Inexpensive production of near‐native engineered stromas

Although the self‐assembly approach is an efficient method for the production of engineered physiological and pathological tissues, avoiding the use of exogenous materials, it nevertheless remains expensive and requires dexterity, which are features incompatible with large‐scale production. We propose a modification to this technique to make easier the production of mesenchymal compartment, to reduce the cost and to improve the histological quality of the self‐assembled tissues. The stroma produced by this novel approach allowed epithelial cell differentiation, resulting in a pseudostratified epithelium that shared several features with native tissues. The incorporation of endothelial cells in the reconstructed mesenchyme formed a three‐dimensional capillary‐like network, positive for CD31 and von Willebrand factor and surrounded by NG2 positive cells. It could limit self‐contraction of the resulting tissue by recruiting α‐Smooth Muscle Actin positive cells. With this new technique, which is relatively inexpensive and easy to use in a research laboratory set‐up, near‐native stromas can now be produced with minimal handling time. Copyright © 2015 John Wiley & Sons, Ltd.     

Bio‐inspired design of a magnetically active trilayered scaffold for cartilage tissue engineering

An important topic in cartilage tissue engineering is the development of biomimetic scaffolds which mimic the depth‐dependent material properties of the native tissue. We describe an advanced trilayered nanocomposite hydrogel (ferrogel) with a gradient in compressive modulus from the top to the bottom layers (p < 0.05) of the construct. Further, the scaffold was able to respond to remote external stimulation, exhibiting an elastic, depth‐dependent strain gradient. When bovine chondrocytes were seeded into the ferrogels and cultured for up to 14 days, there was good cell viability and a biochemical gradient was measured with sulphated glycosaminoglycan increasing with depth from the surface. This novel construct provides tremendous scope for tailoring location‐specific cartilage replacement tissue; by varying the density of magnetic nanoparticles, concentration of base hydrogel and number of cells, physiologically relevant depth‐dependent gradients may be attained. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

Multiphoton microscopy analysis of extracellular collagen I network formation by mesenchymal stem cells

Collagen I is the major fibrous extracellular component of bone responsible for its ultimate tensile strength. In tissue engineering, one of the most important challenges for tissue formation is to get cells interconnected via a strong and functional extracellular matrix (ECM), mimicking as closely as possible the natural ECM geometry. Still missing in tissue engineering are: (a) a versatile, high‐resolution and non‐invasive approach to evaluate and quantify different aspects of ECM development within engineered biomimetic scaffolds online; and (b) deeper insights into the mechanism whereby cellular matrix production is enhanced in 3D cell–scaffold composites, putatively via enhanced focal adhesion linkage, over the 2D setting. In this study, we developed sensitive morphometric detection methods for collagen I‐producing and bone‐forming mesenchymal stem cells (MSCs), based on multiphoton second harmonic generation (SHG) microscopy, and used those techniques to compare collagen I production capabilities in 2D‐ and 3D‐arranged cells. We found that stimulating cells with 1% serum in the presence of ascorbic acid is superior to other medium conditions tested, including classical osteogenic medium. In contrast to conventional 2D culture, having MSCs packed closely in a 3D environment presumably stimulates cells to produce strong and complex collagen I networks with defined network structures (visible in SHG images) and improves collagen production. Copyright © 2015 John Wiley & Sons, Ltd.     

The potential role of tissue‐engineered urethral substitution: clinical and preclinical studies

Urethral strictures and anomalies remain among the difficult problems in urology, with urethroplasty procedures being the most effective treatment options. The two major types of urethroplasty are anastomotic urethroplasty and widening the urethral lumen using flaps or grafts (i.e. substitution urethroplasty). However, no ideal material for the latter has been found so far. Designing and selecting such a material is a necessary and challenging endeavour, driving the need for further bioengineered urethral tissue research. This article reviews currently available studies on the potentialities of tissue engineering in urethral reconstruction, in particular those describing the use of both acellular and recellularized tissue‐engineered constructs in animal and human models. Possible future developments in this field are also discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrospun fibrinogen–PLA nanofibres for vascular tissue engineering

Here we report on the development of a new type of hybrid fibrinogen–polylactic acid (FBG–PLA) nanofibres (NFs) with improved stiffness, combining the good mechanical properties of PLA with the excellent cell recognition properties of native FBG. We were particularly interested in the dorsal and ventral cell response to the nanofibres' organization (random or aligned), using human umbilical endothelial cells (HUVECs) as a model system. Upon ventral contact with random NFs, the cells developed a stellate‐like morphology with multiple projections. The well‐developed focal adhesion complexes suggested a successful cellular interaction. However, time‐lapse analysis shows significantly lowered cell movements, resulting in the cells traversing a relatively short distance in multiple directions. Conversely, an elongated cell shape and significantly increased cell mobility were observed in aligned NFs. To follow the dorsal cell response, artificial wounds were created on confluent cell layers previously grown on glass slides and covered with either random or aligned NFs. Time‐lapse analysis showed significantly faster wound coverage (within 12 h) of HUVECs on aligned samples vs. almost absent directional migration on random ones. However, nitric oxide (NO) release shows that endothelial cells possess lowered functionality on aligned NFs compared to random ones, where significantly higher NO production was found. Collectively, our studies show that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization. Copyright © 2016 John Wiley & Sons, Ltd.     

Contact‐free monitoring of vessel graft stiffness – proof of concept as a tool for vascular tissue engineering

Tissue‐engineered vessel grafts have to mimic the biomechanical properties of native blood vessels. Manufacturing processes often condition grafts to adapt them to the target flow conditions. Graft stiffness is influenced by material properties and dimensions and determines graft compliance. This proof‐of‐concept study evaluated a contact‐free method to monitor biomechanical properties without compromising sterility. Forced vibration response analysis was performed on human umbilical vein (HUV) segments mounted in a buffer‐filled tubing system. A linear motor and a dynamic signal analyser were used to excite the fluid by white noise (0–200 Hz). Vein responses were read out by laser triangulation and analysed by fast Fourier transformation. Modal analysis was performed by monitoring multiple positions of the vessel surface. As an inverse model of graft stiffening during conditioning, HUV were digested proteolytically, and the course of natural frequencies (NFs) was monitored over 120 min. Human umbilical vein showed up to five modes with NFs in the range of 5–100 Hz. The first natural frequencies of HUV did not alter over time while incubated in buffer (p = 0.555), whereas both collagenase (?35%, p = 0.0061) and elastase (?45%, p < 0.001) treatments caused significant decreases of NF within 120 min. Decellularized HUV showed similar results, indicating that changes of the extracellular matrix were responsible for the observed shift in NF. Performing vibration response analysis on vessel grafts is feasible without compromising sterility or integrity of the samples. This technique allows direct measurement of stiffness as an important biomechanical property, obviating the need to monitor surrogate parameters. Copyright © 2016 John Wiley & Sons, Ltd.     

Modulating cell adhesion to polybutylene succinate biotextile constructs for tissue engineering applications

Textile‐based technologies are powerful routes for the production of three‐dimensional porous architectures for tissue engineering applications because of their feasibility and possibility for scaling‐up. Herein, the use of knitting technology to produce polybutylene succinate fibre‐based porous architectures is described. Furthermore, different treatments have been applied to functionalize the surface of the scaffolds developed: sodium hydroxide etching, ultraviolet radiation exposure in an ozone atmosphere and grafting (acrylic acid, vinyl phosphonic acid and vinyl sulphonic acid) after oxygen plasma activation as a way to tailor cell adhesion. A possible effect of the applied treatments on the bulk properties of the textile scaffolds has been considered and thus tensile tests in dry and hydrated states were also carried out. The microscopy results indicated that the surface morphology and roughness were affected by the applied treatments. The X‐ray photoelectron spectroscopy and contact angle measurements showed the incorporation of oxygen‐containing groups and higher surface free energy as result of the surface treatments applied. The DNA quantification and scanning electron microscopy analysis revealed that these modifications enhanced cell adhesion and altered cell morphology. Generally, sodium hydroxide treatment altered most significantly the surface properties, which in turn resulted in a high number of cells adherent to these surfaces. Based on the results obtained, the proposed surface treatments are appropriate to modify polybutylene succinate knitting scaffolds, influencing cell adhesion and its potential for use in tissue engineering applications. Copyright © 2016 John Wiley & Sons, Ltd.     

Can host reaction animal models be used to predict and modulate skin regeneration?

The study of host reactions in the biomedical and tissue engineering (TE) fields is a key issue but somehow set aside where TE constructs are concerned. Every day new biomaterials and TE constructs are being developed and presented to the scientific community. The combination of cells and biomolecules with scaffolding materials, as TE constructs, make the isolation and the understanding of the effect of each one those elements over the overall host reaction difficult. Eventually, all variables influence the host reaction and the performance of the constructs. For this reason, current assessment of the in vivo performance of TE constructs follows individual approaches, using specific animal models to independently provide insights regarding the contribution of the biomaterials/scaffolds towards the host reaction, and of all the constructs regarding their functionality. Skin wound healing progress into tissue regeneration or repair is highly dependent on the specificities of the inflammatory stage, as demonstrated by comparison between fetal and adult mechanisms. Thus, it would be expected that insights acquired from host tissue reaction evaluation to biomaterials/scaffolds would be explored to predict healing progression and improve the functionality of skin TE constructs. The rational of this review is to make a comprehensive analysis of to what extent the knowledge obtained from the evaluation of in vivo host reactions to implantable biomaterials/scaffolds has been used in the design of skin TE strategies, by promoting tissue regeneration rather than repair. Copyright © 2016 John Wiley & Sons, Ltd.     

Fibrin,the preferred scaffold for cell transplantation after myocardial infarction? An old molecule with a new life

Fibrin is a topical haemostat, sealant and tissue glue, which consists of concentrated fibrinogen and thrombin. It has broad medical and research uses. Recently, several studies have shown that engineered patches comprising mixtures of biological or synthetic materials and progenitor cells showed therapeutic promise for regenerating damaged tissues. In that context, fibrin maintains cell adherence at the site of injury, where cells are required for tissue repair, and offers a nurturing environment that protects implanted cells without interfering with their expected benefit. Here we review the past, present and future uses of fibrin, with a focus on its use as a scaffold material for cardiac repair. Fibrin patches filled with regenerative cells can be placed over the scarring myocardium; this methodology avoids many of the drawbacks of conventional cell‐infusion systems. Advantages of using fibrin also include extraction from the patient's blood, an easy readjustment and implantation procedure, increase in viability and early proliferation of delivered cells, and benefits even with the patch alone. In line with this, we discuss the numerous preclinical studies that have used fibrin–cell patches, the practical issues inherent in their generation, and the necessary process of scaling‐up from animal models to patients. In the light of the data presented, fibrin stands out as a valuable biomaterial for delivering cells to damaged tissue and for promoting beneficial effects. However, before the fibrin scaffold can be translated from bench to bedside, many issues must be explored further, including suboptimal survival and limited migration of the implanted cells to underlying ischaemic myocardium. Copyright © 2016 John Wiley & Sons, Ltd.     

Advances and perspectives in tooth tissue engineering

Bio‐engineered teeth that can grow and remodel in a manner similar to that of natural teeth have the potential to serve as permanent replacements to the currently used prosthetic teeth, such as dental implants. A major challenge in designing functional bio‐engineered teeth is to mimic both the structural and anisotropic mechanical characteristics of the native tooth. Therefore, the field of dental and whole tooth regeneration has advanced towards the molecular and nanoscale design of bio‐active, biomimetic systems, using biomaterials, drug delivery systems and stem cells. The focus of this review is to discuss recent advances in tooth tissue engineering, using biomimetic scaffolds that provide proper architectural cues, exhibit the capacity to support dental stem cell proliferation and differentiation and sequester and release bio‐active agents, such as growth factors and nucleic acids, in a spatiotemporal controlled manner. Although many in vitro and in vivo studies on tooth regeneration appear promising, before tooth tissue engineering becomes a reality for humans, additional research is needed to perfect methods that use adult human dental stem cells, as opposed to embryonic dental stem cells, and to devise the means to generate bio‐engineered teeth of predetermined size and shape. Copyright © 2016 John Wiley & Sons, Ltd.