Word of caution: negative impact of mouthwashes on folded Platelet-Rich Fibrin (F- PRF) membrane viability

Backround: The number of clinical application of different platelet-rich fibrin (PRF) membranes has increased in regenerative medicine including regenerative dentistry. Intact platelets, leukocytes and stem cells of PRF play a crucial role in the local bone augmentation releasing cytokines and growth factors. An integral part of the postsurgical management is the application of mouthwashes especially chlorhexidine-digluconate, which is recommended in order to prevent postoperative infections. In some cases there is possibility that there is contact between the mouthwash and PRF membrane. The impact of mouthwashes on cell viability of folded F-PRF was tested. Methods: 3 mouthwash brands were tested: Chorsodyl, Listerine 6 in 1 and Elmex Sensitive Plus using MTT viability assay after 30 seconds treatment and 72 hours treatment (twice daily for 30 seconds). The membrane samples were incubated in cell culture conditions. Results: 30 seconds of mouthwash treatment diminished the fresh F-PRF viability significantly by 15-21% depending on the agent. After 72 hours of treatment the viability loss was ~50%. Conclusion: The decreased number of platelets and other blood cells can not launch optimal bone morphogenesis. The MTT assay is cheap, reliable and simple method to assess the platelet and cellular viability and potential regenerative capacity of F-PRF membrane, or any platelet-rich product. The isolation of the PRF membrane from oral liquids and/or application of less aggressive mouthwashes is recommended for at least 5-7 days after PRF surgery.

Allogeneic chondrocyte implantation: What is stopping it from being a standard of care?

Allogenic chondrocyte implantation refers to harvesting of donor chondrocytes, growing them in culture plates with growth factors and implanting them with/without biocompatible scaffolds into cartilage defects. Despite its huge potential, it suffers several drawbacks with respect to source, biomaterial, preservation, cell-culture conditions as well as clinical utility. Through this letter, we attempt to provide an account of these limitations that are stopping it from being a standard of care.

Genitourinary Tissue Engineering: Reconstruction and Research Models

Tissue engineering is an emerging field of research that initially aimed to produce 3D tissues to bypass the lack of adequate tissues for the repair or replacement of deficient organs. The basis of tissue engineering protocols is to create scaffolds, which can have a synthetic or natural origin, seeded or not with cells. At the same time, more and more studies have indicated the low clinic translation rate of research realised using standard cell culture conditions, i.e., cells on plastic surfaces or using animal models that are too different from humans. New models are needed to mimic the 3D organisation of tissue and the cells themselves and the interaction between cells and the extracellular matrix. In this regard, urology and gynaecology fields are of particular interest. The urethra and vagina can be sites suffering from many pathologies without currently adequate treatment options. Due to the specific organisation of the human urethral/bladder and vaginal epithelium, current research models remain poorly representative. In this review, the anatomy, the current pathologies, and the treatments will be described before focusing on producing tissues and research models using tissue engineering. An emphasis is made on the self-assembly approach, which allows tissue production without the need for biomaterials.

Circumventing Drug Treatment? Intrinsic Lethal Effects of Polyethyleneimine (PEI)-Functionalized Nanoparticles on Glioblastoma Cells Cultured in Stem Cell Conditions

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood–brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.

Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

A Straightforward Hypoxic Cell Culture Method Suitable for Standard Incubators

We present a new and straightforward method by which standard cell culture plates can be sealed off from ambient air and be placed under controlled hypoxic cell culture conditions without costly or highly specialized materials. The method was established on a murine cell culture system using the dendritic cell line JAWS II but can be readily adapted to other cell cultures. The procedure was designed to be easy to implement in cell culture laboratories with standard incubators and requires only readily available materials, resources, and consumables, such as six-well plates, degassed culture medium, CoCl2, a vacuum sealer, etc., and no further complicated laboratory equipment. The simple hypoxic cell culture method presented here is technically reliable and experimentally safe. As it can be performed in any standard incubator, it is suitable for use at both low and higher biosafety levels.

Mitochondrial and metabolic remodeling in human skin fibroblasts in response to glucose availability

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine as well as in the validation of ingredients for cosmetics. Given these cells’ short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in Normal Human Dermal Fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level (LGm), or its complete removal and replacement by galactose (OXPHOSm), always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm it was possible to observe the selective inhibition of mitochondrial ATP production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), oxidation of citric acid cycle substrates, fatty acids, lactate and other substrates, mitochondrial network extension and polarization and changes in several key transcripts related to energy metabolism. We also evaluated the relevance of galactose, glutamine and pyruvate for OXPHOS stimulation, by comparing OCR and ECAR in the presence or absence of these substrates. Galactose and pyruvate seem to be important, but redundant, to promote OXPHOS, whereas glutamine was essential. We concluded that LGm does not promote significant metabolic changes but the short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.Author ContributionsCC, SAP, SLCP and IMS performed experiments. TCO and PJO designed research and acquired funding. JS, and OB analyzed data. CC and TCO analyzed data and wrote the paper. All authors contributed to the final version of the manuscript.