The paracrine neuroprotective effects of Mesenchymal Stem Cells (MSC) are well known, and our understanding of the underlying mechanisms is expanding by the day. From among their diverse secretome, extracellular vesicles (EVs) have gained particular interest lately, owing to their good safety profile, stability, and relative ease of use as a cell-free therapy. The cargo and thus the potential effect of these nano-sized lipid membrane-enclosed vesicles is highly dependent on the type, age, and environment of the donor cells. Therefore, it is paramount to know which cell types are best utilised in any given situation. Glaucoma is a chronic progressive optic neuropathy that is the most common cause of irreversible blindness worldwide, characterised by the loss of Retinal Ganglion Cells (RGC) whose axons make up the optic nerve. Preservation of these neurons via the administration of the right EVs represents a promising approach for slowing down or halting disease progression, thereby preventing vision loss. Here, we evaluate the neuroprotective and neuritogenic potential of EVs isolated from five different cell types using a rodent in vitro model of RGC degeneration. Our findings showed that Adipose Mesenchymal Stem Cells (ADSC) release the most potent EVs, with Bone Marrow (BMSC) being a close second. EVs released by cells of the Umbilical Cord (UCSC), Dental Pulp (DPSC), Dermal Fibroblasts (DF), and an Oral Mucosal Lamina Propria-Progenitor cells (OMLP-PCs) did not have an observable benefit. Thus, our study provides greater insight into how the efficacy of different EVs compare to each other.

Glioblastoma multiforme is characterised by resistance to conventional treatments via sustained pro-inflammatory signalling. This study investigated whether secretomes and small extracellular vesicles (sEVs) from human oral mucosa neural crest-derived stem cells (NCSCs) could disrupt multiple glioblastoma features by rebalancing pro- and anti-inflammatory pathways. NCSCs were characterised, and their secretomes/sEVs isolated, and analysed using a range of state-of-the-art methods. Anti-tumour effects were evaluated using multiple glioblastoma cell lines (U251, U373, U87) through viability, proliferation, migration, and tumourigenicity assays. Mechanistic studies employed dual nuclear factor-κB (NF-κB) and quadruple NF-κB/interferon regulatory factor 3 (IRF3) reporter systems, cytokine arrays, and immunocytochemistry. Therapeutic potential was assessed using temozolomide (TMZ) chemosensitivity assays and organotypic mouse brain slice models. NCSC secretomes and sEVs demonstrated consistent anti-glioblastoma activity across all functional assays. Both products potently suppressed NF-κB activation across multiple pro-inflammatory stimuli whilst simultaneously enhancing IRF3 nuclear translocation and transcriptional activity. This dual pathway modulation reprogrammed glioblastoma cytokine secretion towards anti-inflammatory profiles, and inhibited tumourigenicity in both 3D culture and ex vivo brain slice models. Notably, secretomes and sEVs enhanced the efficacy of TMZ, reducing colony size compared to monotherapy, without compromising tissue viability. These findings demonstrate the first evidence of dual NF-κB/IRF3 pathway rebalancing by NCSC-derived products in glioblastoma. The simultaneous suppression of tumour-promoting inflammation whilst enhancing anti-tumour immune signalling represents a novel therapeutic paradigm that addresses multiple resistance mechanisms simultaneously. This strategy redefines stem cell-based therapy for glioblastoma by integrating immunomodulation with chemosensitisation in a single, Good Manufacturing Practice (GMP)-compatible product.

Direct interrogation of nanoscale chemical features on and within biological structures remains a major frontier challenge in biophysical and biomedical research. These nanoscale features govern molecular organization, structural dynamics, and cellular function, yet conventional non-invasive techniques such as Fourier-transform infrared spectroscopy (FTIR) are fundamentally limited by optical diffraction. Although hybrid approaches, including scattering-type scanning near-field optical microscopy (s-SNOM) and atomic force microscopy infrared spectroscopy (AFM-IR), have advanced spatial resolution, they remain insufficient to resolve individual macromolecular assemblies. Furthermore, precise control over the depth of analysis within biological architectures, where critical molecular information underpinning intra- and inter-cellular communication resides, has yet to be fully achieved. Here, we employ photo-induced force microscopy (PiFM), an atomic force microscopy (AFM) based technique that directly measures forces arising from light-induced polarization in the near-field region. These forces, typically on the order of piconewtons, are localized perpendicular to the sample surface. This localization enables a theoretical spatial resolution approaching 5 nm, with depth sensitivity spanning approximately 2-200 nm. Crucially, PiFM can operate under ambient and environmentally controlled conditions, preserving physiologically relevant architectures in vitro. Our findings demonstrate that aldehyde-based fixing, including formalin treatment, causes substantial chemical modifications and spectral overlap within the nuclear envelopes of oral mucosa lamina propria progenitor cells (OMLP-PCs). These effects highlight the necessity for rigorous validation of sample-preparation protocols in nano-spectroscopy. In contrast, live-cell PiFM imaging under controlled humidity conditions enables visualisation of native biomolecular states and dynamic cellular processes in OMLP-PCs. Our approach captured whole-cell and membrane-level phenomena, including extracellular vesicle (EV) biogenesis and nuclear stress responses. PiFM mapping of isolated human bone marrow stromal cell (hBMSC) EVs further uncovers nanoscale compositional heterogeneity at the single-EV level. This work demonstrates the application of PiFM as a transformative nano-spectroscopic tool for probing the structural and spatial chemical information of biological matter, potentially down to 5 nm resolution. By bridging physical chemistry and biophysics, PiFM enables direct visualisation of compositional heterogeneity under near-physiological conditions, offering a non-invasive and in situ pathway for nanoscale characterisation and mechanistic understanding of complex biological systems.

Abstract Mesenchymal Stem Cells (MSC) possess a diverse secretome with well-established neuroprotective effects. Form among the materiel released by these cells, extracellular vesicles (EVs) have gained particular interest lately, owing to their good safety profile, stability, and relative ease of use as a cell-free therapy. These lipid-enclosed nano-carriers can significantly alter the survival of recipient cells through the delivery of a wide variety of signalling molecules, the exact composition of which is highly dependent on the type, age, and environment of the donor cells. Glaucoma is a chronic progressive optic neuropathy characterised by the loss of Retinal Ganglion Cells whose axons make up the optic nerve. Preservation of these neurons via the administration of the right EVs represents a promising approach for slowing disease progression, thereby preventing vision loss. Here, we evaluate and compare the protective and neuritogenic potential of small extracellular vesicles (sEVs), a subset of EVs with a diameter smaller than 220 nm, from six different cell types using a rodent in vitro model of RGC degeneration. Our findings showed that Adipose Mesenchymal Stem Cells release the most potent sEVs, with Bone Marrow being a close second. EVs released by cells of the Umbilical Cord, Dental Pulp, Dermal Fibroblasts, and an Oral Mucosal Lamina Propria-Progenitor cells did not have an observable benefit. Thus, our study provides greater insight into how the efficacy of different EVs compares to each other.

Chronic wounds often host pathogens like Staphylococcus aureus, prompting interest in developing new antimicrobial and wound healing strategies, including the utilisation of extracellular vesicles (EVs). Whilst there has been a recent emphasis within the EV community to ensure standardization of characterization and isolation techniques, there has been less focus placed on the upstream tissue culture methodologies used for collection of vesicle-containing conditioned medium (CM). Hence, this study investigated the antimicrobial properties of the CM used for EV enrichment. CM exhibited bacteriostatic effects against penicillin-sensitive S. aureus NCTC 6571, but not penicillin-resistant S. aureus 1061 A. Further analysis revealed that the antimicrobial activity was due to residual antibiotics rather than cell-secreted factors, specifically the retention and release of penicillin to tissue culture plastic surfaces. Pre-washing cells and minimizing antibiotic concentrations in basal medium reduced this carry-over effect. These findings emphasize the importance of controlling antibiotic use in tissue culture to avoid misleading conclusions about the antimicrobial potential of CM or EVs. Researchers should carefully consider medium selection and supplementation during method development as accurately determining the antimicrobial mechanisms of any CM is essential for validating future cell-based therapeutic applications.

Unlike skin, oral mucosal wounds are characterized by rapid healing and minimal scarring, attributable to the "enhanced" healing properties of oral mucosal fibroblasts (OMFs). As oxidative stress is increasingly implicated in regulating wound healing outcomes, this study compared oxidative stress biomarker and enzymic antioxidant profiles between patient-matched oral mucosal/skin tissues and OMFs/skin fibroblasts (SFs) to determine whether superior oral mucosal antioxidant capabilities and reduced oxidative stress contributed to these preferential healing properties. Oral mucosa and skin exhibited similar patterns of oxidative protein damage and lipid peroxidation, localized within the lamina propria/dermis and oral/skin epithelia, respectively. SOD1, SOD2, SOD3 and catalase were primarily localized within epithelial tissues overall. However, SOD3 was also widespread within the lamina propria localized to OMFs, vasculature and the extracellular matrix. OMFs were further identified as being more resistant to reactive oxygen species (ROS) generation and oxidative DNA/protein damage than SFs. Despite histological evaluation suggesting that oral mucosa possessed higher SOD3 expression, this was not fully substantiated for all OMFs examined due to inter-patient donor variability. Such findings suggest that enzymic antioxidants have limited roles in mediating privileged wound healing responses in OMFs, implying that other non-enzymic antioxidants could be involved in protecting OMFs from oxidative stress overall.

Orofacial soft tissue wounds caused by surgery for congenital defects, trauma, or disease frequently occur leading to complications affecting patients' quality of life. Scarring and fibrosis prevent proper skin, mucosa and muscle regeneration during wound repair. This may hamper maxillofacial growth and speech development. To promote the regeneration of injured orofacial soft tissue and attenuate scarring and fibrosis, intraoral and extraoral stem cells have been studied for their properties of facilitating maintenance and repair processes. In addition, the administration of stem cell-derived extracellular vesicles (EVs) may prevent fibrosis and promote the regeneration of orofacial soft tissues. Applying stem cells and EVs to treat orofacial defects forms a challenging but promising strategy to optimize treatment. This review provides an overview of the putative pitfalls, promises and the future of stem cells and EV therapy, focused on orofacial soft tissue regeneration.

Abstract Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell–mediated repair in varied settings; hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow–derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings.

We report an analysis of how an interdisciplinary project bringing together biologists, physicists and engineers worked in practice. The authorship team are the Principle Investigator who led the project, and a social scientist who studied the project as it was conducted by interviewing participants and observing practice. We argue it is accurate and productive to think of the interdisciplinary team as an Expert-Network, which means it was a managed set of relationships between disciplinary groups punctuated by specific junctions at which interdisciplinary exchange of materials, knowledge, and in limited cases, practices, occurred. We stress the role of trust in knowledge exchange, and document how hard sharing knowledge – and especially tacit knowledge - between disciplines can be. Key is the flexible management of the network, as the membership and required skill set change. Our analysis is embedded within, and contributes to, the Sociology of Experience and Expertise (SEE) framework. We close by suggesting advice for others seeking to manage a similar interdisciplinary Expert-Network.

Chronic wounds are a significant global problem with an increasing economic and patient welfare impact. How wounds move from an acute to chronic, non-healing, state is not well understood although it is likely that it is driven by a poorly regulated local inflammatory state. Opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa are well known to stimulate a pro-inflammatory response and so their presence may further drive chronicity. Studies have demonstrated that host cell extracellular vesicles (hEVs), in particular exosomes, have multiple roles in both increasing and decreasing chronicity within wounds; however, the role of bacterial extracellular vesicles (bEVs) is still poorly understood. The aim of this review is to evaluate bEV biogenesis and function within chronic wound relevant bacterial species to determine what, if any, role bEVs may have in driving wound chronicity. We determine that bEVs drive chronicity by both increasing persistence of key pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa and stimulating a pro-inflammatory response by the host. Data also suggest that both bEVs and hEVs show therapeutic promise, providing vaccine candidates, decoy targets for bacterial toxins or modulating the bacterial species within chronic wound biofilms. Caution should, however, be used when interpreting findings to date as the bEV field is still in its infancy and as such lacks consistency in bEV isolation and characterization. It is of primary importance that this is addressed, allowing meaningful conclusions to be drawn and increasing reproducibility within the field.

BACKGROUND:Small extracellular vesicles (SEVs) have a diameter between 30 and 150 nm and play a key role in cell-cell communication. As cells cultured in 3D vs 2D behave differently, this project aimed to assess whether there were differences in SEVs derived from human oral mucosa lamina propria-progenitor cells (OMLP-PCs) cultured in a 3D matrix compared with traditional 2D monolayer cultures. METHODS:OMLP-PCs were cultured in 3D type I collagen matrices or on traditional 2D tissue culture plastic. Cell morphology and viability were assessed by light microscopy, actin staining, and trypan blue staining. SEVs secreted by OMLP-PCs were purified and quantitatively analyzed by a BCA assay and nanoparticle tracking analysis (NTA; nanosight™). SEVs were further characterized by flow cytometry. SEV proliferative function was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS:Cells cultured in 3D grew well as observed by light microscopy and phalloidin staining with cells branching in three dimensions (as opposed to the cells grown as monolayers on tissue culture plastic). NTA demonstrated a significantly higher number of SEV-sized particles in the conditioned medium of cells grown in 3D type I collagen matrices vs a 2D monolayer (P < .01). Like SEVs from 2D culture, SEVs from 3D culture demonstrated a particle size within the expected SEV range. Tetraspanin analysis confirmed that 3D-derived SEVs were positive for typical, expected tetraspanins. Cell proliferation analysis demonstrated that SEVs produced through 3D cell culture conditions significantly reduced the proliferation of skin fibroblasts when compared with SEVs from 2D monolayers (P < .05). CONCLUSION:3D culture of OMLP-PCs produced typical SEVs but in a greater amount than when the same cells were cultured in 2D. The downstream proliferative potential of the SEVs was influenced by the initial culture methodology. Future work should now assess the potential effects of 3D SEVs on key wound healing activities.