Conversion of cellulosic materials into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processes

Background: Mannosylerythritol lipids (MEL) are glycolipids with unique biosurfactant properties and are produced by Pseudozyma spp. from different substrates, preferably vegetable oils, but also sugars, glycerol or hydrocarbons. However, solvent intensive downstream processing and the relatively high prices of raw materials currently used for MEL production are drawbacks in its sustainable commercial deployment. The present work aims to demonstrate MEL production from cellulosic materials and investigate the requirements and consequences of combining commercial cellulolytic enzymes and Pseudozyma spp. under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. Results: MEL was produced from cellulosic substrates, Avicel® as reference (>99% cellulose) and hydrothermally pretreated wheat straw, using commercial cellulolytic enzymes (Celluclast 1.5 L® and Novozyme 188®) and Pseudozyma antarctica PYCC 5048T or Pseudozyma aphidis PYCC 5535T. The strategies included SHF, SSF and fed-batch SSF with pre-hydrolysis. While SSF was isothermal at 28°C, in SHF and fed-batch SSF, yeast fermentation was preceded by an enzymatic (pre-)hydrolysis step at 50°C for 48 h. Pseudozyma antarctica showed the highest MEL yields from both cellulosic substrates, reaching titres of 4.0 and 1.4 g/l by SHF of Avicel® and wheat straw (40 g/l glucan), respectively, using enzymes at low dosage (3.6 and 8.5 FPU/gglucan at 28°C and 50°C, respectively) with prior dialysis. Higher MEL titres were obtained by fed-batch SSF with pre-hydrolysis, reaching 4.5 and 2.5 g/l from Avicel® and wheat straw (80 g/l glucan), respectively. Conclusions: This work reports for the first time MEL production from cellulosic materials. The process was successfully performed through SHF, SSF or Fed-batch SSF, requiring, for maximal performance, dialysed commercial cellulolytic enzymes. The use of inexpensive lignocellulosic substrates associated to straightforward downstream processing from sugary broths is expected to have a great impact in the economy of MEL production for the biosurfactant market, inasmuch as low enzyme dosage is sufficient for good systems performance

New adsorbers for the removal of genotoxic impurities from active pharmaceutical ingredients

Active pharmaceutical ingredients (APIs) available in the market are mostly synthesized, in organic solvent media, using highly reactive molecules that may be present in the final product as genotoxic impurities (GTIs). These compounds have the ability to react with DNA, preventing its normal replication, resulting in an associated carcinogenic risk, becoming an increasing concern from pharmaceutical companies and regulatory authorities [1]. Although it is desirable to avoid the use of GTIs in the manufacture of APIs, this is not always possible. Therefore, there is a call to develop simple, robust and economical routes to remove GTIs to limits below the Threshold of Toxicological Concern (1.5 µg/day) [2]. Such adsorbents should be highly selective to reach ultra-low GTI levels with minimal API losses and compatible with organic solvents where the API synthesis takes place [3]. Herein we report two different strategies for the development of new adsorbing materials designed for selective removal of GTIs from API organic solvent solutions. These new materials are: i) molecular imprinted polymers (MIPs), in the particular case designed for removal of an aromatic amine GTI, 4-dymethylaminopyridine) [4]; and ii) a novel functionalized polymer consisting on polybenzimidazole (PBI) modified with a DNA base (PBI-adenine). PBI-Adenine is designed to have a high affinity for a broad range of DNA alkylating agents mimicking the DNA-GTI adduct formation that takes place in vivo [5,6]. These platforms proved to be robust materials being able to remove, in a single stage, more than 95% of the GTIs from organic solvent API mixtures. Both approaches, meet the pharmaceutical industry challenges, by opening new horizons for the use of these adsorbers as a complement to the existing operation units as MIPs, as well as their assembling as membranes for organic solvent nanofiltration (OSN) derived from PBI. References [1] Teasdale A. et al., Org. Process Res. Dev. 17, 2013, 221-230. [2] EMEA Guidelines on the “Limits on Genotoxic Impurities”, EMEA/CHMP/QWP/251344/2006, 2006. [3] Székely G. et al., Green Chem. 15, 2013, 210-225. [4] Esteves T. et al., Sep. Purif. Technol. 163, 2016, 206-214. [5] Ferreira F. C.; Esteves T.; Vicente A. I.; Afonso C. A. M., “Polímeros polibenzimidazolo com cadeia espaçadora funcionalizada e seu método de obtenção para remoção de impurezas genotóxicas.” Patent request Nº 109480, with priority date of 22 June 2016. [6] Vicente A. I. et al., Chem. Mat., 2016, under preparation. Acknowledgements: We thank financial support from Fundação para a Ciência e Tecnologia (FCT) through the Project SelectHost (PTDC/QEQ-PRS/4157/2014) and iBB-Institute for Bioengineering and Biosciences (UID/BIO/04565/2013), from Programa Operacional Regional de Lisboa 2020 (Lisboa-01-0145-FEDER-007317). We thank to Hovione PharmaScience Ltd for supplying the API and technical know-how

Magnetic field dynamic strategies for the improved control of the angiogenic effect of mesenchymal stromal cells

project PTDC/EDM-EDM/30828/2017 SFRH/BD/114043/2015 co-financed by the ERDF under the PT2020 Partnership Agreement (POVI-01-0145-FEDER-007265), as well as from POR Lisboa 2020 grant PRECISE (Project N. 16394). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.This work shows the ability to remotely control the paracrine performance of mesenchymal stromal cells (MSCs) in producing an angiogenesis key molecule, vascular endothelial growth factor (VEGF-A), by modulation of an external magnetic field. This work compares for the first time the application of static and dynamic magnetic fields in angiogenesis in vitro model, exploring the effect of magnetic field intensity and dynamic regimes on the VEGF-A secretion potential of MSCs. Tissue scaffolds of gelatin doped with iron oxide nanoparticles (MNPs) were used as a platform for MSC proliferation. Dynamic magnetic field regimes were imposed by cyclic variation of the magnetic field intensity in different frequencies. The effect of the magnetic field intensity on cell behavior showed that higher intensity of 0.45 T was associated with increased cell death and a poor angiogenic effect. It was observed that static and dynamic magnetic stimulation with higher frequencies led to improved angiogenic performance on endothelial cells in comparison with a lower frequency regime. This work showed the possibility to control VEGF-A secretion by MSCs through modulation of the magnetic field, offering attractive perspectives of a non-invasive therapeutic option for several diseases by revascularizing damaged tissues or inhibiting metastasis formation during cancer progression.publishersversionpublishe

Magnetic stimulation of the angiogenic potential of mesenchymal stromal cells in vascular tissue engineering

The authors acknowledge the financial support from Fundação para a Ciência e a Tecnologia (FCT-MEC), Portugal, through the dedicated project [PTDC/EDM-EDM/30828/2017] (BeLive) and PhD grant [SFRH/BD/114043/2015] and  through the project [EXPL/CTM-POL/1117/1135/2012] Moreover, the authors thanks POR Lisboa 2020 for the research project [PRECISE, Project N. 16394]. We acknowledge Dr. Marta Teixeira and the IPATIMUP facilities for the development of the ex vivo CAM experiments. The authors acknowledge Prof. Reyes Mallada (University of Zaragoza, Spain) for the use of the vibrating sample magnetometer (VSM) equipment and Dr. Pavel Strichovanec (University of Zaragoza, Spain) for the technical assistance provided during the experiments. We also acknowledge the Instituto de Medicina Molecular (IMM, Lisboa) for the services provided concerning the use of the Confocal Scanning Microscopy (Zeiss LSM 710). Publisher Copyright: © 2021 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Group.The growing prevalence of vascular diseases worldwide has emphasized the need for novel tissue-engineered options concerning the development of vascularized 3D constructs. This study reports, for the first time, the use of external magnetic fields to stimulate mesenchymal stromal cells (MSCs) to increase the production of vascular endothelial growth factor-A (VEGF-A). Polyvinylalcohol and gelatin-based scaffolds, containing iron oxide nanoparticles, were designed for optimal cell magnetic stimulation. While the application of static magnetic fields over 24 h did not impact on MSCs proliferation, viability and phenotypic identity, it significantly increased the production of VEGF-A and guided MSCs morphology and alignment. The ability to enhance MSCs angiogenic potential was demonstrated by the increase in the number of new vessels formed in the presence of MSCs conditioned media through in vitro and in vivo models. Ultimately, this study uncovers the potential to manipulate cellular processes through short-term magnetic stimulation.publishersversionpublishe

Effects of culture media and suspension expansion technologies in mesenchymal stem cell manufacturing - A computational bioprocess and bioeconomics study

Mesenchymal stem cell (MSC) based therapies are promising for a large spectrum of unmet medical needs. Despite this promise, the scaling-up of production of clinical grade MSCs is hindered by the use of planar technologies that require intensive labor and are not enough to meet market demands, as well as due to high product and process variability introduced by the use of xenogeneic materials. This work presents a new bioprocess and bioeconomics model of stem cell expansion to support informed decisions for stem cells process scaling up at reduced annual costs. The intrinsic equations and parameters that capture the cell biological features, according with their source and media used, are embedded in the model. A target number of cells per dose of 140 million and a GMP facility of 400 sq mt with 4 BSCs and 8 incubators will be used as the baseline for expansion of both bone marrow MSCs (BM-MSCs) and adipose stem cells (ASCs) using planar expansion technologies. The current standard medium for MSC culture containing fetal bovine serum (FBS) will be compared with the xeno-free alternative of human platelet lysate (hPL). The use of hPL for both cell sources results in an increase of the number of doses produced and a decrease of the cost of goods (CoG) per dose (Table 1). In order to improve the production capacity, 8 bioreactors with capacity up to 50L were input in the model, using xeno-free plastic microcarriers for cell adhesion and hPL as the culture medium. The model results indicate that the investment in the use of suspension cultures is valuable due to a considerable increase in the production and a decrease of CoG/dose. As the number of doses produced per year increases, the reagent costs dominate relatively to the facility costs (Fig. 1). Sensitivity analysis was performed by varying 11 model variables by +/- 33%. The main factors that influence annual capacity and CoGs are related to harvesting density and yield, growth rates and microcarrier area and concentration (Table 2). These findings may be used to improve the design of expansion methods with fully xeno-free materials and highlight the relevance of the optimization of harvesting and downstream processing protocols. Please click Additional Files below to see the full abstract

Effects of glycosaminoglycan supplementation in the chondrogenic differentiation of bone marrow- and synovial- derived mesenchymal stem/stromal cells on 3D-extruded poly (ε-caprolactone) scaffolds

The lack of effective and long-term treatments for articular cartilage defects has increased the interest for innovative tissue engineering strategies. Such approaches, combining cells, biomaterial matrices and external biochemical/physical cues, hold promise for generating fully functional cartilage tissue. Herein, this study aims at exploring the use of the major cartilage glycosaminoglycans (GAGs), chondroitin sulfate (CS) and hyaluronic acid (HA), as external biochemical cues to promote the chondrogenic differentiation of human bone marrow- and synovium-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) on custom-made 3 D porous poly (ε-caprolactone) (PCL) scaffolds. The culture conditions, namely the chondrogenic medium and hypoxic environment (5% O2 tension), were firstly optimized by culturing hBMSCs on PCL scaffolds without GAG supplementation. For both MSC sources, GAG supplemented media, particularly with HA, promoted significantly cartilage-like extracellular matrix (ECM) production (higher sulfated GAG amounts) and chondrogenic gene expression. Remarkably, in contrast to tissues generated using hBMSCs, the hSMSC-based constructs showed decreased expression of hypertrophic marker COL X. Histological, immunohistochemical and transmission electron microscopy (TEM) analysis confirmed the presence of typical articular cartilage ECM components (GAGs, aggrecan, collagen fibers) in all the tissue constructs produced. Overall, our results highlight the potential of integrating GAG supplementation, hSMSCs and customizable 3 D scaffolds toward the fabrication of bioengineered cartilage tissue substitutes with reduced hypertrophy.info:eu-repo/semantics/publishedVersio

Hydroxyapatite-filled osteoinductive and piezoelectric nanofibers for bone tissue engineering

In this study entitled “Hydroxyapatite-filled osteoinductive and piezoelectric nanofibers for bone tissue engineering”, we describe the development of novel hydroxyapatite (HAp)-filled osteoinductive piezoelectric poly(vinylidene fluoride-cotetrafluoroethylene) (PVDF-TrFE) electrospun nanofibers as a potential strategy for supporting bone repair in delayed-union and non-union osteoporotic-related fractures, for which current clinical techniques have proven to be largely inadequate and scaffold-based tissue engineering approaches hold significant promise. While the piezoelectric properties of native bone tissue have been extensively discussed in the literature, including their key role in preserving tissue homeostasis and promoting tissue repair, they have been widely neglected in the design of scaffolds for bone tissue engineering (BTE) applications. Piezoelectric scaffolds can be used not only for mimicking the native piezoelectric features of bone but also to provide a platform for applying electrical or mechanical stimuli to damaged tissue, contributing to an accelerated regeneration process. The nanofibrous scaffolds generated in this study were capable of replicating the main electrical, structural and compositional properties of bone extracellular matrix (ECM). To the best of our knowledge, this was the first time that the combination of HAp with the piezoelectric polymer PVDF-TrFE was found to induce key shifts in the chemical structure of the polymer and promote ß phase nucleation, not only enhancing the piezoelectric features of the constructs but also improving their surface properties, including their ability to support mineralization in vitro. The HAp nanoparticles also provided meaningful bone-like biological cues (osteoinduction), enhancing the osteogenic differentiation of seeded human mesenchymal stem/stromal cells (hMSCs), which was confirmed by an increased ALP activity, cellderived calcium deposition and expression of important osteogenic gene markers. Overall, our findings highlight, for the first time, the potential of combining PVDFTrFE and HAp for developing electroactive and osteoinductive nanofibrous constructs with improved piezoelectric properties, surface features and osteogenic potential capable of improving bone tissue regeneration.Peer ReviewedPostprint (published version

Additive manufactured Poly("-caprolactone)-graphene scaffolds: Lamellar crystal orientation, mechanical properties and biological performance

Understanding the mechano–biological coupling mechanisms of biomaterials for tissue engineering is of major importance to assure proper scaffold performance in situ. Therefore, it is of paramount importance to establish correlations between biomaterials, their processing conditions, and their mechanical behaviour, as well as their biological performance. With this work, it was possible to infer a correlation between the addition of graphene nanoparticles (GPN) in a concentration of 0.25, 0.5, and 0.75% (w/w) (GPN0.25, GPN0.5, and GPN0.75, respectively) in three-dimensional poly("-caprolactone) (PCL)-based scaffolds, the extrusion-based processing parameters, and the lamellar crystal orientation through small-angle X-ray scattering experiments of extruded samples of PCL and PCL/GPN. Results revealed a significant impact on the scaffold’s mechanical properties to a maximum of 0.5% of GPN content, with a significant improvement in the compressive modulus of 59 MPa to 93 MPa. In vitro cell culture experiments showed the scaffold’s ability to support the adhesion and proliferation of L929 fibroblasts (fold increase of 28, 22, 23, and 13 at day 13 (in relation to day 1) for PCL, GPN0.25, GPN0.5, and GPN0.75, respectively) and bone marrow mesenchymal stem/stromal cells (seven-fold increase for all sample groups at day 21 in relation to day 1). Moreover, the cells maintained high viability, regular morphology, and migration capacity in all the different experimental groups, assuring the potential of PCL/GPN scaffolds for tissue engineering (TE) applications.info:eu-repo/semantics/publishedVersio