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Impact statement This review discusses the research done using marine collagens (MCs) on biomaterials for bone, cartilage, and osteochondral tissue regenerative applications with the underlying technologies that enable their development, and explains the methodologies used to characterize MCs highlighting their importance, namely regarding the performance of derived biomaterials, and the inherent properties of such collagens. In the second part, the applicability of MCs as biomaterials for hard tissue applications was studied, focusing on the mostly applied fabrication techniques. In conclusion, this review describes the major challenges to be overcome and the forecast for the upcoming years concerning the use of MCs.

The ability of human tissues to self-repair is limited, which motivates the scientific community to explore new and better therapeutic approaches to tissue regeneration. The present manuscript provides a comparative study between a marine-based composite biomaterial, and another composed of well-established counterparts for bone tissue regeneration. Blue shark skin collagen was combined with bioapatite obtained from blue shark's teeth (mColl:BAp), while bovine collagen was combined with synthetic hydroxyapatite (bColl:Ap) to produce 3D composite scaffolds by freeze-drying. Collagens showed similar profiles, while apatite particles differed in their composition, being the marine bioapatite a fluoride-enriched ceramic. The marine-sourced biomaterials presented higher porosities, improved mechanical properties, and slower degradation rates when compared to synthetic apatite-reinforced bovine collagen. The in vivo performance regarding bone tissue regeneration was evaluated in defects created in femoral condyles in New Zealand rabbits twelve weeks post-surgery. Micro-CT results showed that mColl:BAp implanted condyles had a slower degradation and an higher tissue formation (17.9 ± 6.9 %) when compared with bColl:Ap implanted ones (12.9 ± 7.6 %). The histomorphometry analysis provided supporting evidence, confirming the observed trend by quantifying 13.1 ± 7.9 % of new tissue formation for mColl:BAp composites and 10.4 ± 3.2 % for bColl:Ap composites, suggesting the potential use of marine biomaterials for bone regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the past decade, there has been significant progress in 3D printing research for tissue engineering (TE) using biomaterial inks made from natural and synthetic compounds. These constructs can aid in the regeneration process after tissue loss or injury, but achieving high shape fidelity is a challenge as it affects the construct's physical and biological performance with cells. In parallel with the growth of 3D bioprinting approaches, some marine-origin polymers have been studied due to their biocompatibility, biodegradability, low immunogenicity, and similarities to human extracellular matrix components, making them an excellent alternative to land mammal-origin polymers with reduced disease transmission risk and ethical concerns. In this research, collagen from shark skin, chitosan from squid pens, and fucoidan from brown algae were effectively blended for the manufacturing of an adequate biomaterial ink to achieve a printable, reproducible material with a high shape fidelity and reticulated using four different approaches (phosphate-buffered saline, cell culture medium, 6% CaCl 2 , and 5 mM Genipin). Materials characterization was composed by filament collapse, fusion behavior, swelling behavior, and rheological and compressive tests, which demonstrated favorable shape fidelity resulting in a stable structure without deformations, and interesting shear recovery properties around the 80% mark. Additionally, live/dead assays were conducted in order to assess the cell viability of an immortalized human mesenchymal stem cell line, seeded directly on the 3D printed constructs, which showed over 90% viable cells. Overall, the Roswell Park Memorial Institute cell culture medium promoted the adequate crosslinking of this biopolymer blend to serve the TE approach, taking advantage of its capacity to hamper pH decrease coming from the acidic biomaterial ink. While the crosslinking occurs, the pH can be easily monitored by the presence of the indicator phenol red in the cell culture medium, which reduces costs and time., (Creative Commons Attribution license.)

The central nervous system (CNS) has a limited ability to regenerate after a traumatic injury or a disease due to the low capacity of the neurons to re-grow and the inhibitory environment formed in situ. Current therapies include the use of drugs and rehabilitation, which do not fully restore the CNS functions and only delay the pathology progression. Tissue engineering offers a simple and versatile solution for this problem through the use of bioconstructs that promote nerve tissue repair by bridging cavity spaces. In this approach, the choice of biomaterial is crucial. Herein, we present recent advances in the design and development of adhesive and self-healing materials that support CNS healing. The adhesive materials have the advantage of promoting recovery without the use of needles or sewing, while the self-healing materials have the capacity to restore the tissue integrity without the need for external intervention. These materials can be used alone or in combination with cells and/or bioactive agents to control the inflammation, formation of free radicals, and proteases activity. We discuss the advantages and drawbacks of different systems. The remaining challenges that can bring these materials to clinical reality are also briefly presented., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The engineering of fully functional, biological-like tissues requires biomaterials to direct cellular events to a near-native, 3D niche extent. Natural biomaterials are generally seen as a safe option for cell support, but their biocompatibility and biodegradability can be just as limited as their bioactive/biomimetic performance. Furthermore, integrating different biomaterial cues and their final impact on cellular behavior is a complex equation where the outcome might be very different from the sum of individual parts. This review critically analyses recent progress on biomaterial-induced cellular responses, from simple adhesion to more complex stem cell differentiation, looking at the ever-growing possibilities of natural materials modification. Starting with a discussion on native material formulation and the inclusion of cell-instructive cues, the roles of shape and mechanical stimuli, the susceptibility to cellular remodeling, and the often-overlooked impact of cellular density and cell-cell interactions within constructs, are delved into. Along the way, synergistic and antagonistic combinations reported in vitro and in vivo are singled out, identifying needs and current lessons on the development of natural biomaterial libraries to solve the cell-material puzzle efficiently. This review brings together knowledge from different fields envisioning next-generation, combinatorial biomaterial development toward complex tissue engineering., (© 2022 Wiley-VCH GmbH.)

Aims: Stem cell therapies emerged as treatment modalities with potential to cure neurodegenerative diseases (NDs). However, despite high expectations, their clinical use is still limited. Critical issues in treatment outcomes may be related to stem cells formulation and administration route. We develop a hydrogel as a cell carrier, consisting of compounds (phospholipids and hyaluronic acid-HA) naturally present in the central nervous system (CNS). The HA-based hydrogel physically crosslinked with liposomes is designed for direct injection into the CNS to significantly increase the bone marrow mesenchymal stem cells (BMSCs) bioavailability., Materials and Methods: Hydrogel compatibility is confirmed in vitro with BMSCs and in vivo through its intracerebroventricular injection in rats. To assess its efficacy, the main cause of chronic neurologic disability in young adults is selected, namely multiple sclerosis (MS). The efficacy of the developed formulation containing a lower number of cells than previously reported is demonstrated using an experimental autoimmune encephalomyelitis (EAE) rat model., Key Findings: The distribution of the engineered hydrogel into corpus callosum can be ideal for NDs treatment, since damage of this white matter structure is responsible for important neuronal deficits. Moreover, the BMSCs-laden hydrogel significantly decreases disease severity and maximum clinical score and eliminated the relapse., Significance: The engineering of advanced therapies using this natural carrier can result in efficacious treatments for MS and related debilitating conditions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In bone tissue engineering, the development of advanced biomimetic scaffolds has led to the quest for biomotifs in scaffold design that better recreate the bone matrix structure and composition and hierarchy at different length scales. In this study, an advanced hierarchical scaffold consisting of silk fibroin combined with a decellularized cell-derived extracellular matrix and reinforced with carbon nanotubes was developed. The goal of the carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds is to harvest the individual properties of their constituents to introduce hierarchical capacity in order to improve standard silk fibroin scaffolds. The scaffolds were fabricated using enzymatic cross-linking, freeze modeling, and decellularization methods. The developed scaffolds were assessed for the pore structure and mechanical properties showing satisfying results to be used in bone regeneration. The developed carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds were shown to be bioactive in vitro and expressed no hemolytic effect. Furthermore, cellular in vitro studies on human adipose-derived stem cells (hASCs) showed that scaffolds supported cell proliferation. The hASCs seeded onto these scaffolds evidenced similar metabolic activity to standard silk fibroin scaffolds but increased ALP activity. The histological staining showed cell infiltration into the scaffolds and visible collagen production. The expression of several osteogenic markers was investigated, further supporting the osteogenic potential of the developed carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds. The hemolytic assay demonstrated the hemocompatibility of the hierarchical scaffolds. Overall, the carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds presented the required architecture for bone tissue engineering applications.

The emerging field of tissue engineering and regenerative medicines utilising artificial polymers is facing many problems. Despite having mechanical stability, non-toxicity and biodegradability, most of them lack cytocompatibility and biocompatibility. Natural polymers (such as collagen, hyaluronic acid, fibrin, fibroin, and others), including blends, are introduced to the field to solve some of the relevant issues. Another natural biopolymer: silkworm silk gained special attention primarily due to its specific biophysical, biochemical, and material properties, worldwide availability, and cost-effectiveness. Silk proteins, namely fibroin and sericin extracted from domesticated mulberry silkworm Bombyx mori , are studied extensively in the last few decades for tissue engineering. Wild nonmulberry silkworm species, originated from India and other parts of the world, also produce silk proteins with variations in their nature and properties. Among the nonmulberry silkworm species, Antheraea mylitta (Indian Tropical Tasar), A. assamensis/A. assama (Indian Muga), and Samia ricini/Philosamia ricini (Indian Eri), along with A. pernyi (Chinese temperate Oak Tasar/Tussah) and A. yamamai (Japanese Oak Tasar) exhibit inherent tripeptide motifs of arginyl glycyl aspartic acid in their fibroin amino acid sequences, which support their candidacy as the potential biomaterials. Similarly, sericin isolated from such wild species delivers unique properties and is used as anti-apoptotic and growth-inducing factors in regenerative medicines. Other characteristics such as biodegradability, biocompatibility, and non-inflammatory nature make it suitable for tissue engineering and regenerative medicine based applications. A diverse range of matrices, including but not limited to nano-micro scale structures, nanofibres, thin films, hydrogels, and porous scaffolds, are prepared from the silk proteins (fibroins and sericins) for biomedical and tissue engineering research. This review aims to represent the progress made in medical and non-medical applications in the last couple of years and depict the present status of the investigations on Indian nonmulberry silk-based matrices as a particular reference due to its remarkable potentiality of regeneration of different types of tissues. It also discusses the future perspective in tissue engineering and regenerative medicines in the context of developing cutting-edge techniques such as 3D printing/bioprinting, microfluidics, organ-on-a-chip, and other electronics, optical and thermal property-based applications., (© 2021 IOP Publishing Ltd.)

The application of additive manufacturing in the biomedical field has become a hot topic in the last decade owing to its potential to provide personalized solutions for patients. Different bioinks have been designed trying to obtain a unique concoction that addresses all the needs for tissue engineering and drug delivery purposes, among others. Despite the remarkable progress made, the development of suitable bioinks which combine printability, cytocompatibility, and biofunctionality is still a challenge. In this sense, the well-established synthetic and functionalization routes to prepare nanoparticles with different functionalities make them excellent candidates to be combined with polymeric systems in order to generate suitable multi-functional bioinks. In this review, we briefly discuss the most recent advances in the design of functional nanocomposite hydrogels considering their already evaluated or potential use as bioinks. The scientific development over the last few years is reviewed, focusing the discussion on the wide range of functionalities that can be incorporated into 3D bioprinted constructs through the addition of multifunctional nanoparticles in order to increase their regenerative potential in the field of tissue engineering.

Green solvents such as ionic liquids (ILs) unlock possibilities for developing innovative biomedical and pharmaceutical solutions. ILs are the most investigated solvents for compound extractions, as reaction media and/or catalysts, and a desired eco-friendly solvent to process biomacromolecules for biomaterial production. Investigations demonstrate that the tunable nature and physicochemical features of ILs are also beneficial for building up delivery systems through their combination with bioactive compounds. Bioactive compounds from synthetic origins, like ibuprofen, ketoprofen, and natural sources such as curcumin, flavonoids, and polyphenols are essential starting points as preventive and therapeutic agents for treating diseases. Therefore, the association of those compounds with ILs opens up windows of opportunities in this research field. This Review assesses some of the main and important recent information and the current challenges concerning delivery platforms based on ILs combined with bioactive compounds of both natural and synthetic origins. Moreover, the chemistry, bioavailability, and biological functions of the main bioactive compounds used in the ILs-based delivery platforms are described. These data are presented and are discussed, together with the main delivery routes of the systems.

This review focuses on vascularization and strategies involved in its evaluation and modulation. Clinical issues associated with engineered tissues of an atomically relevant size that require a vascular network to supply their cells with nutrients and oxygen are analyzed in terms of vascular network formation within scaffolds, which can be produced from varying biomaterials, with the capability of connecting to the vasculature of the patient. Developing angiogenesis techniques and monitoring of angiogenesis development as well as how these methods can be further utilized to tailor vascularization within large tissue engineered constructs are also discussed. Finally, we offer a glimpse toward the future by providing an outlook for vascularization and associated emerging bioprinting concepts in tissue engineering applications.

Although it has been repeatedly indicated the importance to develop implantable devices and cell culture substrates with tissue-specific rigidity, current commercially available products, in particular cell culture substrates, have rigidity values well above most tissues in the body. Herein, six resorbable polyester films were fabricated using compression moulding with a thermal presser into films with tailored stiffness by appropriately selecting the ratio of their building up monomers (e.g. lactide, glycolide, trimethylene carbonate, dioxanone, ε-caprolactone). Typical NMR and FTIR spectra were obtained, suggesting that the fabrication process did not have a negative effect on the conformation of the polymers. Surface roughness analysis revealed no apparent differences between the films as a function of polymer composition. Subject to polymer composition, polymeric films were obtained with glass transition temperatures from -52 °C to 61 °C; contact angles in water from 81 ° to 94 °; storage modulus from 108 MPa to 2,756 MPa and loss modulus from 8 MPa to 507 MPa (both in wet state, at 1 Hz frequency and at 37 °C); ultimate tensile strength from 8 MPa to 62 MPa, toughness from 23 MJ/m 3 to 287 MJ/m 3 , strain at break from 3 % to 278 %, macro-scale Young's modulus from 110 MPa to 2,184 MPa (all in wet state); and nano-scale Young's modulus from 6 kPa to 15,019 kPa (in wet state). With respect to in vitro degradation in phosphate buffered saline at 37 °C, some polymeric films [e.g. poly(glycolide-lactide) 30 / 70] started degrading from day 7 (shortest timepoint assessed), whilst others [e.g. poly(glycolide-co-ε-caprolactone) 10 / 90] were more resilient to degradation up to day 21 (longest timepoint assessed). In vitro biological analysis using human dermal fibroblasts and a human monocyte cell line (THP-1) showed the potential of the polymeric films to support cell growth and controlled immune response. Evidently, the selected polymers exhibited properties suitable for a range of clinical indications., Competing Interests: Declaration of Competing Interest Sofia Ribeiro was an early career researcher recruited at Sofradim Production, Medtronic, France and registered for PhD at NUI Galway, Ireland. Yves Bayon is an employee of Sofradim Production, Medtronic, France. Ana M. Carvalho, Emanuel M. Fernandes, Manuela E. Gomes, Rui L. Reis and Dimitrios I. Zeugolis declare no conflicts of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Hyaluronan (HA) is a critical element of the extracellular matrix (ECM). The regulated synthesis and degradation of HA modulates the ECM chemical and physical properties that, in turn, influence cellular behavior. HA triggers signaling pathways associated with the adhesion, proliferation, migration, and differentiation of cells, mediated by its interaction with specific cellular receptors or by tuning the mechanical properties of the ECM. This review summarizes the recent advances on strategies used to mimic the HA present in the ECM to study healthy or pathological cellular behavior. This includes the development of HA-based 2D and 3D in vitro tissue models for the seeding and encapsulation of cells, respectively, and HA particles as carriers for the targeted delivery of therapeutic agents., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

With the worldwide increase of fisheries, fish wastes have had a similar increase, alternatively they can be seen as a source of novel substances for the improvement of society's wellbeing. Elasmobranchs are a subclass fished in high amounts, with some species being mainly bycatch. They possess an endoskeleton composed mainly by cartilage, from which chondroitin sulfate is currently obtained. Their use as a viable source for extraction of type II collagen has been hypothesized with the envisaging of a biomedical application, namely in biomaterials production. In the present work, raw cartilage from shark ( Prionace glauca ) and ray ( Zeachara chilensis and Bathyraja brachyurops ) was obtained from a fish processing company and submitted to acidic and enzymatic extractions, to produce acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). From all the extractions, P. glauca PSC had the highest yield (3.5%), followed by ray ASC (0.92%), ray PSC (0.50%), and P. glauca ASC (0.15%). All the extracts showed similar properties, with the SDS-PAGE profiles being compatible with the presence of both type I and type II collagens. Moreover, the collagen extracts exhibited the competence to maintain their conformation at human basal temperature, presenting a denaturation temperature higher than 37 °C. Hydrogels were produced using P. glauca PSC combined with shark chondroitin sulfate, with the objective of mimicking the human cartilage extracellular matrix. These hydrogels were cohesive and structurally-stable at 37 °C, with rheological measurements exhibiting a conformation of an elastic solid when submitted to shear strain with a frequency up to 4 Hz. This work revealed a sustainable strategy for the valorization of fisheries' by-products, within the concept of a circular economy, consisting of the use of P. glauca , Z. chilensis, and B. brachyurops cartilage for the extraction of collagen, which would be further employed in the development of hydrogels as a proof of concept of its biotechnological potential, ultimately envisaging its use in marine biomaterials to regenerate damaged cartilaginous tissues.

In recent years, there was an abrupt increase in the incidence of renal tumors, which prompt up the appearance of cutting-edge technology, including minimally invasive and organ-preserving approaches, such as laparoscopic partial nephrectomy (LPN). LPN is an innovative technique used to treat small renal masses that have been gaining popularity in the last few decades due to its promissory results. However, the bleeding control remains the main challenge since the majority of currently available hemostatic agents (HAs) used in other surgical specialities are inefficient in LPN. This hurried the search for effective HAs adapted for LPN surgical peculiarities, which resulted on the emergence of different types of topical HAs. The most promising are the natural origin HAs because of their inherent biodegradability, biocompatibility, and lowest toxicity. These properties turn them top interests' candidates as HAs in LPN. In this review, we present a deep overview on the progress achieved in the design of HAs based on natural origin polymers, highlighting their distinguishable characteristics and providing a clear understanding of their hemostat's role in LPN. This way it may be possible to establish a structure-composition properties relation, so that novel HAs for LPN can be designed to explore current unmet medical needs., (© 2020 Wiley Periodicals LLC.)

The combination of marine origin biopolymers for tissue engineering (TE) applications is of high interest, due to their similarities with the proteins and polysaccharides present in the extracellular matrix of different human tissues. This manuscript reports on innovative collagen-chitosan-fucoidan cryogels formed by the simultaneous blending of these three marine polymers in a chemical-free crosslinking approach. The physicochemical characterization of marine biopolymers comprised FTIR, amino acid analysis, circular dichroism and SDS-PAGE, and suggested that the jellyfish collagen used in the cryogels was not denatured (preserved the triple helical structure) and had similarities with type II collagen. The chitosan presented a high deacetylation degree (90.1%) that can strongly influence the polymer physicochemical properties and biomaterial formation. By its turn, rheology, and SEM studies confirmed that these novel cryogels present interesting properties for TE purposes, such as effective blending of biopolymers without visible material segregation, mechanical stability (strong viscoelastic character), as well as adequate porosity to support cell proliferation and exchange of nutrients and waste products. Additionally, in vitro cellular assessments of all cryogel formulations revealed a non-cytotoxic behavior. The MTS test, live/dead assay and cell morphology assessment (phalloidin DAPI) showed that cryogels can provide a proper microenvironment for cell culturing, supporting cell viability and promoting cell proliferation. Overall, the obtained results suggest that the novel collagen-chitosan-fucoidan cryogels herein presented are promising scaffolds envisaging tissue engineering purposes, as both acellular biomaterials or cell-laden cryogels.

Marine resources have considerable potential to develop high-value materials for applications in different fields, namely pharmaceutical, environmental, and biomedical. Despite that, the lack of solubility of marine-derived polymers in water and common organic solvents could restrict their applications. In the last years, ionic liquids (ILs) have emerged as platforms able to overcome those drawbacks, opening many routes to enlarge the use of marine-derived polymers as biomaterials, among other applications. From this perspective, ILs can be used as an efficient extraction media for polysaccharides from marine microalgae and wastes (e.g., crab shells, squid, and skeletons) or as solvents to process them in different shapes, such as films, hydrogels, nano/microparticles, and scaffolds. The resulting architectures can be applied in wound repair, bone regeneration, or gene and drug delivery systems. This review is focused on the recent research on the applications of ILs as processing platforms of biomaterials derived from marine polymers.

Liver diseases contribute markedly to the global burden of mortality and disease. The limited organ disposal for orthotopic liver transplantation results in a continuing need for alternative strategies. Over the past years, important progress has been made in the field of tissue engineering (TE). Many of the early trials to improve the development of an engineered tissue construct are based on seeding cells onto biomaterial scaffolds. Nowadays, several TE approaches have been developed and are applied to one vital organ: the liver. Essential elements must be considered in liver TE-cells and culturing systems, bioactive agents or growth factors (GF), and biomaterials and processing methods. The potential of hepatocytes, mesenchymal stem cells, and others as cell sources is demonstrated. They need engineered biomaterial-based scaffolds with perfect biocompatibility and bioactivity to support cell proliferation and hepatic differentiation as well as allowing extracellular matrix deposition and vascularization. Moreover, they require a microenvironment provided using conventional or advanced processing technologies in order to supply oxygen, nutrients, and GF. Herein the biomaterials and the conventional and advanced processing technologies, including cell-sheets process, 3D bioprinting, and microfluidic systems, as well as the future trends in these major fields are discussed., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Atlantic cod is processed industrially for food purposes, with several by-products being directed to animal feed and other ends. Looking particularly into swim bladders, the extraction of collagen can be a valuable strategy for by-product valorization, explored in the present work for the first time. Collagen was extracted using acetic acid (ASCsb) and pepsin (PSCsb) with yields of 5.72% (w/w) and 11.14% (w/w), respectively. SDS-PAGE profile showed that the extracts were compatible with type I collagen. FTIR, CD and XRD results suggest that the PSCsb structure underwent partial denaturation, with microDSC showing a band at 54 °C probably corresponding to a melting process, while ASCsb structure remained intact, with preserved triple helix and a denaturation temperature of 29.6 °C. Amino acid composition indicates that the total content of proline-like amino acids was 148/1000 residues for ASCsb and 141/1000 residues for PSCsb, with a hydroxylation degree of about 37%. The extracts exhibited a typical shear thinning behavior, interesting property regarding their further processing toward the development of biomaterials. In this regard, assessment of metabolic activity of human fibroblast cells cultured in the presence of collagen extracts with concentrations up to 3 mg/mL revealed the absence of cytotoxic behavior. Collagen extracts obtained from Atlantic cod swim bladders shown attractive properties regarding their use in cosmetic or biomedical applications.

Tissue engineering and regenerative medicine (TERM) are paving the way to the generation of functional and mature biological tissues that closely emulate cellular, biochemical, and mechanical cues. Electrical fields in the human body modulate myriad biological processes, such as synapses, muscle contraction, hearing, and wound healing, which were disregarded in TERM until recently. To preserve and improve tissue electrophysiology, cells can be loaded in electroactive biomaterials and stimulated with exogenous electrical fields. Here, we review how electrical stimulation and electroactive biomaterials can be used to instruct cells to create more mature and functional tissue-engineered constructs. We also highlight the most recent electroactive engineered tissues developed for TERM., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Over the past years, important progresses have been made in the field of tissue engineering. Many of the early trials to improve the development of an engineered tissue construct were centered on the concept of seeding cells onto biomaterial scaffold. By means of innovative manufacturing machineries, the conception of a preformed scaffold became possible. Nowadays, several tissue engineering challenges are associated with applying this scaffold technology to one vital organ construct: liver. The development of microscale tissue ("micro-tissue") constructs to mimic partially the complex structure-function interactions of liver parenchyma have been obtained through the engineering of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. For in vitro applications, micro-tissue constructs are being upgraded into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. The present chapter will focus on the biomaterials currently used for the development of in vitro liver constructs as well as the description of the microfluidic-based models that show great promise for liver regenerative medicine approaches.

Microfluidics techniques can be used to process a wide range of biomaterials, from synthetic to natural origin ones. This chapter describes microfluidic processing of biomaterials, mainly polymeric materials of natural origin, focusing on water-soluble polymers that form non-flowing phases after crosslinking. Some polysaccharides and proteins, including agarose, alginate, chitosan, gellan gum, hyaluronic acid, collagen, gelatin, and silk fibroin are emphasized deu to their relevance in the field. The critical characteristics of these materials are discussed, giving particular consideration to those that directly impact its processability using microfluidics. Furthermore, some microfluidic-based processing techniques are presented, describing their suitability to process materials with different sol-gel transition mechanisms.

Due to increasing life expectancy incidence of neurological disorders is rapidly rising, thus adding urgency to develop effective strategies for treatment. Stem cell-based therapies were considered highly promising and while progress in this field is evident, outcomes of clinical trials are rather disappointing. Suboptimal engraftment, poor cell survival and uncontrolled differentiation may be the reasons behind dismal results. Clearly, new direction is needed and we postulate that with recent progress in biomaterials and bioprinting, regenerative approaches for neurological applications may be finally successful. The use of biomaterials aids engraftment of stem cells, protects them from harmful microenvironment and importantly, it facilitates the incorporation of cell-supporting molecules. The biomaterials used in bioprinting (the bioinks) form a scaffold for embedding the cells/biomolecules of interest, but also could be exploited as a source of endogenous contrast or supplemented with contrast agents for imaging. Additionally, bioprinting enables patient-specific customization with shape/size tailored for actual needs. In stroke or traumatic brain injury for example lesions are localized and focal, and usually progress with significant loss of tissue volume creating space that could be filled with artificial tissue using bioprinting modalities. The value of imaging for bioprinting technology is advantageous on many levels including design of custom shapes scaffolds based on anatomical 3D scans, assessment of performance and integration after scaffold implantation, or to learn about the degradation over time. In this review, we focus on bioprinting technology describing different printing techniques and properties of biomaterials in the context of requirements for neurological applications. We also discuss the need for in vivo imaging of implanted materials and tissue constructs reviewing applicable imaging modalities and type of information they can provide. STATEMENT OF SIGNIFICANCE: Current stem cell-based regenerative strategies for neurological diseases are ineffective due to inaccurate engraftment, low cell viability and suboptimal differentiation. Bioprinting and embedding stem cells within biomaterials at high precision, including building complex multi-material and multi-cell type composites may bring a breakthrough in this field. We provide here comprehensive review of bioinks, bioprinting techniques applicable to application for neurological disorders. Appreciating importance of longitudinal monitoring of implanted scaffolds, we discuss advantages of various imaging modalities available and suitable for imaging biomaterials in the central nervous system. Our goal is to inspire new experimental approaches combining imaging, biomaterials/bioinks, advanced manufacturing and tissue engineering approaches, and stimulate interest in image-guided therapies based on bioprinting., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction : The unique structure of bone and cartilage makes the systemic delivery of free drugs to those connective tissues very challenging. Consequently, effective and targeted delivery for bone and cartilage is of utmost importance. Engineered biodegradable polymers enable designing carriers for a targeted and temporal controlled release of one or more drugs in concentrations within the therapeutic range. Also, tissue engineering strategies can allow drug delivery to advantageously promote the in situ tissue repair. Areas covered : This review article highlights various drug delivery systems (DDS) based on biodegradable biomaterials to treat bone and/or cartilage diseases. We will review their applications in osteoporosis, inflammatory arthritis (namely osteoarthritis and rheumatoid arthritis), cancer and bone and cartilage tissue engineering. Expert opinion : The increased knowledge about biomaterials science and of the pathophysiology of diseases, biomarkers, and targets as well as the development of innovative tools has led to the design of high value-added DDS. However, some challenges persist and are mainly related to an appropriate residence time and a controlled and sustained release over a prolonged period of time of the therapeutic agents. Additionally, the poor prediction value of some preclinical animal models hinders the translation of many formulations into the clinical practice.

Biocompatible ionic liquids (Bio-ILs) are an eco- and bio-friendly family of ionic liquids (ILs) useful in applications ranging from the electrochemical to the biomedical fields. The most promising strategies for their synthesis involve using molecules from bio-renewable sources as a basis for both the anionic and cationic counterparts of the Bio-ILs structure. Several studies have been conducted on Bio-IL properties, including their impact on the environment and health safety. Herein, we review progress and strategies towards the synthesis of Bio-ILs and address their ecotoxicological and biological impact. Furthermore, we discuss the impact of using these compounds in a diverse range of applications, with some insights toward their use in the development of improved technologies.

The menisci have crucial roles in the knee, chondroprotection being the primary. Meniscus repair or substitution is favored in the clinical management of the meniscus lesions with given indications. The outstanding challenges with the meniscal scaffolds include the required biomechanical behavior and features. Suturability is one of the prerequisites for both implantation and implant survival. Therefore, we proposed herein a novel highly interconnected suturable porous scaffolds from regenerated silk fibroin that is reinforced with 3D-printed polycaprolactone (PCL) mesh in the middle, on the transverse plane to enhance the suture-holding capacity. Results showed that the reinforcement of the silk fibroin scaffolds with the PCL mesh increased the suture retention strength up to 400%, with a decrease in the mean porosity and an increase in crystallinity from 51.9 to 55.6%. The wet compression modulus values were significantly different for silk fibroin, and silk fibroin + PCL mesh by being 0.16 ± 0.02, and 0.40 ± 0.06 MPa, respectively. Both scaffolds had excellent interconnectivity (>99%), and a high water uptake feature (>500%). The tissue's infiltration and formation of new blood vessels were assessed by means of performing an in vivo subcutaneous implantation of the silk fibroin + PCL mesh scaffolds that were seeded with primary human meniscocytes or stem cells. Regarding suturability and in vivo biocompatibility, the findings of this study indicate that the silk fibroin + PCL mesh scaffolds are suitable for further studies to be carried out for meniscus tissue engineering applications such as the studies involving orthotopic meniscal models and fabrication of patient-specific implants.

To engineer functional tissue substitutes, it is required a multi-component, multi-scale approach that combines both physical, chemical and biological cues. Fiber-based techniques have been explored in the field of tissue engineering to produce structures recapitulating tissue architecture and mechanical properties. In this work, we engineered biofunctional composite living fibers (CLF) as multi-compartment fibers with a mechanically competent core and a hydrogel layer. For this purpose, commercial silk suture threads were coated with a platelet lysate (PL) hydrogel by first embedding the threads in a thrombin solution and then incubating in PL. The fabrication set-up was optimized and the biological performance was studied by encapsulating human adipose-derived stem cells (hASCs). The developed coating process rendered CLF with a homogenous PL hydrogel layer covering suture threads. Encapsulated hASCs were viable up to 14 d in culture and were able to align at the surface of the core fiber and deposit collagen types I and III. In summary, the study shows that PL-hASCs hydrogel coated suture threads represent a simple multi-compartment and multifunctional system, with PL hydrogel offering biofunctionality to guide the biological activities of encapsulated cells in addition to the replication of tissue-level mechanical support provided by the suture threads.

Collagen is one of the most widely used biomaterials, not only due its biocompatibility, biodegradability and weak antigenic potential, but also due to its role in the structure and function of tissues. Searching for alternative collagen sources, the aim of this study was to extract collagen from the skin of codfish, previously obtained as a by-product of fish industrial plants, and characterize it regarding its use as a biomaterial for biomedical application, according to American Society for Testing and Materials (ASTM) Guidelines. Collagen type I with a high degree of purity was obtained through acid-extraction, as confirmed by colorimetric assays, SDS-PAGE and amino acid composition. Thermal analysis revealed a denaturing temperature around 16 °C. Moreover, collagen showed a concentration-dependent effect in metabolism and on cell adhesion of lung fibroblast MRC-5 cells. In conclusion, this study shows that collagen can be obtained from marine-origin sources, while preserving its bioactivity, supporting its use in biomedical applications.

Surgical site infections (SSI) represent a serious health problem that occur after invasive surgery, thus new antimicrobial biomaterials able to prevent SSI are needed. Silks are natural biopolymers with excellent biocompatibility, low immunogenicity and controllable biodegradability. Spider silk-based materials can be bioengineered and functionalized with specific peptides, such as antimicrobial peptides, creating innovative polymers. Herein, we explored new drug-free multifunctional silk films with antimicrobial properties, specifically tailored to hamper microbial infections. Different spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6mer and 15mer, 27 and 41 kDa proteins, respectively) were fused with the two antimicrobial peptides, Hepcidin (Hep) and Human Neutrophil peptide 1 (HNP1). The self-assembly features of the spider silk domains (β-sheets) were maintained after functionalization. The bioengineered 6mer-HNP1 protein demonstrated inhibitory effects against microbial pathogens. Silk-based films with 6mer-HNP1 and different contents of silk fibroin (SF) significantly reduced bacterial adhesion and biofilm formation, whereas higher bacterial counts were found on the films prepared with 6mer or SF alone. The silk-based films showed no cytotoxic effects on human foreskin fibroblasts. The positive cellular response, together with structural and antimicrobial properties, highlight the potential of these multifunctional silk-based films as new materials for preventing SSI., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The potential of magnetically assisted strategies within the remit of cell-based therapies is increasing, creating new opportunities for biomedical platforms and in the field of tissue engineering and regenerative medicine. Among the magnetic elements approached for building magnetically responsive strategies, superparamagnetic iron oxide nanoparticles (SPIONs) represent tunable and precise tools whose properties can be modelled for detection, diagnosis, targeting and therapy purposes. The most investigated clinical role of SPIONs is as contrast imaging agents for tracking and monitoring cells and tissues. Nevertheless, magnetic detection also includes biomarker mapping, cell labelling and cell/drug targeting to monitor cell events and anticipate the disruption of homeostatic conditions and the progression of disease. Additionally, the isolation and screening techniques of cell subsets in heterogeneous populations or of proteins of interest have been explored in a magnetic sorting context. More recently, SPION-based technologies have been applied to stimulate cell differentiation and mechanotransduction processes and to transport genetic or drug cargo to study biological mechanisms and contribute to improved therapies. Magnetically based strategies significantly contribute to magnetic tissue engineering (magTE), in which magnetically responsive actuators built from magnetic labelled cells or magnetic functionalized systems can be remotely controlled and spatially manipulated upon the actuation of an external magnetic field for the delivery or target of TE solutions. SPION functionalities combined with magnetic responsiveness in multifactorial magnetically assisted platforms can revolutionize diagnosis and therapeutics, providing new diagnosis and theranostic tools, encouraging regenerative medicine approaches and having potential for more effective therapies. This review will address the contribution of SPION-based technologies as multifunctional tools in boosting magnetically assisted cell-based strategies to explore diagnostics and tracking solutions for the detection and analysis of pathologies, and to generate improved treatments and therapies, envisioning precise and customized answers for the management of numerous diseases.

Scaffolds mimicking structural and chemical characteristics of the native bone tissues are critical for bone tissue engineering. Herein, we have developed and characterized epigallocatechin gallate/duck's feet collagen/hydroxyapatite (EGCG/DC/HAp) composite sponges that enhanced the bone tissue regeneration. The three-dimensional composite sponges were synthesized by loading various amounts (i.e. 1, 5 and 10 μM) of EGCG to duck feet derived collagen followed by freeze-drying and then coating with hydroxyapatite. Several measuremental techniques were employed to examine the properties of the as-fabricated composite sponges including morphology and structure, porosity, compressive strength, etc. and as well compared with pristine duck feet derived collagen. SEM observations of EGCG/DC/HAp sponges showed the formation of a highly porous collagen matrix with EGCG embodiment. The porosity and pore size of sponges were found to increase by high EGCG content. The compressive strength was calculated as 3.54 ± 0.04, 3.63 ± 0.03, 3.89 ± 0.05, 4.047 ± 0.05 MPa for 1, 5 and 10 μM EGCG/DC/HAp sponges, respectively. Osteoblast-like cell (BMSCs isolated from rabbit) culture and in vivo experiments with EGCG/DC/HAp sponges implanted in nude mouse followed by histological staining showed enhanced cell internalization and attachment, cell proliferation, alkaline phosphatase expressions, indicating that EGCG/DC/HAp sponges have ahigh biocompatibility. Moreover, highEGCG content in the EGCG/DC/HAp sponges have led to increased cellular behavior. Collectively, the 5 μM of EGCG/DC/HAp sponges were suggested as the potential candidates for bone tissue regeneration.

Blood components play key roles in the modulation of the wound healing process and, together with the provisional fibrin matrix ability to selectively bind bioactive molecules and control its spatial-temporal presentation, define the complex microenvironment that characterize this biological process. As a biomimetic approach, the use of blood derivatives in regenerative strategies has awakened as a source of multiple therapeutic biomolecules. Nevertheless, and despite their clinical relevance, blood derivatives have been showing inconsistent therapeutic results due to several factors, including proper control over their delivery mechanisms. Herein, we highlight recent trends on the use biomaterials to protect, sequester and deliver these pools of biomolecules in tissue engineering and regenerative medicine approaches. Particular emphasis is given to strategies that enable to control their spatiotemporal delivery and improve the selectivity of presentation profiles of the biomolecules derived from blood derivatives rich in platelets. Finally, we discussed possible directions for biomaterials design to potentiate the aimed regenerative effects of blood derivatives and achieve efficient therapies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cancer is a leading cause of mortality and morbidity worldwide. Around 90% of deaths are caused by metastasis and just 10% by primary tumor. The advancement of treatment approaches is not at the same rhythm of the disease; making cancer a focal target of biomedical research. To enhance the understanding and prompts the therapeutic delivery; concepts of tissue engineering are applied in the development of in vitro models that can bridge between 2D cell culture and animal models, mimicking tissue microenvironment. Tumor spheroid represents highly suitable 3D organoid-like framework elucidating the intra and inter cellular signaling of cancer, like that formed in physiological niche. However, spheroids are of limited value in studying critical biological phenomenon such as tumor-stroma interactions involving extra cellular matrix or immune system. Therefore, a compelling need of tailoring spheroid technologies with physiologically relevant biomaterials or in silico models, is ever emerging. The diagnostic and prognostic role of spheroids rearrangements within biomaterials or microfluidic channel is indicative of patient management; particularly for the decision of targeted therapy. Fragmented information on available in vitro spheroid models and lack of critical analysis on transformation aspects of these strategies; pushes the urge to comprehensively overview the recent technological advancements (e.g. bioprinting, micro-fluidic technologies or use of biomaterials to attain the third dimension) in the shed of translationable cancer research. In present article, relationships between current models and their possible exploitation in clinical success is explored with the highlight of existing challenges in defining therapeutic targets and screening of drug efficacy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The survival of a biomaterial or tissue engineered construct is mainly hampered by the deficient microcirculation in its core, and limited nutrients and oxygen availability to the implanted or colonizing host cells. Aiming to address these issues, we herein propose bioresponsive gellan gum (GG) hydrogels that are biodegradable by metalloproteinase 1 (MMP-1) and enable endothelial cells adhesion and proliferation. GG is chemically functionalized with divinyl sulfone (DVS) and then biofunctionalized with thiol cell-adhesive peptides (T1 or C16) to confer GG endothelial cell biorecognition cues. Biodegradable hydrogels are then formed by Michael type addition of GGDVS or/and peptide-functionalized GGDVS with a dithiol peptide crosslinker sensitive to MMP-1. The mechanical properties (6 to 5580 Pa), swelling (17 to 11), MMP-1-driven degradation (up to 70%), and molecules diffusion coefficients of hydrogels are tuned by increasing the polymer amount and crosslinking density. Human umbilical cord vein endothelial cells depict a polarized elongated morphology when encapsulated within T1-containing hydrogels, in contrast to the round morphology observed in C16-containing hydrogels. Cell organization is favored as early as 1 d of cell culture within the T1-modified hydrogels with higher concentration of peptide, while cell proliferation is higher in T1-modified hydrogels with higher modulus. In conclusion, biodegradable and bioresponsive GGDVS hydrogels are promising endothelial cell responsive materials that can be used for vascularization strategies., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of versatile tubular structures is a subject of broad interest in tissue engineering applications. Herein, we demonstrate the production of tubular structures based on chitosan through a combination of dipping, freeze-drying and supercritical technology approaches. The combination of these techniques yields versatile tubes with a perfectly defined hollow imprint, which upon chemical cross-linking with genipin acquire enhanced mechanical properties (Young Modulus ( E) and ultimate tensile stress (σ max )), as well as improved stability in wet conditions. The biological performance reveals that cells remain attached, well-spread and viable on the surface of cross-linked tubes. As so, is envisioned that our methodology opens up new avenues on tissue engineering approaches, where the design of tubular structures with tuned length, diameter and elasticity is required.

The osteochondral defect (OD) comprises the articular cartilage and its subchondral bone. The treatment of these lesions remains as one of the most problematic clinical issues, since these defects include different tissues, requiring distinct healing approaches. Among the growing applications of regenerative medicine, clinical articular cartilage repair has been used for two decades, and it is an effective example of translational medicine; one of the most used cell-based repair strategies includes implantation of autologous cells in degradable scaffolds such as alginate, agarose, collagen, chitosan, chondroitin sulfate, cellulose, silk fibroin, hyaluronic acid, and gelatin, among others. Concerning the repair of osteochondral defects, tissue engineering and regenerative medicine started to design single- or bi-phased scaffold constructs, often containing hydroxyapatite-collagen composites, usually used as a bone substitute. Biomolecules such as natural and synthetic have been explored to recreate the cartilage-bone interface through multilayered biomimetic scaffolds. In this chapter, a succinct description about the most relevant natural and synthetic biomolecules used on cartilage and bone repair, describing the procedures to obtain these biomolecules, their chemical structure, common modifications to improve its characteristics, and also their application in the biomedical fields, is given.

The Central Nervous System (CNS) is a highly complex organ that works as the control centre of the body, managing vital and non-vital functions. Neuro-diseases can lead to the degeneration of neural tissue, breakage of the neuronal networks which can affect vital functions and originate cognitive deficits. The complexity of the neural networks, their components and the low regenerative capacity of the CNS are on the basis for the lack of recovery, having the need for therapies that can promote tissue repair and recovery. Most brain processes are mediated through molecules (e.g. cytokines, neurotransmitters) and cells response accordingly and to surrounding cues, either biological or physical, which offers molecule administration and/or cell transplantation a great potential for use in brain recovery. Biomaterials and in particular, of natural-origin are attractive candidates owed to their intrinsic biological cues and biocompatibility and degradability. Through the use of biomaterials, it is possible to protect the cells/molecules from body clearance, enzymatic degradation while maintaining the components in a place of interest. Moreover, by means of combining several components, it is possible to obtain a more targeted and controlled delivery, to image the biomaterial implantation and its degradation over time and tackling simultaneously occurring events (cell death and inflammation) in brain diseases. In this chapter, it is reviewed some brain-affecting diseases and the current developments on tissue engineering approaches for a functional recovery of the brain from those diseases.

Management of degenerative spine pathologies frequently leads to the need for spinal fusion (SF), where bone growth is induced toward stabilization of the interventioned spine. Autologous bone graft (ABG) remains the gold-standard inducer, whereas new bone graft substitutes attempt to achieve effective de novo bone formation and solid fusion. Limited fusion outcomes have driven motivation for more sophisticated and multidisciplinary solutions, involving new biomaterials and/or biologics, through innovative delivery platforms. The present review will analyze the most recent body of literature that is focused on new approaches for consistent bone fusion of spinal vertebrae, including the development of new biomaterials that pursue physical and chemical aptitudes; the delivery of growth factors (GF) to accelerate new bone formation; and the use of cells to improve functional bone development. Bone graft substitutes currently in clinical practice, such as demineralized bone matrix and ceramics, are still used as a starting point for the study of new bioactive agents. Polyesters such as polycaprolactone and polylactic acid arise as platforms for the development of composites, where a mineral element and cell/GF constitute the delivery system. Exciting fusion outcomes were obtained in several small and large animal models with these. On what regards bioactive agents, mesenchymal stem cells, preferentially derived from the bone marrow or adipose tissue, were studied in this context. Autologous and allogeneic approaches, as well as osteogenically differentiated cells, have been tested. These cell sources have further been genetically engineered for specific GF expression. Nevertheless, results on fusion efficacy with cells have been inconsistent. On the other hand, the delivery of GF (most commonly bone morphogenetic protein-2 [BMP-2]) has provided favorable outcomes. Complications related to burst release and dosing are still the target of research through the development of controlled release systems or alternative GF such as Nel-like molecule-1 (NELL-1), Oxysterols, or COMP-Ang1. Promising solutions with new biomaterial and GF compositions are becoming closer to the human patient, as these evidence high-fusion performance, while offering cost and safety advantages. The use of cells has not yet proved solid benefits, whereas a further understanding of cell behavior remains a challenge.

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., (Copyright © 2016 John Wiley & Sons, Ltd.)

Liquid marbles (LM) are freestanding droplets covered by micro/nanoparticles with hydrophobic/hydrophilic properties, which can be manipulated as a soft solid. The phenomenon that generates these soft structures is regarded as a different method to generate a superhydrophobic behavior in the liquid/solid interface without modifying the surface. Several applications for the LM have been reported in very different fields, however the developments for biomedical applications are very recent. At first, the LM properties are reviewed, namely shell structure, LM shape, evaporation, floatability and robustness. The different strategies for LM manipulation are also described, which make use of magnetic, electrostatic and gravitational forces, ultraviolet and infrared radiation, and approaches that induce LM self-propulsion. Then, very distinctive applications for LM in the biomedical field are presented, namely for diagnostic assays, cell culture, drug screening and cryopreservation of mammalian cells. Finally, a critical outlook about the unexplored potential of LM for biomedical applications is presented, suggesting possible advances on this emergent scientific area., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three-dimensional (3D) printed poly(ε-caprolactone) (PCL) based scaffolds have being proposed for different tissue engineering applications. This study addresses the design and fabrication of 3D PCL constructs with different struts alignments at 90°, 45° and 90° with offset. The morphology and the mechanical behavior under uniaxial compressive load were assessed at different strain percentages. The combination of a new compressionCT device and micro computed tomography (micro-CT) allowed understanding the influence of pore geometry under controlled compressive strain in the mechanical and structural behavior of PCL constructs. Finite element analysis (FEA) was applied using the micro-CT data to modulate the mechanical response and compare with the conventional uniaxial compression tests. Scanning electron microscopic analysis showed a very high level of reproducibility and a low error comparing with the theoretical values, confirming that the alignment and the dimensional features of the printed struts are reliable. The mechanical tests showed that the 90° architecture presented the highest stiffness. With the compressionCT device was observed that the 90° and 90° with offset architectures presented similar values of porosity at same strain and similar pore size, contrary to the 45° architecture. Thus, pore geometric configurations affected significantly the deformability of the all PCL scaffolds under compression. The prediction of the FEA showed a good agreement to the conventional mechanical tests revealing the areas more affected under compression load. The methodology proposed in this study using 3D printed scaffolds with compressionCT device and FEA is a framework that offers great potential in understanding the mechanical and structural behavior of soft systems for different applications, including for the biomedical engineering field.

The objective of this work was to exploit the fibronectin type II (FNII) module from human matrix metalloproteinase-2 as a functional domain for the development of silk-based biopolymer blends that display enhanced cell adhesion properties. The DNA sequence of spider dragline silk protein (6mer) was genetically fused with the FNII coding sequence and expressed in Escherichia coli. The chimeric protein 6mer+FNII was purified by non-chromatographic methods. Films prepared from 6mer+FNII by solvent casting promoted only limited cell adhesion of human skin fibroblasts. However, the performance of the material in terms of cell adhesion was significantly improved when 6mer+FNII was combined with a silk-elastin-like protein in a concentration-dependent behavior. With this work we describe a novel class of biopolymer that promote cell adhesion and potentially useful as biomaterials for tissue engineering and regenerative medicine., Statement of Significance: This work reports the development of biocompatible silk-based composites with enhanced cell adhesion properties suitable for biomedical applications in regenerative medicine. The biocomposites were produced by combining a genetically engineered silk-elastin-like protein with a genetically engineered spider-silk-based polypeptide carrying the three domains of the fibronectin type II module from human metalloproteinase-2. These composites were processed into free-standing films by solvent casting and characterized for their biological behavior. To our knowledge this is the first report of the exploitation of all three FNII domains as a functional domain for the development of bioinspired materials with improved biological performance. The present study highlights the potential of using genetically engineered protein-based composites as a platform for the development of new bioinspired biomaterials., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in vitro and in vivo approaches that leverage the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the past two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due to its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.

Unlabelled: Herein, for the first time, we combined poly-l-lactic acid (PLLA) with a strontium borosilicate bioactive glass (BBG-Sr) using electrospinning to fabricate a composite bioactive PLLA membrane loaded with 10% (w/w) of BBG-Sr glass particles (PLLA-BBG-Sr). The composites were characterised by scanning electron microscopy (SEM) and microcomputer tomography (μ-CT), and the results showed that we successfully fabricated smooth and uniform fibres (1-3μm in width) with a homogeneous distribution of BBG-Sr microparticles (<45μm). Degradation studies (in phosphate buffered saline) demonstrated that the incorporation of BBG-Sr glass particles into the PLLA membranes increased their degradability and water uptake with a continuous release of cations. The addition of BBG-Sr glass particles enhanced the membrane's mechanical properties (69% higher Young modulus and 36% higher tensile strength). Furthermore, cellular in vitro evaluation using bone marrow-derived mesenchymal stem cells (BM-MSCs) demonstrated that PLLA-BBG-Sr membranes promoted the osteogenic differentiation of the cells as demonstrated by increased alkaline phosphatase activity and up-regulated osteogenic gene expression (Alpl, Sp7 and Bglap) in relation to PLLA alone. These results strongly suggest that the composite PLLA membranes reinforced with the BBG-Sr glass particles have potential as an effective biomaterial capable of promoting bone regeneration., Statement of Significance: PLLA membranes were reinforced with 10% (w/w) of strontium-bioactive borosilicate glass microparticles, and their capacity to induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was evaluated. These membranes presented an increased: degradability, water uptake, Young modulus and tensile strength. We also demonstrated that these membranes are non-cytotoxic and promote the attachment of BM-MSCs. The addition of the glass microparticles into the PLLA membranes promoted the increase of ALP activity (under osteogenic conditions), as well as the BM-MSCs osteogenic differentiation as shown by the upregulation of Alpl, Sp7 and Bglap gene expression. Overall, we demonstrated that the reinforcement of PLLA with glass microparticles results in a biomaterial with the appropriate properties for the regeneration of bone tissue., (Copyright © 2016 Acta Materialia Inc. All rights reserved.)

Tissue engineered biomaterials have biodegradable and biocompatible properties. In this study, we have fabricated sponges using duck's feet derived collagen (DC) and gellan gum (GG), and further studied its inflammatory responses. The as-prepared duck's feet DC/GG sponges showed the possibility of application as a tissue engineering material through in vitro and in vivo experiments. The physical and chemical properties of sponges were characterized by compression strength, porosity, and scanning electron microscopy, etc. In vitro cell viability were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. An inflammatory response was studied after seeding RAW264.7 cells on as-fabricated sponges using reverse transcriptase-polymerase chain reaction. In vivo studies were carried out by implanting in subcutaneous nude mouse followed by extraction, histological staining. Collectively, superior results were showed by DC/GG sponges than GG sponge in terms of physical property and cell proliferation and thus can be considered as a potential candidate for future tissue engineering applications.

Unlabelled: Natural biomaterials such as collagen show promise in tissue engineering applications due to their inherent bioactivity. The main limitation of collagen is its low mechanical strength and somewhat unpredictable and rapid degradation rate; however, combining collagen with another material, such as chitosan, can reinforce the scaffold mechanically and may improve the rate of degradation. Additionally, the high cost and the risk of prion transmission associated with mammal-derived collagen has prompted research into alternative sources such as marine-origin collagen. In this context, the overall goal of this study was to determine if the incorporation of chitosan into collagen scaffolds could improve the mechanical and biological properties of the scaffold. In addition the study assessed if collagen, derived from salmon skin (marine), can provide an alternative to collagen derived from bovine tendon (mammal) for tissue engineering applications. Scaffold architecture and mechanical properties were assessed as well as their ability to support mesenchymal stem cell growth and differentiation. Overall, the addition of chitosan to bovine and salmon skin-derived collagen scaffolds improved the mechanical properties, increasing the compressive strength, swelling ratio and prolonged the degradation rate. Mesenchymal stem cell (MSC) attachment and proliferation was most improved on the bovine-derived collagen scaffold containing a 75:25 ratio of collagen:chitosan, and when MSC osteogenic and chondrogenic potential on the scaffold was assessed, a significant increase in calcium production (p<0.001) and sulfated glycosaminoglycan (sGAG) production (p<0.001) was observed respectively. Regardless of chitosan content, the bovine-derived collagen scaffolds out-performed the salmon skin-derived collagen scaffolds, displaying a larger pore size and higher percentage porosity, more regular architecture, higher compressive modulus, a greater capacity for water uptake and allowed for more MSC proliferation and differentiation. This versatile scaffold incorporating the marine biomaterial chitosan show great potential as appropriate platforms for promoting orthopaedic tissue repair while the use of salmon skin-derived collagen may be more suitable in the repair of soft tissues such as skin., Statement of Significance: Collagen is commonly used in tissue engineering due to its biocompatibility; however, it has low mechanical strength and an unpredictable degradation rate. In addition, high cost and risk of prion transmission associated with mammalian-derived collagen has prompted research into alternative collagen sources, namely, marine-derived collagen. In this study, scaffolds made from salmon-skin collagen were compared to the more commonly used bovine-derived collagen with a focus on orthopaedic applications. To improve the mechanical properties of these scaffolds, another marine biomaterial, chitosan, was added to produce scaffolds with increased mechanical stability. The collagen-chitosan composites were also shown to support mesenchymal stem cell differentiation towards both bone and cartilage tissue. This multi-functional scaffold therefore has potential in both bone and cartilage regeneration applications., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Micro- and nanotextured biomaterial surfaces have been widely studied for their capacity to drive the regeneration of organized tissues. Nanotopographical features in the shape of groove-ridge patterns aim at mimicking the extracellular matrix organization. However, to date, a wide array of groove and ridge sizes has been described. In this work, we therefore tested a device composed of a multipatterned array consisting of six patterns of radially arranged parallel nanogrooves, with a pitch ranging from 0 to 1000 nm and a depth ranging from 0 to 170 nm, to be used as a tool for the expeditious and simultaneous screening of surface topographies aiming the regeneration of anisotropically organized tissues such as ligament. The topographies were reproduced in (1) epoxy resin or (2) membranes produced by the crosslinking of platelet lysate (PL) with genipin (gPL). Both materials were seeded with periodontal ligament cells (PDLCs) and the proliferation, migration, as well as cell alignment were assessed. The effect of topography in PDLCs was only evident in terms of cell organization, resulting in a highly anisotropic organization of the cells for the 1000 and 600 nm patterns, and in an increased isotropic organization for shallower topographies. Overall, our results suggest that this multipatterned system can be a valuable diagnostic tool for biomaterials aiming at the regeneration of anisotropically organized tissues, such as periodontal ligament.

In this work, we focused on the potential of bioceramics from different marine sponges-namely Petrosia ficiformis, Agelas oroides and Chondrosia reniformis-for novel biomedical/industrial applications. The bioceramics from these sponges were obtained after calcination at 750 °C for 6 h in a furnace. The morphological characteristics were evaluated by scanning electron microscopy (SEM). The in vitro bioactivity of the bioceramics was evaluated in simulated body fluid (SBF) after 14 and 21 d. Observation of the bioceramics by SEM after immersion in SBF solution, coupled with spectroscopic elemental analysis (EDS), showed that the surface morphology was consistent with a calcium-phosphate (Ca/P) coating, similar to hydroxyapatite crystals (HA). Evaluation of the characteristic peaks of Ca/P crystals by Fourier transform infrared spectroscopy and x-ray diffraction further confirmed the existence of HA. Cytotoxicity studies were carried out with the different ceramics and these were compared with a commercially available Bioglass(®). In vitro tests demonstrated that marine bioceramics from these sponges are non-cytotoxic and have the potential to be used as substitutes for synthetic Bioglass(®).

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (∼0.5-4.7 μm), and mean distance between peaks (RSm) gradually varying from ∼214 μm to 33 μm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra∼1.53 μm/RSm∼79 μm in Dex-deprived OI medium, and Ra∼0.93 μm/RSm∼135 μm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone., Statement of Significance: Biodegradable polymers, such as polycaprolactone (PCL), are promising materials in the field of tissue engineering and regenerative medicine, which aims at creating viable options to replace permanent orthopedic implants. The material, cells, and growth-stimulating factors are often referred to as the key components of engineered tissues. In this article, we studied the hypothesis of specific surface modification of PCL being capable of inducing mesenchymal stem cell differentiation in bone cells in the absence of cell-differentiating factors. The systematic investigation of the linearly varying surface-roughness gradient showed that an average PCL roughness of 0.93 μm alone can serve as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)