Biomimetic PLGA 3D Scaffold Potentiate Amniotic Epithelial Stem Cells Biological Capability for Tendon Tissue Engineering Applications

INTRODUCTION: Tendon tissue engineering represents a promising solution to deal with tendinopathies and aims to develop effective implantable 3D biomimetic scaffolds with ideally native tissue’s physical, mechanical, biological, and functional qualities. These constructs can be engineered with stem cells to potentiate their teno-inductive and immunomodulatory properties (El Khatib, Mauro, Di Mattia, et al., 2020; El Khatib, Mauro, Wyrwa, et al., 2020; Russo et al., 2020). In this context, amniotic epithelial stem cells (AECs) have recently received much attention in the field of regenerative medicine due to their capacity to differentiate into the tenogenic lineage and to their immunomodulatory profile (Barboni et al., 2012, 2018; Mauro et al., 2016). The focus of this research was to create bundle tendon-like PLGA 3D scaffolds, which mimic tendon macro and micro-architecture and biomechanics, and to assess their impacts on AECs’ biological potential. METHODS: PLGA fleeces, with highly aligned fibers, were fabricated via electrospinning technique through a rotatory collector. The obtained fleeces were then wrapped manually to form 3D tendon-like scaffolds, which were evaluated in terms of structure, mechanical characteristics, and biological influence on AECs by conducting in vitro experiments. Indeed, ovine AECs, seeded on the PLGA 3D scaffolds and fleeces, were compared for their morphological changes and for the cytoplasmic expression of TNMD, a mature tendon protein, respect to cells cultured on Petri dishes (CTR), after 48h and 7d of culture through a confocal microscope. Moreover, the teno-differentiative potential and immunomodulatory properties of the produced constructs were assessed by analyzing the gene expression of tendon related markers (early: SCX, late: COL1 and TNMD) and of anti- (IL10) and pro- (IL12) inflammatory cytokines respectively. Moreover, the present research evaluated YAP protein activation in the engineered AECs through immunofluorescence assay by assessing its cellular localization. RESULTS: The produced PLGA 3D scaffolds, analyzed though a scanning electron microscope, showed high fiber alignment, which closely resemble the architecture, both macroscopically and microscopically, and the biomechanical properties of native tendon tissue. AECs seeded on the produced constructs exhibited an elongated tenocyte-like morphology already after 24 hours, while AECs cultivated on petri dishes (CTR) retained their characteristic polygonal morphology. The engineered AECs' phenotypic change was also confirmed by visualizing the cytoplasmic expression of TNMD protein and supported by tendon-related genes (SCX, COL1, and TNMD) upregulation at 7-day culture respect to CTR cells (p<0.05), which showed no TNMD protein expression or significant increase in tendon-related genes. Moreover, AECs seeded on 3D PLGA scaffolds showed an anti-inflammatory profile, with a significant higher IL10/IL12 ratio respect to the CTR (p<0.05). Finally, 3D scaffolds with highly aligned fibers stimulated AECs in terms of cell cytoskeleton stress, activating their mechanosensitive YAP pathway by significantly increasing YAP nuclear localization compared to the CTR (p<0.05), in which YAP was instead localized in the cytoplasm. DISCUSSION & CONCLUSIONS: Overall, these results support the biomimicry of the fabricated scaffolds in terms of structure and biomechanics and reveal their great teno/immuno-inductive potential and mechanosensing stimulus on AECs, thus standing biomimetic PLGA 3D scaffolds as a potential candidate for tendon regeneration

CUSTOMIZATION OF ELECTROSPINNING FOR TISSUE ENGINEERING

This paper deals with two electrospinning technologies: the melt electrospinning with a customized jet head, adapted from the fused deposition modeling (FDM) 3D printer, in comparison with the standard solution electrospinning, aiming at fabrication of tissue engineering scaffolds. The resulting fibers are compared. The influence of the collector properties on those of the fabricated scaffold is investigated. The resulting electrospun fibers exhibit different characteristics such as morphology and thickness, depending on the technology. The micro-fibers are produced by the melt electrospinning with an inbuilt 3D printer jet head, whereas the solution electrospinning has produced nano- and micro-fibers. The scaffolds fabricated on the rotating drum collector exhibit a more ordered structure as well as thinner fibers than those produced on the stationary plate collector. Further investigations should aim at fabrication of porous hollow fibers and tissue engineering scaffolds with controlled porosity and properties

A Novel Resorbable Composite Material Containing Poly(ester-co-urethane) and Precipitated Calcium Carbonate Spherulites for Bone Augmentation—Development and Preclinical Pilot Trials

Polyurethanes have the potential to impart cell-relevant properties like excellent biocompatibility, high and interconnecting porosity and controlled degradability into biomaterials in a relatively simple way. In this context, a biodegradable composite material made of an isocyanate-terminated co-oligoester prepolymer and precipitated calcium carbonated spherulites (up to 60% w/w) was synthesized and investigated with regard to an application as bone substitute in dental and orthodontic application. After foaming the composite material, a predominantly interconnecting porous structure is obtained, which can be easily machined. The compressive strength of the foamed composites increases with raising calcium carbonate content and decreasing calcium carbonate particle size. When stored in an aqueous medium, there is a decrease in pressure stability of the composite, but this decrease is smaller the higher the proportion of the calcium carbonate component is. In vitro cytocompatibility studies of the foamed composites on MC3T3-E1 pre-osteoblasts revealed an excellent cytocompatibility. The in vitro degradation behaviour of foamed composite is characterised by a continuous loss of mass, which is slower with higher calcium carbonate contents. In a first pre-clinical pilot trial the foamed composite bone substitute material (fcm) was successfully evaluated in a model of vertical augmentation in an established animal model on the calvaria and on the lateral mandible of pigs

Electrospun poly(d/l-lactide-co-l-lactide) hybrid matrix: a novel scaffold material for soft tissue engineering

Electrospinning is a long-known polymer processing technique that has received more interest and attention in recent years due to its versatility and potential use in the field of biomedical research. The fabrication of three-dimensional (3D) electrospun matrices for drug delivery and tissue engineering is of particular interest. In the present study, we identified optimal conditions to generate novel electrospun polymeric scaffolds composed of poly-d/l-lactide and poly-l-lactide in the ratio 50:50. Scanning electron microscopic analyses revealed that the generated poly(d/l-lactide-co-l-lactide) electrospun hybrid microfibers possessed a unique porous high surface area mimicking native extracellular matrix (ECM). To assess cytocompatibility, we isolated dermal fibroblasts from human skin biopsies. After 5 days of in vitro culture, the fibroblasts adhered, migrated and proliferated on the newly created 3D scaffolds. Our data demonstrate the applicability of electrospun poly(d/l-lactide-co-l-lactide) scaffolds to serve as substrates for regenerative medicine applications with special focus on skin tissue engineering

Surface Modification of electrospun PLGA Microfibers by Using Cold Atmospheric Plasma: Effect of treatment distance and exposure time

Plasma treatment has been widely applied to improve the hydrophilic properties of hydrophobic materials used in the field of tissue engineering, in particular for scaffold fabrication. The purpose of this treatment is to functionalize the scaffolds by generating polar groups on their surfaces without affecting the bulk whilst reducing their hydrophobic properties that could enhance cell adhesion and biocompatibility [1]. Our aim was to evaluate the effect of Cold Atmospheric Plasma (CAP) on physical and chemical properties of electrospun PLGA microfibers by varying the treatment distance and time

Comparable Studies on Nanoscale Antibacterial Polymer Coatings Based on Different Coating Procedures

The antibacterial activity of different antibiotic and metal-free thin polymer coatings was investigated. The films comprised quaternary ammonium compounds (QAC) based on a vinyl benzyl chloride (VBC) building block. Two monomeric QAC of different alkyl chain lengths were prepared, and then polymerized by two different polymerization processes to apply them onto Ti surfaces. At first, the polymeric layer was generated directly on the surface by atom transfer radical polymerization (ATRP). For comparison purposes, in a classical route a copolymerization of the QAC-containing monomers with a metal adhesion mediating phosphonate (VBPOH) monomers was carried out and the Ti surfaces were coated via drop coating. The different coatings were characterized by X-ray photoelectron spectroscopy (XPS) illustrating a thickness in the nanomolecular range. The cytocompatibility in vitro was confirmed by both live/dead and WST-1 assay. The antimicrobial activity was evaluated by two different assays (CFU and BTG, resp.,), showing for both coating processes similar results to kill bacteria on contact. These antibacterial coatings present a simple method to protect metallic devices against microbial contamination

Electrospun poly(lactide-co-glycolide) scaffold with high grade of fibers alignment mimics tendon extracellular matrix influencing amniotic epithelial stem cells phenotype and orientation

Poly(lactide-co-glycolide) (PLGA) is a copolymer known for its biodegradability and biocompatibility, and when electrospun, it becomes a fibrous device that can be engineered with stem cells. In this study, electrospun PLGA were engineered with amniotic derived stem cells (AECs). These type of stem cells are already known for their easy retrieval, non-ethical concerns, non-tumorigenic and immunomodulatory properties, thus ideal in allo and xenotransplantation settings. Moreover, they are able to differentiate toward the tenocyte linage when co-cultivated in vitro with tendon explants or when transplanted in vivo in a tendon injury model. In fact, when transplanted, oAECs are able to direct tissue remodeling either indirectly, thanks to their ability to release paracrine factors, and directly by producing Collagen Type 1 (COL1), which is the major protein expressed in a tendon. Thus, PLGA electrospun scaffolds were fabricated with a high degree of aligned fibers, in order to mimic tendon extracellular matrix (ECM), and with random fibers (control). Then, these scaffolds were cultured with ovine AECs in order to verify their biocompatibility and if the high degree of fiber alignment could influence cell phenotype and orientation mimicking a tendon tissue structure. To this aim, oAECs were seeded on scaffolds and cultivated for 48h. The results obtained in this study demonstrate that oAECs are biocompatible with the analyzed scaffolds. In fact, Calcein AM and PKH26 vital dyes and Ki67, a cell proliferation marker, immunostaining show that nearly all cells were alive and able to proliferate on electrospun PLGA. Additionally, these fluorescent dyes proved that oAECs spatial distribution and orientation was influenced by scaffold fibers’ alignment. In fact, when oAECs were cultivated on these highly aligned electrospun PLGA fibers they changed their morphology acquiring a spindle tenocyte-like shape, and were able to align along the longitudinal axis of the fibers, whereas in random electrospun PLGA scaffolds oAECs maintained their cuboidal morphology. Moreover, several of these oAECs, were able to express in their cytoplasm COL1 after 48h of culture only on aligned fibers scaffolds and not on the random oriented fibers ones. These findings indicate that when oAECs are seeded on electrospun PLGA scaffolds with highly aligned fibers, their phenotype and orientation are influenced by this artificial tendon ECM structure, thus acquiring an early tenogenic-like phenotype. In conclusion, electrospun PLGA scaffolds engineered with oAECs appears to be a good synergy that can be used for future clinical application in the treatment of tendon disorders

High-Sulfated Glycosaminoglycans Prevent Coronavirus Replication

Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy