Synovial Membrane Is a Major Producer of Extracellular Inorganic Pyrophosphate in Response to Hypoxia.

Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-ß1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-ß1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-ß1 in normoxic or hypoxic (5% O2) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-ß1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-ß1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-ß1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.

Is Vesicular Therapy the Newcomer That Matters for the Medicine of Tomorrow?

Extracellular vesicles (EVs) are membrane-enclosed particles released by cells into their extracellular environment [...].

Neuroprotective Effect of Curcumin-Loaded RGD Peptide-PEGylated Nanoliposomes.

Curcumin is known for its anti-inflammatory, neuroprotective, and antioxidant properties, but its use in biological applications is hindered by its sensitivity to light, oxygen, and temperature. Furthermore, due to its low water solubility, curcumin has a poor pharmacokinetic profile and bioavailability. In this study, we evaluated the potential application of curcumin as a neuroprotective agent encapsulated in RGD peptide-PEGylated nanoliposomes developed from salmon-derived lecithin. Salmon lecithin, rich in polyunsaturated fatty acids, was used to formulate empty or curcumin-loaded nanoliposomes. Transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis characterizations indicated that the marine-derived peptide-PEGylated nanoliposomes were spherical in shape, nanometric in size, and with an overall negative charge. Cytotoxicity tests of curcumin-loaded nanoliposomes revealed an improved tolerance of neurons to curcumin as compared to free curcumin. Wild-type SH-SY5Y were treated for 24 h with curcumin-loaded nanoliposomes, followed by 24 h incubation with conditioned media of SH-SY5Y expressing the Swedish mutation of APP containing a high ratio of Aß40/42 peptides. Our results revealed significantly lower Aß-induced cell toxicity in cells pre-treated with RGD peptide-PEGylated curcumin-loaded nanoliposomes, as compared to controls. Thus, our data highlight the potential use of salmon lecithin-derived RGD peptide PEGylated nanoliposomes for the efficient drug delivery of curcumin as a neuroprotective agent.

Pro-Inflammatory Cytokine Priming and Purification Method Modulate the Impact of Exosomes Derived from Equine Bone Marrow Mesenchymal Stromal Cells on Equine Articular Chondrocytes.

Osteoarthritis (OA) is a widespread osteoarticular pathology characterized by progressive hyaline cartilage degradation, exposing horses to impaired well-being, premature career termination, alongside substantial financial losses for horse owners. Among the new therapeutic strategies for OA, using mesenchymal stromal cell (MSC)-derived exosomes (MSC-exos) appears to be a promising option for conveying MSC therapeutic potential, yet avoiding the limitations inherent to cell therapy. Here, we first purified and characterized exosomes from MSCs by membrane affinity capture (MAC) and size-exclusion chromatography (SEC). We showed that intact MSC-exos are indeed internalized by equine articular chondrocytes (eACs), and then evaluated their functionality on cartilaginous organoids. Compared to SEC, mRNA and protein expression profiles revealed that MAC-exos induced a greater improvement of eAC-neosynthesized hyaline-like matrix by modulating collagen levels, increasing PCNA, and decreasing Htra1 synthesis. However, because the MAC elution buffer induced unexpected effects on eACs, an ultrafiltration step was included to the isolation protocol. Finally, exosomes from MSCs primed with equine pro-inflammatory cytokines (IL-1ß, TNF-α, or IFN-γ) further improved the eAC hyaline-like phenotype, particularly IL-1ß and TNF-α. Altogether, these findings indicate the importance of the exosome purification method and further demonstrate the potential of pro-inflammatory priming in the enhancement of the therapeutic value of MSC-exos for equine OA treatment.

Functionalized liposomes for targeted breast cancer drug delivery.

Despite the exceptional progress in breast cancer pathogenesis, prognosis, diagnosis, and treatment strategies, it remains a prominent cause of female mortality worldwide. Additionally, although chemotherapies are effective, they are associated with critical limitations, most notably their lack of specificity resulting in systemic toxicity and the eventual development of multi-drug resistance (MDR) cancer cells. Liposomes have proven to be an invaluable drug delivery system but of the multitudes of liposomal systems developed every year only a few have been approved for clinical use, none of which employ active targeting. In this review, we summarize the most recent strategies in development for actively targeted liposomal drug delivery systems for surface, transmembrane and internal cell receptors, enzymes, direct cell targeting and dual-targeting of breast cancer and breast cancer-associated cells, e.g., cancer stem cells, cells associated with the tumor microenvironment, etc.

Hop Extract Anti-Inflammatory Effect on Human Chondrocytes Is Potentiated When Encapsulated in Rapeseed Lecithin Nanoliposomes.

Hop (Humulus lupulus L.) is a plant used as an ingredient in beer or employed for its anti-inflammatory properties. The cultivation of hops is currently dedicated to the brewing industry, where mainly female flowers are used, whereas aerial parts, such as leaves, are considered coproducts. Osteoarthritis is the most common musculoskeletal disease associated with low-grade cartilage inflammation. Liposomes have been shown to be promising systems for drug delivery to cartilage cells, called chondrocytes. The aim of our work was to vectorize hop extract valorized from coproducts as a therapeutic agent to alleviate inflammation in human chondrocytes in vitro. Liquid chromatography allowed the identification of oxidized bitter acids in a methanolic extract obtained from the leaves of Cascade hops. The extract was encapsulated in rapeseed lecithin nanoliposomes, and the physicochemical properties of empty or loaded nanoliposomes exhibited no difference. Increasing concentrations of the hop extract alone, empty nanoliposomes, and loaded nanoliposomes were tested on human chondrocytes to assess biocompatibility. The appropriate conditions were applied to chondrocytes stimulated with interleukin-1ß to evaluate their effect on inflammation. The results reveal that encapsulation potentiates the hop extract anti-inflammatory effect and that it might be able to improve joint inflammation in osteoarthritis. Furthermore, these results also show that a "zero waste" chain is something that can be achieved in hop cultivation.

Bone Marrow MSC Secretome Increases Equine Articular Chondrocyte Collagen Accumulation and Their Migratory Capacities.

Equine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to the cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternatives to overcome those limitations while preserving MSC restorative properties. The effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) was analyzed with indirect co-culture and/or MSC-conditioned media (CM). The expression of healthy cartilage/OA and proliferation markers was evaluated in eACs (monolayers or organoids). In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. The presence of nanosized EVs in MSC-CM was appraised with nanoparticle tracking assay and transmission electron microscopy. Our results demonstrated that the MSC secretome influences eAC phenotype by increasing cartilage functionality markers and cell migration in a greater way than MSCs, which could delay OA final outcomes. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, the MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects.

Encapsulation of Salmon Peptides in Marine Liposomes: Physico-Chemical Properties, Antiradical Activities and Biocompatibility Assays.

Salmon byproducts (Salmo salar) generated by the food chain represent a source of long-chain polyunsaturated fatty acids (eicosapentaenoic acid (EPA): 20:5n-3; docosahexaenoic acid (DHA): 22:6n-3) and peptides that can be used as supplements in food for nutraceutical or health applications, such as in the prevention of certain pathologies (e.g., Alzheimer's and cardiovascular diseases). The extraction of polar lipids naturally rich in PUFAs by enzymatic processes without organic solvent (controlled by pH-Stat method), coupled with the production of 1 kDa salmon peptides by membrane filtration, allowed the formulation of nanocarriers. The physicochemical properties of the nanoliposomes (size ranging from 120 to 140 nm, PDI of 0.27, zeta potential between -32 and -46 mV and encapsulation efficiency) were measured, and the bioactivity of salmon hydrolysate peptides was assessed (antioxidant and antiradical activity: ABTS, ORAC, DPPH; iron metal chelation). Salmon peptides exhibited good angiotensin-conversion-enzyme (ACE) inhibition activity, with an IC50 value of 413.43 ± 13.12 µg/mL. Cytotoxicity, metabolic activity and proliferation experiments demonstrated the harmlessness of the nanostructures in these experimental conditions.

Efficient TGF-ß1 Delivery to Articular Chondrocytes In Vitro Using Agro-Based Liposomes.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-ß1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-ß1 to rat chondrocytes. Results showed that TGF-ß1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-ß1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Is Extracellular Vesicle-Based Therapy the Next Answer for Cartilage Regeneration?

"Extracellular vesicles" (EVs) is a term gathering biological particles released from cells that act as messengers for cell-to-cell communication. Like cells, EVs have a membrane with a lipid bilayer, but unlike these latter, they have no nucleus and consequently cannot replicate. Several EV subtypes (e.g., exosomes, microvesicles) are described in the literature. However, the remaining lack of consensus on their specific markers prevents sometimes the full knowledge of their biogenesis pathway, causing the authors to focus on their biological effects and not their origins. EV signals depend on their cargo, which can be naturally sourced or altered (e.g., cell engineering). The ability for regeneration of adult articular cartilage is limited because this avascular tissue is partly made of chondrocytes with a poor proliferation rate and migration capacity. Mesenchymal stem cells (MSCs) had been extensively used in numerous in vitro and preclinical animal models for cartilage regeneration, and it has been demonstrated that their therapeutic effects are due to paracrine mechanisms involving EVs. Hence, using MSC-derived EVs as cell-free therapy tools has become a new therapeutic approach to improve regenerative medicine. EV-based therapy seems to show similar cartilage regenerative potential compared with stem cell transplantation without the associated hindrances (e.g., chromosomal aberrations, immunogenicity). The aim of this short review is to take stock of occurring EV-based treatments for cartilage regeneration according to their healing effects. The article focuses on cartilage regeneration through various sources used to isolate EVs (mature or stem cells among others) and beneficial effects depending on cargos produced from natural or tuned EVs.

Physicochemical Properties and Liposomal Formulations of Hydrolysate Fractions of Four Sea Cucumbers (Holothuroidea: Echinodermata) from the Northwestern Algerian Coast.

To promote the nutritional and pharmacological values of four sea cucumber species (Holothuria poli, H. tubulosa, H. arguinensis, and H. sanctori), harvested from the Algerian coast, we aimed to study their proximate composition, fatty acid profile and angiotensin-converting enzyme (ACE) inhibitory activity. Their phospholipids were also used to elaborate nanoliposomes and to encapsulate peptides obtained from the same source. After the physico-chemical characterization of nanoliposomes and peptides, in vitro analyses were realized. The four holothurian species showed a high amount of protein (49.26-69.34%), and an impressive lipid profile of 27 fatty acids, mainly composed of polar fatty acids (91.16-93.85%), with a high polyunsaturated fatty acids (PUFA) content (50.90-71.80%), particularly eicosapentaenoic acid (EPA) (5.07-8.76%) and docosahexaenoic acid (DHA) (4.86-7.25%). A high phospholipids amount was also found (55.20-69.85%), mainly composed of phosphatidylcholine (PC) (51.48-58.56%). Their peptide fractions exhibited a high ACE inhibitory activity (IC50 0.30 to 0.51 mg/mL). The results also showed that the nanoliposomes do not induce cytotoxicity and cell death in human MSCs and no perturbation of proliferation for all the times and the tested concentrations, as well as the combined nanoliposomes and hydrolysates (HTS) at a concentration of 0.1 mg/mL. All four sea cucumbers show potential as a new source for omega-3, omega-6, and bioactive peptides.

Nanoliposomes from Agro-Resources as Promising Delivery Systems for Chondrocytes.

Investigations in cartilage biology have been hampered by the limited capacity of chondrocytes, especially in rats and humans, to be efficiently transfected. Liposomes are a promising delivery system due to their lipid bilayer structure similar to a biological membrane. Here we used natural rapeseed lecithin, which contains a high level of mono- and poly-unsaturated fatty acids, to evaluate the cytocompatibility of these phospholipids as future potential carriers of biomolecules in joint regenerative medicine. Results show that appropriate concentrations of nanoliposome rapeseed lecithin under 500 µg/mL were safe for chondrocytes and did not induce any alterations of their phenotype. Altogether, these results sustain that they could represent a novel natural carrier to deliver active substances into cartilage cells.

Diversity and heterogeneity of extracellular RNA in human plasma.

Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. In humans, exRNA populations are found in nearly any physiological liquid and are attracting growing interest as a potential source for biomarker discovery. Human plasma, a readily available sample for biomedical analysis, reported to contain various subpopulations of exRNA, some of which are most likely components of plasma ribonucleoproteins (RNPs), while others are encapsulated into extracellular vesicles (EVs) of different size, origin and composition. This variation explains the extreme complexity of the human exRNA fraction in plasma. In this work, we aimed to characterize exRNA species from blood samples of healthy human donors to achieve the most comprehensive overview of the species, sizes and origins of the exRNA present in plasma fractions. Unbiased analysis of exRNA composition was performed with prefractionation of plasma exRNA followed by library preparation, sequencing and bioinformatics analysis. Our results demonstrate that, in addition to "mature", adaptor ligation-competent RNA species (5'-P/3'-OH), human plasma contains a substantial proportion of degraded RNA fragments (5'-OH/3'-P or cycloP), which can be made competent for ligation using appropriate treatments. These degraded RNAs represent the major fraction in the overall population and mostly correspond to rRNA, in contrast to mature products, which mostly contain miRNAs and hY4 RNA fragments. Precipitation polyethylene glycol (PEG)-based kits for EV isolation yield a fraction that is highly contaminated by large RNPs and by RNA loosely bound to EVs. Purer EV preparations are obtained by using proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC). These samples have rather distinct RNA compositions compared to PEG-precipitated EV preparations and contain a substantial proportion of exRNA of non-human origin, arising from human skin and gut microbiota, including viral microbiota. These exogenous exRNAs represent up to 75-80% of total RNA reads in highly purified extracellular vesicles, paving the way for biomedical exploitation of these non-human biomarkers.

The effect of nacre extract on cord blood-derived endothelial progenitor cells: A natural stimulus to promote angiogenesis?

Angiogenesis is a critical parameter to consider for the development of tissue-engineered bone substitutes. The challenge is to promote sufficient vascularization in the bone substitute to prevent cell death and to allow its efficient integration. The capacity of nacre extract to restore the osteogenic activity of osteoarthritis osteoblasts has already been demonstrated. However, their angiogenic potential on endothelial progenitor cells (EPCs) was not yet explored. Therefore, the current study aimed at investigating if nacreous molecules affect EPC behavior. The gene and protein expression levels of endothelial cell (EC)-specific markers were determined in EPCs cultivated in presence of a nacre extract (ethanol soluble matrix [ESM] at two concentrations: 100 µg/mL and 200 µg/mL (respectively abbreviated ESM100 and ESM200)). Cell functionality was explored by proangiogenic factors production and in vitro tube formation assay. ESM200 increased the expression of some EC-specific genes. The in vitro tube formation assay demonstrated that ESM200 stimulated tubulogenesis affecting angiogenic parameters. We demonstrated that a stimulation with 200 µg/mL of ESM increased angiogenesis key elements. This in vitro study strongly highlights the proangiogenic effect of ESM. Due to its osteogenic properties, previously demonstrated, ESM could constitute the key element to develop an ideal prevascularized bone substitute. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Dental Pulp Stem Cell-Derived Conditioned Medium: An Attractive Alternative for Regenerative Therapy.

Mesenchymal stem cells (MSC) have a lot of potential in regenerative medicine, and MSC-based therapies are currently explored in numerous research fields. Among these cells, deciduous or permanent dental pulp-MSC represent a promising option in tissue engineering. This expectation is based on their capacity to self-renew, to repair various damaged tissues and organs due to their multipotency, as well as their ability to modulate immune system. They present other advantages such as the harvesting by a simple, painless, and noninvasive procedure and the absence of ethical considerations. The role played by these cells in the reparative process is mainly attributed to paracrine mechanisms mediated by their secreted factors, namely the secretome. The secreted factors can be found in the cell culture medium, called conditioned medium (CM). Moreover, CM presents many advantages compared with cells such as possible use in allogeneic therapies. This minireview aims at investigating the therapeutic use of dental pulp MSC-derived CM to develop cell-free therapies. The analysis of the available literature illustrates its massive panel of potential applications: mainly reduction of inflammation, promotion of angiogenesis and neurogenesis, reduction of stroke or ischemia, and organ regeneration. Furthermore, studies often highlight its superiority over the other sources of CM derived from other stem cells for the same applications. Dental pulp MSC-derived CM is an attractive, noninvasive, and acellular tool for therapeutic approaches in regenerative medicine. This promising novel approach should be further explored for clinical applications.

Biomaterials and Gene Therapy: A Smart Combination for MSC Musculoskeletal Engineering.

Musculoskeletal pathologies, especially those affecting bones and joints, remain a challenge for regenerative medicine. The main difficulties affecting bone tissue engineering are the size of the defects, the need for blood vessels and the synthesis of appropriate matrix elements in the engineered tissue. Indeed, the cartilage is an avascular tissue and consequently has limited regenerative abilities. Thanks to their self-renewal, plasticity and immunomodulatory properties, mesenchymal stem cells (MSCs) became a central player in tissue engineering, and have already been shown to be able to differentiate towards chondrogenic or osteogenic phenotypes. Whether synthetic (e.g. tricalcium phosphate) or from natural sources (e.g. hyaluronic acid), biomaterials can be shaped to fit into bone and cartilage defects to ensure mechanical resistance and may also be designed to control cell spatial distribution or differentiation. Soluble factors are classically used to promote cell differentiation and to stimulate extracellular matrix synthesis to achieve the desired tissue production. But as they have a limited lifetime, transfection using plasmid DNA or transduction via a viral vector of therapeutic genes to induce the cell secretion of these factors allows to have more lasting effects. Also, the chondrocyte phenotype may be difficult to control over time, with for example the production of hypertrophic or osteogenic markers that is undesirable in hyaline cartilage. Thus, tissue regeneration strategies became more elaborate, with an attempt at associating the benefits of MSCs, biomaterials, and gene therapy to achieve a proper tissue repair. This minireview focuses on in vitro and in vivo studies combining biomaterials and gene therapy associated with MSCs for bone and cartilage engineering.

Human umbilical cord derived matrix: A scaffold suitable for tissue engineering application.

BACKGROUND: Human tissue derived natural extracellular matrix (ECM) has great potential in tissue engineering. OBJECTIVE: We sought to isolate extracellular matrix derived from human umbilical cord and test its potential in tissue engineering. METHODS: An enzymatic method was applied to isolate and solubilized complete human umbilical cord derived matrix (hUCM). The obtained solution was analyzed for growth factors, collagen and residual DNA contents, then used to coat 2D and 3D surfaces for cell culture application. RESULTS: The hUCM was successfully isolated with trypsin digestion to acquire a solution containing various growth factors and collagen but no residual DNA. This hUCM solution can form a coating on 2D and 3D substrates suitable cell culture. CONCLUSION: We developed a new matrix derived from human source that can be further used in tissue engineering.