Extracellular Nanovesicles of Mesenchymal Stem Cells Induced to Glycolytic Metabolism for the Treatment of Osteoarthritis
Patent Information
- Application Number
- AE202603118
- Authority / Receiving Office
- AE · AE
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-03-18
- Filing Date
- 2025-03-18
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Abstract
Description
Extracellular Nanovesicles of Mesenchymal Stem Cells Induced to Glycolytic Metabolism for the Treatment of Osteoarthritis DescriptionBackgroundThe WHO considers osteoarthritis (OA) to be a disease that affects the quality of life of the population and that by 2050, more than 579 million people in the world will suffer from it. It is the fourth disease that takes away the highest quality of life from patients for each year lived.According to global statistics, more than 50% of adults over the age of 60 suffer from OA. There are currently no effective FDA / EMA-approved treatments available for patients with OA that can halt or reverse the progression of the disease by being more than pain-palliatives. From a therapeutic point of view, there are no treatments that modify, slow or restore the function of the articular cartilage, so the current strategy is based on palliative medications of low effectiveness that seek to control pain, inflammation and stiffness. According to the guidelines provided by the Ministry of Health, through its "2009 Clinical Guide Medical Treatment in People 55 years of age and older with Mild or Moderate Hip and / or Knee Osteoarthritis", the management of OA focuses on a multimodal treatment that includes patient education and self-management, non-pharmacological management and pharmacological treatments consisting of the administration of simple analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, analgesic opioids and intra-articular drugs (IA). The last-line therapeutic resource continues to be prosthetic surgery for the replacement of the native joint with an artificial prosthesis. This therapeutic problem represents an opportunity for the introduction of new therapies that pursue a structural purpose with an emphasis on the regenerative or chondroprotective and not only the symptomatic. In this regard, the use of gene therapy has been proposed as an alternative solution, however, viral strategies entail high immunogenicity, high production costs and problems associated with genetic alterations in other cell targets, while non-viral strategies have low transfection efficiencies and poor cell specificity. In this regard, the delivery of ex vivo genes offers more advantages, such as TissueGene-C (TG-C), which consists of the use of human allogeneic chondrocytes and irradiated GP2-293 cells (derived from HEK-293) that overexpress TGF-β1. This procedure has been shown to be effective and safe in phase 1 and 2 clinical studies, approved for the treatment of patients with moderate knee osteoarthritis in South Korea, and recently in phase 3 in the U.S. However, despite the remarkable results of these clinical trials, the molecular mechanisms underlying the effect of TG-C, as well as the potential side effects associated with long-term cell line injection, remain unknown.On the other hand, mesenchymal stem cells (MSCs) have been the focus of attention of physicians and scientists for more than a decade, as a potential biological therapy for the treatment of OA due to their chondrogenic potential, their immunosuppressive and anti-inflammatory effect, and their safety in autologous and allogeneic administration. However, although preclinical data has shown that intra-articular (IA) treatment with MSCs can prevent, or even reverse, cartilage degradation in animal models of OA, clinical results in patients have been inconclusive and market approvals have been limited. Recently, our group has successfully conducted a Phase I / II Randomized Controlled Clinical Trial (NCT02580695) of the IA administration of allogeneic umbilical cord-derived MSCs (UC-MSCs) in patients with OA, reporting a long-standing symptomatic benefit at 12 months of follow-up, a finding also replicated in recent studies. Currently, the treatment for OA is being addressed through research that develops and studies potential compounds capable of operating as DMOAD (disease-modifying osteoarthritis drugs). In general terms, these experimental compounds act on one of the 3 axes of the pathogenesis of the disease: Regeneration of articular cartilage, remodeling of subchondral bone or management of synovial inflammation, thus limiting its therapeutic potential. In contrast, MSC therapy has been shown to act on at least 2 axes – cartilage regeneration and inflammation – which gives an advantage over other therapies. Despite the above, the most recent systematic reviews of clinical trials with MSCs conclude that, although the therapy is beneficial in some patients with OA, the regenerative effectiveness of MSCs as a treatment method is still uncertain and market authorizations have been limited. From the above, the researchers deduce the idea of enhancing MSCs in their regenerative properties in order to provide an effective treatment for OA. This hypothesis of "cell potentiation" has been worked on in recent years in their laboratory, using oligomycin as an inhibitor of oxidative phosphorylation to increase glycolysis and thus enhance its regenerative and anti-inflammatory effects. The findings that the stimulation of glycolytic metabolism in UC-MSCs -labelled by the inventors as GlycoStem- increase their in vitro anti-inflammatory and regenerative potential in an animal model of OA have been robustly demonstrated, evidence that has generated both intellectual property (PCT / IB2020 / 061190), and scientific articles that are already under review in different international journals. Since it is impossible to consider the development of a new medicinal product without a precise understanding of its mechanism of action (MoA), we have established that the invention exerts at least part of its biological effect through its small extracellular vesicles (sEV), mainly exosomes, which constitutes this invention. As previously described by the inventors and other research groups, sEV are nanovesicles secreted by almost all cells that act as important mediators of intercellular communication capable of generating phenotypic changes in the acceptor cell, thus influencing different physiological and pathological conditions. It is now known that sEV can enhance tissue regeneration, participate in immune modulation, and function as potential alternatives to stem cell therapy. The inventors have shown that the induction of glycolytic metabolism in UC-MSC using oligomycin and its analogues promotes its regenerative (PCT / IB2020 / 061190), immunosuppressive and anti-inflammatory53 potential in vitro and in vivo. Recently, the inventors have established that the mechanism of action by which the cell thus treated is exerting its biological effects in vivo, would be mediated by its secreted sEV. This is not at all obvious or expected, since sEV constitute less than 10% of the exchange products with the medium generated by cells, so if a cell has an effect, it cannot necessarily be established that the same effect is obtained by using its exosomes (sEV).The inventors' results have shown that an sEV-based product can be developed for a new class of biological therapy, such as an acellular biodrug, capable of generating a transformative impact on the way OA is treated. The previous results of the inventors show that the use of a pre-treatment with oligomycin in MSCs, short or long term, does not have toxic effects on cells, does not alter the immunophenotype of MSCs and also maintains glycolytic activity for at least 3 days. This last piece of information is very important from the point of view of the manufacture of the sEV-based product, since the vesicles are isolated from the cellular supernatant after 48 hours. Likewise, the pre-incubation of MSCs with oligomycin does not alter the rate of direct secretion of sEV into the cell culture medium, also preserving their size distribution. Moreover, after the isolation of the -sEVs from the MSC cells treated with oligomycin, it was corroborated that, in terms of concentration and identity, there are no great variations with respect to the sEVs derived from the native MSCs, and importantly, in terms of size as described by the ISEV (International Society of Extracellular Vesicles), it was observed that there is an enrichment of the exosomal fraction (> 200nm) contained in the total fraction of sEV. Likewise, we have observed that soluble factors of MSC cells treated with oligomycin as in the present invention, promote the expression of hyaline cartilage markers and the survival of chondrocytes from patients with OA. These results allowed us to propose as a bioactive mechanism -sEV of the invention, which shows a very similar profile to UC-MSC-sEV in terms of secretion rate, size distribution, as well as size and post-isolation characterization, using a standard protocol validated by our group. In addition, our data shows that UC-MSC-sEV have a standard morphology by electron microscopy and are capable of internalizing in target cells such as chondrocytes from patients with OA. An interesting fact that impacts the production of -sEV according to the invention, is that there are no great differences in performance, size and characterization between fresh and frozen cells, which are thawed directly for the purpose of obtaining sEV. On the other hand, at the functional level, in order to compare the anti-inflammatory effect of UC-MSC and its sEV derivatives, we used as an experimental approach an in vitro assay on murine macrophages activated with lipopolysaccharide (LPS), to obtain pro-inflammatory M1 macrophages. The results of the inventors show that both Glycostem and GlycoStem-sEV possess a greater anti-inflammatory capacity than UC-MSC or its derivative-sEV, in terms of promoting the generation of anti-inflammatory macrophages of the M2 type and inhibiting pro-inflammatory phenotypes of the M1 type. Interestingly, the effects exerted by -sEV according to the invention were very similar to its cellular counterpart, a result that allows us to propose to the inventors that a large part of the effect obtained by these cells is based on their sEV. In the previous art, no document has been found that associates the use of oligomycin as an enhancer of MSC cells or its extracellular derivatives for the treatment of OA. Also, as explained above, to date there are no documents that disclose the use of oligomycin as an MSC enhancer for the production of sEV or exosomes for the treatment of OA. These new results complement previous developments by the same inventors, such as those protected in the PCT application (PCT / IB2020 / 061190), concerning the use of oligomycin-pretreated MSCs for the treatment of OA. The advantages of an acellular therapy, which is a new technology protected in this invention, will be evident to the person versed in the technique, based on the determination that the treatment effect of OA can also be obtained with only the sEV of oligomycin-treated MSCs, which was not anticipated in the previous art. sEV, including exosomes, are secreted by almost all cells as part of their normal physiology or during the acquisition of abnormalities. Depending on their cell of origin, sEV may contain many of the cell's constituents such as nucleic acids, lipids, metabolites, and cytosolic and cell-surface proteins. As a result of its efficient ability to transfer the encoded charge inside it and trigger phenotypic changes in acceptor cells, substantial interest has been generated from the scientific and medical community as a new therapeutic strategy. Without a doubt, one of the main clinical advantages of sEV is that being "naturally" secreted by cells, the composition of lipids and proteins of their membrane is very similar to that of their parent cell, which makes them inherently non-immunogenic. Another advantage that sEV have over their cells of origin is that they are less likely to be trapped in the microvasculature due to their nanometric size, their practically zero risk of malignancy per se, since they are inert compounds they do not have the ability to divide, and their greater effectiveness in vivo as a result of their greater stability. Finally, from a marketing point of view, they are preservable at -80°C, or if they are freeze-dried, they can be kept at room temperature for several months, without loss of their therapeutic efficiency, which gives them an indisputable advantage as they are immediately available for clinical use (off-the-shelf therapy).Brief description of the figures. Figure 1. EVs from glycostem (EVs-Glyco) exhibit a microRNA signature that is significantly different from their sEVS-Native counterpart in lyophilized samples. Using the EdgeR tool, 178 genes were identified with a significant difference with respect to the native sEVs condition of the total, 175 were found to be increased with respect to native sEVS (Log2 fold change >0.5 and <-0.5; Adjusted p-value ≤ 0.05) and 3 decreased from the native sEVs condition (Log2 fold change >0.5 and <-0.5; Adjusted p-value ≤ 0.05). N = 4 biological samples. Figure 2. Diagram of the experimental design of the murine model of osteoarthritis. C57 BL6 mice were injected with 5μL with collagenase type VII or with saline solution (SHAM) intra-articular (IA). After 7 and 14 days post-induction, the animals were treated with 5μL of saline solution or with naïve sEVs or glyco sEVs. On the day, 42 mice were euthanized and the joints were removed for image analysis by microCT or histology. The clinical score for microCT and histology was analyzed in the different areas of the joint as shown on the left, in order to obtain the values of bone density and histological joint damage score.Figure 3. EVs according to the invention decrease bone mineralization, increasing the therapeutic potential of UC-MSC-EVs in a mouse model of collagenase-induced osteoarthritis (OA). C57 BL6 mice were injected with 5μL with collagenase type VII or with saline solution (SHAM) intra-articular (AI). After 7 and 14 days post-induction, the animals were treated with 5μL of saline solution or with UC-MSCs or UC-MSCs-oligomycin cells and their respective extracellular sEVs vesicles. Graphs represent the + / -SD mean of 3 independent experiments using at least 5 mice per experimental group. P values associated with a significant difference p < 0.05 are indicated.Figure 4. shows the degree of joint degeneration of the analyzed areas associated with OA according to histological score. All analyses were performed in double-blind. The data show that Glycostem-sEV significantly reduces the progression of OA, highlighting an increase in hyaline cartilage in the glycostem-sEV group, presenting a greater therapeutic efficacy associated with a regeneration of the damaged cartilage area in OA. Graphs represents the + / -SD mean of 3 independent experiments using at least 8 mice per experimental group. P values were calculated with respect to the OA experimental group or when indicated between groups. Red indicates p-values associated with a significant difference p < 0.05 Figure 5: Lyophilized GlycoStem-derived EVs (glyco sEVs) preserve their therapeutic potential in a mouse model of collagenase-induced osteoarthritis (OA). (A) Graph showing the bone density values in the different areas of analysis. The data show that lyophilized glycostem-sEVs present the same significant improvement to chondral mineralization as their frozen counterpart, suggesting that it preserves its therapeutic capacity. (B) Graph showing the histological score values according to the modified Pritzker OARSI scale in the different areas of analysis. The data shows that both frozen and lyophilized glycostem-sEVs have the same therapeutic activity associated with a regeneration of the area, with no significant differences between the two groups. Graphs represent the + / -SD mean of 1 experiment, using 5-8 mice per experimental group. P values were calculated with respect to the OA experimental group or when indicated between groups. * p < 0.05; ** p < 0.01; p < 0.005; p < 0.001.Description of the Invention The invention points to a solution to the problem of osteoarthritis, with an acellular bioproduct based on extracellular nanovesicles derived from MSC, UC-MSC-sEV, which overexpress the proteins Keratin, type II cuticular Hb5 or P78386 (KRT85 gene); Agrine or A0A494 C0 G5 (AGRN gene); and Basement membrane-specific heparan sulfate proteoglycan core protein or P98160 (HSPG2 gene). These specific nanovesicles, sEVsglyco or Glycostem-sEV, are experimentally obtained in a higher proportion from induced MSCs to glycolytic metabolism with Oligomycin. These nano vesicles are useful for the treatment of OA, where the inventors have validated their efficacy and safety profile in normal and OA cartilage and in synovial tissues in vitro and in vivo. Additionally, this product, being acellular, maintains its functional efficiency both in its frozen format at -80 and freeze-dried for 48 hours, which gives great versatility to the product, which can be transported in ambient temperature conditions maintaining its integrity and therefore its effectiveness. The inventors have evaluated the biodistribution of the product of the invention administered locally (intra-articular) in the knee of healthy mice and with OA. It is worth mentioning that one of the main challenges of acellular therapy is the time the product remains in the area of interest. To answer this question, the inventors also conducted a preliminary study, using the in vivo model of murine collagenase-induced osteoartirtis (CIOA). The results showed that UC-MSC-sEV injected intra-articularally are able to remain at the injection site for more than 96h, enough time to be internalized by the different resident acceptor cells. Additionally, preliminary results indicated that sEV of the invention significantly decreases mineralization and histological score in the joints of treated animals, as well as fibrosis in the joint, compared to UC-MSC-sEV, which demonstrates a better therapeutic and regenerative effect in this animal model. In terms of protein signature and miRNA, we were able to identify specific proteins associated with cartilage regeneration such as Agrin, HSPG2; these together with KTR85 are overexpressed in the condition of sEVs derived from MSC pretreated with oligomycin (Glycostem). Similarly, we describe a microRNA signature that was found to be increased only in the condition of sEVs derived from MSCs pretreated with oligomycin. In this way, the invention refers to a composition enriched in extracellular nanovesicles (sEV) of umbilical cord mesenchymal cells, where the composition consists of - a set of sEV in which the expression of the AGRN and HSPG2 proteins has increased, - and a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Additionally, the nanovesicles contain at least the following miRNAs: miR365a-5p, miR4257 and miR4535 overexpressed. Optionally, KRT85 overexpression can also be evaluated. In this composition, the nano vesicles are in a concentration of between 5x107 to 1x1013 total particles per mL and have a size of between 30 and 300 nm.Conveniently, the AGRN and HSPG2 proteins are elevated between 1.5 to 8 times in their concentration with respect to the concentration in the native condition, that is, of sEV of UC-MSCs without oligomycin treatment. Normally the concentration rises between 1.8 and 4 times. Optionally the same happens with the expression of KRT85In a second embodiment, the invention points to the method of obtaining the composition which includes the following stagesa) culturing umbilical cord mesenchymal cells (UC-MSCs), in an induction medium supplemented with 0.01 to 100 μg / mL of Oligomycin, for 2 to 48 hours, and thereafterb) isolating the nanovesicles secreted by MSCs into the environment, c) checking the increased expression of the AGRN and HSPG2 proteins; and d) resuspending the nanovesicles in a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Where, in step b) the nanovesicles are isolated from the supernatant of the culture by ultrafiltration, ultracentrifugation and / or centrifugation, are washed, at least 2 times with a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0, and finally are resuspended in a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Specifically in stage c) the overexpression of proteins is evaluated with respect to the native condition, nanovesicles of UC-MSCs without incubation with oligomycin. Additionally, the nanovesicles contain at least the following miRNAs: miR365a-5p, miR4257 and miR4535 overexpressed with respect to a control of nanovesicles obtained from UC-MSCs without oligomycin incubation. Optionally, KRT85 overexpression is evaluated. Where the composition obtained in step d) is subjected to freeze-drying or freezing at -80°C. In another embodiment, the invention points to a pharmaceutical composition comprising the composition of claim 1 and pharmaceutically acceptable vehicles for intra-articular injections; which is conveniently freeze-dried.Additionally, the invention refers to the use of the pharmaceutical composition to prepare a drug useful in the treatment of osteoarticular diseases or osteoarticular trauma. Especially for the regeneration of damaged cartilage, prevention of cartilage degradation, or in the generation of new cartilage. The pharmaceutical composition is especially useful for the treatment of osteoarthritis, arthritis, osteoarthritis.Finally, the invention points to a method for the regeneration of damaged cartilage, prevention of cartilage degradation, or for the generation of new cartilage, which involves the administration of a therapeutically effective amount of the pharmaceutical composition of the invention, which contains the composition enriched in nanovesicles as described. Examples Example 1. Obtaining and characterizing nanovesicles.Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were cultured in DMEM medium supplemented with 10% FBS, Pen / Strept and glutamine (DMEM-10) until 80% confluence was reached when the culture medium was removed and washed once with buffered saline (PBS). Then the "glycolytic stimulation" was prepared by adding DMEM-10 of the UC-MSCs supplemented with an oligomycin solution containing the A, B, C isomers to a final concentration of 1μg / ml of oligomycin, the cells were incubated with glycolytic stimulation for 24 h. Subsequently, the oligomycin-containing medium was removed and the cells were washed with buffered saline (PBS). In this way, UC-MSC s-glyco (by glycolytic metabolism. These cells were cultured for 48 h in a medium free of animal components, specifically in DMEM medium high in glucose; supplemented only with 1% L-Glut, for 48 h, proceeding, after that time, to collect the supernatant from the culture. In parallel, the same procedure was performed, as a control, without supplementation with oligomycin from the culture, in order to observe the initial or native condition. The collected supernatant was centrifuged 600 g 4°C for 10 minutes to remove impurities, which precipitate. The supernatants from the previous centrifugation were processed in an ultracentrifuge at 100,000 g for 1 hour and 10 minutes at 4°C, this step was repeated until the total volume of the supernatant was processed, where the nanovesicles or exosomes are obtained in the pellet. The supernatant was removed and the pellets were released using a vortex. Subsequently, the pellets were subjected to two washes in order to eliminate possible contaminants. To do this, the pellets were resuspended in filtered PBS reaching a final volume of 10 ml, and centrifuged at 100,000g at 4°C over night. Subsequently, the last wash was carried out, for which the supernatants were removed and the pellets were resuspended in a final volume of 10ml Ringer Lactate (pH 6.5, 273mOsm / L) and processed for 1 hour and 10 minutes at 4°C in an ultracentrifuge at 100,000 g. Finally, the supernatant was removed, the pellets were passed through a vortex and aliquoted for storage.The obtained nanovesicles were evaluated, evaluating the overexpressed proteins in the nanovesicles of glycolytic cells, especially the results of the proteins of interest, KRT85, AGRN and HSPG2 with respect to the control condition, without the addition of oligomycin, the results are shown in Table 1. Where AGRN and HSPG2 are proteins associated with cartilage regeneration.Based on these results, the inventors decided to determine, as a validation signature of these nanovesicles, the concentration of agrine and HSP G2, in addition to the concentration of KRT85. sEVsglyco vs sEVs nativeGene NameProtein IDProtein name Log 2Times of changeKTR85P78386Keratin, type II cuticle Hb52.084.2KTR86O43790Keratin, type II cuticle Hb61.843.6KTR16P08779Keratin, cytoskeletal type 161.53KTR31Q15323Keratin, cuticle type Ha11.142.2AGRNA0A494C0G5Agrine1.122.2HSP G2P98160Core protein of the basement membrane-specific heparan sulfate proteoglycan1.062.1KRT6AP02538Keratin, cytoskeletal type 6A0.921.9RP59P4678140S Ribosomal Protein 590.7191.6DSPP15924Desmoplakin0.7051.6Table 1. Overexpressed proteins in MSC-oligomycin -sEVs condition. N = 4 biological samples. On the other hand, sEVs from glycostem exhibit a microRNA signature that is significantly different from their sEVS-Nativa counterpart in lyophilized samples. A total of 2067 miRNAs from lyophilized samples of sEVS glycostem and UC-MSC-sEV were analyzed, of which 990 were found in the 4 biological replicates analyzed. Using the EdgeR tool, 178 genes were obtained with a significant difference with respect to the native sEVs condition. Of the total, 175 were found to be increased with respect to native sEVS (Log2 fold change >0.5 and <-0.5; Adjusted p-value ≤ 0.05) and 3 decreased with respect to the native sEVs condition (Log2 fold change >0.5 and <-0.5; Adjusted p-value ≤ 0.05). These results are shown in Figure 1. For the signature validation of sEVs-Glyco, the miR with the highest times of change with respect to the native sEVs condition were selected, which correspond to miR365a-5p, miR4257 and miR4535. Example 2. In vivo studies.To establish the effect of the GlycoStem-sEV nanovesicles of the invention, the evolution of an osteoarthritis model was evaluated when treated according to the invention versus, the vehicle-only control and the control of cell nanovesicles without pretreatment with oligimycin. Healthy C57BL6 / j mice in the collagenase-induced OA model of both sexes, 10-12 weeks of age, appropriately randomized in the different study groups: untreated, cells and nanovesicles of UC-MSC-sEV in native state and pretreated with oligomycin, GlycoStem-sEV and single administration of the vehicle (of the nanovesicular product formulation) (5 μl in PBS). The diagram of the experimental design is shown in Figure 2. The parameters monitored included: mortality (with a determined cause of mortality if possible), clinical observation, body weight, physical examination, and clinical pathology parameters (blood biochemistry [liver, renal, pancreatic function, glycemia, different electrolytes], hematology, coagulation, urinalysis), organ weight and histopathology, and cellular and humoral immune response. Specifically, the mice were injected with 5μL with collagenase type VII or with saline solution (SHAM) intra-articular (IA). After 7 and 14 days post-induction, the animals were treated with 5μL of saline solution or with native sEVs or glyco sEVs. On day 42 the mice were euthanized and the joints were removed for microCT image analysis or histology. The clinical score for microCT and histology was analyzed in the different areas of the joint, in order to obtain the values of bone density and histological joint damage score. The results are shown in Figures 3 and 4. Figure 3 shows the bone density values in the different areas of analysis. The data show that UC-MSCs-Glycostem-sEV significantly improve chondral mineralization, compared to native UC-MSC-sEVS. Figure 4 shows the degree of joint degeneration of the analyzed areas associated with OA according to histological score. All analyses were performed in double-blind. The data show that Glycostem-sEV significantly reduces the progression of OA, highlighting an increase in hyaline cartilage in the glycostem-sEV group, presenting a greater therapeutic efficacy associated with a regeneration of the damaged cartilage area in OA. These results show that the nanovesicles of the invention have a direct effect on osteoarthritis (OA) both at the level of chondral mineralization and by decreasing joint degeneration. Example 3. Post-freeze-drying stability.For this last experimental design, glycostem-derived sEVs were frozen in PBS for at least 48H and then divided into two samples, one that was maintained at -80° for later use and the other was lyophilized and maintained at 4°C for 24h. For freeze-drying, the samples were placed in an operon brand freeze dryer at an average temperature between -80 to -90°C. using a pressure of 200 mmtor, then the sample (which is completely frozen at -80) is placed in the equipment. The vacuum is performed and the sample is left for two hours inside the equipment. Prior to intra-articular injection, the lyophilized glycostem-sEVs were resuspended in sterile miliQ water and the equivalent of 2x108 particles of both frozen and lyophilized glycostem-sEVs were injected into the animals after resuspension.Specifically, an intra-articular injection was performed in the joint of OA-induced mice, following the experimental design shown in Figure 2. Interestingly, as shown in Figure 5, the therapeutic capacity is maintained with the freeze-dried product. Figure 5 A shows the bone density values in the different areas of analysis. The data shows that lyophilized glycostem-sEVs present the same significant improvement to chondral mineralization as their frozen counterpart, suggesting that it preserves its therapeutic capacity. Likewise, Figure 5 B shows the histological score values according to the modified Pritzker OARSI scale in the different areas of analysis. The data shows that both frozen and lyophilized glycostem-sEVs have the same therapeutic activity associated with a regeneration of the area, with no significant differences between the two groups Therefore, the nano vesicles of the invention maintain their therapeutic activity even after being freeze-dried and reconstituted.
Claims
Claim 1.Composition enriched in extracellular nano vesicles (sEV) of umbilical cord mesenchymal cells, CHARACTERIZED in that the composition consists of - a set of sEV that have increased expression of AGRN and HSPG2 proteins, - and a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Claim 2. Composition according to claim 1 CHARACTERIZED in that the nanovesicles contain at least the following miRNA: miR365a-5p, miR4257 and miR4535 overexpressed Claim 3. Composition according to claim 1 CHARACTERIZED in that the nanovesicles have an increased expression of the KRT 85 protein. Claim 4. Composition according to claim 1 CHARACTERIZED in that the nano vesicles are in a concentration of between 5x107 to 1x1013 total particles per mL . Claim 5. Composition according to claim 3 CHARACTERIZED in that nanovesicles have a size between 30 and 300 nm Claim 6. Method of obtaining the composition of claim 1 CHARACTERIZED in that it comprises a) culturing umbilical cord mesenchymal cells (UC-MSCs), in an induction medium supplemented with 0.01 to 100 μg / mL of Oligomycin, for 2 to 48 hours, and thereafterb) isolating the nanovesicles secreted by the MSCs into the medium, c) checking the increased expression of the AGRN and HSPG2 proteins; and d) resuspending the nanovesicles in a medium or vehicle, free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0.Claim 7. Method according to claim 6 CHARACTERIZED in that in step b) the nanovesicles are isolated from the supernatant of the culture by ultrafiltration, ultracentrifugation and / or centrifugation, they are washed at least 2 times with a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0, and finally they are resuspended in a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Claim 8. Method according to claim 6 CHARACTERIZED in that in step c) the overexpression of the proteins is evaluated with respect to the native condition, nanovesicles of UC-MSCs without incubation with oligomycin.Claim 9. Method according to claim 8 CHARACTERIZED in that the nanovesicles have an increased expression of the KRT 85 protein. Claim 10. Method according to claim 6 CHARACTERIZED in that the nanovesicles contain at least the following miRNAs: miR365a-5p, miR4257 and miR4535 overexpressed with respect to a control of nanovesicles obtained from UC-MSCs without oligomycin incubation. Claim 11. Method according to claim 6 CHARACTERIZED in that the composition obtained in step d) is subjected to freeze-drying or freezing at -80°C. Claim 12. Pharmaceutical composition CHARACTERIZED in that it comprises the composition of claim 1 and pharmaceutically acceptable vehicles for intra-articular injections. Claim 13. Pharmaceutical composition of claim 12 CHARACTERIZED in that it is freeze-dried. Claim 14. Use of the pharmaceutical composition of claim 10 CHARACTERIZED in that it serves to prepare a medicinal product useful in the treatment of osteoarticular diseases or traumas.Claim 15. Use, according to claim 12 CHARACTERIZED in that the composition is useful in the regeneration of damaged cartilage, prevention of cartilage degradation or in the generation of new cartilage. Claim 16. Use, according to claim 12 CHARACTERIZED in that the composition is useful for the treatment of arthrosis, arthritis, osteoarthritis.