Recent advances on open fluidic systems for biomedical applications: A review

Microfluidics has become an important tool to engineer microenvironments with high precision, comprising devices and methods for controlling and manipulating fluids at the submillimeter scale. A specific branch of microfluidics comprises open fluidic systems, which is mainly characterized by displaying a higher air/liquid interface when compared with traditional closed-channel setups. The use of open channel systems has enabled the design of singular architectures in devices that are simple to fabricate and to clean. Enhanced functionality and accessibility for liquid handling are additional advantages inputted to technologies based on open fluidics. While benchmarked against closed fluidics approaches, the use of directly accessible channels decreases the risk of clogging and bubble-driven flow perturbation. In this review, we discuss the advantages of open fluidics systems when compared to their closed fluidics counterparts. Platforms are analyzed in two separated groups based on different confinement principles: wall-based physical confinement and wettability-contrast confinement. The physical confinement group comprises both open and traditional microfluidics; examples based on open channels with rectangular and triangular cross-section, suspended microfluidics, and the use of narrow edge of a solid surface for fluid confinement are addressed. The second group covers (super)hydrophilic/(super)hydrophobic patterned surfaces, and examples based on polymer-, textile- and paper-based microfluidic devices are explored. The technologies described in this review are critically discussed concerning devices' performance and versatility, manufacturing techniques and fluid transport/manipulation methods. A gather-up of recent biomedical applications of open fluidics devices is also presented.European Research Council grant agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE and ERC-2014-ADG-669858 for project “ATLAS”. N. M. Oliveira acknowledges the financial support from Portuguese Foundation for Science and Technology − FCT (Grant SFRH/BD/73172/2010), from the financial program POPH/FSE from QREN. The work was developed within the scope of the project CICECO Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013). Sara Vilabril acknowledges the financial support from national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreementinfo:eu-repo/semantics/publishedVersio

Dendrimers and derivatives as a potential therapeutic tool in regenerative medicine strategies : a review

Since the pioneering work dealing with the synthesis and physicochemical aspects of dendrimers, a predictable and tunable set of compositions for therapeutic, scaffolding and imaging systems has been reported. These are well documented, butmanyhot issues should be examined and reviewed. Herein, a review is given on dendritic nanopolymers and their applications that show promise in the field of regenerative medicine. This review begins with a brief overview on research merging nanotechnology and regenerative medicine. Fundamentals of the synthesis and macromolecular structure of dendritic polymers are provided. Dendrimers fulfill the requirements as carriers for gene, nucleic acids, bioactive molecules and peptide/protein delivery aimed at modulate the cells functions, in vitro and in vivo. However, to make use of this potential, toxicological, drug-loading capacity, surface engineering and host–guest chemistries in dendrimers must be addressed and thus are also discussed. We focus on recent work involving dendrimers with applications in tissue engineering and the central nervous system. Due to their innovative character, applications beyond drug delivery systems became possible, namely as scaffolding and chemoattractants for tissue regeneration, and implantable biodegradable nanomaterialbased medical devices integrated with drug delivery functions (theranostics). Finally, we highlight promising areas for further research and comment on how and why dendrimer and dendron technology should be viewed as the next generation of biomaterials for the 21st century.The authors wish to acknowledge the funding provided by the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programs, and FCT project (SMARTCARBO). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and European Union HIPPOCRATES STREP Project (NMP3-CT-2003-505758). Canon Foundation in Europe is also gratefully acknowledged

Fast decellularization process using supercritical carbon dioxide for trabecular bone

Decellularization is a process that consists on the removal of immunogenic cellular material from a tissue, so that it can be safely implanted as a functional and bioactive scaffold. Most decellularization protocols rely on the use of harsh chemicals and very long washing processes, leading to severe changes in the ultrastructure and loss of mechanical integrity. To tackle these challenges, supercritical carbon dioxide (scCO2) is herein proposed as an alternative methodology for assisting decellularization of porcine trabecular bone tissue and is combined, for the first time, with Tri(n-butyl) phosphate (TnBP). Histological and DNA analysis revealed that both TnBP and scCO2 were able to extract the DNA content from the scaffolds, being this effect more pronounced in treatments that used TnBP as a co-solvent. The combined protocol led to a decrease in DNA content by at least 90%, demon- strating the potential of this methodology and opening new possibilities for future optimizations.info:eu-repo/semantics/publishedVersio

Angiogenic potential of gellan gum-based hydrogels for application in nucleus pulposus regeneration : in vivo study

Hydrogels for nucleus pulposus (NP) regeneration should be able to comprise a nonangiogenic or even antiangiogenic feature. Gellan gum (GG)–based hydrogels have been reported to possess adequate properties for being used as NP substitutes in acellular and cellular strategies, due to its ability to support cell encapsulation, adequate mechanical properties, and noncytotoxicity. In this study, the angiogenic response of GG-based hydrogels was investigated by performing the chorioallantoic membrane assay. The convergence of macroscopic blood vessels toward the GG, ionic-crosslinked methacrylated GG (iGG-MA), and photo-crosslinked methacrylated GG (phGGMA) hydrogel discs was quantified. Gelatin sponge (GSp) and filter paper (FP) alone and with vascular endothelial growth factor were used as controls of angiogenesis. The images obtained were digitally processed and analyzed by three independent observers. The macroscopic blood vessel quantification demonstrated that the GG-based hydrogels are not angiogenic as compared with FP controls. No statistical differences between the GG-based hydrogels tested in respect to its angiogenic ability were observed. Hematoxylin and eosin staining and SNA-lectin immunohistochemistry assay indicated that the iGG-MA and phGG-MA hydrogels do not allow the ingrowth of chick endothelial cells, following 4 days of implantation. On the contrary, GG, GSp, and FP controls allowed cell infiltration. The histological data also indicated that the GG-based hydrogels do not elicit any acute inflammatory response. The results showed that the GG, iGG-MA, and phGG-MA hydrogels present different permeability to cells but functioned as a physical barrier for vascular invasion. These hydrogels present promising and tunable properties for being used as NP substitutes in the treatment of degenerative intervertebral disc.The authors thank the funds provided by Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programs (SFRH/BI/33503/2008). This work was also carried out with the support of the European Union funded Collaborative Project Disc Regeneration (NMP3-LA-2008-213904)

Gemcitabine delivered by fucoidan/chitosan nanoparticles presents increased toxicity over human breast cancer cells

Aim: To produce marine-origin nanoparticles (NPs) aiming to develop more effective and tolerated therapies for breast cancer. Materials & methods: NPs based in two marine-origin polymers (fucoidan and chitosan) were prepared by polyelectrolyte complexation, for the delivery of an antitumor drug model (gemcitabine [Gem]). Results: Final formulation resulted in stable NPs around 115â 140 nm in size and with a polydispersity index less than 0.2. Gem was encapsulated at a maximum entrapment efficiency of 35â 42%. Drug-release studies demonstrated that around 84% of Gem is released within 4 h. Cytotoxicity results of Gem-loaded NPs showed increased toxicity (around 25%) when compared with free Gem. Conclusion: The drug-loaded NPs present increased toxicity over human breast cancer cells without increasing toxic effects over endothelial cells.The authors thank the PhD scholarship of C Oliveira for ‘NORTE-08-5369-000037’ financed by NORTE 2020, Portuguese Foundation for Science and Tecnology (FCT) for the investigator grant of A Martins for (IF/00376/2014) and the support from European Research Council under the Advanced Grant ComplexiTE. The work here reported also received financial support from the European Regional Development Fund (ERDF) under the Structured Project ‘Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marine-derived biomaterials and stem cells’ (NORTE-01-0145FEDER-000021),supported by Norte Portugal Regional Operational Program(NORTE2020), under the PORTUGAL2020 Partnership Agreement. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.info:eu-repo/semantics/publishedVersio

A design of experiments (DoE) approach to optimize cryogel manufacturing for tissue engineering applications

Marine origin polymers represent a sustainable and natural alternative to mammal counterparts regarding the biomedical application due to their similarities with proteins and polysaccharides present in extracellular matrix (ECM) in humans and can reduce the risks associated with zoonosis and overcoming social- and religious-related constraints. In particular, collagen-based biomaterials have been widely explored in tissue engineering scaffolding applications, where cryogels are of particular interest as low temperature avoids protein denaturation. However, little is known about the influence of the parameters regarding their behavior, i.e., how they can influence each other toward improving their physical and chemical properties. Factorial design of experiments (DoE) and response surface methodology (RSM) emerge as tools to overcome these difficulties, which are statistical tools to find the most influential parameter and optimize processes. In this work, we hypothesized that a design of experiments (DoE) model would be able to support the optimization of the collagen-chitosan-fucoidan cryogel manufacturing. Therefore, the parameters temperature (A), collagen concentration (B), and fucoidan concentration (C) were carefully considered to be applied to the Boxâ Behnken design (three factors and three levels). Data obtained on rheological oscillatory measurements, as well as on the evaluation of antioxidant concentration and adenosine triphosphate (ATP) concentration, showed that fucoidan concentration could significantly influence collagen-chitosan-fucoidan cryogel formation, creating a stable internal polymeric network promoted by ionic crosslinking bonds. Additionally, the effect of temperature significantly contributed to rheological oscillatory properties. Overall, the condition that allowed us to have better results, from an optimization point of view according to the DoE, were the gels produced at −80ºC and composed of 5% of collagen, 3% of chitosan, and 10% fucoidan. Therefore, the proposed DoE model was considered suitable for predicting the best parameter combinations needed to develop these cryogels.This research was funded by the Portuguese Foundation for Science and Technology (FCT) for Ph.D. fellowship (D.N.C.) under the scope of the doctoral program Tissue Engineering, Regenerative Medicine and Stem Cells, ref. PD/BD/143044/2018, for postdoctoral fellowship (C.G.), ref. SFRH/BPD/94277/2013. This work has been partially funded by ERDF under the scope of the Atlantic Area Program through project EAPA_151/2016 (BLUEHUMAN)

Continuous production of highly tuned silk/calcium-based composites: exploring new pathways for skin regeneration

Calcium plays an important role in barrier function repair and skin homeostasis. In particular, calcium phosphates (CaPs) are well established materials for biomedical engineering due to their biocompatibility. To generate biomaterials with a more complete set of biological properties, previously discarded silk sericin (SS) has been recovered and used as a template to grow CaPs. Crucial characteristics for skin applications, such as antibacterial activity, can be further enhanced by doping CaPs with cerium (Ce) ions. The effectiveness of cell attachment and growth on the materials highly depends on their morphology, particle size distribution, and chemical composition. These characteristics can be tailored through the application of oscillatory flow technology, which provides precise mixing control of the reaction medium. Thus, in the present work, CaP/SS and CaP/SS/Ce particles were fabricated for the first time using a modular oscillatory flow plate reactor (MOFPR) in a continuous mode. Furthermore, the biological behavior of both these composites and of previously produced pure CaPs was assessed using human dermal fibroblasts (HDFs). It was demonstrated that both CaP based with plate-shaped nanoparticles and CaP-SS-based composites significantly improved cell viability and proliferation over time. The results obtained represent a first step towards the reinvention of CaPs for skin engineering.info:eu-repo/semantics/publishedVersio

Development of gellan gum-based microparticles/hydrogel matrices for application in the intervertebral disc regeneration

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where intervertebral disc degeneration begins. Gellan gum-based (GG) hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MPs/hydrogels discs formulations were prepared by means of mixing high (HAGG 0.75% (w/v)) and low acyl (LAGG 2% (w/v)) GG aqueous solutions at different ratios, namely 75%:25% (v/v), 50%:50% (v/v), 25%:75% (v/v), respectively. The GG MPs size was measured using a stereo microscope and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physico-chemically characterized by Fourier-transform infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy. The swelling behaviour and degradation rate were assessed by immersion in a phosphate buffer saline solution for the period of 14 days. The morphology and mechanical behaviour were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogels discs were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). In order to investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48 and 72 hours of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are non-cytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 hours of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration.Fundação para a Ciência e a Tecnologia (FCT) throught the POCTI and FEDER, including ProteoLigh