Connexin 43 Modulation in Human Chondrocytes, Osteoblasts and Cartilage Explants: Implications for Inflammatory Joint Disorders.

Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1ß), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (-73% and -32%, respectively), while IL-1ß showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (-65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders.

Serum starvation affects mitochondrial metabolism of adipose-derived stem/stromal cells.

BACKGROUND AIMS: A large part of mesenchymal stromal cell (MSC) regenerative and immunomodulatory action is mediated by paracrine signaling. Hence, an increasing body of evidence acknowledges the potential of MSC secretome in a variety of preclinical and clinical scenarios. Mid-term serum deprivation is a common approach in the pipeline of MSC secretome production. Nevertheless, up to now, little is known about the impact of this procedure on the metabolic status of donor cells. METHODS: Here, through untargeted differential metabolomics, we revealed an impairment of mitochondrial metabolism in adipose-derived MSCs exposed for 72 h to serum deprivation. RESULTS: This evidence was further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. Probably as a repair mechanism, an upregulation of mitochondrial superoxide dismutase was also induced. CONCLUSIONS: Of note, the analysis of mitochondrial functionality indicated that, despite a significant reduction of basal respiration and ATP production, serum-starved MSCs still responded to changes in energy demand. This metabolic phenotype correlates with the obtained evidence of mitochondrial elongation and branching upon starvation.

Dynamics of Connexin 43 Down Modulation in Human Articular Chondrocytes Stimulated by Tumor Necrosis Factor Alpha.

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (-46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-ß1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-ß1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.

Bioactive Lipids in MSCs Biology: State of the Art and Role in Inflammation.

Lipidomics is a lipid-targeted metabolomics approach that aims to the comprehensive analysis of lipids in biological systems in order to highlight the specific functions of lipid species in health and disease. Lipids play pivotal roles as they are major structural components of the cellular membranes and energy storage molecules but also, as most recently shown, they act as functional and regulatory components of intra- and intercellular signaling. Herein, emphasis is given to the recently highlighted roles of specific bioactive lipids species, as polyunsaturated fatty acids (PUFA)-derived mediators (generally known as eicosanoids), endocannabinoids (eCBs), and lysophospholipids (LPLs), and their involvement in the mesenchymal stem cells (MSCs)-related inflammatory scenario. Indeed, MSCs are a heterogenous population of multipotent cells that have attracted much attention for their potential in regulating inflammation, immunomodulatory capabilities, and reparative roles. The lipidomics of the inflammatory disease osteoarthritis (OA) and the influence of MSCs-derived lipids have also been addressed.

Nitrogen Containing Bisphosphonates Impair the Release of Bone Homeostasis Mediators and Matrix Production by Human Primary Pre-Osteoblasts.

Bisphosphonates (BPs) represent the first-line treatment for a wide array of bone disorders. Despite their well-known action on osteoclasts, the effects they induce on osteoblasts are still unclear. In order to shed light on this aspect we evaluated the impact of two nitrogen containing bisphosphonates, Alendronate (ALN) and Zoledronate (ZOL), on human primary pre-osteoblasts. At first, we showed an inhibitory effect on cell viability and alkaline phosphatase activity starting from µM concentrations of both drugs. In addition, an inhibitory trend on mineralized nodules deposition was observed. Then low doses of both ALN and ZOL rapidly increased the release of the pro-inflammatory mediators TNFα and IL-1ß, while increased DKK-1 and Sclerostin, both inhibitors of osteoblastogenesis. Finally, ALN and 10-7M ZOL decreased the expression of type I Collagen and Osteopontin, while both drugs slightly stimulated SPARC production. With these results, we would like to suggest a direct inhibitory action on bone-forming cells by nitrogen containing bisphosphonates.

17ß-estradiol differently affects osteogenic differentiation of mesenchymal stem/stromal cells from adipose tissue and bone marrow.

Adipose-derived and bone marrow stem/stromal cells (ASCs and BMSCs) have been often compared for their application in regenerative medicine, and several factors sustaining their differentiation and efficacy have been investigated. 17 ß-estradiol (E2) has been reported to influence some functions of progenitor cells. Here we studied the effects of 10 and 100nM E2 on ASC and BMSC vitality, proliferation and differentiation towards osteogenic and adipogenic lineages. E2 did not modulate ASC and BMSC vitality and growth rate, while the hormone produced a pro-adipogenic effect on both mesenchymal stem/stromal cells (MSCs). In particular, the synergy between 7-day pre-treatment and 100nM E2 led to the most evident result, increasing lipid vacuoles formation in ASCs and BMSCs of +44% and +82%, respectively. Despite the fact that E2 did not alter collagen deposition of osteo-induced MSCs, we observed a different modulation of ASC and BMSC alkaline phosphatase (ALP) activity. Indeed, this osteogenic marker was always enhanced by 17 ß-estradiol in BMSCs, and 7-day pre-treatment with 100nM E2 increased it of about 70%. In contrast, E2 weakened ASC osteogenic potential, reducing their ALP activity of about 20%, with the most evident effect on ASCs isolated from pre-menopausal women (-30%). Finally, we identified an estrogen receptor α (ERα) variant of about 37kDa expressed in both MSCs. Interestingly, adipogenic stimuli drastically reduced its expression, while osteogenic ones mildly increased this isoform in BMSCs only. In conclusion, E2 positively affected the adipogenic process of both MSCs while it favored osteogenic induction in BMSCs only, and both mesenchymal progenitors expressed a novel 37kDa ER-α variant whose expression was modulated during differentiation.

Does Freeze-Thawing Influence the Effects of Platelet Concentrates? An In Vitro Study on Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (hASCs) have been proposed as a possible therapy for tissue regeneration in aesthetic, plastic, and reconstructive surgery. Today, platelet concentrates are used in a wide range of disciplines, but their storage has become a controversial aspect. The purpose of this in vitro study was to evaluate the effect of plasma rich in growth factors (PRGF), after a freeze-thawing cycle, on the proliferation and biological activity of progenitor cells involved in soft tissue healing. Different formulations of activated PRGF were added to hASCs cultured in serum-free medium. Cell proliferation was assessed by MTT test and cell count up to 7 and 12-day incubation. Osteo-differentiation ability of hASCs was also tested after 7 and 14-day incubation by alkaline phosphatase assay. The effects of 4 PRGF preparations (fresh/frozen and with/without platelets) were compared with corresponding formulations of plasma poor in growth factors and with standard medium. hASCs cultured in the presence of platelet concentrates increased proliferation rate with respect to cells grown in standard medium without significant differences among all the tested plasma formulations on cell viability up to 12 days of culture. PRGF activity is preserved after cryopreservation and platelet-rich preparations promoted osteo-differentiation of hASCs at day 7. In conclusion, PRGF supports the proliferation and the differentiation of progenitor cells in vitro also when applied after cryopreservation. Platelet concentrates, either alone or in combination with mesenchymal stem cells, might be a valuable tool in the field of tissue regeneration.

Effect of an Activated Platelet Concentrate on Differentiated Cells Involved in Tissue Healing.

Tissue healing is a complex process involving several players such as cells and growth factors released from platelets upon activation. Today, platelet concentrates (PCs) are used in many different medical fields including oral, orthopaedic, and reconstructive surgery since they allow growth factors delivery to the injured site, aiming at enhancing tissue regeneration. The purpose of this in vitro study was to evaluate the effect of the acellular plasma of an activated platelet concentrate obtained using a manual protocol, on the proliferation, and biological activity of differentiated cells involved in tissue healing. Human osteoblasts and dermal fibroblasts were grown in serum-free medium supplemented with PC derived from several donors. Human osteoblast and human dermal fibroblast proliferation was assessed by MTT test after 7 days and cells were count up to 12-day incubation. Human osteoblast osteo-differentiation was tested after 7 and 14-day incubation by alkaline phosphatase assay. The addition of PC to the culture medium caused an increased proliferation with respect to cells grown in standard condition. The results of the present study suggest that PC supports the proliferation of terminally differentiated cells involved in wound healing and tissue regeneration, confirming its beneficial clinical application in regenerative therapies.

Human Adipose-Derived Stem Cells on Rapid Prototyped Three-Dimensional Hydroxyapatite/Beta-Tricalcium Phosphate Scaffold.

In the study, we assess a rapid prototyped scaffold composed of 30/70 hydroxyapatite (HA) and beta-tricalcium-phosphate (ß-TCP) loaded with human adipose-derived stem cells (hASCs) to determine cell proliferation, differentiation toward osteogenic lineage, adhesion and penetration on/into the scaffold.In this in vitro study, hASCs isolated from fat tissue discarded after plastic surgery were expanded, characterized, and then loaded onto the scaffold. Cells were tested for: viability assay (Alamar Blue at days 3, 7 and Live/Dead at day 32), differentiation index (alkaline phosphatase activity at day 14), scaffold adhesion (standard error of the mean analysis at days 5 and 18), and penetration (ground sections at day 32).All the hASC populations displayed stemness markers and the ability to differentiate toward adipogenic and osteogenic lineages.Cellular vitality increased between 3 and 7 days, and no inhibitory effect by HA/ß-TCP was observed. Under osteogenic stimuli, scaffold increased alkaline phosphatase activity of +243% compared with undifferentiated samples. Human adipose-derived stem cells adhered on HA/ß-TCP surface through citoplasmatic extensions that occupied the macropores and built networks among them. Human adipose derived stem cells were observed in the core of HA/ß-TCP. The current combination of hASCs and HA/ß-TCP scaffold provided encouraging results. If authors' data will be confirmed in preclinical models, the present engineering approach could represent an interesting tool in treating large bone defects.

Cartilage responses to inflammatory stimuli and adipose stem/stromal cell-derived conditioned medium: Results from an ex vivo model.

Introduction: Osteoarthritis (OA), a chronic inflammatory joint disorder, still lacks effective therapeutic interventions. Consequently, the development of convenient experimental models is crucial. Recently, research has focused on the plasticity of Mesenchymal Stem/stromal Cells, particularly adipose-derived ones (ASCs), in halting OA progression. This study investigates the therapeutic potential of a cell-free approach, ASC-derived conditioned medium (CM), in reversing cytokine-induced OA markers in an ex vivo model of human cartilage explants. Methods: 4 mm cartilage punches, derived from the femoral heads of patients undergoing total hip replacement, were treated with 10 ng/ml TNFα, 1 ng/ml IL-1ß, or a combination of both, over a 3-day period. Analysis of OA-related markers, such as MMP activity, the release of NO and GAGs, and the expression of PTGS2, allowed for the selection of the most effective inflammatory stimulus. Subsequently, explants challenged with TNFα+IL-1ß were exposed to CM, consisting of a pool of concentrated supernatants from 72-h cultured ASCs, in order to evaluate its effect on cartilage catabolism and inflammation. Results: The 3-day treatment with both 10ng/ml TNFα and 1ng/ml IL-1ß significantly increased MMP activity and NO release, without affecting GAG release. The addition of CM significantly downregulated the abnormal MMP activity induced by the inflammatory stimuli, while also mildly reducing MMP3, MMP13, and PTGS2 gene expression. Finally, SOX9 and COL2A1 were downregulated by the cytokines, and further decreased by CM. Conclusion: The proposed cartilage explant model offers encouraging evidence of the therapeutic potential of ASC-derived CM against OA, and it could serve as a convenient ex vivo platform for drug screening.

Boosting the therapeutic potential of cell secretome against osteoarthritis: Comparison of cytokine-based priming strategies.

The secretome, or conditioned medium (CM), from Mesenchymal Stem/stromal Cells (MSCs) has recently emerged as a promising cell-free therapeutic against osteoarthritis (OA), capable of promoting cartilage regeneration and immunoregulation. Priming MSCs with 10 ng/ml tumor necrosis factor α (TNFα) and/or 10 ng/ml interleukin 1ß (IL-1ß) aims at mimicking the pathological milieu of OA joints in order to target their secretion towards a pathology-tailored phenotype. Here we compare the composition of the CM obtained after 24 or 72 h from untreated and cytokine-treated adipose-derived MSCs (ASCs). The 72-hour double-primed CM presents a higher total protein yield, a larger number of extracellular vesicles, and a greater concentration of bioactive lipids, in particular sphingolipids, fatty acids, and eicosanoids. Moreover, the levels of several factors involved in immunomodulation and regeneration, such as TGF-ß1, PGE2, and CCL-2, are strongly upregulated. Additionally, the differential profiling of 80 bioactive molecules indicates that primed CM is enriched in immune cell chemotaxis and migration factors. Our results indicate that pre-conditioning ASCs with inflammatory cytokines can modulate the composition of their CM, promoting the release of factors with recognized anti-inflammatory, chondroprotective, and immunoregulatory properties.

Significant association between FGFR1 mutation frequency and age in central giant cell granuloma.

Central giant cell granulomas (CGCG) are rare intraosseous osteolytic lesions of uncertain aetiology. Despite the benign nature of this neoplasia, the lesions can rapidly grow and become large, painful, invasive, and destructive. The identification of molecular drivers could help in the selection of targeted therapies for specific cases. TRPV4, KRAS and FGFR1 mutations have been associated with these lesions but no correlation between the mutations and patient features was observed so far. In this study, we analysed 17 CGCG cases of an Italian cohort and identified an interesting and significant (p=0.0021) correlation between FGFR1 mutations and age. In detail, FGFR1 mutations were observed frequently and exclusively in CGCG from young (<18 years old) patients (4/5 lesions, 80%). Furthermore, the combination between ours and previously published data confirmed a significant difference in the frequency of FGFR1 mutations in CGCG from patients younger than 18 years at the time of diagnosis (9/23 lesions, 39%) when compared to older patients (1/31 lesions, 0.03%; p=0.0011), thus corroborating our observation in a cohort of 54 patients. FGFR1 variants in young CGCG patients could favour fast lesion growth, implying that they seek medical attention earlier. Our observation might help prioritise candidates for FGFR1 testing, thus opening treatment options with FGFR inhibitors.

Lipidomics of Cell Secretome Combined with the Study of Selected Bioactive Lipids in an In Vitro Model of Osteoarthritis.

Analytical advancements in lipidomics have enabled large-scale investigations of lipid biology. Herein, we focused on four bioactive lipid families, namely polyunsaturated fatty acids, eicosanoids, endocannabinoids, and N-acylethanolamines, and their involvement in the mesenchymal stem cells (MSC)-related inflammatory scenario. Since MSC secretome may represent a valid therapeutic alternative, here, the complete secretome and its vesicular component from adipose- and bone marrow-derived MSC and dermal fibroblasts were characterized by targeted mass spectrometry lipidomics. The 2-arachidonoylglycerol (2AG) and the palmitoylethanolamide (PEA), previously quantified in the MSC's secretome, were further investigated by assessing hypothetical effects in an in vitro model of osteoarthritis (OA) based on human primary articular chondrocytes (CH) stimulated with tumor necrosis factor alpha (TNFα). TNFα enhances the release of the inflammatory lipid prostaglandin E2 (PGE2), and an additional increment was observed when CH were treated with both TNFα and 2AG. In contrast, PEA downmodulates the PGE2 release to the levels of unstimulated CH suggesting a protective effect. TNFα also increases the expression of cyclooxygenase 2 (COX2), in particular when combined with 2AG, while PEA partly blunts TNFα-induced COX2 expression. In addition, TNFα-stimulated CH produce significantly higher levels of the inflammatory mediator nitric oxide (NO) both in the presence and in the absence of 2AG, and PEA was able to partially reduce NO release. Our results show a first partial lipidomic profile of MSC and DF secretome and suggest a possible implication of bioactive lipids in the OA scenario and in the future use of these cell-free products as innovative therapeutics.

Polythiophene-mediated light modulation of membrane potential and calcium signalling in human adipose-derived stem/stromal cells.

Recent progress in the fields of regenerative medicine and tissue engineering has been strongly fostered both by the investigation of crucial cues, able to trigger the regeneration of damaged tissues, and by the development of ad hoc functional materials, capable of selectively (re-)activating relevant physiological pathways. In parallel to the successful realization of biochemical cues and the optimization of delivery protocols, the use of biophysical stimuli has been emerging as an alternative, highly effective strategy. Techniques based on electrical, magnetic and mechanical stimulation have been reported to efficiently direct differentiation of stem cells and modulate cell physiology at different developmental stages. In this framework, the use of optical stimulation represents a valuable approach, possibly overcoming current limitations of chemical cues, like limited spatial and temporal resolution and poor control over the extracellular environment. Surprisingly, the effects of light on the physiological properties (light toxicity, cell membrane potential, and cell ionic trafficking) of undifferentiated cells, as well as on their differentiation pathways, were investigated to a very limited extent and rarely quantified in a systematic way. In this work, we aim at clarifying the effects of optical excitation on the physiological behaviour of undifferentiated human adipose-derived stem cells (hASC), cultured on top of a light-sensitive conjugated polymer, region-regular poly-3-hexyl-thiophene (P3HT). Interestingly, we observe statistically significant modulation of the cell membrane potential, as well as noticeable effects on intracellular calcium signalling, triggered by P3HT excitation upon green light stimuli. Possible mechanisms involved in the signal transduction pathways are considered and critically discussed. The capability to modulate the physiological response of hASC upon photoexcitation, in a highly controlled and selective manner, provides a promptly available and non invasive diagnostic tool, thus contributing to the understanding of the complex machinery behind stem cells and material interfaces. Moreover, it may open the route to novel techniques to drive the differentiation path with unprecedented versatility and operational easiness.

Human Osteochondral Explants as an Ex Vivo Model of Osteoarthritis for the Assessment of a Novel Class of Orthobiologics.

Osteoarthritis (OA) is a highly prevalent joint disease still lacking effective treatments. Its multifactorial etiology hampers the development of relevant preclinical models to evaluate innovative therapeutic solutions. In the last decade, the potential of Mesenchymal Stem Cell (MSC) secretome, or conditioned medium (CM), has emerged as an alternative to cell therapy. Here, we investigated the effects of the CM from adipose MSCs (ASCs), accounting for both soluble factors and extracellular vesicles, on human osteochondral explants. Biopsies, isolated from total knee replacement surgery, were cultured without additional treatment or with the CM from 106 ASCs, both in the absence and in the presence of 10 ng/mL TNFα. Tissue viability and several OA-related hallmarks were monitored at 1, 3 and 6 days. Specimen viability was maintained over culture. After 3 days, TNFα induced the enhancement of matrix metalloproteinase activity and glycosaminoglycan release, both efficiently counteracted by CM. The screening of inflammatory lipids, proteases and cytokines outlined interesting modulations, driving the attention to new players in the OA process. Here, we confirmed the promising beneficial action of ASC secretome in the OA context and profiled several bioactive factors involved in its progression, in the perspective of accelerating an answer to its unmet clinical needs.

Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts.

Conditioned medium (CM) and extracellular vesicles (EV) from Adipose-derived Stem/stromal cells (ASC) and Dermal fibroblasts (DF) represent promising tools for therapeutic applications. Which one should be preferred is still under debate and no direct comparison of their proteome has been reported yet. Here, we apply quantitative proteomics to explore the protein composition of CM and EV from the two cell types. Data are available via ProteomeXchange (identifier PXD020219). We identified 1977 proteins by LC-MS/MS proteomic analysis. Unsupervised clustering analysis and PCA recognized CM and EV as separate groups. We identified 68 and 201 CM and EV specific factors. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation factors. The analysis of ASC and DF secretomes revealed the presence of cell type-specific proteins. ASC-CM and -EV carried factors involved in ECM organization and immunological regulation, respectively. Conversely, DF-CM and -EV were enriched in epithelium development associated factors and -EV in Wnt signaling factors. In conclusion, this analysis provides evidence of a different protein composition between CM and EV and of the presence of cell type-specific bioactive mediators suggesting their specific future use as advanced therapy medicinal products. SIGNIFICANCE: The use of cell secretome presents several advantages over cell therapy such as the lower risks associated to the administration step and the avoidance of any potential risk of malignant transformation. The main secretome preparations consist in concentrated conditioned medium (CM) and extracellular vesicles (EV). Both of them showed well-documented therapeutic potentials. However, it is still not clear in which case it should be better to use one preparation over the other and an exhaustive comparison between their proteome has not been performed yet. The choice of the cell source is another relevant aspect that still needs to be addressed. In order to shed light on these questions we explored the protein composition of CM and EV obtained from Adipose-derived Stem/stromal Cells (ASC) and Dermal Fibroblasts (DF), by a comprehensive quantitative proteomics approach. The analysis showed a clear distinction between CM and EV proteome. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation-related factors. Furthermore, the analysis of ASC and DF secretomes revealed specific biological processes for the different cell products. ASC secretome presented factors involved in ECM organization (hyaluronan and glycosaminoglycan metabolism) and immunological regulation (e.g. macrophage and IkB/NFkB signaling regulation), respectively. On the other hand, DF-CM and -EV were both enriched in epithelium development associated factors, whilst DF-CM in proteins involved in cellular processes regulation and -EV in Wnt signaling factors. In conclusion, our study shed a light on the different protein composition of CM and EV of two promising cell types, spanning from basic processes involved in secretion to specific pathways supporting their therapeutic potential and their possible future use as advanced therapy medicinal products.

Intracranial meningioma in two coeval adult cats from the same litter.

CASE SUMMARY: In this report we describe the occurrence of intracranial meningioma in two adult cats from the same litter. The location of the meningioma varied: one tumour was at the level of the brainstem, and the other was affecting the temporal and piriform lobes. The cat with the brainstem meningioma was treated with radiotherapy and the littermate had a rostrotentorial craniectomy for tumour removal. Both cats had a histopathological diagnosis of grade I meningioma of a predominantly fibrous subtype. RELEVANCE AND NOVEL INFORMATION: Cases of familial meningioma in cats have not previously been described in the veterinary literature. However, familial meningioma is well described in humans and it is possible that cases are underestimated in animals. We discuss the possible genetic background and other causes, as well as challenges we may face in veterinary medicine in identifying these associations.

Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics.

Extracellular Vesicles (EVs) and Conditioned Medium (CM) are promising cell-free approaches to repair damaged and diseased tissues for regenerative rehabilitation purposes. They both entail several advantages, mostly in terms of safety and handling, compared to the cell-based treatment. Despite the growing interest in both EVs and CM preparations, in the light of a clinical translation, a number of aspects still need to be addressed mainly because of limits in the reproducibility and reliability of the proposed protocols. Raman spectroscopy (RS) is a non-destructive vibrational investigation method that provides detailed information about the biochemical composition of a sample, with reported ability in bulk characterization of clusters of EVs from different cell types. In the present brief report, we acquired and compared the Raman spectra of the two most promising cell-free therapeutics, i.e., EVs and CM, derived from two cytotypes with a history in the field of regenerative medicine, adipose-derived mesenchymal stem/stromal cells (ASCs) and dermal fibroblasts (DFs). Our results show how RS can verify the reproducibility not only of EV isolation, but also of the whole CM, thus accounting for both the soluble and the vesicular components of cell secretion. RS can provide hints for the identification of the soluble factors that synergistically cooperate with EVs in the regenerative effect of CM. Still, we believe that the application of RS in the pipeline of cell-free products preparation for therapeutic purposes could help in accelerating translation to clinics and regulatory approval.

Towards Secretome Standardization: Identifying Key Ingredients of MSC-Derived Therapeutic Cocktail.

The therapeutic potential of the conditioned medium (CM) derived from MSCs (mesenchymal stem/stromal cells) in disparate medical fields, from immunology to orthopedics, has been widely suggested by in vitro and in vivo evidences. Prior to MSC-CM use in clinical applications, appropriate quality controls are needed in order to assess its reproducibility. Here, we evaluated different CM characteristics, including general features and precise protein and lipid concentrations, in 3 representative samples from adipose-derived MSCs (ASCs). In details, we first investigated the size and distribution of the contained extracellular vesicles (EVs), lipid bilayer-delimited particles whose pivotal role in intercellular communication has been extensively demonstrated. Then, we acquired Raman signatures, providing an overlook of ASC-CM composition in terms of proteins, lipids, and nucleic acids. At last, we analyzed a panel of 200 molecules including chemokines, cytokines, receptors, and inflammatory and growth factors and searched for 32 lipids involved in cell signalling and inflammation. All ASC-CM contained a homogeneous and relevant number of EVs (1.0 × 109 ± 1.1 × 108 particles per million donor ASCs) with a mean size of 190 ± 5.2 nm, suggesting the appropriateness of the method for EV retaining and concentration. Furthermore, also Raman spectra confirmed a high homogeneity among samples, allowing the visualization of specific peaks for nucleic acids, proteins, and lipids. An in depth investigation that focused on 200 proteins involved in relevant biological pathways revealed the presence in all specimens of 104 factors. Of these, 26 analytes presented a high degree of uniformity, suggesting that the samples were particularly homogenous for a quarter of the quantified molecules. At last, lipidomic analysis allowed the quantification of 7 lipids and indicated prostaglandin-E2 and N-stearoylethanolamide as the most homogenous factors. In this study, we assessed that ASC-CM samples obtained with a standardized protocol present stable features spanning from Raman fingerprint to specific marker concentrations. In conclusion, we identified key ingredients that may be involved in ASC-CM therapeutic action and whose consistent levels may represent a promising quality control in the pipeline of its preparation for clinical applications.

Endogenous 1-H-Pyrrole-2,3,5-Tricarboxylic Acid (PTCA) in Hair and Its Forensic Applications: A Pilot Study on a Wide Multi-Ethnic Population.

Over the years, several studies have shown that many factors are likely to affect the results of forensic hair analyses and complicate their interpretation. Among these factors, one of the major drawbacks in hair analysis is the affectability of deposited xenobiotics by cosmetic treatments, which could be eventually used to adulterate the sample. It is well known that some cosmetic treatments containing hydrogen peroxide, such as permanent dyeing or bleaching, lead to the formation of 1-H-pyrrole-2,3,5-tricarboxylic acid (PTCA), a melanin degradation product. Considering that PTCA is also an endogenous compound, spontaneously formed by natural oxidation of melanin, its only detection in hair is not enough to confirm a cosmetic oxidative treatment. For this reason, the aim of the present work was to develop and validate a reliable liquid-liquid extraction method in ultra-high-performance liquid chromatographic-tandem mass spectrometry for the determination of endogenous PTCA in hair from a wide multi-ethnic population (African, Arab, Asian-Pacific, Caucasian, Hispanic and Indian). According to previous studies, untreated hair samples showed a PTCA content of 8.54 ± 5.72 ng/mg (mean ± standard deviation [SD]), ranging between 0.44 and 23.7 ng/mg; after in vitro cosmetic bleaching, PTCA increased to 16.8 ± 6.95 ng/mg (range: 4.16-32.3 ng/mg). Comparing baseline PTCA levels of each subgroup with the others, we could not observe any statistically significant difference, except for Caucasians (P < 0.05), wherein the concentrations were lower. Further studies and a wider sampling are necessary to elucidate the role of PTCA as diagnostic marker of cosmetic hair treatment in forensic field.