The first tissue-engineered airway transplantation: 5-year follow-up results.

BACKGROUND: In 2008, the first transplantation of a tissue-engineered trachea in a human being was done to replace an end-staged left main bronchus with malacia in a 30-year-old woman. We report 5 year follow-up results. METHODS: The patient was followed up approximately every 3 months with multidetector CT scan and bronchoscopic assessment. We obtained mucosal biopsy samples every 6 months for histological, immunohistochemical, and electron microscopy assessment. We also assessed quality of life, respiratory function, cough reflex test, and production and specificity of recipient antibodies against donor human leucocyte antigen. FINDINGS: By 12 months after transplantation, a progressive cicatricial stenosis had developed in the native trachea close to the tissue-engineered trachea anastomosis, which needed repeated endoluminal stenting. However, the tissue-engineered trachea itself remained open over its entire length, well vascularised, completely re-cellularised with respiratory epithelium, and had normal ciliary function and mucus clearance. Lung function and cough reflex were normal. No stem-cell-related teratoma formed and no anti-donor antibodies developed. Aside from intermittent bronchoscopic interventions, the patient had a normal social and working life. INTERPRETATION: These clinical results provide evidence that a tissue-engineering strategy including decellularisation of a human trachea, autologous epithelial and stem-cell culture and differentiation, and cell-scaffold seeding with a bioreactor is safe and promising. FUNDING: European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, ALF Medicine.

Endothelialization approaches for viable engineered tissues.

One of the main limitation in obtaining thick, 3-dimensional viable engineered constructs is the inability to provide a sufficient and functional blood vessel system essential for the in vitro survival and the in vivo integration of the construct. Different strategies have been proposed to simulate the ingrowth of new blood vessels into engineered tissue, such as the use of growth factors, fabrication scaffold technologies, in vivo prevascularization and cell-based strategies, and it has been demonstrated that endothelial cells play a central role in the neovascularization process and in the control of blood vessel function. In particular, different "environmental" settings (origin, presence of supporting cells, biomaterial surface, presence of hemodynamic forces) strongly influence endothelial cell function, angiogenic potential and the in vivo formation of durable vessels. This review provides an overview of the different techniques developed so far for the vascularization of tissue-engineered constructs (with their advantages and pitfalls), focusing the attention on the recent development in the cell-based vascularization strategy and the in vivo applications.

Aspirin-loaded electrospun poly(ε-caprolactone) tubular scaffolds: potential small-diameter vascular grafts for thrombosis prevention.

Thrombosis is the main cause of failure of small-diameter synthetic vascular grafts when used for by-pass procedures. The development of bioresorbable vascular scaffolds with localized and sustained intra-luminal antithrombotic drug release could be considered a desirable improvement towards a valuable solution for this relevant clinical need. For this aim, we present the fabrication and characterization of aspirin-loaded electrospun poly(ε-caprolactone) tubular scaffolds as a vascular drug-delivery graft. Three different drug concentrations were considered (i.e., 1, 5 or 10 % w/w). Although a fibrous structure was clearly observed for all the collected scaffolds, aspirin content was directly implied in the final microstructure leading to a bimodal fiber diameter distribution and fused fibers at crossing-points (5 or 10 % w/w). Mechanical response highlighted a direct relationship for modulus and stress at break with the aspirin content, while the elongation at break was not remarkably different for the investigated cases. The temporal drug release was strongly dependent from the amount of loaded aspirin, reaching a steady state release after about 50 h. Finally, the adhesion assay confirmed the capability of the electrospun scaffolds to reduce platelet adhesion/aggregation onto aspirin loaded polymeric fibers. Aspirin-loaded electrospun tubular scaffold could represent a feasible candidate to develop a novel bioresorbable drug-releasing graft for small-diameter vessel replacements.

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study.

BACKGROUND: Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS: A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 µg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS: We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION: Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING: European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Mesenchymal stromal cells for tissue-engineered tissue and organ replacements.

Mesenchymal stromal cells (MSCs), a rare heterogeneous subset of pluripotent stromal cells that can be easily isolated from different adult tissues, in vitro expanded and differentiated into multiple lineages, are immune privileged and, more important, display immunomodulatory capacities. Because of this, they are the preferred cell source in tissue-engineered replacements, not only in autogeneic conditions, where they do not evoke any immune response, but especially in the setting of allogeneic organ and tissue replacements. However, more preclinical and clinical studies are requested to completely understand MSC's immune biology and possible clinical applications. We herein review the immunogenicity and immunomodulatory properties of MSCs, their possible mechanisms and potential clinical use for tissue-engineered organ and tissue replacement.

Translating tissue-engineered tracheal replacement from bench to bedside.

There are a variety of airway diseases with different clinical settings, which may extend from a surgical approach to total organ replacement. Tissue engineering involves modifying cells or tissues in order to repair, regenerate, or replace tissue in the body and seems to be a promising approach for airway replacement. The successful implantation of stem-cell-based tissue-engineered trachea in a young woman with end-stage post-tuberculosis left main bronchus collapse serves as a prototype for the airway tissue-engineered-based approach. The trachea indeed could represent a perfect model system to investigate the translational aspects of tissue engineering, largely due to its low-oxygen needs. This review highlights the anatomy of the airways, the various disease conditions that cause damage to the airways, elaborates on the essential components of the tissue-engineering approach, and discusses the success of the revolutionary trachea transplantation approach.

In vitro astrocyte and cerebral endothelial cell response to electrospun poly(epsilon-caprolactone) mats of different architecture.

This work focuses on the evaluation of the potential use of electrospun poly(epsilon-caprolactone) (PCL) micrometric and/or sub-micrometric fibrous membranes for rat hippocampal astrocyte (HA) and rat cerebro-microvascular endothelial cell (CEC) cultures. Both mats supported cell adhesion, proliferation, cellular phenotype and spreading. Microfibrous mats allowed cellular infiltration, while both HAs and CECs were unable to migrate within the sub-micrometric fibrous mat, leaving an acellularized inner region. This finding was correlated to the presence of larger voids within electrospun PCL microfibrous mats, suggesting that the morphology should be accurately selected for the realization of a cell environment-mimicking mat. Based on our results, the proper fiber architecture can be regarded as a crucial issue to be considered in order to deal with suitable polymeric mats tailored for specific in vitro application.

Microstructure and cytocompatibility of electrospun nanocomposites based on poly(epsilon-caprolactone) and carbon nanostructures.

Carbon nanostructures (CNSs) are attractive and promising nanomaterials for the next generation of tissue engineering scaffolds, especially in neural prosthesis. Optimizing scaffold vascularization may be an important strategy to promote the repair of damaged brain tissue. In this context, the idea was to evaluate the cell response of electrospun nanohybrid scaffolds loaded with CNSs. Fibrous composites based on poly(epsilon-caprolactone) (PCL) and CNSs were fabricated by means of electrospinning technique. High-purity carbon nanofibers (CNFs) and single-wall carbon nanotubes (SWNTs) were studied. A detailed microstructural characterization was performed to evaluate the most favorable experimental conditions for the realization of fibrous PCL/CNS fabrics. Electrospun mats comprised of rather uniform and homogeneous submicrometric fibers were obtained starting from 1:1 v/v mixture of tetrahydrofuran (THF) and N,N dimethylformamide (DMF). In vitro cytocompatibility tests were performed using rat cerebro-microvascular endothelial cells (CECs). Acquired results showed an increased cell viability for PCL/CNS nanocomposites, suggesting these materials as a suitable environment for endothelial cells. These results are indicative of the promising potential of CNS-based nanocomposites in biomedical devices for tissue engineering applications where endothelial functional properties are required.

3D Printing Decellularized Extracellular Matrix to Design Biomimetic Scaffolds for Skeletal Muscle Tissue Engineering.

Functional engineered muscles are still a critical clinical issue to be addressed, although different strategies have been considered so far for the treatment of severe muscular injuries. Indeed, the regenerative capacity of skeletal muscle (SM) results inadequate for large-scale defects, and currently, SM reconstruction remains a complex and unsolved task. For this aim, tissue engineered muscles should provide a proper biomimetic extracellular matrix (ECM) alternative, characterized by an aligned/microtopographical structure and a myogenic microenvironment, in order to promote muscle regeneration. As a consequence, both materials and fabrication techniques play a key role to plan an effective therapeutic approach. Tissue-specific decellularized ECM (dECM) seems to be one of the most promising material to support muscle regeneration and repair. 3D printing technologies, on the other side, enable the fabrication of scaffolds with a fine and detailed microarchitecture and patient-specific implants with high structural complexity. To identify innovative biomimetic solutions to develop engineered muscular constructs for the treatment of SM loss, the more recent (last 5 years) reports focused on SM dECM-based scaffolds and 3D printing technologies for SM regeneration are herein reviewed. Possible design inputs for 3D printed SM dECM-based scaffolds for muscular regeneration are also suggested.

Information-Driven Design as a Potential Approach for 3D Printing of Skeletal Muscle Biomimetic Scaffolds.

Severe muscle injuries are a real clinical issue that still needs to be successfully addressed. Tissue engineering can represent a potential approach for this aim, but effective healing solutions have not been developed yet. In this regard, novel experimental protocols tailored to a biomimetic approach can thus be defined by properly systematizing the findings acquired so far in the biomaterials and scaffold manufacturing fields. In order to plan a more comprehensive strategy, the extracellular matrix (ECM), with its properties stimulating neomyogenesis and vascularization, should be considered as a valuable biomaterial to be used to fabricate the tissue-specific three-dimensional structure of interest. The skeletal muscle decellularized ECM can be processed and printed, e.g., by means of stereolithography, to prepare bioactive and biomimetic 3D scaffolds, including both biochemical and topographical features specifically oriented to skeletal muscle regenerative applications. This paper aims to focus on the skeletal muscle tissue engineering sector, suggesting a possible approach to develop instructive scaffolds for a guided healing process.

3D printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and characterization.

Additive manufacturing for tissue engineering applications offers the possibility to design scaffolds characterized by a fine and detailed microarchitecture. Several fabrication technologies are currently available which allow to prepare tailored structures with a large selection of materials for restoring and healing tissues. However, 3D printed scaffolds are generally collected by assembling repetitive geometrical units or reproducing specific patterns in the layering direction, leading to a highly ordered architecture that does not mimic the morphology of the natural extracellular matrix (ECM), one of the main goals to be reached for an effective therapeutic approach. It is usually stated in the tissue engineering field that a scaffold has to be considered a temporary ECM, resembling all the peculiar features as close as possible and, in this regard, an ordered microstructure cannot be usually observed within biological tissues and organs. With the aim to overcame this limitation and offer a potential approach for bone tissue applications, the present study proposes a design methodology to fabricate 3D printed scaffolds characterized by a random microarchitecture which can be repeatedly reproduced thanks to the intrinsic controllable process of additive manufacturing. In this framework, four different models in polylactic acid were fabricated by means of fused deposition modelling, including a three-dimensional random distribution of spherical pores of 400, 500, and 600 µm for the first three cases, and a randomly varied distribution in the range 400-600 µm for the fourth case. A detailed assessment by means of microcomputed tomography and mechanical evaluation was then carried out in order to fully analyse the resulting scaffolds, providing both morphological and quantitative data.

Involvement of the receptor for advanced glycation-end products (RAGE) in beta-amyloid-induced toxic effects in rat cerebromicrovascular endothelial cells cultured in vitro.

To ascertain whether the potential biological effects of beta amyloid (betaA) on the endothelium are partly mediated by the receptor for advanced glycation-end products (RAGE), we performed a series of experiments which analyzed the effects of the betaA(1-42) peptide on in vitro cerebromicrovascular endothelial cells (CECs). Our results suggest that RAGE is directly responsible for betaA(1-42) actions on CECs, such as its toxic effect on cell survival, viability and angiogenic capability. We observed that a 6-h incubation period exposing CECs to betaA(1-42) increased the extracellular levels of nitrite. Furthermore, the presence of a nitric oxide synthase inhibitor, L-NAME, was able to enhance CEC survival and viability. Immunocytochemical analyses demonstrated that the peptide induced expression of the inducible form of NOS, iNOS, typically synthesized in response to immune/inflammatory stimuli. Upon blocking the interaction of betaA(1-42) and RAGE, we observed significantly decreased levels of NO and suppression of iNOS immunoreactivity. In conclusion, our data suggest the involvement of RAGE, at least partly, in mediating the effects of betaA(1-42) on CECs. In particular, the decrease of in vitro cell viability and functionality and nitrosative stress activation was inhibited by blocking betaA(1-42)-RAGE interaction.

Raman Spectroscopy and Aptamers for a Label-Free Approach: Diagnostic and Application Tools.

Raman spectroscopy is a powerful optical technique based on the inelastic scattering of incident light to assess the chemical composition of a sample, including biological ones. Medical diagnostic applications of Raman spectroscopy are constantly increasing to provide biochemical and structural information on several specimens, being not affected by water interference, and potentially avoiding the constraint of additional labelling procedures. New strategies have been recently developed to overcome some Raman limitations related, for instance, to the need to deal with an adequate quantity of the sample to perform a reliable analysis. In this regard, the use of metallic nanoparticles, the optimization of fiber optic probes, and other approaches can actually enhance the signal intensity compared to spontaneous Raman scattering. Moreover, to further increase the potential of this investigation technique, aptamers can be considered as a valuable means, being synthetic, short, single, or double-stranded oligonucleotides (RNAs or DNAs) that fold up into unique 3D structures to specifically bind to selected molecules, even at very low concentrations, and thus allowing an early diagnosis of a possible disease. Due to the paramount relevance of the topic, this review focuses on the main Raman spectroscopy techniques combined with aptamer arrays in the label-free mode, providing an overview on different applications to support healthcare management.

Publisher Correction: Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats.

This corrects the article DOI: 10.1038/ncomms4562.

Comparative effects of Abeta(1-42)-Al complex from rat and human amyloid on rat endothelial cell cultures.

Metal ions are widely recognized as a key factor for the conformational changes and aggregation of the Alzheimer's disease amyloid (Abeta). So far Al(3+) has received much less attention than other biometals in terms of interaction with Abeta. Brain endothelial cells have been identified as important regulators of the neuronal microenvironment, including Abeta levels. The purpose of this study is to compare the effects of the complex amyloid (Abeta(1-42))-Al, from human and rat, with the effects produced by metal-free Abeta on rat neuroendothelial cells (NECs). To establish Abeta and Abeta-Al toxicity on NECs, survival, vitality, and angiogenesis are evaluated. Cell survival is reduced by human and rat Abeta in a time-dependent manner. This toxic effect is remarkably pronounced in the presence of human Abeta-Al. Moreover, rat Abeta has anti-angiogenic properties on NECs, and this effect is aggravated dramatically by using both human and rat Abeta-Al complexes. The data and arguments presented herein clearly demonstrate the involvement of Al(3+) in Abeta aggregation and, consequently, increasing endothelial cell toxicity.

Electrospun polyphosphazene nanofibers for in vitro rat endothelial cells proliferation.

A large variety of natural and synthetic polymers have been explored as scaffolds for the seeding and growth of different types of cells. To fabricate a scaffold that can be used as a synthetic extracellular matrix (ECM), it is important to replicate the nanoscale dimensions of natural ECM. The electrospinning process allows to produce ultrathin fibers so that this method represents a suitable approach to scaffold fabrication for tissue engineering applications. In this work, the feasibility of obtaining flat or tubular matrices from biocompatible poly[(ethyl phenylalanato)(1.4) (ethyl glycinato)(0.6) phosphazene] by electrospinning was evaluated and the effect of process parameters on the diameter of nanofibers was examined. The adhesion and growth of rat neuromicrovascular endothelial cells cultured on sheets and tubes composed by the polymer with an average fiber diameter of 850 +/- 150 nm were also reported. Microscopic examination of the seeded tubes demonstrated that, after 16 days of incubation, endothelial cells formed a monolayer on the whole surface. These results are the first step to demonstrate that tubes of biodegradable polyphosphazenes might be a feasible model to construct human tissues such as vessels or cardiac valves.

Caspase-8 activation and oxidative stress are involved in the cytotoxic effect of beta-amyloid on rat brain microvascular endothelial cells.

Several studies have demonstrated that cerebrovascular dysfunction and damage play a significant role in the pathogenesis of Alzheimer disease (AD). In fact, beta-amyloid peptides (Abetas), the major component of the senile plaques and cerebrovascular amyloid deposits in AD, were shown to be cytotoxic to endothelial cells. We have recently observed that Abetas exert a toxic effect on neuromicrovascular endothelial cells (NECs) in a time- and concentration-dependent manner, apoptosis playing a pivotal role in this process. Hence, it seemed worthwhile to investigate the Abeta-mediated apoptosis mechanism in NECs. Abetas were found to induce, after a short incubation period, apoptosis throughout caspase-8 activation. Moreover, Abetas elicited a highly significant (p < 0.001) increase in superoxide dismutase (SOD) levels after a 3-h exposure period, while SOD concentration was not affected after a 24-h incubation. The time-dependent increase in SOD concentration is probably correlated with the production of an excess of reactive oxygen species. Collectively, our findings allow us to conclude that: i) Abetas may induce apoptosis via the activation of caspase-8, presumably by cross-linking and activating receptors of the death-receptor family; ii) oxidative stress is possibly involved in the Abeta-induced cytotoxic effect; and iii) these two mechanisms do not act sequentially but, probably, are independent of each other.

Apolipoprotein-E modulates the cytotoxic effect of beta-amyloid on rat brain endothelium in an isoform-dependent specific manner.

Several studies support the hypothesis that apolipoprotein-E (ApoE) acts as a pathological chaperone protein that promotes the beta-plated sheet conformation of beta-amyloid (Abeta) peptides into amyloid fibers. In vitro evidence is also available that ApoE inhibits the neurotoxic effect of Abeta in an allele-specific manner (E2 > or = E3 > E4). We have recently shown that Abeta peptides exert a time- and concentration-dependent toxic effect on rat neuromicrovascular endothelial cells (NECs), and this study aimed to ascertain whether ApoE isoforms are able to modulate this effect. ApoE2 and ApoE4 decreased and increased, respectively, the cytotoxic effect of Abeta(1-40) and Abeta(1-42) on NECs, as evaluated by their survival and viability rates. The toxic effect of both Abeta peptides and ApoE4 was associated with the rise in the necrosis rate of NECs within a 24-h incubation period. Moreover, ApoE2 prevented and ApoE4 magnified the inhibitory effect of Abeta on the capability of NECs cultured on Matrigel to form a capillary-like network. The opposite effects of ApoE isoforms could be due to their different interactions with the C-terminal domain of Abeta. ApoE2, at variance with ApoE4, is thought to form sodium dodecyl sulphate-stable complexes with Abeta and, as a consequence, it could block the interactions of the non-fibrillar Abeta peptide with the plasma membrane, Abeta peptide aggregation and the ensuing cytotoxicity. Collectively, our findings confirm the view that ApoE plays a relevant role in the pathogenesis of Alzheimer's disease.

Vinblastine inhibits the angiogenic response induced by adrenomedullin in vitro and in vivo.

Adrenomedullin (ADM) is protumorigenic by stimulating tumor cell growth and angiogenesis. In this context, ADM is identified as a novel target for antiangiogenic therapy. In this study, we addressed the possibility that vinblastine (VBL), as demonstrated in other experimental conditions, may act as an angiostatic molecule in the angiogenic response induced by ADM in two assays, such as Matrigel tube formation in vitro and angiogenesis in the chick embryo chorioallantoic membrane (CAM) in vivo. When tested on Matrigel, ADM caused a morphogenetic effect. In fact, endothelial cells spread and aligned with each other to form branching anastomosing tubes with multicentric junctions that gave rise to a meshwork of capillary-like structures. When ADM was administered in the presence of VBL, the capillary-like tubes were interrupted, most cells were spherical, either isolated or aggregated in small clumps. In the CAM assay, ADM induced a strong angiogenic response, which was counteracted by the treatment with VBL. Overall, these observations implicate ADM as a promoter of tumor growth and a possible target for anticancer strategies, such as the use of VBL at very low, nontoxic doses. Nevertheless, the antiangiogenic activity of low-dose VBL deserves further investigation, alone or together with other antiangiogenic agents for the treatment of tumors characterized by enhanced angiogenesis.