Oral mucosa keratinocytes and their exosomes for epithelial tissue regeneration

Early tumors, including high grade dysplasia and intramucosal invasive cancer, of the esophagus can today be removed using endoscopic resection, often using a technique called endoscopic submucosal dissection (ESD). This treatment is better tolerated and has considerably less mortality and morbidity compared to conventional, more invasive surgery, which usually entails esophagectomy. However, after larger endoscopic dissections, stricture formation is a common complication. Such strictures are usually treated with balloon dilatations, but the procedure often has to be repeated several times and is associated with risks such as perforations. In recent years, Japanese researchers developed a new method to reduce the risk of strictures. About two weeks before the treatment, an oral mucosa biopsy is taken from the patient from which epithelial cells are isolated and grown on special temperature-responsive polymer-coated surfaces. The polymer changes morphology and wettability properties depending on temperature, which enables non-enzymatic cell harvesting – the cells can be detached as contiguous sheets and with a large amount of extracellular matrix (ECM) maintained. After the ESD, cell sheets can be transplanted to the wound bed without the need for suture or other fixative, it is thought that the remaining ECM acts as a glue. Nine patients were treated in Tokyo and we subsequently transferred the technology to Stockholm where five additional patients were treated. Although one of nine patients in the Japanese cohort and three of five patients in the Swedish cohort still developed strictures, they appeared to be milder and easier to treat than expected. However, the aim of these projects was to evaluate safety and feasibility, further studies are needed to evaluate efficacy of the treatment. Since some patients still developed strictures despite the cell sheet transplantation, we next aimed to evaluate if exosomes from the cell culture media could be used as a pro-regenerative agent, perhaps in combination with cell sheet therapy. Media was collected from clinical- grade production of cell sheets from eight healthy donors. The media was concentrated by ultra-filtration and exosomes were isolated by size-exclusion chromatography. The exosomes were characterized by western blot (CD9+, Flotillin-1+, GRP94-), electron microscopy and nanoparticle tracking analysis (~125 nm). They reduced the proliferation of skin fibroblasts and stimulated upregulation of gene expression of growth factors relevant for wound healing. We studied the exosomes’ adhesion to esophageal wound bed by topical application to porcine esophageal wounds ex vivo and could detect signal after as little as one minute adhesion time. We also found that the exosomes stimulated wound healing of full-thickness skin wounds in immunocompetent rats, both at the 6th day and 17th day time point. In conclusion we found that exosomes could be isolated from cell sheet media and that they exhibited pro-regenerative properties even in a xenogeneic setting. Further studies are necessary to evaluate their potential to stimulate mucosal wound healing and reduce stricture formation of the esophagus

Quantitative uptake of colloidal particles by cell cultures

The use of nanotechnologies involving nano- and microparticles has increased tremendously in the recent past. There are various beneficial characteristics that make particles attractive for a wide range of technologies. However, colloidal particles on the other hand can potentially be harmful for humans and environment. Today, complete understanding of the interaction of colloidal particles with biological systems still remains a challenge. Indeed, their uptake, effects, and final cell cycle including their life span fate and degradation in biological systems are not fully understood. This is mainly due to the complexity of multiple parameters which need to be taken in consideration to perform the nanosafety research. Therefore, we will provide an overview of the common denominators and ideas to achieve universal metrics to assess their safety. The review discusses aspects including how biological media could change the physicochemical properties of colloids, how colloids are endocytosed by cells, how to distinguish between internalized versus membrane-attached colloids, possible correlation of cellular uptake of colloids with their physicochemical properties, and how the colloidal stability of colloids may vary upon cell internalization. In conclusion three main statements are given. First, in typically exposure scenarios only part of the colloids associated with cells are internalized while a significant part remain outside cells attached to their membrane. For quantitative uptake studies false positive counts in the form of only adherent but not internalized colloids have to be avoided. pH sensitive fluorophores attached to the colloids, which can discriminate between acidic endosomal/lysosomal and neutral extracellular environment around colloids offer a possible solution. Second, the metrics selected for uptake studies is of utmost importance. Counting the internalized colloids by number or by volume may lead to significantly different results. Third, colloids may change their physicochemical properties along their life cycle, and appropriate characterization is required during the different stages.This work was supported by the European Commission (grant FutureNanoNeeds) grant agreement no. 604602 to WJP. NF acknowledges funding from the Lars Hiertas Minne Fundation (Sweden), SA, BP and IC acknowledge a fellowship from the Alexander von Humboldt Fundation (Germany). AE acknowledges Junta de Andalucía (Spain) for a Talentia Postdoc Fellowship, co-financed by the European Union Seventh Framework Programme, grant agreement no 267226. AHS acknowledges the Egyptian government (Ministry of Higher Education, Mission). The project was also supported by the Dr. Dorka-Stiftung (Germany) to PJ

RNA-sequencing reveals positional memory of multipotent mesenchymal stromal cells from oral and maxillofacial tissue transcriptomes

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Diverse Applications of Nanomedicine

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic. \ua9 2017 American Chemical Society

Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing

The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer