical testing. Outcomes showed scaffolds with no cellular or nuclear remnants and a well-preserved tissue structure, corroborated by mechanical properties similar to native tissue. Decellularized scaffolds were seeded on the inner layer with human endothelial cells and implanted as iliac artery replacement in 4 pharmacologically immune-compromised pigs. This chimeric model was performed as a very preliminary evaluation to investigate the performances of these scaffolds in vivo, and to investigate the fate of seeded cells. Recipients were sacrificed on day 14 and day 70 after surgery, and vessels were found to be patent and with no evidence of thrombi formation. The inner layer was covered by endothelial cells, and the migration of cells positive for α-smooth-muscle actin was observed from the outer layer towards the tunica media. Intriguingly, the endothelial cells on explanted vessels were entirely derived from the host while the seeded cells were lost. In conclusion, this work presents a novel tool for a safe and controlled production of arterial scaffolds, with good decellularization outcomes and a good performance in a short-term, large-animal implantation.

Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering.

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at -80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Young's modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.

A novel device for the automatic decellularization of biological tissues.

OBJECTIVES: Decellularized biological scaffolds represent a promising solution for tissue engineering. They offer a good substrate for cells in terms of biochemical composition, ultrastructure and mechanical properties without generating an immunogenic response. The aim of this study was to design and develop a device for the automatic decellularization of biological tissues to overcome manual operation limits, toward a good manufacturing practice-compliant process. METHODS: A versatile, modular and easy-to-use device was designed, able to automatically exchange decellularization fluids and to provide mechanical shaking according to a user-defined protocol. Preliminary decellularization tests were made on porcine abdominal aortas comparing results between conventional process and device-operated process using water, sodium deoxycholate and DNase. Vessels were processed up to 4 cycles of the protocol and after each decellularization cycle histological analyses (hematoxylin-eosin, Movat pentachrome and DAPI stainings) were observed. Preliminary mechanical tests were also performed to compare the mechanical behavior of blood vessels processed with the 2 methods mentioned above. RESULTS: Briefly, the device consists of decellularization chambers, a shaking system and hydraulic modules for the exchange of fluids. The device was bench-tested for functionality and reliability with positive outcomes. The protocol used revealed to be effective, with a progressive tissue decellularization through repeated cycles. No difference between manual and automated operation was observed in histological or mechanical analyses. CONCLUSIONS: The developed device is able to automate the decellularization process lowering operator-related risks, and is a reliable and functional tool for clinical use.