Objectives. Circulating exosomes represent novel biomarkers for multiple diseases. In this study, we investigated whether circulating exosome levels could be used as a diagnostic biomarker for steroid-induced osteonecrosis of the femoral head (ONFH). Methods. We assessed the serum exosome level of 85 patients with steroid-induced ONFH and 115 healthy donors by Nanosight detection. We then assessed the diagnostic accuracy of serum exosomes by receiver operating characteristic curve analysis. Results. The circulating exosome level of the ONFH group was significantly lower than that of control group. The area under the curve was 0.72, suggesting that the level of serum exosomes has moderate diagnostic accuracy for steroid-induced ONFH. Conclusion. Circulating exosome levels are valuable in the diagnosis of steroid-induced ONFH. Cite this article: H-Y. Zhu, Y-C. Gao, Y. Wang, C-Q. Zhang. Circulating exosome levels in the diagnosis of steroid-induced osteonecrosis of the femoral head. Bone Joint Res 2016;5:276–279. DOI: 10.1302/2046-3758.56.BJR-2015-0014.R1

Osteoarthritis (OA) is a common age-related degenerative joint disease, affecting 7% of the global population, more than 500 million people worldwide. Exosomes from mesenchymal stem cells (MSCs) showed promise for OA treatment, but the insufficient biological targeting weakens its efficacy and might bring side effects. Here, we report the chondrocyte-targeted exosomes synthesized via click chemistry as a novel treatment for OA. Exosomes are isolated from human umbilical cord-derived MSCs (hUC-MSCs) using multistep ultracentrifugation process, and identified by electron microscope and nanoparticle tracking analysis (NTA). Chondrocyte affinity peptide (CAP) is conjugated on the surface of exosomes using click chemistry. For tracking, nontagged exosomes and CAP-exosomes are labeled by Dil, a fluorescent dye that highlights the lipid membrane of exosomes. To verify the effects of CAP-exosomes, nontagged exosomes and CAP-exosomes are added into the culture medium of interleukin (IL)-1β-induced chondrocytes. Immunofluorescence are used to test the expression of matrix metalloproteinase (MMP)-13. CAP-exosomes, compared with nontagged exosomes, are more easily absorbed by chondrocytes. What's more, CAP-exosomes induced lower MMP-13 expression of chondrocytes when compared with nontagged exosomes (p<0.001). CAP-exosomes show chondrocyte-targeting and exert better protective effect than nontagged exosomes on chondrocyte extracellular matrix. Histological and in vivo validation are now being conducted

Osteoarthritis (OA), the most prevalent chronic joint disease, represents a relevant social and economic burden worldwide. Human umbilical cord mesenchymal stem cells (HUCMSCs) have been used for injection into the joint cavity to treat OA. The aim of this article is to clarify whether Huc-MSCs derived exosomes could inhibit the progression of OA and the mechanism in this process. A rabbit OA model was established by the transection of the anterior cruciate ligament. The effects of HUCMSCs or exosomes derived from HUCMSCs on repairing articular cartilage of knee osteoarthritis was examined by micro-CT. Immunohistochemical experiments were used to confirm the expression of relevant inflammatory molecules in OA. In vitro experiments, Transwell assay was used to assess the migration of macrophages induced by TNF-a. Results showed that a large number of macrophages migrated in arthcular cavity in OA model in vivo, while local injection of HUCMSCs and exosomes did repair the articular cartilage. Immunohistochemical results suggested that the expression of CCL2 and CD68 in the OA rabbit model increased significantly, but was significantly reduced by HUCMSCs or exosomes. Transwell assay showed that both HUCMSCs and exosomes can effectively inhibit the migration of macrophage. In conclusion, the exosomes derived by HUCMSCs might might rescue cartilage defects in rabbit through its anti-inflammatory effects through inhibiting CCL2

Introduction and Objective. Exosomal miRNA have been shown to regulate many myogenic and osteogenic pathways involved in injury repair and healing. It is also known that rehabilitation and exercise can improve muscle mass and bone growth. The mechanisms by which this occurs in vivo are well studied, but the impact exosomes and their associated miRNA cargo have is unclear. With this knowledge and question in mind, we hypothesized that C2C12 myoblasts subjected to in vitro mechanical stimulus (“exercise”) would exhibit improved exosome production and differentially expressed miRNA cargo when compared to their static (“unexercised”) counterparts. Materials and Methods. C2C12 myoblasts were cultured using the FlexCell FX-5000TT bioreactor. Two exercise regimens were programmed: 1) low intensity regimen (LIR) (0–15% strain at 0.5 Hz for 24 hours) 2) high intensity interval regimen (HIIR) (12–22% strain at 1 Hz for 10 minutes followed by 50 minutes of rest repeated for 24 hours). Unexercised (static) cells were cultured in parallel. Exosomes were isolated using the Invitrogen Total Exosome Isolation Reagent. The Pierce BCA Protein Assay, System Bioscience's ExoELISA-ULTRA CD81 Kit and, SBI's ExoFlow-ONE EV labeling kit were used to confirm and quantify exosome number and protein concentration. The SBI Exo-NGS service was used to perform miRNA sequencing on isolated exosomes. Results. All exercise regimens resulted in increased exosome concentrations as determined by CD81 exosome ELISA and flow-cytometry based exosome quantification. The LIR interestingly produced significantly more exosomes than static and HIIR. Within the exosomes from mechanically stimulated cells, 35 miRNAs were found to be differentially expressed when compared to exosomes from unexercised cells. Interestingly, this significance was only found within exosomes from the HIIR group. Specifically, upon investigation 8 of these miRNAs were found to be involved in myogenic and osteogenic proliferation and differentiation. These results correlate with our previous findings that exosomes from exercised cells improve the proliferation and myogenic differentiation of C2C12 myoblasts. Conclusions. Our results indicate that exercise can be optimized to improve the production and regenerative capacity of exosomes. These results also indicate that exosomes may be intimately involved in systemic health and repair during rehabilitation and exercise. To examine these results in vivo, mouse studies using a crush injury model and exosomes from mechanically stimulated cells are currently planned

Mesenchymal stem cell (MSC) exosomes are intracellular vesicles, which can regulate transcription and control gene expression through the molecules they carry, easily enter into the target cell, contain no regenerative effect, and do not produce an immune response. There are different methods in the literature to obtain these vesicles. However, studies on the isolation of MSC-derived exosomes and their comparative characterization using magnetically active cell sorting (MACS) and ultracentrifugation methods are lacking. The most appropriate isolation method for MSC-derived exosomes can be determined by comparing the isolation and characterization parameters of mesenchymal stem cells using magnetically active cell sorting and ultracentrifugation methods. The aim of this study was to define the advantages and disadvantages of the methods used for determining the purpose-oriented method. Human bone marrow-derived mesenchymal stem cells were cultured in standard MSC culture conditions (37ºC and 5% CO 2). Exosomal contamination was prevented by removal of exosomes from the serum that used in the standard growth medium. For exosome isolation of the cells reaching sufficient density, the media were replaced with new ones every two days, the old media were collected in liquid refrigerated with liquid nitrogen and stored at −80ºC. Part of the accumulated exosomes were isolated by using the MACS method, while the other was isolated by using the ultracentrifugation method, which included serial centrifugation steps. The amount of protein contained in the phosphate buffer solution in which the exosomes were reconstituted was determined by microplate reader using the BCA kit. Based on the protein concentration obtained, exosomes were read by means of a dye flow cytometer with fluorescent antibodies attached to surface markers specific to CD9, CD63, and CD81 specific for exosomes by latex beads. Finally, the exosomes were stained with uranyl acetate and phosphotungstic acid and then placed on 200 mesh and formvar-carbon film coated grids. Exosomes were isolated using both ultracentrifugation and MACS methods. While ultra-large amounts of exosomes can be isolated by ultracentrifugation method, MACS method provides a lower amount of isolation. Exosomes with magnetically active cell sorting are selected with specific surface markers, therefore, exosomal purity is thought to be higher. Exosomes which were isolated by both ultracentrifugation and MACS methods were monitored by using transmission electron microscopy and they were not found to be morphologically different. In conclusion, MACS and ultracentrifugation are effective methods for the isolation of human bone marrow-derived MSC exosomes. Both methods have advantages and disadvantages. Exosomes can be isolated together with magnetic beads using the MACS method. In the ultracentrifuge method, cleaner exosomes can be isolated. While the exosomes are isolated by MACS, they can also be characterized by beads

Intervertebral disc degeneration (IDD) affects more than 80% of the population all over the world. Current strategies for the treatment of IDD are based on conservative or surgical procedures with the aim of relieving pain. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in recent decades, but studies showed that the particularly hostile microenvironment in the intervertebral disc (IVD) can compromise cells survival rate. The use of exosomes, extracellular vesicles released by various cell types, possess considerable economic advantages including low immunogenicity and toxicity. Exosomes allow intercellular communication by conveying functional proteins, RNA, miRNA and lipids between cells. The purpose of this study is to assess the therapeutic effects of exosomes derived from Wharton Jelly mesenchymal stromal cells (WJ-MSC) on human nucleuspulposus cells (hNPC) in an in vitro 3D culture model. Exosomes (exos) were isolated by tangential flow filtration of WJ-MSC conditioned media and characterized by: quantification with BCA test; morphological observation with TEM, surface marker expression by WB and size evaluation by NTA. Confocal microscopy has been used to identify exosomes marked with PKH26 and monitor fusion and/or incorporation in hNPC. hNPC were isolated from waste surgical material from patients undergoing discectomy (n = 5), expanded, encapsulated in alginate beads and treated with: culture medium (control group); WJ-MSC exos (WJ-exos) at different concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml). They were then analysed for: cell proliferation (Trypan Blu); viability (Live/Dead Assay); quantification of nitrites (Griess) and glycosaminoglycans, GAG (DMBB). The hNPC in alginate beads treated for 7 days were included in paraffin and histologically analysed to determine the presence of extracellular matrix (ECM) components. Finally, the expression levels of catabolic and anabolic genes were evaluated through real-time polymerase chain reaction (qPCR). All concentrations of WJ-exos under exam were capable to induce a significant increase in cell proliferation after 10 and 14 days of treatment (p < 0.01 and p < 0.001, respectively). Live/Dead assay showed a decrease in cell death at 50 μg/ml of WJ-exos (p < 0.05). While cellular oxidative stress indicator, nitrite production, was reduced in a dose-dependent way and statistically significant only with 100 μg/ml of WJ-exos (p < 0.05). WJ-exos at 10 and 100 μg/ml induced a significant increase in GAG content (p < 0.05; p < 0.01, respectively) confirmed by Alcian Blu staining. Exos derived from WJ-MSC modulated gene expression levels by increasing expression of ACAN and SOX-9 genes and reducing significantly of IL-6, MMP-1, MMP-13 and ADAMTS-5 levels (p < 0.05; p < 0.01) compared to the control group. Our results supported the potential use of exosomes from WJ-MSC for the treatment of IDD. Exosomes improved hNPC growth, attenuated ECM degradation and reduced oxidative stress and inflammation. This study offers a new scenario in IVD regeneration, promoting the potential use of extracellular vesicles as an alternative strategy to cell therapy

Intervertebral disc degeneration (IDD) affects more than 80% of the population and is often linked to a reduction of the proteoglycan content within the nucleus pulposus (NP). The nutritional decline and accumulation of degraded matrix products promote the inflammatory process favoring the onset of disease. Several regenerative approaches based on cell therapy have been explored. Recently, paracrine factors and extracellular vesicles (EVs) such as exosomes have been described to play a fundamental role in the cross-talk between mesenchymal stem cells (MSCs) and NP in the microenvironment. EVs vehicule different molecules: proteins, nucleic acids and lipids involved in intercellular communication regulating the homeostasis of recipient cells. Therefore, MSCs-derived exosomes are an interesting emerging tool for cell-free therapies in IDD. The aim of this study was to evaluate the in vitro effects of MSCs derived exosomes on human NP cells (hNPCs). Exosomes were isolated through a multistep ultracentrifugation of bone marrow-MSCs (BM-MSCs) conditioned media (CM), obtained by culturing BM-MSCs without fetal bovine serum (FBS) for 48 hours. Exosomal morphology was characterized by transmission electron microscope (TEM). The exosomes were quantified by bicinchoninic acid assay (BCA) and cryopreserved at –80 °C. hNPCs derived from surgical speciments digested with type II collagenase. After culture expansion in vitro, hNPCs in alginate beads (three-dimensional culture system) were treated with growth medium (controls), exosomes, CM, interleukin-1 beta (IL-1b), IL-1b plus exosomes, IL-1b plus CM. After 24 hours, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). TEM analysis confirmed the cup-shaped vescicles in our preparations. Gene expression levels resulted to be modulated by both exosomes and CM compared to controls. In addition, both treatments were capable to alter the inflammatory stimuli of IL-1b. Interestingly, exosomes were able to change anabolic and catabolic gene expression levels differently from CM. In our experimental conditions, both exosomes and CM from BM-MSCs could be an interesting alternative strategy in intervertebral disc regeneration, overcoming the costs and translational limits of cell therapy to the clinical practice

The aim of this study was to investigate the regenerative effects of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) derived exosomes (WJ-Exos) on human nucleus pulposus cells (hNPCs) in an in vitro 3D model. WJ-Exos were isolated by tangent flow filtration of WJ-MSCs conditioned media and characterized by TEM, WB for markers expression and quantified with NTA. WJ-Exos PKH26-labeled uptake in hNPCs was detected by confocal microscopy. hNPCs, isolated from surgical specimens (n=4), culture expanded in vitro and encapsulated in alginate beads, were pre-treated with IL1β (10 ng/ml) for 24 hours, then with WJ-Exos at 10, 50 and 100 µg/ml. Cells with growth medium were used as control. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess) iii) glycosaminoglycan (GAG) amount (DMBB), iv) histological staining for extracellular matrix (ECM) analysis and v) gene expression levels of catabolic and anabolic genes (qPCR). The investigations were performed in triplicate for each donor. One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D. A dose dependent increase in hNPCs proliferation was noticed at all exos concentrations under study. Cell death decreased significantly in WJ-Exos 50 µg/ml samples (p ≤ 0,05) compared to IL1β treated hNPCs. Nitrite production was significantly attenuated at 10µg/ml of WJ-Exos (p ≤ 0,01). GAG content and histological analysis showed a difference in ECM synthesis between treated and untreated hNPCs (p ≤ 0,05). Catabolic and inflammatory markers were modulated by WJ-Exos at 100 µg/ml concentration (p ≤ 0,05) whereas 10 µg/ml group increased anabolic gene expression levels (p ≤ 0,05). These findings offer new opportunities for the potential use of exosomes as an attractive alternative cell-free strategy of IDD. WJ-MSC exosomes ameliorate hNPCs growth and viability, attenuate ECM degradation and oxidative stress-related IDD progression after IL1β stimulation. Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Disease modifying approaches are commonly applied in OA patients. An aging society with better life expectancies is increasing in Europe and the globe. Orthobiologics cover intraarticular hyaluronan injections and also cellular therapies. Cellular therapies range from platelet rich plasma (PRP) applications to exosomes. Short term follow-up of limited number of patients revealed favorable results in clinical cellular therapies. Most of these studies evaluated decrease of pain and increase in function. Recent basic science studies focused on the action mechanism of orthobiologic therapies however patient perspective is less studied. Our research team has recently performed a qualitative study on the patient perspective of hyaluronan injection of the knee joint. Findings of that study will be shared and future patient knowledge based options on orthobiologics will be discussed

Mesenchymal stem cells (MSC) are multipotent cells that possess regenerative functions that are of interest for in osteoarticular diseases such as osteoarthritis (OA). These functions are thought to be primarily mediated by mediators released within extracellular vesicles (EV). The aim of this study was to compare the immunomodulatory effects of two major types of EV, exosomes and microparticles, secreted by MSCs. EV subsets were isolated from murine primary MSCs by ultracentrifugation. Size and structure were evaluated by Dynamic Light Scattering and electron microscopy. Expression of membrane and endosomal markers was tested by flow cytometry. Proliferation of murine splenocytes was quantified after 72h of incubation with EVs after CFSE-labelling. Phenotypic analysis of T lymphocyte subpopulations was also performed by flow cytometry. In vivo, EVs were injected in the knee joint in the collagenase-induced osteoarthritis (CIOA) model and histological score was performed. In vitro functional analysis indicated that addition of microparticles or exosomes in proliferative assays inhibited the proliferation of total splenocytes in a dose-dependent manner. Analysis of T cell subpopulations revealed a decrease in CD8. +. IFNγ. +. lymphocytes and an increase in both CD4. +. IL10. +. Tr1 and CD4. +. CD25. +. FOXP3. +. Treg cells. This immunomodulatory function of EVs was also observed in vivo in the CIOA model. In summary, our data indicated that the immunosuppressive effect of MSCs is in part mediated by exosomes and microparticles that play in vivo a major role in MSC-mediated therapeutic effect by reducing osteoarthritic symptoms

Matrix-bound vesicles (MBVs) are embedded within osteoid and function as the site of initial mineral formation. However, they remain insufficiently characterised in terms of biogenesis, composition and function while their relationship with secreted culture medium EVs (sEVs) such as exosomes remains debated. We aimed to define the biogenesis and pro-mineralisation capacity of MBVs and sEVs to understand their potential in regenerative orthopaedics. sEVs and MBVs isolated from conditioned medium (differential ultracentrifugation) and ECM (collagenase digestion and differential ultracentrifugation) of mineralising MC3T3 pre-osteoblast and human bone marrow MSC cultures were characterised by nanoparticle tracking analysis, western blotting, nano-flow cytometry, super resolution microscopy (ONI) and TEM. Immunoprecipitated populations positive for alkaline phosphatase (ALP), a putative marker of mineralisation capacity, were also characterised. Collagen binding efficiency was evaluated using MemGlow staining. Results reported were comparative across both cell lines. Western blots indicated MBV fractions were positive for markers of endosomal biogenesis (CD9, CD81, ALIX, TSG101) and pro-mineralising proteins (ALP, Pit1, Annexin II, Annexin V), with Annexin V and CD9 present in immunoprecipitated ALP-positive fractions. MBVs were significantly larger than sEVs (p<0.05) and contained a higher amount of ALP (p<0.05) with a significant increase from day 7 to day 14 of cellular mineralisation (p<0.05). This mirrored the pattern of electron-dense vesicles seen via TEM. Super resolution single vesicle analysis revealed for the first-time co-expression of ALP with markers of endosomal biogenesis (CD9, CD63, CD81, ALIX) and Annexin II in both vesicle types, with higher co-expression percentage in MBVs than sEVs. MBVs also exhibited preferential collagen binding. Advanced imaging methods demonstrated that contrary to opinions in the field, MBVs appear to possess exosomal markers and may arise via endosomal biogenesis. However, it was evident that a higher proportion of MBVs possessed machinery to induce mineralisation and were enriched in mineral-dense material

Tendon injuries in both the human and horse represent a challenge due to persistent inflammation combined with inadequate reparative cells and a poorly organised extracellular matrix. The potential of mesenchymal stem cells (MSCs) in regenerating tendon injuries remains to be fully realised. The main mechanism of action by MSCs is considered to be primarily mediated via paracrine mechanisms. This may involve the production and release of extracellular vesicles (EVs) by stem cells with a sub-fraction of these EVs (<100 nm diameter) called exosomes that appear to be the main paracrine effectors. EVs can be readily prepared from MSCs and offer a clinically relevant therapy. However, EVs for tendon repair need to be fully characterised. The horse represents a highly relevant model of tendon and ligament injuries as it shares many features of mechanical loading, function and aetiopathology with the human. We have isolated and characterised EVs from equine MSCs for modulating tendon cell phenotype in an in vitro tendon injury model using IL-1ß. EVs can be isolated from IL-1ß stimulated MSCs although their levels are not significantly increased over controls suggesting that the nature of the stimulated EV cargo may be more important than absolute levels of released EVs

Multiple myeloma (MM) is an incurable hematological tumor stemming from malignant plasma cells. MM cells accumulate in the bone marrow (BM) and shape the BM niche by establishing complex interactions with normal BM cells, boosting osteoclasts (OCLs) differentiation and causing bone disease. This unbalance in bone resorption promotes tumor survival and the development of drug resistance. The communication between tumor cells and stromal cells may be mediated by: 1) direct cell-cell contact; 2) secretion of soluble factors, i.e. chemokines and growth factors; 3) release of extracellular vesicles/exosomes (EVs) which are able to deliver mRNAs, miRNAs, proteins and metabolites in different body district. Primary CD138+ MM cells were isolated from patients BM aspirates. MM cell lines were cultured alone in complete RPMI-1640 medium or co-cultured with murine (NIH3T3) or human (HS5) BMSC cell lines or murine Raw264.7 monocytes in DMEM medium supplemented with 10% V/V FBS. Silencing of Jagged1 and Jagged2 was obtained by transient expression of specific siRNAs or by lentiviral transduction using a Dox-inducible system (pTRIPZ). EVs were isolated using differential ultracentrifugation. EVs concentration and size were analyzed using Nano Track Analysis (NTA) system. The uptake of PKH26-labelled MM-derived EVs by HS5 or Raw264.7 was measured after 48 hours by confocal microscopy and flow cytometry. Osteoclast (OCL) differentiation of Raw264.7 cells was induced by 50ng/ml mRANKL, co-culturing with MM cells, CM or EVs. OCLs were stained by TRAP Kit and counted. Bone resorption was assessed by Osteo Assay Surface plates. Flow cytometric detection of apoptotic cells was performed after staining with Annexin V. Gene expression was analyzed by qRT-PCR, while protein levels were determined using flow cytometry ELISA or WB. Notch oncogenic signaling is dysregulated in several hematological and solid malignancies. Notch receptors and ligands are key players in the crosstalk between tumor cells and BM cells. We have demonstrated that: 1) the dysregulated Jagged ligands on MM cells trigger the activation of Notch receptors in the nearby stromal cells by cell-cell contact. This results in the release of anti-apoptotic and growth stimulating factors, i.e. IL6 and SDF1; 2) MM cells promote the development of bone lesions boosting osteoclast differentiation by secreting soluble factors (i.e. RANKL) and by the activation of Notch signaling mediated by direct contact with osteoclast precursors; 3) Finally, we present evidences that EVs play a crucial role in the dysregulated interactions of MM cells with the microenvironment and that Notch signaling regulates their release and participate in this cross-talk. These evidences supports the hypothesis that Jagged targeting on MM cells may interrupt the communication between tumor cells and the surrounding milieu, blocking the activation of the oncogenic Notch pathway and finally resulting in the a reduction of MM-associated bone disease and drug resistance

Summary Statement. Umbilical cord derived stem cell secretion could enhance the osteogenic differentiation of human bone marrow stem cells. It may promote bone, cartilage and tendon regeneration in rat models, but the effect was not significant up to now. Introduction. Mesenchymal stem cells (MSCs) are multipotent cells that have extensive proliferative capacity. MSCs synthesise various exosomes, growth factors and cytokines. Stem cell secretions were made from serum free conditioned medium of stem cells collected from different human tissues, such as adipose tissue and dental pulp. Our hypothesis is umbilical cord stem cell secretion could promote multiple proliferation and differentiation of MSCs, also enhance the regeneration of musculoskeletal tissues. Methods. In vitro: Human bone marrow mesenchymal stem cells (hBMSCs) were cultured in high glucose dulbecco's modified eagle medium with 10% serum. hBMSCs were treated by differential medium for osteogenic, tenogenic and chondrogenic differentiation. Alizarin red S staining, alcian blue staining and sirius red staining were used to test osteogenesis, chondrogenesis and tenogenesis of hBMSCs after treated by secretion. RNA expression level of hBMSCs were detected by real-time reverse transcriptase polymerase chain reaction. In vivo: 10 weeks male Sprague-Dawley rats were used in all the animal studies. Rat calvarial bone defect model, rat femoral closed fracture model with internal fixation, rat articular cartilage defect model and rat patella tendon window defect model were used in animal experiments. Radiography analysis, micro-computed tomography imaging analysis, mechanical test, ultrasound test and histology analysis were used to evaluate the regeneration of bone, cartilage and tendon. Results. Alizarin red S staining showed the minimal effective concentration of 20ug/ml umbilical cord stem cells secretion could promote strong osteogenesis of hBMSCs, with enhanced expression of osteogenic markers runx2 and ocn. 20ug/ml umbilical cord stem cells secretion could promote tenogenic differentiation. The bone defect healing study using rat calvarial defect model indicated no significant difference (p»0.05) between 0.5ug/1ug umbilical cord secretion treated group (agarose gel with secretion was implanted in defect) and control (PBS) in 4 weeks or 8 weeks time points. In the rat femoral closed fracture model, the difference of bone repair between 10ug umbilical cord secretion local injection group (injected 10ug in callus after surgery) and control (PBS injected) was not significant (p»0.05) in 4 weeks or 8 weeks. In the rat articular cartilage defect model, 1ug umbilical stem cell secretion with 20ul alginate gel group recovered better than alginate gel only group in 6 weeks(p<0.05), but the difference of cartilage healing was not significant (p»0.05) between other groups (alginate gel with BMSCs) in 6 weeks or 9 weeks. In the rat patella tendon window defect model, there were more compact collagen fibers in 1ug umbilical cord secretion group (secretion with fibrin glue), but the alignment of new tissue was not better than control (PBS with fibrin glue). Also the stress of defected area was not significantly different (p»0.05) between treated and control in 6 weeks and 9 weeks. Discussion/Conclusion. The umbilical cord stem cell secretion demonstrated osteogenic, and tenogenic effect in vitro, but the result in the healing of bone, cartilage and tendon was not significant. The optimal dosage and slow release method will be considered to improve the experiment. The mechanism of stem cell secretions will be studied in further research