Comprehensive micropollutant characterization of wastewater during Covid-19 crisis in 2020: Suspect screening and environmental risk prioritization strategy.

Micropollutants monitoring in wastewater can serve as a picture of what is consuming society and how it can impact the aquatic environment. In this work, a suspect screening approach was used to detect the known and unknown contaminants in wastewater samples collected from two wastewater treatment plants (WWTPs) located in the Basque Country (Crispijana in Alava, and Galindo in Vizcaya) during two weekly sampling campaigns, which included the months from April to July 2020, part of the confinement period caused by COVID-19. To that aim, high-resolution mass spectrometry was used to collect full-scan data-dependent tandem mass spectra from the water samples using a suspect database containing >40,000 chemical substances. The presence of > 80 contaminants was confirmed (level 1) and quantified in both WWTP samples, while at least 47 compounds were tentatively identified (2a). Among the contaminants of concern, an increase in the occurrence of some compounds used for COVID-19 disease treatment, such as lopinavir and hydroxychloroquine, was observed during the lockdown. A prioritization strategy for environmental risk assessment was carried out considering only the compounds quantified in the effluents of Crispijana and Galindo WWTPs. The compounds were scored based on the removal efficiency, estimated persistency, bioconcentration factor, mobility, toxicity potential and frequency of detection in the samples. With this approach, 33 compounds (e.g. amantadine, clozapine or lopinavir) were found to be considered key contaminants in the analyzed samples based on their concentration, occurrence and potential toxicity. Additionally, antimicrobial (RQ-AR) and antiviral (EDRP) risk of certain compounds was evaluated, where ciprofloxacin and fluconazole represented medium risk for antibiotic resistance (1 > RQ-AR > 0.1) in the aquatic ecosystems. Regarding mixture toxicity, the computed sum of toxic unit values of the different effluents (> 1) suggest that interactions between the compounds need to be considered for future environmental risk assessments.

Analyzing the potential environmental impact of NIOSH list of hazardous drugs (group 2).

For the first time, several pharmaceuticals have been defined as priority substances in the new proposal of the revision of the Water Framework Directive (WFD). Consequently, environmental quality standards have been determined for several drugs. This is the case with the antiepileptic carbamazepine, which is considered as hazardous in healthcare settings by The National Institute for Occupational Safety and Health (NIOSH). This organism considers as such drugs that have shown teratogenicity, carcinogenicity, genotoxicity or other developmental, reproductive, or organ toxicity at low doses in studies with animals or humans. This study has been focused on the non-carcinogenic drugs classified in group 2, and their presence in the environment. This group contains many different therapeutic agents such as antineoplastics, psychoactive drugs, immunosuppressants and antivirals, among others. Of the 116 drugs included in the list, 26 have been found in aquatic environmental matrices. Certain drugs have received most attention (e.g., the antiepileptic carbamazepine, progesterone and the antidepressant paroxetine) while others completely lack environmental monitoring. Carbamazepine, fluconazole, paroxetine and warfarin have been found in invertebrates' tissues, whereas carbamazepine, oxazepam and paroxetine have been found in fish tissues. The main aim of the NIOSH's hazardous drug list is to inform healthcare professionals about adequate protection measures to prevent occupational exposure to these pharmaceuticals. However, this list contains useful information for other professionals and researchers such as environmental scientists. The paucity of relevant environmental data of certain hazardous pharmaceuticals might be important to help in the prioritization of compounds that may demand further research.

Bioinspired gelatin/bioceramic composites loaded with bone morphogenetic protein-2 (BMP-2) promote osteoporotic bone repair.

There are currently several commercialized products approved by the Food and Drug Administration and the European Medicines Agency based on the use of recombinant human BMP-2 for the treatment of non-unions long fractures and spinal fusion. However, the adverse effects recorded with the use of BMPs suggest the need for drug delivery carriers that allow reducing the required doses and improve their cost-effectiveness. Herein, we have developed a new osteoconductive scaffold that reduces the required doses of BMP-2 for promoting bone regeneration in an osteoporotic defect model. The composite is, in brief, a gelatin-based 3D scaffold reinforced with either calcium sulfate or hydroxyapatite as an inorganic osteoconductive biomaterial. To this end, the organic/inorganic composite systems showed high hydration capacity and good in vitro degradability. The incorporation of 7.5% (m/v) ceramic compounds resulted in scaffolds with stiffer Young modulus (179 and 75 kPa for CaSO4_7 and HA_7, respectively) than bare gelatin hydrogels (48 kPa). Studies with human bone-marrow derived mesenchymal stem cells (hBM-MSCs) revealed that the 3D scaffolds promote cell adhesion and proliferation along with osteogenic differentiation capabilities. Specifically, downregulation of stemness (Nanog, Oct4) genes and upregulation of osteogenic markers (ALP, Col1a1, Fmod) by two fold were observed over 10 days under basal culture conditions. Promisingly, the sustained in vitro release of BMP-2 observed from the porous reinforced scaffolds allowed us to address the critical-sized osteoporotic mice calvarial defects with a relatively low growth factor doses (600 ng BMP-2/scaffold) compared to conventional doses at 2-15 micrograms. Overall, this study demonstrates the promising potential of osteoconductive gelatin/calcium bioceramics composites as osteogenic growth factors delivery carriers for bone-regeneration via ultra-low growth factor doses.

Drugs used during the COVID-19 first wave in Vitoria-Gasteiz (Spain) and their presence in the environment.

The city of Vitoria-Gasteiz was one of the probable first entrances of the SARS-CoV2 in Spain, one of the worst affected countries in the world during the first COVID 19 wave. Driven by the urgency of the situation, multiple drugs with antiviral activity were used off label. Sadly, most of these treatments were of little or no benefit and thus, the number of patients suffering from COVID-19 attended in intensive care units (ICUs) multiplied. After being administered to patients, a variable proportion of these drugs reach the environment where they may have detrimental effects, although this aspect is usually ignored by healthcare professionals. In this study we measured the patterns of hospital drug use in the city of Vitoria-Gasteiz (Spain) during the first COVID-19 wave pandemic, focusing on those with antiviral activity and those used in the ICUs. Subsequently, we measured concentrations of selected drugs in the city's wastewater treatment plant influent and effluent and estimated the potential risk for the environment. The hospital use of certain antivirals and drugs used for sedo-analgesia were dramatically increased during the first wave (cisatracurium was multiplied by 25 and lopinavir/ritonavir by 20). A mean of 1.632 daily defined doses of hydroxychloroquine were used during the period of February-May 2020. In this study we report the first positive detection of hydroxychloroquine ever in the environment. We also show the second positive report of lopinavir. Low risk was estimated for hydroxychloroquine, lopinavir and ritonavir (Risk quotients (RQ) <1), and medium risk for azithromycin (RQ 0f 0.146).

Hazardous drugs (NIOSH's list-group 1) in healthcare settings: Also a hazard for the environment?

Healthcare workers can be exposed to dangerous drugs during their daily practice. The National Institute for Occupational Safety and Health (NIOSH) considers "hazardous drugs" as those that had shown one or more of the following characteristic in studies with animals, humans or in vitro systems: carcinogenicity, teratogenicity or other toxicity for development, reproductive toxicity, organ toxicity at low doses, or genotoxicity. In the actual list (draft list 2020), drugs classified in group 1 are those with carcinogenic effects. Moreover, the global human and veterinary cancer is expected to grow, so antineoplastic drug consumption may consequently grow, leading to an increase of anticancer pharmaceuticals in the environment. Not all drugs pertaining to group 1 can be classified as "antineoplastic" or "cytostatic". Since most of the research on environment presence and ecotoxicological effects of pharmaceuticals has been focused on this therapeutic class, other carcinogenic drugs belonging to different therapeutic groups may have been omitted in previous studies. In this study we aim to review the presence in the environment of the hazardous drugs (NIOSH group 1) and their possible environmental impact. Of the 90 drugs considered, there is evidence of presence in the environment for 19. Drugs with more studies reporting positive detections are: the antibiotic chloramphenicol (55), the alkylating agents cyclophosphamide (39) and ifosfamide (30), and the estrogen receptor modulator tamoxifen (18). Although the original purpose of the NIOSH list and related documents is to provide guidance to healthcare professionals in order to adequately protect them from the hazards posed by these drugs in healthcare settings, we believe they can be useful for environmentalists too. Absence of data regarding the potential of environmental risk of certain hazardous drugs might tell us which drugs ought to be prioritized in the future.

Progress in the use of plasma rich in growth factors in ophthalmology: from ocular surface to ocular fundus.

INTRODUCTION: The use of blood derivatives and especially Plasma rich in growth factors (PRGF), for regenerative purposes has been a common trend along the last decades in the field of oral surgery, dermatology, orthopedics, and more recently in ophthalmology. AREAS COVERED: PRGF is a type of platelet-rich plasma that is being explored for the treatment of ocular injuries. The present review article highlights 50 ophthalmology-related publications about the application of PRGF in the treatment of acute and chronic pathologies in ophthalmology as well as most relevant challenges and future prospects. EXPERT OPINION: PRGF technology provides a wide range of formulations that can be used therapeutically in many different acute and chronic ocular pathologies. In addition to eye drops enriched with autologous growth factors, PRGF enables the preparation of both immunologically safe and fibrin-based formulations. Recent advances in the field have promoted PRGF storage for 12 months under freezing conditions, its daily use for 7 days at room temperature and the freeze-dried formulation. The thermally treated immunosafe formulation has shown promising clinical results for the treatment of several diseases such as Sjögren syndrome, graft versus host disease or cicatrizing conjunctivitis. In addition, several fibrin formulations have been preclinically evaluated and clinically incorporated as an adjuvant to ocular surface or glaucoma surgeries, dermal fat graft procedures, limbal stem cell expansion and retinal surgeries. The present review explores the latest scientific and clinical data, current challenges, and main prospects of this technology for the treatment of several ocular injuries.

Drug pollution & Sustainable Development Goals.

The United Nations set "The 2030 Agenda for Sustainable Development," which includes the Sustainable Development Goals (SDGs), a collection of 17 global goals designed to be a "blueprint to achieve a better and more sustainable future for all". Although only mentioned in one of the seventeen goals (goal 3), we argue that drugs in general, and growing drug pollution in particular, affects the SDGs in deeper, not readily apparent ways. So far, the emerging problem of drug pollution has not been sufficiently addressed. Here, we outline and discuss how drug pollution can affect SDGs and even threaten their achievement.

Antipsychotics as environmental pollutants: An underrated threat?

The heterogeneous class of what we nowadays call antipsychotics was born almost 70 years ago with the serendipitous discovery of chlorpromazine. Their utilization is constantly growing because they are used to treat a diverse group of diseases and patients across all age groups: schizophrenia, bipolar disease, depression, autism, attention deficit hyperactivity disorder, behavioural and psychological symptoms in dementia, among others. They possess a complex pharmacological profile, acting on multiple receptors: dopaminergic, serotoninergic, histaminergic, adrenergic, and cholinergic, leading scientists to call them "agents with rich pharmacology" or "dirty drugs". Serotonin, dopamine, acetylcholine, noradrenaline, histamine and their respective receptors are evolutionary ancient compounds, and as such, are found in many different living beings in the environment. Antipsychotics do not disappear once excreted by patient's urine or faeces and are transported to wastewater treatment plants. But as these plant's technology is not designed to eliminate drugs and their metabolites, a variable proportion of the administered dose ends up in the environment, where they have been found in almost every matrix: municipal wastewater, hospital sewage, rivers, lakes, sea and even drinking water. We believe that reported concentrations found in the environment might be high enough to exert significant effect to aquatic wildlife. Besides, recent studies suggest antipsychotics, among others, are very likely bioaccumulating through the web food. Crucially, psychotropics may provoke behavioural changes affecting populations' dynamics at lower concentrations. We believe that so far, antipsychotics have not received the attention they deserve with regards to drug pollution, and that their role as environmental pollutants has been underrated.

Type 1 Diabetes Mellitus reversal via implantation of magnetically purified microencapsulated pseudoislets.

Microencapsulation of pancreatic islets for the treatment of Type I Diabetes Mellitus (T1DM) generates a high quantity of empty microcapsules, resulting in high therapeutic graft volumes that can enhance the host's immune response. We report a 3D printed microfluidic magnetic sorting device for microcapsules purification with the objective to reduce the number of empty microcapsules prior transplantation. In this study, INS1E pseudoislets were microencapsulated within alginate (A) and alginate-poly-L-lysine-alginate (APA) microcapsules and purified through the microfluidic device. APA microcapsules demonstrated higher mechanical integrity and stability than A microcapsules, showing better pseudoislets viability and biological function. Importantly, we obtained a reduction of the graft volume of 77.5% for A microcapsules and 78.6% for APA microcapsules. After subcutaneous implantation of induced diabetic Wistar rats with magnetically purified APA microencapsulated pseudoislets, blood glucose levels were restored into normoglycemia (<200 mg/dL) for almost 17 weeks. In conclusion, our described microfluidic magnetic sorting device represents a great alternative approach for the graft volume reduction of microencapsulated pseudoislets and its application in T1DM disease.

Cell Microencapsulation and Cryopreservation with Low Molecular Weight Hyaluronan and Dimethyl Sulfoxide.

Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.

Low molecular-weight hyaluronan as a cryoprotectant for the storage of microencapsulated cells.

The low-temperature storage of therapeutic cell-based products plays a crucial role in their clinical translation for the treatment of diverse diseases. Although dimethylsulfoxide (DMSO) is the most successful cryoprotectant in slow freezing of microencapsulated cells, it has shown adverse effects after cryopreserved cell-based products implantation. Therefore, the search of alternative non-toxic cryoprotectants for encapsulated cells is continuously investigated to move from bench to the clinic. In this work, we investigated the low molecular-weight hyaluronan (low MW-HA), a natural non-toxic and non-sulfated glycosaminoglycan, as an alternative non-permeant cryoprotectant for the slow freezing cryopreservation of encapsulated cells. Cryopreservation with low MW-HA provided similar metabolic activity, cell dead and early apoptotic cell percentage and membrane integrity after thawing, than encapsulated cells stored with either DMSO 10% or Cryostor 10. However, the beneficial outcomes with low MW-HA were not comparable to DMSO with some encapsulated cell types, such as the human insulin secreting cell line, 1.1B4, maybe explained by the different expression of the CD44 surface receptor. Altogether, we can conclude that low MW-HA represents a non-toxic natural alternative cryoprotectant to DMSO for the cryopreservation of encapsulated cells.

Advances in the slow freezing cryopreservation of microencapsulated cells.

Over the past few decades, the use of cell microencapsulation technology has been promoted for a wide range of applications as sustained drug delivery systems or as cells containing biosystems for regenerative medicine. However, difficulty in their preservation and storage has limited their availability to healthcare centers. Because the preservation in cryogenic temperatures poses many biological and biophysical challenges and that the technology has not been well understood, the slow cooling cryopreservation, which is the most used technique worldwide, has not given full measure of its full potential application yet. This review will discuss the different steps that should be understood and taken into account to preserve microencapsulated cells by slow freezing in a successful and simple manner. Moreover, it will review the slow freezing preservation of alginate-based microencapsulated cells and discuss some recommendations that the research community may pursue to optimize the preservation of microencapsulated cells, enabling the therapy translate from bench to the clinic.

Biologically active and biomimetic dual gelatin scaffolds for tissue engineering.

We have designed, developed and optimized Genipin cross-linked 3D gelatin scaffolds that were biologically active and biomimetic, show a dual activity both for growth factor and cell delivery. Type B gelatin powder was dissolved in DI water. 100mg of genipin was dissolved in 10ml of DI water. Three genipin concentrations were prepared: 0.1%, 0.2% and 0.3% (w/v). Solutions were mixed at 40°C and under stirring and then left crosslinking for 72h. Scaffolds were obtained by punching 8 mm-cylinders into ethanol 70% solution for 10min and then freeze-drying. Scaffolds were biologically, biomechanically and morphologically evaluated. Cell adhesion and morphology of D1-Mesenchymal stem cells (MSCs) and L-929 fibroblast was studied. Vascular endothelial grwoth factor (VEGF) and Sonic hedgehog (SHH) were used as model proteins. Swelling ratio increased and younǵs module decreased along with the concentration of genipin. All scaffolds were biocompatible according to the toxicity test. MSC and L-929 cell adhesion improved in 0.2% of genipin, obtaining better results with MSCs. VEGF and SHH were released from the gels. This preliminary study suggest that the biologically active and dual gelatin scaffolds may be used for tissue engineering approaches like bone regeneration.

Autologous method for ex vivo expansion of human limbal epithelial progenitor cells based on plasma rich in growth factors technology.

PURPOSE: Develop an autologous culture method for ex vivo expansion of human limbal epithelial progenitor cells (LEPCs) using Plasma Rich in Growth Factors (PRGF) as a growth supplement and as a scaffold for the culture of LEPCs. METHODS: LEPCs were cultivated in different media supplemented with 10% fetal bovine serum (FBS) or 10% PRGF. The outgrowths, total number of cells, colony forming efficiency (CFE), morphology and immunocytochemistry against p63- α and cytokeratins 3 and 12 (CK3-CK12) were analyzed. PRGF was also used to elaborate a fibrin membrane. The effects of the scaffold on the preservation of stemness and the phenotypic characterization of LEPCs were investigated through analysis of CK3-CK12, ABCG-2 and p63. RESULTS: LEPCs cultivated with PRGF showed a significantly higher growth area than FBS cultures. Moreover, the number of cells were also higher in PRGF than FBS, while displaying a better morphology overall. CFE was found to be also higher in PRGF groups compared to FBS, and the p63-α expression also differed between groups. LEPCs cultivated on PRGF membranes appeared as a confluent monolayer of cells and still retained p63 and ABCG-2 expression, being negative for CK3-CK12. CONCLUSIONS: PRGF can be used in corneal tissue engineering, supplementing the culture media, even in a basal media without any other additives, as well as providing a scaffold for the culture.

Ultrasound-guided plasma rich in growth factors injections and scaffolds hasten motor nerve functional recovery in an ovine model of nerve crush injury.

In the present study we evaluated the motor recovery process of peripheral nerve injury (PNI), based on electrophysiological and histomorphometric criteria, after treatment with plasma rich in growth factors (PRGF) injections and scaffolds in an ovine model. Three groups of sheep underwent a nerve crush lesion: the first group (n = 3) was left to recover spontaneously (SR); the second group was administered saline injections (SI; n = 5) and a third group (n = 6) received PRGF injections and scaffolds immediately after the crush injury. At post-intervention week 8, 70% of sheep in the PRGF group were CMAP-positive, with no electrophysiological response in the rest of the groups. Histomorphometric analysis 12 weeks after the surgical intervention revealed that the average axonal density of the SR (1184 ± 864 axons/µm2 ) and SI (3109 ± 2450 axons/µm2 ) groups was significantly inferior to the control (8427 ± 2433 axons/µm2 ) and also inferior to the PRGF group (5276 ± 4148 axons/µm2 ), showing no significant differences between the control and PRGF groups. The axonal size of the SR and SI groups was significantly smaller compared with the control group (18 ± 4 µm2 ), whereas the axonal size of the PRGF group (6 ± 5 µm2 ) did not show statistical differences from the control. Morphometry of the target muscles indicated that the PRGF group had the lowest percentage volume reduction 12 weeks after the crush injury. The PRGF group had larger muscle fibre areas than the SI and SR groups, although the differences did not reach statistical significance. Overall, these data suggest that the PRGF injections and scaffolds hastened functional axon recovery and dampened atrophy of the target muscles in an ovine model. Copyright © 2015 John Wiley & Sons, Ltd.

Use of Flow Focusing Technique for Microencapsulation of Myoblasts.

Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.