Open Access

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs.

Due to their strong biomimetic potential, silk fibroin (SF) hydrogels are impressive candidates for tissue engineering, due to their tunable mechanical properties, biocompatibility, low immunotoxicity, controllable biodegradability, and a remarkable capacity for biomaterial modification and the realization of a specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms available for the generation of SF hydrogels (SFHs). These methods, either chemical or physical, change the structure of SF and improve its mechanical stability, although each method has its advantages and disadvantages. While chemical crosslinking agents guarantee the mechanical strength of SFH through the generation of covalent bonds, they could cause some toxicity, and their usage is not compatible with a cell-friendly technology. On the other hand, physical crosslinking approaches have been implemented in the absence of chemical solvents by the induction of β-sheet conformation in the SF structure. Unfortunately, it is not easy to control the shape and properties of SFHs when using this method. The current review discusses the different crosslinking mechanisms of SFH in detail, in order to support the development of engineered SFHs for biomedical applications.

Background: In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. Methods: Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. Findings: Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. Interpretation: Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. Funding: Austrian Research Promotion Agency FFG ("CartiScaff" #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08). Keywords: Cartilage regeneration; Decellularisation; Ectopic animal model; Laser engraving; Mechanical testing; Repopulation.