| Objective: Osteoarthritis（OA）is the most common clinical arthritis characterized by knee pain,cartilage degradation and osteophyte formation.OA is a complex chronic arthritis that.OA is a complex chronic arthritis that affects the entire joint structure.After the activation of matrix metalloproteinase（MMP）,cartilage,subchondral bone and synovium are all involved in the pathogenesis of OA,of which synovitis is one of the key factors.There are two main subtypes of synovial cells,namely macrophages and fibroblast-like synovial cells.Macrophages can be activated and polarized,and can quickly transition from one phenotype to another in different microenvironment.M1 macrophages secrete inflammatory cytokines such as TNF-α,IL-1 β,IL-6 and iNOS,while M2 macrophages maintain their anti-inflammatory phenotype and secrete anti-inflammatory cytokines such as IL-10.The abnormal polarization of macrophages exists in the affected joints of OA patients,and plays an important role in the occurrence and development of OA.Various signaling pathways regulate the differentiation of macrophages,but the specific mechanism has not yet been fully elucidated.Urine-derived stem cells（USC）are easy to obtain,safe and non-invasive,low cost,easy to be transported and stored,without risk of immune rejection.Compared with other sources of MSC,USC has longer telomeres and higher telomerase activity.USC has a clear advantage and therefore has a broad market prospect than other stem cells.MV has the ability to inhibit the release of inflammatory factors and regulate the occurrence of inflammatory reactions.MSC-MV can also lower levels of inflammatory factors such as TNF-α,IL-6,IFN-γ in serum,while raising the anti-inflammatory factor IL-10.Relevant experimental and clinical studies have confirmed the exact efficacy of MSC therapy for OA,but there are no further reports about whether USC derived-MV could alleviate surgically induced OA by regulating the polarization of macrophages.Therefore,based on the current theoretical background and the research basis of our group,we put forward a thesis that USC-MV may treat OA by regulating M1/M2 macrophage differentiation.In vivo and in vitro experiments were conducted,a variety of detection methods were used to explore the efficacy and related mechanisms underlying MSC and EV treat OA.Further exploration of the mechanism of extracellular（EV）on macrophage polarization in OA can provide new ideas for seeking effective strategies to prevent the development of OA.And provide a basis for the clinical transformation of autologous USC-MV as an OA therapeutic method.Materials and Methods: USC was extracted from the clean middle urine of healthy adults by multiple centrifugation,and a stable culture system of USC proliferation in vitro was established.We observe the cell morphology by microscope and draw the cell proliferation curve.The in vitro osteogenic,adipogenic,and chondrogenic differentiation capabilities of MSC and USC were identified by alizarin red,oil red O staining,and alcian blue staining/safranin O staining,respectively.Flow cytometry（FCM）was used to identify USC and MSC surface antigen.MV derived from USC and MSC were extracted after 48 h serum-free culture.NanoDrop,NTA and transmission electron microscopy（TEM）were used to measured MV protein concentration,particle size and concentration,and ultra-microstructure.WB was used to identify classic exosome surface markers of EV.We conducted surgically induced knee OA models of the rabbits using the modified Hulth surgery.30 male rabbits were randomly divided into Sham group,PBS group,USC-MV group,MSC-MV group and USC group,with a total of five groups of 6 each.After OA was successful induced,intra-articular injection was administered once a week,a total of 3 times.（1）Sham group: placebo surgery and intra-articular injection of 100 μL PBS;（2）PBS group: modified Hulth surgery and 100 μL PBS injection;（3）MSC-MV group: modified Hulth surgery and 100 μL（0.12 mg）UC-MSC-MV suspension injection;（4）USC-MV group: modified Hulth surgery and 100 μL USC-MV（0.12 mg）suspension injection;（5）USC group: modified Hulth surgery and intra-articular injection of 100 μL USCs suspension（2.0×106 cells）.The rabbits were euthanized a week after the third administration,and samples of peripheral blood,synovial fluid,urine and knees were collected.The articular cartilage of rabbits’ knees was graded using the Macroscopic score without india ink-stained.Left knee was scaned by micro CT,and three-dimensional image of the joints were evaluated using K-L grade.50-layer area above the metaphysis scanned by micro CT were quantitatively analyzed using Caliper Analyze software,and their BV/TV,BS/TV,BS/BV,Tb.Th,Tb.N,Tb.SP et al.data were analyzed.IL-1 β and TNF-α levels in serum and synovial fluid,as well as metabolite levels of bone and cartilage in serum and urine were tested by ELISA.Gene expression level associated with inflammation factors,MMP and macrophage polarization in the right knee were assessed by qPCR.HE staining was used to evaluate inflammation of joint cartilage and synovitis.MRC-1 immunohistochemistry staining evaluates the M2 macrophage expression.Mankin system and the OACH scoring system were adopted to score joint cartilage damage stained by safranin O-fast green and toluidine blue.Morphology,FCM and qPCR assess the effects of USC-MV and MSC-MV on the differentiation of THP-1-derived macrophages.Confocal microscope verifies the ability of macrophages to internalize MV.Effects of USC-MV and MSC-MV on cell proliferation capacity were detected with CCK8.Cell scratch experiments were conducted to detect the effects of USC-MV and MSC-MV on the migration capacity of M0,M1,M2 macrophages.Enzymatic digestion method established chondrocyte cell line.WB detected signaling pathway proteins;Transwell system were used to co-culture chondrocytes and macrophages.We use IL-1β to simulate the inflammatory state in OA joints,and detect the immunomodulatory effect of MV in the inflammatory inch by qPCR.Results:Primary USC culture can be attached after 3 to 7 days,three common forms can be observed: grain type,silk-linked type and shuttle type.The proliferation of primary USC was slow,but the proliferation rate increased significantly after passage,and the cell morphology was stable after multiple passage.USC and MSC can differentiate into adipocytes,osteoblasts and chondrocytes in vitro,express CD73,CD90 and CD105 antigens,and do not express CD11 b,CD14,CD34,CD45,CD19,CD79 a or HLA-DR.MSC-MV and USC-MV appeared as saucer-shaped depressed vesicles on one side,uniform in size,with a diameter between 100 and 300 nm under TEM.Both USC-MV and MSC-MV can express classic exosome markers such as Anti-CD9,Anti-FLOT1,and Anti-TSG101 proteins.The OA rabbit model was successfully constructed.The articular surface of the PBS group was rougher than that of the Sham group,the cartilage surface was severely fibrotic,and the color turned gray.The cartilage surface is obviously thinned,and subchondral bone was exposed locally.After treatment with MSC-MV,USC-MV and USC,the cartilage was significantly thicker than the PBS group,and the articular surface was smooth and complete,showing milky white.The cartilage destruction on the articular surface of the three treatment groups was improved and repaired to varying degrees.Macroscopic score had been decreased after treatment,and the difference was statistically significant,the therapeutic effect of USC-MV is comparable to that of MSC-MV and USC.Micro CT results showed that the joints in the Sham group were basically normal,with smooth joint surface,uniform joint space,no obvious joint stenosis,no obvious osteophyte formation and joint deformity.In the PBS group,there were obvious joint stenosis,rough and thin cartilage surface,asymmetrical joint space,osteoporosis and cystic degeneration of the subchondral bone,and island-shaped callus formation.The articular cartilage was slightly thickened,the tibial plateau became smooth,the trabecular bone density increased,and osteoporosis was also improved to varying degrees after treatment.K-L grade indicated that MSC-MV,USC-MV and USC treatments were all effective,and the USC-MV group reduced the K-L score most significantly.Analyzing the bone mass parameters and trabecular bone structure parameters of each group,we found that the Tb.N and Tb.Th values of the PBS group were lower,and the Tb.Sp value was higher than the Sham group.The values of Tb.N and Tb.Th increased slightly,and the value of Tb.Sp decreased after treatment,but there was no statistically significant difference except for the MSC-MV treatment.The results of ELISA indicated that levels of CTX Ⅰ and CTX Ⅱ in the urine of the PBS group were significantly higher than those in the Sham group.USC-MV can significantly down-regulate the urine levels of CTX Ⅰ and CTX Ⅱ,and MSC-MV can reduce the CTX in the urine,the difference is statistically significant.The P1 NP concentration in the urine of PBS group was lower than that of the Sham group.All three treatments could increase the P1 NP level.Among them,USC was better than MSC-MV and better than USC-MV.But only the difference of urine P1 NP levels between the USC treatment group and the PBS model group was statistically significant.However,the CTX Ⅰ,CTX Ⅱ and P1 NP in serum did not find a consistent trend in each group.Levels of serum IL-1β and TNF-α in the PBS group were higher than those in the Sham group,and the difference was statistically significant.After treatment with MSC-MV,USC-MV and USC,the levels of inflammatory factors decreased,but only serum IL-1β showed statistically difference.A similar trend was observed in synovial fluid,but the difference was not statistically significant.The qPCR results indicated that the expression of IL-1β,TNF-α and other inflammatory factors in the PBS group was higher than that in the Sham group and decreased after treatment,but only the USC treatment had statistical significance in down-regulating IL-1β.We also observed that the level of TGF-β in the PBS group decreased and increased after treatment.The effect of USC was better than MV treatment.Levels of MMP-13 and MMP-9 in the articular cartilage in the PBS group was significantly higher than that in the Sham group.USC-MV,MSC-MV and USC treatments can all down-regulate the levels of MMP-13 and MMP-9,the difference was statistically significant.Polarization state of macrophages in articular cartilage was detected by qPCR.The results suggest that the expression level of M1 macrophage marker IL-6 in the PBS group was significantly higher than that in the Sham group,and was significantly down-regulated after treatment with USC-MV and MSC-MV.MSC-MV can also significantly up-regulate the M2 macrophage marker MRC-1 level.It shows that the PBS group has a significant M1 polarization compared with the Sham group,and it shows a trend of polarization to M2 after treatment.Left knee joints of each group were stained by safranin O-fast green,toluidine blue and HE staining.Cartilage surface of Sham group was smooth and complete,the structure of each layer of cartilage was clear,and there was a complete tide line.Cartilage staining was uniform,the subchondral bone structure was normal,and the bone trabecula was uniform in Sham group.In the PBS group,uneven cartilage surface,light staining of cartilage matrix,fractures perpendicular to the bone surface appeared on the cartilage surface,reaching to the whole layer of cartilage,tide line defect or even disappeared,partial surface of articular cartilage necrosis and exfoliation,residual chondrocytes showed villous appearance,severe regional cartilage layer disappeared,and exposed bare bone surface.In the MSC-MV group,the cartilage surface was not smooth,the surface cartilage appeared fibrosis,abnormal staining,superficial ulcers and cartilage tears were seen.The surface chondrocytes are abnormal in morphology,with cell hypertrophy and cell necrosis.The cartilage layer becomes thinner,the tide line is disordered,and new capillaries grow from the subchondral bone into the cartilage layer.In the USC-MV group,the cartilage surface was uneven,the surface cartilage appeared to be cracked,and the staining was abnormal.There are new cartilage clusters formed on the surface,which are irregular or spherical masses.The layers of cartilage are arranged neatly,and the tide line moves forward.The overall cartilage thickness in the USC group was acceptable and the staining was even.The surface cartilage layer is not smooth,the superficial and transitional layers of cartilage can be seen to form shallow fissures,and new capillaries can be seen to grow into the deep cartilage.MSC-MV,USC-MV and USC treatment can significantly reduce OACH score and Mankin score,and the difference is statistically significant.And the therapeutic effect of MSC-MV is better than USC and better than USC-MV.HE staining was used to evaluate the inflammatory infiltration of the synovium.It can be seen that the synovium in the Sham group is composed of 2 to 3 layers of cells,attached to the osteochondral junction,surrounded by loose connective tissue,and no obvious mononuclear cell infiltration was found.In the PBS group,the synovium near the damaged cartilage was thickened,hyperemia,and villus formation,and a large number of inflammatory cells were seen in the interstitium.Treatment of MSC-MV,USC-MV and USC can significantly reduce inflammatory cell infiltration and synovial congestion and edema.Results of immunohistochemistry showed that MRC-1 was positively expressed on the cell membrane and cytoplasm of chondrocytes,and it appeared as brownish-yellow or tan granular deposits under the microscope.MRC-1 in the PBS group was mainly expressed on the surface of cartilage and deposited linearly along the edge of the cartilage.The cartilage in the PBS group was severely damaged,and MRC-1 was scattered on the edges of the remaining cartilage.The expression of MRC-1 in the MSC-MV group,USC-MV group and USC group was significantly increased,which was distributed in the superficial layer,middle layer,cartilage surface and the edge of the fissure.THP-1 cells showed uniform spherical suspension cells under the microscope.Stimulation of 100 ng/mL PMA for 48 h can induce THP-1 cells to differentiate into adherent M0 macrophages,and their morphology becomes flat.Wash away the non-adherent cells,and stimulate the cells with 100 ng/mL LPS and 20 ng/mL IFN-γ for 24 hours to differentiate the cells into M1.Under the microscope,there are many protrusions in the cells,which are slender and shuttle-shaped.FCM showed that more than 50% of the cells were CD80 positive,indicating that they successfully differentiated into M1 macrophages.20 ng/mL IL-4 and 20 ng/mL IL-13 could induce the cells to differentiate into polygonal or round-like M2 macrophages.The mRNA expression of polarization-related genes in macrophages was verified by qPCR.At about 6 h,the expression of most M1 and M2 polarization genes increased significantly,and some reached their peaks.The qPCR results showed that the expression of M1-related genes CD80,i NOS,IL-12 P40,IL-6 were significantly higher after inducing macrophages to polarize to M1,as well as the expression of inflammation-related genes such as IL-1β,TNF-α,COX-2,NF-κB.Lilely,levels of M2 related genes CD206,Fizz-1,DC-Sign increased significantly after M2 polarization.MVs were stained with the fluorescent dye PKH26 and incubated with CFSE-stained macrophages for 3 hours.The CFSE-treated macrophages showed green fluorescence,and EVs showed red fluorescence after PKH67 staining.No red fluorescence was found in macrophages that were not co-cultured with EV.For macrophages added with MSC-EV or USC-EV,red fluorescence was found in the cells.CCK8 results suggest that MSC-EV or USC-EV could increase the proliferation rate of THP-1 cells as the concentration of MV increases within the concentration range of 1*1031*1010/mL.In the MV concentration range of 5*1082*109/mL,compared with untreated cells,the proliferation rate of RAW264.7 cells increased with the increase of MV concentration.Under the microscope,it can be seen that after 24 hours of treatment with MSC-MV or USC-MV,M0 cells appeared many protrusions,the cell morphology was polygonal,and brown particles appeared in the cells,which are characteristics of M2 macrophages.When stimulated by LPS and IFN-γ,the cells protruded from the pseudopodia and became slender and shuttle-shaped,showing the characteristics of M1 macrophages.When M0 was incubated with MSC-MV or USC-MV for 1 h and then stimulated by LPS and IFN-γ,both spindle cells and polygonal cells appeared after 24 h.Results of qPCR indicated that pretreatment with different concentrations of MSC-EV or USC-EV could block the M1 polarization process,and up-regulated the expression of M2 polarization related genes,such as CD206,Arginase-1,LOX-1 and IL-10.MSC-MV at 109/mL and 1010/mL concentrations and USC-MV at 107/mL,108/mL and 109/mL concentrations had the best effect.The scratch test results indicated that USC-MV and MSC-MV at a concentration of 1*109/mL could promote the migration of M0,M1 and M2 cells,and USC-MV was superior to MSC-MV.The primary chondrocytes were successfully cultured,and the morphology of the cells after adherence was polygonal.As the number of passages increased,the cells gradually became long spindle-shaped.Chondrocytes were identified by the methods of alcian blue and safranin O staining.M0 or M1 macrophages were co-cultured with chondrocytes with or without 10 ng/mL IL-1β.The results showed that levels of IL-1β,TNF-α,MMP-13 and IL-6 were increased and the levels of IL-10 were decreased after the addition of IL-1β stimulation.In the culture system of USC-MV and MSC-MV at the concentration of 2*109/mL,the changes in the levels of these inflammatory factors could be reversed.WB detects PI3K-Akt-mTOR and NF-κB pathway proteins.NF-κB pathway is activated in the M1 polarization state,and P65 protein is phosphorylated.Both MSC-EV and USC-EV can down-regulate NF-κB by inhibiting the phosphorylation of P65,and the inhibiting effect is concentration-dependent.Akt pathway is activated in the M2 polarization state,and the Akt is phosphorylated.Both MSC-EV and USC-EV can activate the P-Akt pathway by up-regulating the phosphorylation of Akt,and it is concentration-dependent.However,it has no obvious regulatory effect on the expression and phosphorylation of PI3 K and mTOR pathway proteins.Conclusion:1.USC can be isolated and extracted from the clean mid-stage urine of healthy adults,and stably expanded in vitro,expressing the surface markers of stem cells and having the potential for multidirectional differentiation.USC-MV and MSC-MV are shown as saucer-shaped vesicles with uniform size and diameter between 100 and 300 nm,expressing classic exosomal markers.2.MSC-MV,USC-MV and USC treatment can reduce the serum inflammation level of OA,regulate cartilage metabolism and bone metabolism,reduce and repair cartilage damage on the articular surface,reduce synovial inflammation,reduce OACH score and Mankin score at the same time.The macrophages in OA articular cartilage showed a tendency of M1 polarization and could polarize to M2 after treatment.3.MV can be successfully internalized by macrophages.M1-related genes CD80,iNOS,IL-12 P40,IL-6 and inflammatory related genes such as IL-1β,TNF-α,COX-2,NF-κB were significantly increased when M1 polarization.While after M2 polarization,the expression levels of M2-related genes CD206,FIZZ-1 and DC-Sign were significantly increased.4.MSC-EV and USC-EV can promote the proliferation and migration of macrophages in vitro,block the M1 polarization induced by LPS and IFN-γ,and induce macrophages polarized to M2.5.MSC-EV and USC-EV may increase the phosphorylation of Akt,activate the PI3K-Akt-mTOR pathway,and/or block the phosphorylation of P65 protein to inhibit the activation of the NF-κB pathway,thereby blocking the M1 polarization process and polarizing macrophages towards M2.6.We used the Transwell system to co-culture chondrocytes and macrophages,and used IL-1β to stimulate chondrocytes in a simulated OA joint inflammation.We found that USC-MV and MSC-MV can down-regulate the secretion of IL-1β-activated chondrocytes.The level of inflammatory factors indicates that MV can still play a normal anti-inflammatory effect in the inflammatory microenvironment. |
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