Human Platelet Lysate-Derived Nanofibrils as Building Blocks to Produce Free-Standing Membranes for Cell Self-Aggregation.

Amyloid-like fibrils are garnering keen interest in biotechnology as supramolecular nanofunctional units to be used as biomimetic platforms to control cell behavior. Recent insights into fibril functionality have highlighted their importance in tissue structure, mechanical properties, and improved cell adhesion, emphasizing the need for scalable and high-kinetics fibril synthesis. In this study, we present the instantaneous and bulk formation of amyloid-like nanofibrils from human platelet lysate (PL) using the ionic liquid cholinium tosylate as a fibrillating agent. The instant fibrillation of PL proteins upon supramolecular protein-ionic liquid interactions was confirmed from the protein conformational transition toward cross-ß-sheet-rich structures. These nanofibrils were utilized as building blocks for the formation of thin and flexible free-standing membranes via solvent casting to support cell self-aggregation. These PL-derived fibril membranes reveal a nanotopographically rough surface and high stability over 14 days under cell culture conditions. The culture of mesenchymal stem cells or tumor cells on the top of the membrane demonstrated that cells are able to adhere and self-organize in a three-dimensional (3D) spheroid-like microtissue while tightly folding the fibril membrane. Results suggest that nanofibril membrane incorporation in cell aggregates can improve cell viability and metabolic activity, recreating native tissues' organization. Altogether, these PL-derived nanofibril membranes are suitable bioactive platforms to generate 3D cell-guided microtissues, which can be explored as bottom-up strategies to faithfully emulate native tissues in a fully human microenvironment.

Photocrosslinkable microgels derived from human platelet lysates: injectable biomaterials for cardiac cell culture.

Cardiovascular diseases are a major global cause of morbidity and mortality, and they are often characterized by cardiomyocytes dead that ultimately leads to myocardial ischemia (MI). This condition replaces functional cardiac tissue with fibrotic scar tissue compromising heart function. Injectable systems for the in situ delivery of cells or molecules to assist during tissue repair have emerged as promising approaches for tissue engineering, particularly for myocardial repair. Methacryloyl platelet lysates (PLMA) have been employed for constructing full human-based 3D cell culture matrices and demonstrated potential for xeno-free applications. In this study, we propose using PLMA to produce microparticles (MPs) serving as anchors for cardiac and endothelial cells and ultimately as injectable systems for cardiac tissue repair. The herein reported PLMA MPs were produced by droplet microfluidics and showed great properties for cell attachment. More importantly, it is possible to show the capacity of PLMA MPs to serve as cell microcarriers even in the absence of animal-derived serum supplementation in the culture media.

Influence of excessive daytime sleepiness on the treatment adherence of obstructive sleep apnea.

OBJECTIVES: To compare positive airway pressure (PAP) adherence between patients with or without excessive daytime sleepiness (EDS) in mild, moderate and severe obstructive sleep apnea (OSA). METHODS: Patients ≥18 years diagnosed with OSA in 2018 and 2019, without previous history of PAP usage and with adherence registration in the first medical consultation after treatment initiation, were included. EDS was defined as a score of ≥10 on the Epworth Scale. Patients were divided into two groups according to the adherence to PAP: "Adherent" if using the device for ≥4 h for ≥70% of the nights and "Nonadherent" otherwise. Simple and multiple logistic regression models for adherence were determined. RESULTS: 321 patients were included, most male (64.2%), with mean age 56.56 years. Most patients had severe OSA (n = 159; 49.5%), and median AHI was 29.3/h [16.8; 47.5]. Being older or having a severe OSA resulted in an increased adherence (OR = 1.020, CI95% = [1.002; 1.039] and OR = 2.299, CI95% = [1.273; 4.191], respectively). In patients without EDS a statistically significant difference was found in adherence between those with severe OSA and both mild and moderate OSA categories (OR = 0.285, p = 0.023 and OR = 0.387, p = 0.026, respectively), with patients with severe OSA being adherent. There was no statistical difference in adherence between patients with or without EDS (OR 1.083; p = 0.876), nor in the different degrees of severity in those with EDS. CONCLUSION: In our study there were no differences in PAP therapy adherence between patients with or without excessive daytime sleepiness. Older age and higher OSA severity resulted in higher adherence rates.

Self-Assembly of Platelet Lysates Proteins into Microparticles by Unnatural Disulfide Bonds for Bottom-Up Tissue Engineering.

There is a demand to design microparticles holding surface topography while presenting inherent bioactive cues for applications in the biomedical and biotechnological fields. Using the pool of proteins present in human-derived platelet lysates (PLs), the production of protein-based microparticles via a simple and cost-effective method is reported, exploring the prone redox behavior of cysteine (Cy-SH) amino acid residues. The forced formation of new intermolecular disulfide bonds results in the precipitation of the proteins as spherical, pompom-like microparticles with adjustable sizes (15-50 µm in diameter) and surface topography consisting of grooves and ridges. These PL microparticles exhibit extraordinary cytocompatibility, allowing cell-guided microaggregates to form, while also working as injectable systems for cell support. Early studies also suggest that the surface topography provided by these PL microparticles can support osteogenic behavior. Consequently, these PL microparticles may find use to create live tissues via bottom-up procedures or injectable tissue-defect fillers, particularly for bone regeneration, with the prospect of working under xeno-free conditions.

Elexacaftor/tezacaftor/ivacaftor-real-world clinical effectiveness and safety. A single-center Portuguese study.

OBJECTIVE: To evaluate the effectiveness of treatment with elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) and to characterize its safety profile in cystic fibrosis (CF) patients in a real-world clinical setting. METHODS: This was a prospective observational study carried out in a CF referral center in Portugal involving adult CF patients who started treatment with ELX/TEZ/IVA. Clinical characteristics of the patients were collected, and effectiveness and safety data were evaluated. RESULTS: Of the 56 patients followed in the center at the time of the study, 28 were eligible for ELX/TEZ/IVA treatment in accordance with the Portuguese National Authority for Medicines and Health Products at the time of the study. Of these, 24 met the follow-up time requirement to be included in the clinical effectiveness analysis. The mean follow-up time was 167.3 ± 96.4 days. Adverse events were generally mild and self-limited. Significant improvements in lung function, BMI, sweat chloride concentration, and number of pulmonary exacerbations were observed. No significant differences in outcomes between F508del homozygous and heterozygous patients were found. The effectiveness of this new CFTR modulator combination also applied to patients with advanced lung disease. CONCLUSIONS: Treatment with ELX/TEZ/IVA showed effective improvement in real-world clinical practice, namely in lung function, BMI, sweat chloride concentration, and number of pulmonary exacerbations, with no safety concerns.

Elexacaftor/tezacaftor/ivacaftor-real-world clinical effectiveness and safety. A single-center Portuguese study / Elexacaftor/tezacaftor/ivacaftor-efetividade clínica e segurança no mundo real. Estudo unicêntrico português

ABSTRACT Objective: To evaluate the effectiveness of treatment with elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) and to characterize its safety profile in cystic fibrosis (CF) patients in a real-world clinical setting. Methods: This was a prospective observational study carried out in a CF referral center in Portugal involving adult CF patients who started treatment with ELX/TEZ/IVA. Clinical characteristics of the patients were collected, and effectiveness and safety data were evaluated. Results: Of the 56 patients followed in the center at the time of the study, 28 were eligible for ELX/TEZ/IVA treatment in accordance with the Portuguese National Authority for Medicines and Health Products at the time of the study. Of these, 24 met the follow-up time requirement to be included in the clinical effectiveness analysis. The mean follow-up time was 167.3 ± 96.4 days. Adverse events were generally mild and self-limited. Significant improvements in lung function, BMI, sweat chloride concentration, and number of pulmonary exacerbations were observed. No significant differences in outcomes between F508del homozygous and heterozygous patients were found. The effectiveness of this new CFTR modulator combination also applied to patients with advanced lung disease. Conclusions: Treatment with ELX/TEZ/IVA showed effective improvement in real-world clinical practice, namely in lung function, BMI, sweat chloride concentration, and number of pulmonary exacerbations, with no safety concerns.

RESUMO Objetivo: Avaliar a efetividade do tratamento com elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) e caracterizar seu perfil de segurança em pacientes com fibrose cística (FC) em um cenário clínico de mundo real. Métodos: Estudo observacional prospectivo realizado em um centro de referência em FC de Portugal com pacientes adultos com FC que iniciaram o tratamento com ELX/TEZ/IVA. As características clínicas dos pacientes foram coletadas, e os dados de efetividade e segurança, avaliados. Resultados: Dos 56 pacientes acompanhados no centro na época do estudo, 28 eram elegíveis para o tratamento com ELX/TEZ/IVA de acordo com a Autoridade Nacional do Medicamento e Produtos de Saúde. Destes, 24 atenderam ao requisito de tempo de acompanhamento para inclusão na análise de efetividade clínica. O tempo médio de acompanhamento foi de 167,3 ± 96,4 dias. Os eventos adversos foram geralmente leves e autolimitados. Foram observadas melhoras significativas na função pulmonar, no IMC, na concentração de cloreto no suor e no número de exacerbações pulmonares. Não foram encontradas diferenças significativas nos resultados entre os pacientes homozigotos e heterozigotos para F508del. A efetividade dessa nova combinação de moduladores da CFRT em fibrose cística também se aplica a pacientes com doença pulmonar avançada. Conclusões: O tratamento com ELX/TEZ/IVA demonstrou melhora efetiva na prática clínica real, a saber, na função pulmonar, no IMC, na concentração de cloreto no suor e no número de exacerbações pulmonares, sem preocupações de segurança.

Nutritional Intake and Training Load of Professional Female Football Players during a Mid-Season Microcycle.

Football (soccer) is a high-intensity intermittent sport with large energy demands. In a repeated-measures design, we analysed the nutritional intake and training load of fourteen female football players (22.50 ± 4.38 y; 57.23 ± 8.61 kg; 164 ± 6.00 cm; 18.33 ± 2.48% of fat mass and 23.71 ± 2.51 kg of muscle mass) competing in the highest female Football Portuguese League across a typical mid-season microcycle. The microcycle had one match day (MD), one recovery session (two days after the MD, MD+2), three training sessions (MD-3, MD-2, MD-1) and two rest days (MD+1). Energy intake and CHO (g.kg.BW−1) intake were lower on the days before the competition (MD+2, MD-3, MD-2 and MD-1 vs. MD; p < 0.05; ES: 0.60−1.30). Total distance, distance covered at high-speed running (HSRD) and the high metabolic distance load (HMLD) were lower on MD+2, MD-3 and MD-1 compared with MD (p < 0.05; ES: <0.2−5.70). The internal training load was lower in all training sessions before the competition (MD+2, MD-3, MD-2 and MD-1 vs. MD; p ≤ 0.01; ES: 1.28−5.47). Despite the small sample size and a single assessment in time, the results suggest that caloric and CHO intake were below the recommendations and were not structured based on the physical requirements for training sessions or match days.

Designing highly customizable human based platforms for cell culture using proteins from the amniotic membrane.

In the past few years researchers have witnessed a paradigm shift in the development of biomaterials for drug discovery, tissue engineering, and regenerative medicine. After the great advances resulting from the transition of the 2D to the 3D, the new focus has been to increase the clinical relevance of such systems, as well as avoid the use of animals, by developing platforms that better replicate the human physiology in vitro. In this sense, we envisage the use of human matrices extracted from ethically sourced and readily available tissues as an optimal and promising alternative to currently used approaches. Hereupon, we report for the first time the chemical modification of human ECM proteins from the amniotic membrane (AM) with photoresponsive groups to produce bioinks and hydrogel precursors to engineer customizable platforms that are representative of native tissues and capable of supporting long-term cell culture. Our results demonstrated an efficient decellularization, liquefaction and functionalization of AM-derived ECM with methacryloyl domains (AMMA), with production of stable and versatile hydrogels. Mechanical characterization evidenced an increased compression strength as a function of methacrylation degree and decellularized ECM concentration. Three-dimensional (3D) stem cell culture in the AMMA hydrogels resulted in viable and proliferative cells up to 7 days; moreover, the mouldable character of the hydrogel precursors permits the processing of patterned hydrogel constructs allowing the control over cellular alignment and elongation, or microgels with highly tunable shape.

Core-shell microcapsules: biofabrication and potential applications in tissue engineering and regenerative medicine.

The fabrication of scaffolds that accurately recreate the architecture of living tissues is a major challenge in the field of tissue engineering and regenerative medicine. Core-shell microcapsules hold great potential in this regard, as they can recreate the hierarchical structure of biological systems. The independent modulation of the composition of both core and shell layers allows the design of compartmentalized platforms tailored to the recreation of specific cell niches. Emergent technologies such as superhydrophobic surfaces, microfluidics, electrospray, and layer-by-layer assembly have been successful in producing core-shell microcapsules for the encapsulation of cells and bioactive factors. This review provides an overview of available materials and techniques used in the generation of core-shell microcapsules, while also highlighting some of their potential applications in the design of innovative and effective tissue engineering and regenerative medicine strategies.

High-Throughput Production of Microsponges from Platelet Lysate for Tissue Engineering Applications.

Cell-based therapies require a large number of cells, as well as appropriate methods to deliver the cells to damaged tissue. Microcarriers provide an optimal platform for large-scale cell culture while also improving cell retention during cell delivery. However, this technology still presents significant challenges due to low-throughput fabrication methods and an inability of the microcarriers to recreate the properties of human tissue. This work proposes, for the first time, the use of methacryloyl platelet lysates (PLMA), a photocrosslinkable material derived from human platelet lysates, to produce porous microcarriers. Initially, high quantities of PLMA/alginate core-shell microcapsules are produced using coaxial electrospray. Subsequently, the microcapsules are collected, irradiated with ultraviolet light, washed, and freeze dried yielding PLMA microsponges. These microsponges are able to support the adhesion and proliferation of human adipose-derived stem cells, while also displaying potential in the assembly of autologous microtissues. Cell-laden microsponges were shown to self-organize into aggregates, suggesting possible applications in bottom-up tissue engineering applications. Impact Statement Microcarriers have increasingly been used as delivery platforms in cell therapy. Herein, the encapsulation of human-derived proteins in alginate microcapsules is proposed as a method to produce microcarriers from photopolymerizable materials. The capsules function as a template structure, which is then processed into spherical microparticles, which can be used in cell culture, cell delivery, and bottom-up assembly. As a proof of concept, this method was combined with lyophilization to process methacryloyl platelet lysates into injectable microsponges for cell delivery.

Human Protein-Based Porous Scaffolds as Platforms for Xeno-Free 3D Cell Culture.

Extracellular matrix and protein-based biomaterials emerge as attractive sources to produce scaffolds due to their great properties regarding biocompatibility and bioactivity. In addition, there are concerns regarding the use of animal-derived supplements in cell culture not only due to the risk of transmission of xenogeneic contaminants and antigens but also due to ethical issues associated with collection methods. Herein, a novel human protein-derived porous scaffold produced from platelet lysates (PL) as platform for xeno-free 3D cell culture has been proposed. Human PL are chemically modified with methacryloyl groups (PLMA) to make them photocrosslinkable and used as precursor material to produce PLMA-based sponges. The herein reported human-based sponges have highly tunable morphology and mechanical properties, with an internal porous structure and Young's modulus dependent on the concentration of the polymer. Human adipose-derived stem cells (hASCs) are cultured on top of PLMA sponges to validate their use for 3D cell culture in xeno-free conditions. After 14 days hASCs remained viable, and results show that cells are able to proliferate during time even in the absence of animal-derived supplementation. This study reveals for the first time that such scaffolds can be promising platforms for culture of human cells avoiding the use of any animal-derived supplement.

Self-glucose feeding hydrogels by enzyme empowered degradation for 3D cell culture.

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.

Bioengineering a humanized 3D tri-culture osteosarcoma model to assess tumor invasiveness and therapy response.

To date, anticancer therapies with evidenced efficacy in preclinical models fail during clinical trials. The shortage of robust drug screening platforms that accurately predict patient's response underlie these misleading results. To provide a reliable platform for tumor drug discovery, we herein propose a relevant humanized 3D osteosarcoma (OS) model exploring the potential of methacryloyl platelet lysates (PLMA)-based hydrogels to sustain spheroid growth and invasion. The architecture and synergistic cell-microenvironment interaction of an invading tumor was recapitulated encapsulating spheroids in PLMA hydrogels, alone or co-cultured with osteoblasts and mesenchymal stem cells. The stem cells alignment toward OS spheroid suggested that tumor cells chemotactically attracted the surrounding stromal cells, which supported tumor growth and invasion into the hydrogels. The exposure of established models to doxorubicin revealed an improved drug resistance of PLMA-based models, comparing with scaffold-free spheroids. The proposed OS models highlighted the feasibility of PLMA hydrogels to support tumor invasion and recapitulate tumor-stromal cell crosstalk, demonstrating the potential of this 3D platform for complex tumor modelling. STATEMENT OF SIGNIFICANCE: Cell invasion mechanisms involved in tumor progression have been recapitulated in the field of 3D in vitro modeling, leveraging the great advance in biomimetic materials. In line with the growing interest in human-derived biomaterials, the aim of this study is to explore for the first time the potential of methacryloyl platelet lysates (PLMA)-based hydrogels to develop a humanized 3D osteosarcoma model to assess tumor invasiveness and drug sensitivity. By co-culturing tumor spheroids with human osteoblasts and human mesenchymal stem cells, this study demonstrated the importance of the synergistic tumor cell-microenvironment interaction in tumor growth, invasion and drug resistance. The established 3D osteosarcoma model highlighted the feasibility of PLMA hydrogels as a relevant 3D platform for complex tumor modelling.

Perinatal tissues and cells in tissue engineering and regenerative medicine.

Perinatal tissues are an abundant source of human extracellular matrix proteins, growth factors and stem cells with proved potential use in a wide range of therapeutic applications. Due to their placental origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Additionally, as a temporary organ, placenta is usually discarded as a medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. Although some of these tissues, such as the amniotic membrane and umbilical cord, have been used in clinical practices, most of them continue to be highly under explored. This review aims to outline the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM), as well as highlight how these solutions can be used to overcome the shortage of adequate scaffolds and cell sources that currently hampers the translation of TERM strategies towards clinical settings. STATEMENT OF SIGNIFICANCE: Stem cells and extracellular matrix derived from perinatal tissues such as placenta and umbilical cord, have drawn great attention for use in a wide variety of applications in the biomedical field. Due to their origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Also they are typically considered medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. This work aims to present and discuss the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM).

Human Platelet Lysates-Based Hydrogels: A Novel Personalized 3D Platform for Spheroid Invasion Assessment.

Fundamental physiologic and pathologic phenomena such as wound healing and cancer metastasis are typically associated with the migration of cells through adjacent extracellular matrix. In recent years, advances in biomimetic materials have supported the progress in 3D cell culture and provided biomedical tools for the development of models to study spheroid invasiveness. Despite this, the exceptional biochemical and biomechanical properties of human-derived materials are poorly explored. Human methacryloyl platelet lysates (PLMA)-based hydrogels are herein proposed as reliable 3D platforms to sustain in vivo-like cell invasion mechanisms. A systematic analysis of spheroid viability, size, and invasiveness is performed in three biomimetic materials: PLMA hydrogels at three different concentrations, poly(ethylene glycol) diacrylate, and Matrigel. Results demonstrate that PLMA hydrogels perfectly support the recapitulation of the tumor invasion behavior of cancer cell lines (MG-63, SaOS-2, and A549) and human bone-marrow mesenchymal stem cell spheroids. The distinct invasiveness ability of each cell type is reflected in the PLMA hydrogels and, furthermore, different mechanical properties produce an altered invasive behavior. The herein presented human PLMA-based hydrogels could represent an opportunity to develop accurate cell invasiveness models and open up new possibilities for humanized and personalized high-throughput screening and validation of anticancer drugs.

Biomedical applications of laminarin.

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 ß-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.

Photopolymerizable Platelet Lysate Hydrogels for Customizable 3D Cell Culture Platforms.

3D cell culture platforms have emerged as a setting that resembles in vivo environments replacing the traditional 2D platforms. Over the recent years, an extensive effort has been made on the development of more physiologically relevant 3D cell culture platforms. Extracellular matrix-based materials have been reported as a bioactive and biocompatible support for cell culture. For example, human plasma derivatives have been extensively used in cell culture. Despite all the promising results, in most cases these types of materials have poor mechanical properties and poor stability in vitro. Here plasma-based hydrogels with increased stability are proposed. Platelet lysates are modified by addition of methacryloyl groups (PLMA) that polymerize in controlled geometries upon UV light exposure. The hydrogels could also generate porous scaffolds after lyophilization. The results show that PLMA materials have increased mechanical properties that can be easily adjusted by changing PLMA concentration or modification degree. Cells readily adhere, proliferate, and migrate, exhibiting high viability when encapsulated in PLMA hydrogels. The innovation potential of PLMA materials is based on the fact that it is a complete xeno-free solution for human cell culture, thus an effective alternative to the current gold standards for 3D cell culture based on animal products.

Blood Plasma Derivatives for Tissue Engineering and Regenerative Medicine Therapies.

Platelet-rich plasma (PRP) and its derivatives have been investigated and applied in regenerative medicine. The use of PRP as a supplement of cell culture media has consistently shown to potentiate stem cell proliferation, migration, and differentiation. In addition, the clinical utility of PRP is supported by evidence that PRP contains high concentrations of growth factors (GFs) and proteins which contribute to the regenerative process. PRP based therapies are cost effective and also benefit from the accessibility and safety of using the patient's own GFs. In the last years, a great development has been witnessed on PRP based biomaterials, with both structural and functional purposes. In this study we overview the most relevant PRP applications encompassing PRP based materials for tissue engineering and regenerative medicine. This review also summarizes the challenges in the fields of tissue engineering and regenerative medicine and provides a perspective on future directions.