Effects Of Stem Cells Mediated With Xanthan Gum In The Treatment Of Osteoarthritis

ObjectivesOsteoarthritis(OA)is a chronic articular disorder that is pathologically characterized by progressive cartilage destruction and inflammation of synovium and synovial fluid.Articular cartilage does not heal spontaneously under physiological circumstances due to innate avascularity,lowcellularity and low cell turnover.The epidemiology of OA is multifactorial and complex,with age,obesity,career,genetic,hormone level.The current therapeutic options for OA include the use of pain medications,nonsteroidal anti-inflammatory drugs(NSAIDs),lubricating supplements,and surgical interventions.However,these approaches only focus on temporarily alleviating the symptoms rather than treating the pathogenesis of the disease or reversing the process of OA.Recent years,stem cells used in the treatment of OA due to their ability of chondrogenic differentiation and immunomodulatory effect.Adipose tissue derived stem cells(ADSCs)are abundant and easily acquired by liposuction with minimal donor-site morbidity.In particular,various animal studies and clinical trials have demonstrated promising results following ADSC therapy for OA.Although there has been some promising progress toward the clinical use of ADSCs,a number of problems have been identified.All clinical trials that have reported the use of ADSCs for the treatment of OA have relied on the use of autologous cells from the stromal vascular fraction(SFV),and most animal studies have also focused on autologous ADSCs.The application of allogenic ADSCs for the treatment of OA is limited.However,the expression of MHC-class-II molecules in ADSCs is low or absent,making it possible to apply allogenic ADSCs for the treatment of degenerative diseases and immune disorders.Additionally,allogenic cells can be a practical 'off the shelf' therapeutic agent because they can be isolated from healthy donors and cultured in advance.The therapeuticeffect of ADSCs treatment on OA has been hindered due to various limitations such as low viability in articularcavity.Currently,the most commonly used vehicles for articular stem cell transplantation are fibrin glue,natural polymers such as hyaluronic acid(HA),and nutritional body fluid such as platelet-rich plasma(PRP).Although many studies support the use of HA for OAtreatment,the use of HA is controversial because HA is not stable in vivo and is quickly degraded through hydrolytic or enzymatic reactions and its degradation products(e.g.,low-molecular-weight HA)have been shown to mediate inflammatory effects.Xanthan gum(XG)has a similar viscoelasticity to and is more stable than HA and is not easily degraded in vivo.Our previous studies demonstrated that intra-articular(IA)injection of XG could protect articular cartilage during OA progression.We hypothesizethat XG can be used as an effective vehicle for ADSCs for OA treatment.In the present study,we evaluated the feasibility of XG combined with ADSCs(XG-ADSCs)for OA treatment.Methods1 Isolation,culture and identification of ADSCsRat ADSCs was isolated from inguinal and abdominal subcutaneous fat by type ?collagenasedigestion.Morphology of ADSCs was observed by nverted phase contrast microscope.Surface markers were analyzed by flow cytometry.ADSCs were performed adipogenic,osteogenic and chondrogenic differentiation to detect Multilineage differentiation potential.2 Effects of XG on proliferation of ADSCs in vitroADSCs were treated with different concentration of XG,morphology of ADSCs was observed by nverted phase contrast microscope.Cell proliferation was detected by Cell Counting Kit-8(CCK-8).Cloning formation was detected by crystalvioletstaining.Cell cycles were analyzed by Muse Cell Cycle Detection Kit.3 Protection effects of XG on hydrogen peroxide-treated ADSCs in vivoADSCs were pre-treated with different concentrations of XG,followed by treatment with hydrogen peroxide and XG.morphology of ADSCs was observed by SEM.DNA fragmentation was detected by TUNAL.Cell apoptosisrate was assayed by Annexin V and Hochest 33258 staining.The level of intracellular reactive oxygen species(ROS)was assayed by DCFH-DA.Mitochondrial permeability transition(MPT)was detected by MitoTracker Red CMXRos.4 Study of culture-expanded allogenic adipose tissue-derived stem cellstreatment on experimental osteoarthritisModel making ofanterior cruciate ligament transaction(ACLT)induced rat OA:ACL of rat right knee was transected to induce OA model.Rats were sacrificed at 2 weeks,4 weeks,8 weeks and 12 weeks.The damage of articular cartilage was detected by macroscopic and histologicalanalysis.4 weeks after surgery,the OA rats were randomLy divided into an ADSCs group and a control group.Approximately 1 × 106 culture-expanded allogenic ADSCs suspended in 60 ?L PBS were intra-articularly injected into the OA joints of the rats in the ADSC group,whereas the control group received 60?L PBS without cells.Behavior,body weight,and food intake were detected throughout the experimental period.Animals were sacrificed at 8 weeks and 12 weeks after ACLT surgery.Knee width was measured by vermiercaliper.The damage of articular cartilage was detected by macroscopic and histological analysis.Hematology and blood biochemical indexes were detected at 12 weeks.Chondrocytes were treated with IL-1? to mimic OAchondrocytes.The OA chondrocytes were co-cultured with ADSCs for 7 d.The mRNA expression levels of the matrix-degrading enzymes MMP-3 and MMP-13 and the pro-inflammatory cytokines TNF-?,IL-6,and IL-10 were detected by PT-PCR.5 Study of ADSCs in combination with xanthan gum treatment on experimentalosteoarthritisAt 4 weeks after surgery,the OA rats were randomLy divided into four groups:the XG-ADSCs group,the ADSCs group,the XG group,and the PBS group.Rats in the XG-ADSCs group and the ADSCs group received an IA injection of 1×106 ADSCs suspended in 60 ?L of 1%XG or PBS,while rats in the XG group and the PBS group received 60 ?L of 1%XG or PBS without cells,respectively.Changes in hind paw weight distribution was measured at 1 week,2 weeks,3 weeks and 4 weeks.The damage of articular cartilage was detected by macroscopic and histological analysis.Expression level of type II collagen and MMP-13 were examined by immunohistochemistry.Protein levels of MMP-3,MMP-13,TNF-? and IL-1? in synovial fluid were measured by ELISA.For tracing the viability of ADSCs in articular cavity,ADSCs were labeled by RFP-LucR.The total flux of various numbers of RFP-Luc-ADSCs was examined by Xenogen IVIS Lumina Imaging System in vitro.At 4 weeks after surgery,the OA rats were randomLy divided into tow groups:XG-ADSCs group and ADSCs group.Rats in XG-ADSCs groupreceived anIA injected 1×106 RFP-Luc-ADSCs were suspended into 60 ?L1%XG.Rats in ADSCs group received an IA injection of 1×106RFP-Luc-ADSCs suspended in 60 ?L of PBS.Intra-articular bioluminescence imaging of RFP-Luc-ADSCs was performed 1,7,14 and 28 d after injection.Results1 Successfully isolated and cultured rat ADSCsADSCs were successfully isolated and cultured.The ADSCs exhibited a typical fibroblast-like morphology.ADSCs werepositive for CD90 and CD44 and negative for CD45 and CD11b.The dipogenic differentiated ADSCs formated ytoplasmic lipid droplets and were positively stained with oil red O.The osteogenic induction of ADSCs resulted in positive staining by alizarin red S.Chondrogenic induction resulted in increased expression of type ?collagen,2 Xanthan gum promoted the proliferation of ADSCs and protected ADSCs from H2O2XG has no toxic effect on ADSCs.There was a significant increase of OD450 in ADSCs that were treated with XG respect to the control group.The numbers and size of cell cloning were significantly increased in XG treated group compared with control group.XG treatment promoted ADSCs cell cycle from G0/G1 phase into S phase and G2/M phase.XG pre-treatment decreased the H2O2 induced apoptosis and intracellular ROS of ADSCs.XG pre-treatment inhibited the H2O2 induced MTP of ADSCs.3 Successfully induced rat OA modelMacroscopic and histological analysis indicated that severity of articular cartilage damage and macroscopic and hitological scores of articular cartilageincreased as time passed away.The scores of articular cartilage at 4 weeks,8 weeks and 12 weeks group were significantly higher than the sham operated group.4 Culture expanded allogenic ADSCs inhibited cartilage degeneration of experimental OABehavior,body weight,and food intake were all normal for all experimental rats.ADSC treatment did not cause any local adverse reactions,such as swelling or joint redness.The hematological and hemato-biochemical parameters and histology of the main organs were normal for all experimental rats.At 8 weeks and 12 weeks,the knee width of ADSCs group was significantly narrow than the control group.The degeneration of articular cartilage was significantly weaker in the ADSC group compared to that in the control group at both 8 and 12 weeks.Co-culture with ADSCs counteracted the IL-1? induced mRNA upregulation of the extracellular matrix-degrading enzymes MMP-3 and MMP-13 and the pro-inflammatory cytokines TNF-a and IL-6 in chondrocytes.Importantly,ADSCs increased the expression of the anti-inflammatory cytokine IL-10 in chondrocytes.5 ADSCs in combination with XG attenuated osteoarthritis progression in an experimental OAFor the rats in the XG-ADSC-s and ADSCs-treated groups,the weight-bearing percentage of the right hind limb was significantly increased compared to that in the PBS group and was sustained over 4 weeks.However,the positive effect in the XG-ADSCs group was significantly greater than that in the ADSCs group.For the rats in the XG group,the efficacy decreased during the third week after surgery.The articular cartilage was relatively normal in the XG-ADSCs group,and moderate degeneration was observed in the ADSCs and XG groups.The expression level of type II collagen was higher in sham group and XG-ADSCs group,slight loss in expression was observed in ADSCs and XG group,severe loss in expression was observed in PBS group.Low expressionof MMP-13 were observed in sham group and XG-ADSCs group,slight expression were observed in ADSCs and XG group,and the high expression was observed in PBS group.ADSCs and XG-ADSC treatments significantly decreased the concentrations of IL-1?,TNF-?,MMP-3 and MMP-13 in synovial fluid;however,the attenuating effect of the XG-ADSCs treatment was significantly enhanced compared with that of the ADSCs treatment alone.Total flux of various numbers of RFP-Luc-ADSCs was positivecorrelation with cell numbers.The total flux of ADSCs group and XG-ADSCs group had no significant differences at 1 d.The total flux of XG-ADSCs group was significantly stronger than the ADSCs group at 7 d(P<0.05)and 14d(P<0.01).The total flux of XG-ADSCs group was stronger than the ADSCs group at 28 d,but not significantly(P>0.05).The total flux of both groups increased before 14 d and decreased after 14 d.Conclusions1.Successfully isolated and cultured rat ADSCs.2 XG has no toxic effect on ADSCs.XG could increase proliferation and cloning formation of ADSCs and promote ADSCs cell cycle from G0/G1 phase into S phase and G2/M phase.3.XG pre-treatment decreased the H2O2 induced apoptosis and intracellular ROS of ADSCs.XG pre-treatment inhibited the H2O2 induced MTP of ADSCs.4.ADSCs treatment did not cause any adverse local or systemic reactions.IA injection of culture-expanded allogenic ADSCs attenuated cartilage degeneration of rat OA.The paracrine effects of ADSCs on OA chondrocytes are at least part of the mechanism by which ADSCs exert their therapeutic activity.5.IA injection of allogeneic ADSCs combined with XG efficiently attenuated OA progression with a therapeutic effect that was significantly greater than that of either ADSCs or XG alone.IA injection of XG-ADSCs might be an effective treatment for OA in humans.