No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Self-Glucose Feeding Hydrogels by Enzyme Empowered Degradation for 3D Cell Culture
Abstract
Hydrogels have been used in combination with cells for several biomedical and biotechnological applications. Nevertheless, the use of bulk hydrogels has exhibited severe limitations in diffusion of oxygen, nutrients, and metabolites. Here, a support for cell culture is reported where glucose is generated in situ by the own hydrogel degradation, allowing cell survival and function while promoting tissue growth. For this purpose, laminaran (or laminarin)-based hydrogels were fabricated, immobilizing the adequate enzymes to obtain structural platforms for 3D cell culture and providing glucose feeding for metabolic activity of cells through polysaccharide degradation. We demonstrate that tumor A549 cells and human mesenchymal stem cells (hMSCs) can use the glucose resultant from the hydrogel degradation to survive and grow in non-added glucose cell culture medium. Additionally, in vivo biocompatibility and biodegradability of laminaran-based hydrogels were explored for the first time. The self-feeding hydrogels exhibited high potential in cell survival compared to native cell-laden laminaran hydrogels over two weeks of sub-cutaneous implantation. Such bioscaffolds with enzyme-empowered degradation capacity can be applied in diverse biotechnological contexts such as tissue regeneration devices, biofactories, disease models, and cell delivery systems.
... Additionally, FU can also be extracted from some marine invertebrates (sea urchins and sea cucumbers) (Lee and Mooney, 2012;Quitain et al., 2013;Cunha and Grenha, 2016). Although LAM is still relatively unexplored, it has recently attracted the attention of the scientific community in the biomedical field due to its low viscosity, cytocompatibility and biocompatibility (Zargarzadeh et al., 2022). ...
... The insertion of methacrylate groups (MA) into the polymer backbone to be further photocrosslinked is a widely applied chemical route in natural polysaccharides and used in the fabrication of several biomedical devices, such as 3D hydrogel structures (Bjørge et al., 2018;Martins et al., 2018), cell encapsulation systems (Khademhosseini et al., 2006), and in drug delivery platforms (Jeon et al., 2009). The introduction of photo-crosslinkable moieties into polysaccharide backbones can occur across two distinct functional groups: i) primary hydroxyl groups (Custódio et al., 2016;Martins et al., 2018;Zargarzadeh et al., 2022) and ii) amine groups Bjørge et al., 2018). The methacrylation reaction via hydroxyl and amine groups is carried out through substitution reaction, achieving a higher substitution degree for the latter one due to its nucleophilic strength (higher for amines than hydroxyl groups) (Wang H. et al., 2018;Kolawole et al., 2018). ...
Over the past few decades, natural-origin polysaccharides have received increasing attention across different fields of application, including biomedicine and biotechnology, because of their specific physicochemical and biological properties that have afforded the fabrication of a plethora of multifunctional devices for healthcare applications. More recently, marine raw materials from fisheries and aquaculture have emerged as a highly sustainable approach to convert marine biomass into added-value polysaccharides for human benefit. Nowadays, significant efforts have been made to combine such circular bio-based approach with cost-effective and environmentally-friendly technologies that enable the isolation of marine-origin polysaccharides up to the final construction of a biomedical device, thus developing an entirely sustainable pipeline. In this regard, the present review intends to provide an up-to-date outlook on the current green extraction methodologies of marine-origin polysaccharides and their molecular engineering toolbox for designing a multitude of biomaterial platforms for healthcare. Furthermore, we discuss how to foster circular bio-based approaches to pursue the further development of added-value biomedical devices, while preserving the marine ecosystem.
... Such scaffolds, equipped with capabilities of enzymatic degradation, have broad applications, including tissue regeneration and cell delivery systems. 69 For 3D bioprinting, the preparation of hydrogel bioinks, which is dynamically crosslinked, is obtaining importance as a groundbreaking approach to improve the production of mechanically adjustable cell-laden frameworks for diverse applications in 3D in vitro disease modeling and tissue engineering. In this context, Amaral et al. explored a dynamic bioink containing boronic acid-functionalized laminarin and alginate for the bioprinting of 3D structures under a physiologically appropriate environment. ...
Laminarin, a complicated polysaccharide originating from brown algae, has emerged as a compelling candidate in the domain of biomedical research. This enigmatic molecule, composed of glucose units associated with both β-1,3 and β-1,6 glycosidic bonds, possesses an array of remarkable characteristics that render it auspicious for multifaceted biomedical applications. This review investigates the comprehensive potential of laminarin in the biomedical domain, emphasizing its remarkable biocompatibility, low cytotoxicity, and cell proliferation support. Laminarin's immunomodulatory attributes position it as an encouraging contender in immunotherapy and the development of vaccines. Moreover, its anti-inflammatory and antioxidant characteristics provide a promising avenue for combatting conditions associated with oxidative stress. In particular, laminarin excels as a drug delivery vehicle owing to its exceptional encapsulation capabilities emerging from its porous framework. Integrating pH and redox responsiveness in laminarin-based drug delivery systems is poised to redefine targeted therapies. Laminarin substantially contributes to tissue engineering by improving adhesion, migration of cells, and deposition of extracellular matrix. This augmentation magnifies the regenerative capability of tissue-engineered constructs, substantiated by the advancement of laminarin-based wound dressings and tissue scaffolds, marking considerable progress in the domain of wound healing and tissue regeneration. While laminarin exhibits substantial potential in biomedical applications, it remains in the initial phases of exploration. Comprehensive preclinical and clinical research is warranted to verify its effectiveness and safety across various applications. In essence, laminarin, a marine marvel, has the capability to remodel biomedical research, offering inventive solutions to complex difficulties.
... However, to the best of our knowledge, the assembly of neutral marine polysaccharides into LbL biostructures has not been exploited to date, despite their wide abundance and variety, easy functionality, and invaluable potential in the biomedical and biotechnological fields. For instance, laminarin (LAM) -a low-molecular-weight, branched, and neutral marine polysaccharide found in brown algae, which is composed of glucose units linked by β-1,3 glycosidic bonds, as well as by some β-1,6 side-chain branches -has been recently functionalized with a wide variety of chemical functional groups for being processed into either photocrosslinkable hydrogels or microparticles with high stability for being used in 3D cell culture, tissue engineering, and regenerative medicine [34][35][36][37][38]. Pullulan (PUL) is a high-molecular-weight, linear, and neutral polysaccharide composed of maltotriose units linked by α-1,4-bonds produced by the yeast-like fungus (Aureobasidium pullulans), which can be found in several marine environments, from marshes to marine sediments and estuarine waters [39]. ...
Marine-origin polysaccharides, in particular cationic and anionic ones, have been widely explored as building blocks in fully natural or hybrid electrostatic-driven Layer-by-Layer (LbL) assemblies for bioapplications. However, the low chemical versatility imparted by neutral polysaccharides has been limiting their assembly into LbL biodevices, despite their wide availability in sources such as the marine environment, easy functionality, and very appealing features for addressing multiple biomedical and biotechnological applications. In this work, we report the chemical functionalization of laminarin (LAM) and pullulan (PUL) marine polysaccharides with peptides bearing either six lysine (K6) or aspartic acid (D6) amino acids via Cu(I)-catalyzed azide-alkyne cycloaddition to synthesize positively and negatively charged polysaccharide-peptide conjugates. The successful conjugation of the peptides into the polysaccharide’s backbone was confirmed by proton nuclear magnetic resonance and attenuated total reflectance Fourier-transform infrared spectroscopy, and the positive and negative charges of the LAM-K6/PUL-K6 and LAM-D6/PUL-D6 conjugates, respectively, were assessed by zeta-potential measurements. The electrostatic-driven LbL build-up of either the LAM-D6/LAM-K6 or PUL-D6/PUL-K6 multilayered thin film was monitored in situ by quartz crystal microbalance with dissipation monitoring, revealing the successful multilayered film growth and the enhanced stability of the PUL-based film. The construction of the PUL-peptide multilayered thin film was also assessed by scanning electron microscopy and its biocompatibility was demonstrated in vitro towards L929 mouse fibroblasts. The herein proposed approach could enable the inclusion of virtually any kind of small molecules in the multilayered assemblies, including bioactive moieties, and be translated into more convoluted structures of any size and geometry, thus extending the usefulness of neutral polysaccharides and opening new avenues in the biomedical field, including in controlled drug/therapeutics delivery, tissue engineering, and regenerative medicine strategies.
... In an attempt to overcome these limitations, self-feeding laminarin-based hydrogels with immobilized β-glucanases were assembled and used to support the growth of 3D tumor and human stem cell cultures, while simultaneously providing a continuous source of glucose for the metabolic activity of cells, through laminarin degradation [245]. ...
β-glucans are a diverse group of polysaccharides composed of β-1,3 or β-(1,3-1,4) linked glucose monomers. They are mainly synthesized by fungi, plants, seaweed and bacteria, where they carry out structural, protective and energy storage roles. Because of their unique physicochemical properties, they have important applications in several industrial, biomedical and biotechnological processes. β-glucans are also major bioactive molecules with marked immunomodulatory and metabolic properties. As such, they have been the focus of many studies attesting to their ability to, among other roles, fight cancer, reduce the risk of cardiovascular diseases and control diabetes. The physicochemical and functional profiles of β-glucans are deeply influenced by their molecular structure. This structure governs β-glucan interaction with multiple β-glucan binding proteins, triggering myriad biological responses. It is then imperative to understand the structural properties of β-glucans to fully reveal their biological roles and potential applications. The deconstruction of β-glucans is a result of β-glucanase activity. In addition to being invaluable tools for the study of β-glucans, these enzymes have applications in numerous biotechnological and industrial processes, both alone and in conjunction with their natural substrates. Here, we review potential applications for β-glucans and β-glucanases, and explore how their functionalities are dictated by their structure.
During two-dimensional (2D) culture, stem cells gradually lose their proliferative activity and multipotency due to various physicochemical conditions, which significantly hinder the large-scale clinical application of stem cell therapy. In...
More recently, single‐cell encapsulation emerged as a promising field in biomedicine due to its potential applications, in cell analysis and therapy. Traditional techniques involve embedding cells in crosslinked polymers to create continuous microgels, suitable mainly for adherent cells, or encapsulating them in droplets for only short‐term analysis, due to their instability. In this study, we developed a method for encapsulating single cells in liquid‐core microcapsules to address these limitations. The liquid encapsulation system is generated in an all aqueous environment through polymeric electrostatic interactions. Additionally, we design an innovative and low cost sorting system utilizing magnetic nanoparticles (MNPs) to efficiently select single‐cell encapsulated units for further analysis and applications. This system is tested with both suspension and adherent cell types, demonstrating cytocompatibility and no abnormal effects on cell behavior. The MNP‐based sorting achieved nearly 80% purity of the single‐cell population. Overall, this technology provides a highly efficient method for single‐cell applications, such as cell screening, by enabling precise short to medium‐term analysis, real time monitoring, and high resolution imaging of cellular behavior. Furthermore, the semipermeable membrane unlocks new potential for advancing cell therapy by offering protection for encapsulated cells while ensuring the efficient diffusion of therapeutic factors, paving the way for innovative therapeutic strategies.
Injectable hydrogels represent a promising strategy for the extended release of biological molecules, thereby reducing the frequency of injections. This study introduces a novel system based on Michael addition of dextran and polyethylene glycol (PEG) polymers functionalized with oxanorbornadiene (OND) and thiol groups, respectively. Reliable control over gelation speed allows administration by injection using a simple syringe‐to‐syringe mixing protocol that entrains more than 95% of virus‐like particle (VLP) cargo. A combination of retro‐Diels‐Alder and hydrolytic ester bond cleavage gives rise to programmable release of the VLPs. Different release profiles, including burst, linear, and delayed release over a two‐week period, are engineered using different OND linkages, and rheological characterization shows the hydrogels to be well within the desired range of stiffness for subcutaneous use. The modular nature of this system offers a generalizable platform for developing degradable materials aimed at sustained release biomedical applications.
Photo‐crosslinkable platelet lysate (PL)‐based hydrogels have been proven to support human‐derived cell cultures owing to their high content of bioactive molecules, such as cytokines and growth factors. As a unique self‐maintained and biocompatible 3D scaffold, the recently reported self‐feeding hydrogels with enzyme‐empowered degradation capacity have shown high biological performance in vitro and in vivo. To take advantage of all features of both PL and self‐feeding hydrogels, here UV responsive laminaran‐methacrylate (LamMA) and PL‐methacrylate (PLMA) derivatives plus glucoamylase (GA), which significantly improve the overall features of a 3D system, is coupled. This self‐sustaining hybrid hydrogel emerges as a unique scaffold due to the sustained delivery of glucose produced via enzymatic degradation of laminaran while granting the release of growth factors through the presence of PL. This biomaterial is applied to fabricate high‐throughput freestanding microgels with controlled geometric shapes. Furthermore, this multicomponent hybrid hydrogel is successfully implemented as the first reported glucose supplier bioink to manufacture intricate and precisely defined cell‐laden structures using a support matrix. Finally, such hydrogels are utilized as a proof of concept to serve as 3D in vitro cancer models, with the aim of recapitulating the tumor microenvironment.
The optimized physical adhesion between bees' leg hairs and pollen grains - whereby the latter's diameter aligns with the spacing between the hairs - has previously inspired the development of a biomimetic drug dressing. Combining this optimized process with the improved natural mussels' adhesion in wet environments in a dual biomimetic process, it is herein proposed the fabrication of a natural-derived micropatterned hydrogel patch of methacrylated laminarin (LAM-MET), with enriched drug content and improved adhesiveness, suitable for applications like wound healing. Enhanced adhesion is accomplished by modifying LAM-MET with hydroxypyridinone groups, following the patch microfabrication by soft lithography and UV/visible-irradiation, resulting in a membrane with micropillars with high aspect-ratio. Following the biomimetics rational, a drug patch is engineered by combining the microfabricated dressing with drug particles milled to fit the spaces between pillars. Controlled drug release is achieved, together with inherent antibacterial activity against Escherichia coli and Pseudomonas aeruginosa, and enhanced biocompatibility using the bare micropatterned patches. This new class of biomimetic dressings overcomes the challenges of current patches like poor mechanical properties and biocompatibility, limited adhesiveness and drug dosage, and lack of prolonged antimicrobial activity, opening new insights for the development of high drug-loaded dressings with improved patient compliance. This article is protected by copyright. All rights reserved.
Research has advanced considerably since the first clinical trial of human mesenchymal stem cells (MSCs) in the early 1990s. During this period, our understanding of MSC biology and our ability to expand and manipulate these cells have provided hope for the repair of damaged tissues due to illness or injury. MSCs have conventionally been injected systemically or locally into target tissue; however, inconsistent cell homing and engraftment efficiencies represent a major bottleneck that has led to mixed results in clinical studies. To overcome these issues, MSCs have been pre-conditioned with biomolecules, genetically altered, or surface engineered to enhance their homing and engraftment capabilities. In parallel, a variety of cell-encapsulating materials have been designed to improve cell delivery and post-transplantation survival and function. In this review, we discuss the current strategies that have been employed on cultured MSCs to improve targeted cell delivery and retention for tissue repair. We also discuss the advances in injectable and implantable biomaterial technologies that drive the success of MSC-based therapies in regenerative medicine. Multi-faceted approaches combining cellular modification and cell-instructive material design can pave the way for efficient and robust stem cell transplantation for superior therapeutic outcomes.
The natural design and coupling of biological structures are the root of realizing the high strength, toughness, and unique functional properties of biomaterials. Advanced architecture design is applied to many materials, including metal materials, inorganic nonmetallic materials, polymer materials, and so on. To improve the performance of advanced materials, the designed architecture can be enhanced by bionics of biological structure, optimization of structural parameters, and coupling of multiple types of structures. Herein, the progress of structural materials is reviewed, the strengthening mechanisms of different types of structures are highlighted, and the impact of architecture design on the performance of advanced materials is discussed. Architecture design can improve the properties of materials at the micro level, such as mechanical, electrical, and thermal conductivity. The synergistic effect of structure makes traditional materials move toward advanced functional materials, thus enriching the macroproperties of materials. Finally, the challenges and opportunities of structural innovation of advanced materials in improving material properties are discussed.
Injectable hydrogels are potential local drug delivery systems since they contain plenty of water and soft like biological tissues. Such hydrogels could be injected directly into the tumor site where the drug is released under the tumor microenvironment. However, drug loaded hydrogels for cancer treatment based on lipoic acid (natural small molecule) have not been exploited. Here, a novel polylipoic acid-polyethylene glycol (PEG-PTA) hydrogels were prepared through a two-step reaction. The hydrogels contained disulfide bonds, so they could be degraded via the thiol exchange reaction with the abundant GSH in the tumor microenvironment, and subsequently release the drug. The results in vitro and at cellular level showed that the hydrogels were degraded and released the drugs only in the presence of GSH. Therefore, the injectable GSH-responsive hydrogels are promising to be served as an intelligent drug delivery system for cancer treatment.
3D cell cultures are becoming the new standard for cell-based in vitro research, due to their higher transferrability toward in vivo biology. The lack of established techniques for the non-destructive quantification of relevant variables, however, constitutes a major barrier to the adoption of these technologies, as it increases the resources needed for the experimentation and reduces its accuracy. In this review, we aim at addressing this limitation by providing an overview of different non-destructive approaches for the evaluation of biological features commonly quantified in a number of studies and applications. In this regard, we will cover cell viability, gene expression, population distribution, cell morphology and interactions between the cells and the environment. This analysis is expected to promote the use of the showcased technologies, together with the further development of these and other monitoring methods for 3D cell cultures. Overall, an extensive technology shift is required, in order for monolayer cultures to be superseded, but the potential benefit derived from an increased accuracy of in vitro studies, justifies the effort and the investment.
Solid state fermentation (SsF) is recognized as a suitable process for the production of enzymes using organic residues as substrates. However, only a few studies have integrated an evaluation of the feasibility of applying enzymes produced by SsF into subsequent hydrolyses followed by the production of target compounds, e.g., lactic acid (LA), through submerged-liquid fermentations (SmF). In this study, wheat bran (WB) was used as the substrate for the production of enzymes via SsF by Aspergillus awamori DSM No. 63272. Following optimization, cellulase and glucoamylase activities were 73.63 ± 5.47 FPU/gds and 107.10 ± 2.63 U/gdb after 7 days and 5 days of fermentation, respectively. Enzymes were then used for the hydrolysis of the organic fraction of municipal solid waste (OFMSW). During hydrolysis, glucose increased considerably with a final value of 19.77 ± 1.56 g/L. Subsequently, hydrolysates were fermented in SmF by Bacillus coagulans A166 increasing the LA concentration by 15.59 g/L. The data reported in this study provides an example of how SsF and SmF technologies can be combined for the valorization of WB and OFMSW.
In the primordial cell encapsulation systems, the main goal is to treat endocrine diseases avoiding the action of the immune system. Although lessons afforded by such systems are of outmost importance for the demands of tissue engineering and regenerative medicine, the paradigm has recently completely changed. If before the most important feature was to mask the encapsulated cells from the immune system, now it is known that the synergetic interplay between immune cells and the engineered niche is responsible for an adequate regenerative process. Combined with such immuno‐awareness, novel or nonconventional emerging techniques are being proposed to develop the new generation of cell encapsulation systems, namely layer‐by‐layer, microfluidics, superhydrophobic surfaces, and bioprinting technologies. Alongside the desire to create more realistic cell encapsulation systems, cell‐laden hydrogels are being explored as building blocks for bottom‐up strategies, within the concept of modular tissue engineering. The idea is to use the well‐established cell‐friendly environment provided by hydrogels and create more close‐to‐native systems that possess high heterogeneity, while providing multifunctional and adaptive inputs. Cell encapsulation systems recreating the heterogeneity of native tissues are of utmost importance in tissue healing. Different new/nonconventional techniques are presented to produce microstructural functional units for bottom‐up assembly, while their immunomodulatory properties are debated.
Vascularization is among the top challenges that impede the clinical application of engineered tissues. This challenge has spurred tremendous research endeavor, defined as vascular tissue engineering (VTE) in this article, to establish a pre-existing vascular network inside the tissue engineered graft prior to implantation. Ideally, the engineered vasculature can be integrated into the host vasculature via anastomosis to supply nutrient to all cells instantaneously after surgery. Moreover, sufficient vascularization is of great significance in regenerative medicine from many other perspectives. Due to the critical role of vascularization in successful tissue engineering, we aim to provide an up-to-date overview of the fundamentals and VTE strategies in this article, including angiogenic cells, biomaterial/bio-scaffold design and bio-fabrication approaches, along with the reported utility of vascularized tissue complex in regenerative medicine. We will also share our opinion on the future perspective of this field.
Engineering three-dimensional (3D) tissues in clinically relevant sizes have demonstrated to be an effective solution to bridge the gap between organ demand and the dearth of compatible organ donors. A major challenge to the clinical translation of tissue-engineered constructs is the lack of vasculature to support an adequate supply of oxygen and nutrients post-implantation. Previous efforts to improve the vascularization of engineered tissues have not been commensurate to meeting the oxygen demands of implanted constructs during the process of homogeneous integration with the host. Maintaining cell viability and metabolic activity during this period is imperative to the survival and functionality of the engineered tissues. As a corollary, there has been a shift in the scientific impetus beyond improving vascularization. Strategies to engineer biomaterials that encapsulate cells and provide the sustained release of oxygen over time are now being explored. This review summarizes different types of oxygen-releasing biomaterials, strategies for their fabrication, and approaches to meet the oxygen requirements in various tissue engineering applications, including cardiac, skin, bone, cartilage, pancreas, and muscle regeneration.
Injectable hydrogels can fill irregular defects and promote in situ tissue regrowth and regeneration. The ability of directing stem cell differentiation in a three-dimensional microenvironment for bone regeneration remains a challenge. In this study, we successfully nanoengineer an interconnected microporous networked photocrosslinkable chitosan in situ-forming hydrogel by introducing two-dimensional nanoclay particles with intercalation chemistry. The presence of the nanosilicates increases the Young's modulus and stalls the degradation rate of the resulting hydrogels. We demonstrate that the reinforced hydrogels promote the proliferation as well as the attachment and induced the differentiation of encapsulated mesenchymal stem cells in vitro. Furthermore, we explore the effects of nanoengineered hydrogels in vivo with the critical-sized mouse calvarial defect model. Our results confirm that chitosan-montmorillonite hydrogels are able to recruit native cells and promote calvarial healing without delivery of additional therapeutic agents or stem cells, indicating their tissue engineering potential.
The field of tissue engineering is making great strides in developing replacement tissue grafts for clinical use, marked by the rapid development of novel biomaterials, their improved integration with cells, better‐directed growth and differentiation of cells, and improved 3D tissue mass culturing. One major obstacle that remains, however, is the lack of graft vascularization, which in turn renders many grafts to fail upon clinical application. With that, graft vascularization has turned into one of the holy grails of tissue engineering, and for the majority of tissues it will be imperative to achieve adequate vascularization if tissue graft implantation is to succeed. Many different approaches have been developed to induce or augment graft vascularization, both in vitro and in vivo. In this review, we highlight the importance of vascularization in tissue engineering, and outline various approaches inspired by both biology and engineering to achieve and augment graft vascularization.
Rapid progress in tissue engineering research in past decades has opened up vast possibilities to tackle the challenges of generating tissues or organs that mimic native structures. The success of tissue engineered constructs largely depends on the incorporation a stable vascular network that eventually anastomoses with the host vasculature to support the various biological functions of embedded cells. In recent years, significant progress has been achieved with respect to extrusion, laser, micro-molding, and electrospinning-based techniques that allow the fabrication of any geometry in a layer-by-layer fashion. Moreover, decellularized matrix, self-assembled structures, and cell sheets have been explored to replace the biopolymers needed for scaffold fabrication. While the techniques have evolved to create specific tissues or organs with outstanding geometric precision, formation of interconnected, functional, and perfused vascular networks remains a challenge. This article briefly reviews recent progress in 3D fabrication approaches used to fabricate vascular networks with incorporated cells, angiogenic factors, proteins, and/or peptides. The influence of the fabricated network on blood vessel formation, and the various features, merits, and shortcomings of the various fabrication techniques are discussed and summarized.
Background
Laminarin is a potential biomass feedstock for the production of glucose, which is the most preferable fermentable sugar in many microorganisms by which it can be converted to biofuels and bio-based chemicals. Also, laminarin is a good resource as functional materials because it consists of β-1,3-glucosidic linkages in its backbone and β-1,6-glucosidic linkages in its branches so that its oligosaccharides driven from laminarin have a variety of biological activities. It is industrially important to be able to produce laminarioligosaccharides as well as glucose from laminarin by a single enzyme because the enzyme cost accounts for a large part of bio-based products. In this study, we investigated the industrial applicability of Bgl1B, a unique β-glucosidase from Saccharophagus degradans 2-40T, belonging to the glycoside hydrolase family 1 (GH1) by characterizing its activity of hydrolyzing laminarin under various conditions. ResultsBgl1B was cloned and overexpressed in Escherichia coli from S. degradans 2-40T, and its enzymatic activity was characterized. Similar to most of β-glucosidases in GH1, Bgl1B was able to hydrolyze a variety of disaccharides having different β-linkages, such as laminaribiose, cellobiose, gentiobiose, lactose, and agarobiose, by cleaving β-1,3-, β-1,4-, and β-1,6-glycosidic linkages. However, Bgl1B showed the highest specific activity toward laminaribiose with a β-1,3-glycosidic linkage. In addition, it was able to hydrolyze laminarin, one of the major polysaccharides in brown macroalgae, into glucose with a conversion yield of 75% of theoretical maximum. Bgl1B also showed transglycosylation activity by producing oligosaccharides from laminarin and laminaribiose under a high mass ratio of substrate to enzyme. Furthermore, Bgl1B was found to be psychrophilic, exhibiting relative activity of 59–85% in the low-temperature range of 2–20 °C. Conclusions
Bgl1B can directly hydrolyze laminarin into glucose with a high conversion yield without leaving any oligosaccharides. Bgl1B can exhibit high enzymatic activity in a broad range of low temperatures (2–20 °C), which is advantageous for establishing energy-efficient bioprocesses. In addition, under high substrate to enzyme ratios, Bgl1B can produce high-value laminarioligosaccharides via its transglycosylation activity. These results show that Bgl1B can be an industrially important enzyme for the production of biofuels and bio-based chemicals from brown macroalgae.
Biosynthesis of nanoparticles using isolated compounds from various sources is accepting interest due to their broad array of biological activities and biocompatibility. This paper presents a simple; cost effective and green synthesis of silver nanoparticles (AgNPs) using the polysaccharide, laminarin a storage compound obtained from the brown algae Turbinaria ornata (T. ornata). Initially, the water soluble polysaccharide, laminarin was extracted, purified and analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectroscopy (MALDI-TOF MS) and Proton Nuclear Magnetic Resonance (¹H NMR). Further, the silver nanoparticles (AgNPs) were synthesized using the isolated laminarin and were characterized by Ultraviolet - visible (UV-vis) spectrophotometer, colour value analysis, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD) and High Resolution Transmission Electron Microscopy (HR-TEM). The free radical scavenging activities were performed and the effect of cytotoxicity against retinoblastoma Y79 cell lines was also evaluated by in vitro studies. Induction of apoptosis was evident by the percentage of cells arrested in G2/M phase using flow cytometry analysis and was further confirmed by DNA fragmentation study which identified the presence of double strand break.
Hydrogels are hydrophilic, three-dimensional networks that are able to absorb large quantities of water or biological fluids, and thus have the potential to be used as prime candidates for biosensors, drug delivery vectors, and carriers or matrices for cells in tissue engineering. In this critical review article, advantages of the hydrogels that overcome the limitations from other types of biomaterials will be discussed. Hydrogels, depending on their chemical composition, are responsive to various stimuli including heating, pH, light, and chemicals. Two swelling mechanisms will be discussed to give a detailed understanding of how the structure parameters affect swelling properties, followed by the gelation mechanism and mesh size calculation. Hydrogels prepared from natural materials such as polysaccharides and polypeptides, along with different types of synthetic hydrogels from the recent reported literature, will be discussed in detail. Finally, attention will be given to biomedical applications of different kinds of hydrogels including cell culture, self-healing, and drug delivery.
Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55ºC, and, in the substratum absence, the thermostability was for 1h at 50ºC. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65ºC was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme's preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.
Importance:
In this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40(T) Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in the GH5 family. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucan and β-1,6-glucan. Gly5M also possesses a transglycolyase activity towards β-1,3-oligosacchrides. Due to this broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.
Cell-laden hydrogels can regenerate lost, damaged or malfunctioning tissues. Clinical success of such hydrogels is strongly dependent on the ability to tune their chemical, physico-mechanical, and biological properties to a specific application. In particular, mimicking the intricate arrangement of cell interactive ligands of natural tissues is crucial to proper tissue function. Natural extracellular matrix elements represent a unique source for generating such interactions. A plethora of extracellular matrix based approaches have been explored to augment the regenerative potential of hydrogels. These efforts include the development of matrix-like hydrogels, hydrogels containing matrix-like molecules, hydrogels containing decellularized matrix, hydrogels derived from decellularized matrix, and decellularized tissues as reimplantable matrix hydrogels. Here we review the evolution, strengths and weaknesses of these developments from the perspective of creating tissue regenerating hydrogels.
Polysaccharides are used extensively in various industrial applications, such as food, adhesives, coatings, construction, paper, pharmaceuticals, and personal care. Many polysaccharide structures need to be modified in order to improve their end-use properties; this is mostly done through chemical reactions. In the past 20 years many enzyme-catalyzed modifications have been developed to supplement chemical derivatization methods. Typical reactions include enzymatic oxidation, ester formation, amidation, glycosylation, and molecular weight reduction. These reactions are reviewed in this paper, with emphasis placed on the work done by the authors. The polymers covered in this review include cellulosic derivatives, starch, guar, pectin, and poly(ethylene glycol).
Hydrogel products constitute a group of polymeric materials, the hydrophilic structure of which renders them capable of holding large amounts of water in their three-dimensional networks. Extensive employment of these products in a number of industrial and environmental areas of application is considered to be of prime importance. As expected, natural hydrogels were gradually replaced by synthetic types due to their higher water absorption capacity, long service life, and wide varieties of raw chemical resources. Literature on this subject was found to be expanding, especially in the scientific areas of research. However, a number of publications and technical reports dealing with hydrogel products from the engineering points of view were examined to overview technological aspects covering this growing multidisciplinary field of research. The primary objective of this article is to review the literature concerning classification of hydrogels on different bases, physical and chemical characteristics of these products, and technical feasibility of their utilization. It also involved technologies adopted for hydrogel production together with process design implications, block diagrams, and optimized conditions of the preparation process. An innovated category of recent generations of hydrogel materials was also presented in some details.
Dietary fibres consist of edible plant polysaccharides that are resistant to digestion and absorption in the human small intestine but undergo complete or partial fermentation in the colon. Seaweeds, notably Laminaria spp, are particularly rich in polysaccharides resistant to hydrolysis in the upper gastrointestinal tract and are, in consequence, considered as dietary fibres. Most of the carbohydrates from Laminaria spp are thought to be indigestible by humans. The main storage polysaccharide of these algae is laminarin, a -polymer of glucose. The aims of this work were, on the one hand, to compare various methods of extraction of laminarin by partial characterisation of the product obtained and, on the other hand, to study the fate of this polysaccharide and its effects in the gastrointestinal tract in order to determine its potential as a dietary fibre in human nutrition. Among four methods tested to extract laminarin, the best appeared to be a hot HCl-based method. Human digestive enzymes did not hydrolyse laminarin, so this polysaccharide can be considered as a dietary fibre. After ingestion by rats, this polysaccharide was not found in faeces of these animals. It did not increase the intestinal transit and stool output in vivo, but it increased the contractile response of the stomach to acetylcholine in vitro. Copyright © 2004 Society of Chemical Industry
Most therapeutic applications of bone marrow stromal cells (MSCs), or mesenchymal stem cells, require expansion of these cells. This study aimed to obtain more information about human MSCs regarding their expansion characteristics: growth, metabolism, and growth inhibitors. In addition, the same expansion factors were examined for (model species) goat and rat MSCs to evaluate differences between MSCs of mammalian species. MSC proliferation, nutrient consumption, and metabolite production were determined for five donors per species. In addition, the growth inhibitory concentrations of lactate and ammonia (NH3) were established. Results showed that goat MSCs grew significantly faster than human and rat MSCs and that goat cells metabolized glucose more efficiently into energy (Ylac/glc=0.8) than human (Ylac/glc=2.0) and rat MSCs (Ylac/glc=1.9). In addition, human (qGlc= -9.2pmol cell(-1) day(-1) and rat MSCs (qGlc= -5.9pmol cell(-1) day(-1)) consumed more glucose than goat MSCs (qGlc= -2.6pmol cell(-1) day(-1)). Glutamine was shown not to be important as energy source for human, goat, and rat MSCs. Regarding growth inhibition by metabolites, rat MSCs were more sensitive to lactate and NH3 (growth inhibiting at 16mM lactate and at 1.9mM NH3) than goat (lactate: 28.4mM, NH3: 2.9mM) and human MSCs (lactate: 35.4mM, NH3: 2.4mM). Human MSCs did not lose their differentiation potential when their growth was inhibited by lactate or NH3.
The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.
The ocean is par excellence a fertile territory of biodiversity on our planet. Marine-derived polysaccharides have been applied as functional materials in biomedicine due to their attractive bioactive properties, safety, high availability and low-cost production. Laminarin (or laminaran), a low molecular weight β-glucan storage polysaccharide present in brown algae, can be (bio-) chemically modified to enhance its biological activity and employed in cancer therapies, drug/gene delivery, tissue engineering, antioxidant and anti-inflammatory functions. This review provides a brief overview on laminarin characteristics, modification strategies and highlights its pivotal biomedical applications.
Hydrogel scaffolds that can repair or regrow damaged biological tissue have great potential for the treatment of injury and disease. These biomaterials are widely used in the tissue engineering field due to their ability to support cell proliferation, migration and differentiation, to permit oxygen and nutrient transport, and mimic native soft tissue. Careful design of the underlying polymer scaffold is therefore critical, dictating both the physical and biological properties of a hydrogel. In this review, we will provide a critical overview of hydrogel design from the perspective of the polymer chemistry, highlighting both the advantages and limitations of particular polymer structures, properties, and architectures. By doing so, we hope to equip researchers with the tools to design new polymer systems and hydrogel scaffolds that address current limitations in the field and limit clinical translation.
Enzymatically-degradable materials recapitulate the dynamic and reciprocal interactions between cells and their native microenvironment by allowing cells to actively shape the degradation process. In order to engineer a synthetic 3D environment enabling cells to orchestrate the degradation of the surrounding material, norbornene-modified alginate was crosslinked with two different peptide crosslinkers susceptible to cleavage by matrix metalloproteinases using UV-initiated thiol-ene chemistry. Resulting hydrogels were characterized for their initial mechanical and rheological properties, and their degradation behavior was measured by tracking changes in wet weight upon enzyme incubation. This process was found to be a function of the crosslinker type and enzyme concentration, indicating that degradation kinetics could be controlled and tuned. When mouse embryonic fibroblasts were encapsulated in 3D, cell number remained constant and viability was high in all materials, while cell spreading and extensive filopodia formation was observed only in the degradable gels, not in non-degradable controls. After implanting hydrogels into the backs of C57/Bl6 mice for 8 weeks, histological stainings of recovered gel remnants and surrounding tissue revealed higher tissue and cell infiltration into degradable materials compared to non-degradable controls. This alginate-based material platform with cell-empowered enzymatic degradation could prove useful in diverse tissue engineering contexts, such as regeneration and drug delivery.
Microfabrication technologies have been widely explored to produce microgels that can be assembled in functional constructs for tissue engineering and regenerative medicine applications. Here, we propose microfluidics coupled to a source of UV light to produce multifunctional methacrylated laminarin microparticles with narrow distribution of sizes using photopolymerization. The multifunctional microparticles were loaded with platelet lysates and further conjugated with an adhesive peptide. The adhesive peptides dictated cell adhesiveness to the laminarin microparticles, the incorporation of platelet lysates have resulted in improved cell expansion compared to clear microparticles. Overall, our findings demonstrate that multifunctional methacrylated laminarin microparticles provide an effective support for cell attachment and expansion. Moreover, expanded cells provide the link for microparticles aggregation resulting in robust 3D structures. This suggest the potential for using the methacrylated laminarin microplatforms capable to be assembled by the action of cells to rapidly produce large tissue engineered constructs.
With the advancement in biomaterial sciences, tissue engineered scaffolds are developing as a promising strategy for the regeneration of damaged tissues. However, only a few of these scaffolds have been translated into clinical applications. One of the primary drawbacks of the existing scaffolds is the lack of adequate oxygen supply within the scaffolds. Oxygen producing biomaterials have been developed as an alternate strategy but are faced with two major concerns. One is controlling the rate of oxygen generation, and the other is the production of reactive oxygen species. To address these concerns, here, we report the development of an oxygen releasing antioxidant polymeric cryogel scaffold (PUAO-CPO) for sustained oxygen delivery. PUAO-CPO was fabricated using the cryogelation technique by incorporation of calcium peroxide in the antioxidant polyurethane scaffolds. PUAO-CPO cryogels attenuated reactive oxygen species and showed a sustained release of oxygen over a period of 10 days. An in vitro analysis of the PUAO-CPO cryogels showed their ability to sustain H9C2 cardiomyoblast cells under hypoxic conditions, with cell viability being significantly better than normal polyurethane scaffolds. Furthermore, in vivo studies using an ischemic flap model showed the ability of the oxygen releasing cryogel scaffolds to prevent tissue necrosis upto 9 days. Histological examination indicated maintenance of tissue architecture and collagen content, whereas immunostaining for proliferating cell nuclear antigen (PCNA) confirmed the viability of the ischemic tissue with oxygen delivery. Our study demonstrated an advanced approach for the development of oxygen releasing biomaterials with sustained oxygen delivery as well as attenuated production of residual reactive oxygen species and free radicals either due to ischemia or oxygen generation. Hence, the oxygen releasing PUAO-CPO cryogel scaffolds may be used with cell based therapeutic approaches for regeneration of damaged tissue, particularly with ischemic conditions such as myocardial infarction and chronic wound healing.
Hydrogels have captivated the attention of several research and industry segments, including bioengineering, tissue engineering, implantable/wearable sensors and actuators, bioactive agent delivery, food processing, and industrial processes optimization. A common limitation of these systems is their fixed shape. The concept of hydrogel moldability is often assigned to the injectability potential of liquid precursors, and this feature is often lost right after hydrogel formation. Hydrogel modulation is a recent trend that advocates the importance of designing materials with shape fitting ability targeting on-demand responses or defect filling purposes. Here, we present a compliant and cell encapsulation-compatible hydrogel prepared from unmodified natural origin polymers with the ability to undergo extreme sequential shape alterations with high recovery of its mechanical properties. Different fragments of these hydrogels could be bonded together in spatiotemporally-controlled shape- and formulation-morphing structures. This material is prepared with affordable off-the-shelf polysaccharides of natural origin using a mild and safe processing strategy, based solely on polyelectrolyte complexation followed by an innovative partial coacervate compactation and dehydration step. These unique hydrogels hold potential for multifield industrial and healthcare applications. In particular, they may find application as defect filling agents or highly compliant wound healing patches for cargo release and/or cell delivery for tissue regeneration and cell-based therapies.
Mesenchymal stem cells (MSCs) hold considerable promise in tissue engineering (TE). Their poor survival when exogenously administered, however, limits their therapeutic potential. Previous studies from our group demonstrated that lack of glucose (but not of oxygen) is fatal to human MSCs because it serves as a pro-survival and pro-angiogenic molecule for hMSC upon transplantation. However, which energy-providing pathways MSCs use to metabolize glucose upon transplantation? Are there alternative energetic nutrients to replace glucose? And most importantly, do hMSCs possess significant intracellular glucose reserves for ensuring their survival upon transplantation? These remain open questions at the forefront of TE based-therapies. In the present study, we established for the first time that the in vivo environment experienced by hMSCs is best reflected by near-anoxia (0.1% O2) rather than hypoxia (1% -5% O2) in vitro. Under these near-anoxia conditions, hMSCs rely almost exclusively on glucose through anaerobic glycolysis for ATP production and are unable to use either exogenous glutamine, serine or pyruvate as energy substrates. Most importantly, hMSCs are unable to adapt their metabolism to the lack of exogenous glucose, possess a very limited internal stock of glucose and virtually no ATP reserves. This lack of downregulation of energy turnover as a function of exogenous glucose level results in a rapid depletion of hMSC energy reserves that explains their poor survival rate. These new insights prompt for the development of glucose-releasing scaffolds to overcome this roadblock plaguing the field of TE based-therapies. This article is protected by copyright. All rights reserved.
Biomaterials for tissue engineering provide scaffolds to support cells and guide tissue regeneration. Despite significant advances in biomaterials design and fabrication techniques, engineered tissue constructs remain functionally inferior to native tissues. This is largely due to the inability to recreate the complex and dynamic hierarchical organization of the extracellular matrix components, which is intimately linked to a tissue's biological function. This review discusses current state-of-the-art strategies to control the spatial presentation of physical and biochemical cues within a biomaterial to recapitulate native tissue organization and function.
Laminarin is a low molecular weight (<10kDa) glucan found in brown algae made up of β(1→3)-glucan with β(1→6)-branches. This is one of the most abundant carbon sources in the marine ecosystem. Laminarin has been found to possess various biological interesting properties, such as antioxidant and antimicrobial activities. An attractive feature of laminarin is its inherently low viscosity and high solubility in organic and aqueous solvents that facilitates processing. This makes laminarin an appealing material for the development of new hydrogels that can be easily injected through minimally invasive procedures or used for microfabrication of hydrogels. An approach for synthesizing photocrosslinkable laminarin hydrogels is presented in this work for the first time. Photocrosslinkable laminarin was prepared by chemical modification with acrylate groups. The synthesized photocrosslinkable laminarin material provides the basis for the development of a new injectable system for biomedical purposes that could be used alone, or with encapsulated cells or biological molecules. The crosslinking of the methacrylated laminarin is straightforward via photo-initiated polymerization. The possibility to control the methacrylation degree of laminarin and to prepare solutions up to at least 15% w/v permits to obtain hydrogels with tuned and wide-range of stiffness and swelling. Furthermore, the encapsulation of human adipose stem cells encapsulated in the photocrosslinked hydrogels demonstrated in vitro biocompatibility.
Increasingly, pharmaceutical research and development is based on a detailed understanding of molecular interactions in diseased and healthy states of the human body. Over the past 50 years, most drug research has concentrated on the effects of small molecules on naturally occurring entities called enzymes and receptors. Hence, this chapter commences with an overview of the interactions of low-molecular-weight compounds (some natural [e.g. neurotransmitters] and some non-natural [e.g. drugs that inhibit certain enzymes]) with these natural macromolecules. This high-level introduction is followed by a more detailed inspection of the structures of some typical enzymes and receptors, emphasizing the complex shapes and subtle intermolecular interactions of these high-molecular-weight proteins. In addition, the importance of understanding the 'on-off' interaction between a small molecule and the target protein is illustrated by introducing the rate equations which dictate the kinetics of these episodes. The concluding section provides the first insight into the problems that have to be faced and overcome in moving from the point of having a compound with the desired effect on an enzyme or receptor in vitro to the position of introducing a useful drug to the marketplace.
Brown algae are rich sources of bioactive compounds such as polysaccharides, peptides, omega-3 fatty acids, carotenoids, phenolics, vitamins and minerals. Laminarin is low-molecular-weight polysaccharide and bioactive compound present in brown algae. Laminarin is found in the fronds of Laminaria and Saccharina species. Laminarin, a storage β-glucan, is composed of (1,3)-β-d-glucan and some β-(1,6)-intrachain links. The reported content of laminarin from brown algae is up to levels of 35% on dry basis, which varies depending on species, harvesting season, habitat and method of extraction. Laminarin has many reported biofunctional activities including antitumour, anti-apoptotic, anti-inflammatory, anticoagulant and antioxidant activity. Biofunctional activities of laminarin can be enhanced after suitable chemical modifications, sulphation and novel processing techniques. Studies on feeding of laminarin-rich extracts to animals indicate it's suitability as functional ingredient for food applications. This paper reviews the main sources, structure and extraction of laminarin with its biofunctional activities.
Biosensing and diagnostic platforms with high sensitivity, specificity, and fast response time are based on immobilized biomolecules such as antibodies (Abs), aptamers, enzymes, nucleic acids, receptors, and whole cells for the detection of target analytes. Such sensing biomolecules should be bound to the surface of a signal transducer with a required specific chemical, electrical, or optical property. The biological recognition event generates a quantifiable signal, which is equated to the amount or concentration of the analyte. APTES can be deposited on solid materials, electrode materials, nanomaterials, and nanocomposites under variable conditions of concentration, solvent, temperature, and time. In addition, curing conditions such as air/heat drying might be necessary depending upon the intended application. Pertinent information on the thickness, morphology, and conformation of the APTES layer reported in the literature is often different and conflicting.
Water-soluble polysaccharides were isolated from the brown alga Eisenia bicyclis, which was collected near the coast of the Republic of Korea. The structures of laminaran and fucoidan were investigated. Laminaran from E. bicyclis was determined to be a glucan with β-(1 → 6) side chains linked to a β-(1 → 3) backbone with relatively few branch points. Based on nuclear magnetic resonance (NMR) data, the ratio of the β-(1 → 3) and β-(1 → 6) linkages was estimated as 2.6:1. Fucoidan from E. bicyclis was found to contain 1,3-linked fucose residues, some 1,6-, 1,2,6-, 1,4,6-linked galactose residues and traces of mannose and xylose. In addition, the amount of sulfate in fucoidan was 13.2%. Those polysaccharides were non-cytotoxic to human melanoma SK-MEL-28 and colon cancer DLD-1 cells. Laminaran and fucoidan from E. bicyclis inhibited the colony formation of those cells. Therefore, they may have potential as antitumor agents.
A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.
A proper supply of nutrients to cells in engineered tissues is paramount for an optimal development and survival of these tissues. However, especially in tissues with clinically relevant sizes, the mass transport of nutrients into the tissue is often insufficient to sustain all the cells within the tissue. This is not only the case during in vitro culture. After implantation of an engineered tissue, a vascular network is not directly established. Therefore, the mass transport of nutrients is also critical during the initial period after implantation.
This review introduces the basics of mass transport, leading to the conclusion that three main concepts can be used to increase nutrient supply in tissue engineering. These are; increasing the overall diffusion coefficient, decreasing the diffusion distance, or increasing convective transport. Based on these concepts, the main strategies that have been developed to enhance the supply of nutrients to cells in engineered tissues will be discussed.
Sphere templating is an attractive method to produce porous polymeric scaffolds with well-defined and uniform pore structures for applications in tissue engineering. While high porosity is desired to facilitate cell seeding and enhance nutrient transport, the incorporation of pores will impact gross mechanical properties of tissue scaffolds and will likely be dependent on pore size. The goals of this study were to evaluate the effect of pores, pore diameter, and polymer composition on gross mechanical properties of hydrogels prepared from crosslinked poly(ethylene glycol) (PEG) and poly(2-hydroxyethyl methacrylate) (pHEMA). Sphere templates were fabricated from uncrosslinked poly(methyl methacrylate) spheres sieved between 53-63 and 150-180 μm. Incorporating pores into hydrogels significantly decreased the quasi-static modulus and ultimate tensile stress, but increased the ultimate tensile strain. For pHEMA, decreases in gel crosslinking density and increases in pore diameters followed similar trends. Interestingly, the mechanical properties of porous PEG hydrogels were less sensitive to changes in pore diameter for a given polymer composition. Additionally, pore diameter was shown to affect skeletal myoblast adhesion whereby many cells cultured in porous hydrogels with smaller pores were seen spanning across multiple pores, but lined the inside of larger pores. In summary, incorporation of pores and changes in pore diameter significantly affect the gross mechanical properties, but in a manner that is dependent on gel chemistry, structure, and composition. Together, these findings will help to design better hydrogel scaffolds for applications where gross mechanical properties and porosity are critical.
Use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for various diseases but has generated marginally successful results. A consistent finding of most studies is massive death of transplanted cells. The present study examined the respective roles of glucose and continuous severe hypoxia on MSC viability and function with respect to bone tissue engineering. We hereby demonstrate for the first time that MSCs survive exposure to long-term (12 days), severe (pO(2) < 1.5 mmHg) hypoxia, provided glucose is available. To this end, an in vitro model that mimics the hypoxic environment and cell-driven metabolic changes encountered by grafted sheep cells was established. In this model, the hallmarks of hypoxia (low pO(2) , hypoxia inducible factor-1α expression and anaerobic metabolism) were present. When conditions switched from hypoxic (low pO(2) ) to ischemic (low pO(2) and glucose depletion), MSCs exhibited shrinking, decreased cell viability and ATP content due to complete exhaustion of glucose at day 6; these results provided evidence that ischemia led to the observed massive cell death. Moreover, MSCs exposed to severe, continuous hypoxia, but without any glucose shortage, remained viable and maintained both their in vitro proliferative ability after simulation with blood reperfusion at day 12 and their in vivo osteogenic ability. These findings challenge the traditional view according to which severe hypoxia per se is responsible for the massive MSC death observed upon transplantation of these cells and provide evidence that MSCs are able to withstand exposure to severe, continuous hypoxia provided that a glucose supply is available.
Glucoamylase is one of the oldest and widely used biocatalysts in food industry. The major application of glucoamylase is the saccharification of partially processed starch/dextrin to glucose, which is an essential substrate for numerous fermentation processes and a range of food and beverage industries. Glucoamylase for commercial purposes has traditionally been produced employing filamentous fungi, although a diverse group of microorganisms is reported to produce glucoamylase, since they secrete large quantities of the enzyme extracellularly. The commercially used fungal glucoamylases have certain limitations such as moderate thermostability, acidic pH requirement, and slow catalytic activity that increase the process cost. Consequently, the search for newer glucoamylases and protein engineering to improve pH and temperature optima leading to amelioration in catalytic efficiency of existing enzymes have been the major areas of research over the years. The present review focuses attention on the recent advances in molecular biology and protein engineering of glucoamylase to improve its production and functional properties including the so far success achieved in isolating mutants with enhanced thermostability and selectivity, higher pH optimum and improved catalytic activity. A comprehensive account is included on the diversity, regulation of production, classification, purification and properties, and potential applications of microbial glucoamylases to provide an overview on all the important aspects of the enzyme.
Hydrogels have been extensively used in various biomedical applications such as drug delivery and biosensing. More recently the ability to engineer the size and shape of biologically relevant hydrogels has generated new opportunities in addressing challenges in tissue engineering such as vascularization, tissue architecture and cell seeding. Here, we discuss the use of microengineered hydrogels for tissue engineering applications. We will initially provide an overview of the various approaches that can be used to synthesize hydrogels with controlled features and will subsequently discuss the emerging applications of these hydrogels.
High glucose (HG) concentrations impair cellular functions and induce apoptosis. Exposition of mesenchymal stem cells (MSC) to HG was reported to reduce colony forming activity and induce premature senescence. We characterized the effects of HG on human MSC in vitro using telomerase-immortalized MSC (hMSC-TERT) and primary MSC (hMSC). HG (25mM) enhanced hMSC-TERT proliferation in long-term studies in contrast to hMSC where proliferation was unchanged. Thioredoxin-interacting protein, which is involved in apoptosis regulation, was stimulated by glucose in hMSC-TERT. However, apoptosis was not influenced by HG in both cell types. MSC treatment with HG favored osteogenic differentiation. MSC are resistant to HG toxicity, depending on the stemness of MSC. Proliferation and osteogenic differentiation are stimulated by HG. Effects of HG on the transient amplifying compartment of MSC may differ from those in mature cells. Further research is needed to unravel the molecular mechanisms of HG resistance of MSC.
- Lei