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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.

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.

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.

The repair and reconstruction of bile ducts damaged by disease or trauma remains a vexing medical problem. In particular, surgeons have few options when it comes to a long segment reconstruction of the bile duct. Tissue-engineered substitutes, with properties similar to the native tissue, might represent a solution for the functional reconstruction of bile ducts. In particular, decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the structural and residual biochemical properties of the extracellular matrix are still to be found. This paper presents the characterization of rat bile ducts after implementing an established detergent-enzymatic decellularization method. One cycle was enough to generate a complete decellularized bile duct matrix, histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers, found in the native bile duct matrix was well preserved. Moreover, the decellularization approach did not affect the elastin content.

A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial- and muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi.

Nature Communications 5: Article number: 3562 (2014); Published 15 April 2014; Updated 21 March 2017 This Article is retracted by the authors. Nature Communications previously issued an Editorial Expression of Concern (http://www.nature.com/articles/ncomms13310) related to this Article, following the publication of a report commissioned by The Karolinska Institute and prepared by the Expert Group for Misconduct in Research at the Swedish Central Ethical Review Board.

Tissue-engineered tracheal transplants have been successfully performed clinically. However, before becoming a routine clinical procedure, further preclinical studies are necessary to determine the underlying mechanisms of in situ tissue regeneration. Here we describe a protocol using a tissue engineering strategy and orthotopic transplantation of either natural decellularized donor tracheae or artificial electrospun nanofiber scaffolds into a rat model. The protocol includes details regarding how to assess the scaffolds' biomechanical properties and cell viability before implantation. It is a reliable and reproducible model that can be used to investigate the crucial aspects and pathways of in situ tracheal tissue restoration and regeneration. The model can be established in

Decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the biochemical and structural properties of the extracellular matrix are still to be found. For this aim, bioreactors could be an alternative means to dynamically treat the biological samples, automatically controlling all the variables involved in the process and speeding up the entire procedure in order to deal with a suitable scaffold within a limited time period. This paper presents the characterization of rat tracheae decellularized in dynamic conditions, implementing a detergent-enzymatic method, previously considered. Only 6 cycles were enough to generate a tracheal matrix that was histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers was well preserved, such as the epithelial cilia, the luminal basement membrane and the main matrix components. The elastin content decreased, even if not significantly, after the decellularization protocol. Mechanical properties of the treated tissues were slightly affected by the procedure, and were partially recovered after crosslinking with genipin, a naturally-derived agent. The use of bioreactors could enhance the decellularization procedure of tissues/organs, but a careful selection of the processing parameters is needed in order to prevent large modifications compared to the native condition.

The development of tracheal scaffolds fabricated based on electrospinning technique by applying different ratios of polyethylene terephthalate (PET) and polyurethane (PU) is introduced here. Prior to clinical implantation, evaluations of biomechanical and morphological properties, as well as biocompatibility and cell adhesion verifications are required and extensively performed on each scaffold type. However, the need for bioreactors and large cell numbers may delay the verification process during the early assessment phase. Hence, we investigated the feasibility of performing biocompatibility verification using static instead of dynamic culture. We performed bioreactor seeding on 3-dimensional (3-D) tracheal scaffolds (PET/PU and PET) and correlated the quantitative and qualitative results with 2-dimensional (2-D) sheets seeded under static conditions. We found that an 8-fold reduction for 2-D static seeding density can essentially provide validation on the qualitative and quantitative evaluations for 3-D scaffolds. In vitro studies revealed that there was notably better cell attachment on PET sheets/scaffolds than with the polyblend. However, the in vivo outcomes of cell seeded PET/PU and PET scaffolds in an orthotopic transplantation model in rodents were similar. They showed that both the scaffold types satisfied biocompatibility requirements and integrated well with the adjacent tissue without any observation of necrosis within 30 days of implantation.

Summary 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.

Transplantation of tissues and organs is currently the only available treatment for patients with end-stage diseases. However, its feasibility is limited by the chronic shortage of suitable donors, the need for life-long immunosuppression, and by socioeconomical and religious concerns. Recently, tissue engineering has garnered interest as a means to generate cell-seeded three-dimensional scaffolds that could replace diseased organs without requiring immunosuppression. Using a regenerative approach, scaffolds made by synthetic, nonimmunogenic, and biocompatible materials have been developed and successfully clinically implanted. This strategy, based on a viable and ready-to-use bioengineered scaffold, able to promote novel tissue formation, favoring cell adhesion and proliferation, could become a reliable alternative to allotransplatation in the next future. In this article, tissue-engineered synthetic substitutes for tubular organs (such as trachea, esophagus, bile ducts, and bowel) are reviewed, including a discussion on their morphological and functional properties. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 2427–2447, 2014

The clinical outcome of transplantations of bioartificial tissues and organs depends on the presence of living cells. There are still no standard operative protocols that are simple, fast and reliable for confirming the presence of viable cells on bioartificial scaffolds prior to transplantation. By using mathematical modeling, we have developed a colorimetric-based system (colorimetric scale bar) to predict the cell viability and density for sufficient surface coverage. First, we refined a method which can provide information about cell viability and numbers in an in vitro setting: i) immunohistological staining by Phalloidin/DAPI and ii) a modified colorimetric cell viability assay. These laboratory-based methods and the developed colorimetric-based system were then validated in rat transplantation studies of unseeded and seeded tracheal grafts. This was done to provide critical information on whether the graft would be suitable for transplantation or if additional cell seeding was necessary. The potential clinical impact of the colorimetric scale bar was confirmed using patient samples. In conclusion, we have developed a robust, fast and reproducible colorimetric tool that can verify and warrant viability and integrity of an engineered tissue/organ prior to transplantation. This should facilitate a successful transplantation outcome and ensure patient safety.

Editorial Expression of Concern: Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats

Background. We prospectively evaluated a clinical and endoscopic score, the tracheal endoscopic clinical score (TECS), developed as a disease-specified outcome measure in adult patients undergoing operation for subglottic stenosis. We also performed a retrospective chart review to identify preoperative and intraoperative risk factors for worse TECS. Methods. The TECS includes endoscopic (vocal cord and glottic function, anastomotic healing, and patency) and interview (respiration, voice, swallow) variables, and was administered at 6-month follow-up. Endoscopic and subjective domains were weighted to obtain a continuous TECS index ranging from 0 (best) to 1 (worse). The TECS and preoperative variables relationships were evaluated by univariate and multivariate analysis. Results. We collected data (January 2009 to December 2010) from 30 patients (mean age, 48.3 19 years) undergoing subglottic resection and primary reconstruction. Stenosis etiology was postintubation (n 8), idiopathic (n 2), tracheostomy (n 18), and malignant (n 2). Surgery included Pearson operation with (n 7) or without (n 23) a Liberman-Mathisen cricoplasty. Mean length of resected trachea was 30.5 13.5 mm, and mean hospital stay was 7.4 days. Mortality rate was 1 patient (3.3%). The univariate analysis showed positive correlation between 6-month TECS and degree of stenosis (McCaffrey and Cotton scale 0 to 4) stage 4, tracheostomy or T-tube at surgery, bottleneck-type transition stenosis, and resection length. At multivariate analysis, the presence of tracheostomy, bottleneck-type transition stenosis and resection length were indicators of worse postoperative functional result. Conclusions. The TECS seems to be a valid and simple instrument to identify preoperative variables predicting worse results and to assess postoperative outcome. Validation on larger series is necessary. (Ann Thorac Surg 2012;94:1065‐72) © 2012 by The Society of Thoracic Surgeons

We investigated whether decellularized pig tracheas could regenerate in vivo, without being recellularized before transplantation, using the own body as bioreactor. Decellularized pig tracheal scaffolds were intraoperative conditioned with mononuclear cells and growth and differentiation factors. During the postoperative period, the in situ regeneration was boosted by administering bioactive molecules to promote peripheral mobilization and differentiation of stem/progenitor cells and ultimately the regenerative process. Results revealed, after 2 weeks, a nearly normal trachea, with respiratory epithelium and a double-banded cartilage but without any mechanical differences compared to the native tissue. The growth factor administration resulted in a mobilization of progenitor and stem cells into the peripheral circulation and in an up-regulation of anti-apoptotic genes. Isolated stem/progenitor cells could be differentiated in vitro into several cell types, proving their multipotency. We provide evidence that the own body can be used as bioreactor to promote in vivo tissue engineering replacement. Moreover, we demonstrated the beneficial effect of additional pharmaceutical intervention for an improved engraftment of the transplant.

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro . Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture – in particular of collagenous and elastic fiber structure –and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo , having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.

In tissue engineering approach, the scaffold plays a key role for a suitable outcome of cell-scaffold interactions and for the success of tissue healing and regeneration. As a consequence, the characterization of scaffold properties and the in vivo evaluation of tissue responses and effects result to be essential in the development of suitable implantable device. Among the in vivo methods, the chick embryo chorioallantoic membrane (CAM) assay represents a rather simple and cost-effective procedure to study the biocompatibility responses of graft materials. CAM is indeed characterized by low experiment costs, simplicity, relative speed in obtaining the expected results, limited ethical concern, no need of high-level technical skill, and the absence of a mature immune system, resulting in an inexpensive, simple, and practical method to evaluate and characterize tissue-engineered constructs. The results till now obtained suggest that CAM assay can be used as a pre-screening assay, before in vivo animal studies, to determine whether the scaffold is liable to cause an adverse reaction and to evaluate its future enhancement of existing materials for tissue engineering. A review of the more recent results related to the use of CAM for in vivo biomaterial property evaluation is herein reported.

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.

The use of synthetic degradable or permanent polymers and biomaterials has not yet helped to achieve successful clinical whole-airway replacement. A novel, clinically successful approach involves tissue engineering (TE) replacement using three-dimensional biologic scaffolds composed of allogeneic extracellular scaffolds derived from nonautologous sources and recellularized with autologous stem cells or differentiated cells. In this paper, we discuss this novel approach and review information that can lead to a better understanding of stem cell recruitment and/or mobilization and site-specific tissue protection, which can be pharmacologically boosted in humans.

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.

The present study was undertaken to examine the effect of Homocysteine (Hcy) and nitric oxide (NO) levels on specific Computed Axial Tomography (CAT) measurements, as global brain atrophy and brain vascular lesion in Alzheimer Disease (AD) and in Vascular Dementia (VD) patients. We have analysed serum Hcy and NO levels in AD patients and compared the findings with those in VD patients and control subjects. Moreover we have studied the correlation of Hcy and NO levels with cognitive impairment and brain atrophy determined by Computed Axial Tomography. Hcy serum levels significantly increased in all demented patients compared to control group, independently from the dementia type. On the contrary, no differences were observed in NO serum levels between groups. Moreover, we found significant correlation between Hcy and brain atrophy in both demented groups; whereas NO levels correlated only in AD, but not in VD patients. The pathogenic effect of Hcy either in AD and VD patients appears to confirm a definitive vascular component in AD. As regards NO, our results highlight the role of NO as a beneficial molecule in AD and support the use of NO mimetics as an antineurodegenerative therapy for AD patients.

Tissue engineering for reconstruction/regeneration of airways can be regarded as a potential approach for the treatment of severe pathologies that can dramatically impair the correct function of the involved organs. Total or partial transplantation can be the surgical solution for these occurrences, but several limitations are generally associated with this option, as it is dependent on donor shortage and host response to the implanted material. A viable and ready-to-use substitute, able to promote novel tissue formation, would therefore be the desirable scaffold to deal with. Synthetic materials can represent an alternative for this task. Obviously, several drawbacks can be listed to their use, but they can be potentially tailored and modified to minimally reduce the risk of a negative outcome. Herein, the investigated synthetic materials tested so far in order to develop a viable tissue-engineered tracheal scaffold are presented and discussed.

Repetitive measurement with neuroimaging techniques could be useful instruments permitting to differentiate between Alzheimer disease (AD) and vascular dementia (VD). The major genetic risk factor for the development of late-onset AD is the allele epsilon4 of the apolipoprotein E (ApoE). Moreover nitric oxide (NO) and homocysteine (Hcy) seems to be correlated with the degree of cognitive impairment in demented subjects. The aim of this study was to investigate the association between serum NO and Hcy levels, global brain atrophy and brain vascular lesion in AD and VD patients. We report that high plasma levels of homocysteine resulted associated with AD and VD, suggesting that in AD elevated plasma Hcy might be a consequence of concomitant vascular dementia. Otherwise, plasma NO levels were not significantly different in any of the groups. Moreover, neuroimaging measures of vascular lesion level could be of usefulness to differentiate between AD and VD.

Polyphosphazenes are polymers possessing a skeleton composed of alternating phosphorous and nitrogen atoms, and two side-moieties linked to each phosphorous atom. Polyphosphazenes with amino acid esters as side-moieties are biocompatible and biodegradable polymers. Two polyphosphazenes, poly[bis(ethyl alanate) phosphazene] and poly[(ethyl phenylalanate)0.8(ethyl alanate)0.8(ethyl glycinate)0.4 phosphazene] (PPAGP) were synthesized, and processed to form small fibers. Their ability to support rat neuromicrovascular endothelial cell (EC) adhesion and growth has been studied, using poly(D,L-lactic acid) as reference compound. Scanning electron microscopy revealed that both poly[bis(ethyl alanate) phosphazene] and PPAGP fibers were thinner than poly(D,L-lactic acid) fibers, and possessed a more irregular and porous surface. All polymers increased EC adhesion, compared with polystyrene, but only polyphosphazenes were able to improve EC growth. The highest increase in EC proliferation was induced by PPAGP, which, as revealed by environmental scanning electron microscopy, was also able to induce ECs to arrange into tubular structures. The conclusion is drawn that PPAGP may provide the best scaffold for engineered blood vessels, because it promotes adhesion, growth, and organization of ECs into capillary-like structures. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 669–674, 2004

The repair and regeneration of injured tissues and organs depend on the re-establishment of the blood flow needed for cellular infiltration and metabolic support. Among the various materials used in tissue reconstruction, acellular scaffolds have recently been utilized. In this study, we investigated the angiogenic response induced by acellular brain scaffolds implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigations. The results show that acellular brain scaffolds are able to induce a strong angiogenic response, comparable to that of fibroblast growth factor-2 (FGF-2), a well known angiogenic cytokine. The response may be considered dependent on a direct angiogenic effect exerted by the scaffold, because no inflammatory infiltrate was detectable in CAM’s mesenchyme beneath the implant. Acellular brain scaffolds might induce the release of endogenous angiogenic factors, such as FGF-2 and vascular endothelial growth factor (VEGF) released from the extracellular matrix of the developing CAM. In addition, the angiogenic response may depend, in part, also on the presence in the acellular matrix of transforming growth factor beta 1 (TGFβ1).

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development.

A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial- and muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi., Patients with oesophageal diseases may require surgical removal and replacement of the oesophagus. Here the authors seed mesenchymal stromal cells on a decellularized rat oesophagus and show that this bioengineered tissue construct restores swallowing function after transplantation into rats.

It is commonly stated that tissue engineering is the most promising approach to treat or replace failing tissues/organs. For this aim, a specific strategy should be planned including proper selection of biomaterials, fabrication techniques, cell lines, and signaling cues. A great effort has been pursued to develop suitable scaffolds for the restoration of a variety of tissues and a huge number of protocols ranging fromin vitrotoin vivostudies, the latter further differentiating into several procedures depending on the type of implantation (i.e., subcutaneous or orthotopic) and the model adopted (i.e., animal or human), have been developed. All together, the published reports demonstrate that the proposed tissue engineering approaches spread toward multiple directions. The critical review of this scenario might suggest, at the same time, that a limited number of studies gave a real improvement to the field, especially referring toin vivoinvestigations. In this regard, the present paper aims to review the results ofin vivotissue engineering experimentations, focusing on the role of the scaffold and its specificity with respect to the tissue to be regenerated, in order to verify whether an extracellular matrix-like device, as usually stated, could promote an expected positive outcome.

Any irreversible damage to the airway compromises different important functions, such as speech, swallowing, respiration, mucociliary clearance, and immune protection, from inhaled or ingested antigens. No predictably functional replacement for patients with advanced laryngeal and tracheal disorders has been established. Tissue engineering offers, virtually, a viable and effective platform to develop a functional and completely nonimmunogenic and biocompatible airway substitute whose pliability, elasticity, and mucosal lining could be similar to that of the native tissue. Unfortunately, and despite recent clinical advances, the airway is very difficult to replace because, ideally, the scaffold should be able to: provide adequate dynamics for voice production, sphincter action, and structural support to maintain airway patency; be airtight, completely biocompatible and not evoke immune reaction; rapidly epithelialized; and readily available and easy to implant. Herein, the tissue-engineered airway strategies, evaluated in preclinical studies and translated (or are to be) to clinical practice, will be reported.

Diseases which compromise airways alter breathing, speech and swallowing and the consequences are severe effects on psychology, socialization and employment. Around the world, the incidence of primary tracheal cancer is approximately 0.1 in every 100,000 persons per year (Honings et al. 2010) and there are 136,000 new cases of laryngeal cancer each year (Duque et al. 2007), and a smaller, but severely impaired, group of patients with nonfunctioning airways due to benign disease and trauma (Nouraei et al. 2007). Presently, there are no good conventional solutions to treat patients with advanced structural disorders of the trachea and larynx.

The fabrication of an instructive bioabsorbable scaffold is one of the main goals for tissue engineering applications. In this regard, genipin cross-linked gelatin scaffolds, produced by electrospinning, were tested as a platform to include decellularized rat brain extracellular matrix as an active agent to provide fundamental biochemical cues to the seeded cells. This approach is expected to furnish a suitable natural-based polymeric scaffold with sufficient temporal stability to support cell attachment and spreading, also providing tissue-specific signals that can contribute to the expression of the requested cellular phenotype. We first demonstrated the effectiveness of the proposed decellularization protocol and the cytocompatibility of the resulting brain matrix. Then, the in vitro biological assays of the conditioned electrospun scaffolds, using rat allogeneic mesenchymal stromal cells, confirmed their biocompatibility and showed a differentiative potential in presence of just 1% w/w decellularized rat brain extracellular matrix.

No preferred treatment, which could offer a functional solution, has been so far developed for patients affected by extensive airway damages. As the field of tissue engineering attempts to develop tracheal replacements, multiple types and combinations of cells, scaffold materials, and/or culture conditions have been used. Interest has been evoked by decellularized natural matrices, which affecting cell proliferation, migration and differentiation, could play an active part in tissue regeneration and remodeling. Using the detergent-enzymatic method, we were able to obtain decellularized human tracheal matrices lacking MHC antigens (bypassing rejection), having structural, mechanical and in vivo pro-angiogenic properties similar to that of native airways and supporting in vivo recellularization. Starting from these results, we have developed an in vivo tissue engineered strategy, based on airway bioengineered grafts combined with autologous stem cells and pharmacological intervention (to boost progenitor cell recruitment commitment), which resulted to be a clinically successful alternative for patients with serious airway disorders.

Rapid and controlled vascularization of engineered tissues remains one of the key limitations in tissue engineering applications. This study investigates the possible use of natural extracellular matrix-like scaffolds made of gelatin loaded with human vascular endothelial growth factor (VEGF), as a bioresorbable platform for long-term release and consequent angiogenic boosting. For this aim, gelatin was firstly electrospun and then cross-linked at two different concentrations (0.1% and 0.5% w/v) by using genipin, a low toxic agent, in order to fabricate a suitable substrate to be loaded with VEGF. Collected fibers were homogeneous and free of beads, the fibrous structure was retained after cross-linking. Mechanical properties were deeply affected by the chemical treatment showing a different behavior, depending on the testing conditions (i.e., dry or wet state). VEGF release was assessed by means of ELISA assay: a cumulative release of about 90% (0.1% w/v) and 60% (0.5% w/v) at 28 days was measured. Both VEGF loaded mats induced cell viability, endothelial differentiation and showed chemoattractive properties when tested on human mesenchymal stromal cells (hMSCs). In vitro and in vivo angiogenic assays demonstrated that the VEGF loaded mats induced an angiogenic potential in stimulating new vessel formation similar, if not superior, to fresh VEGF. VEGF retains bioactive and pro-angiogenic potential for up to 14 days. The results demonstrated that genipin cross-linked electrospun gelatin mats loaded with VEGF could be part of a useful strategy to stimulate and induce angiogenesis in tissue engineered applications.

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(e-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.

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.