An Preliminary Study Of Mesenchymal Stem Cells Combined With TGF-β3,HA And PTHrP In Repairing Cartilage Defects Of Osteoarthritis

BackgroundOsteoarthritis is a common degenerative disease,often occurring in middle-aged and elderly people,mainly involving weight-bearing joints-knee joint,hip joint,cervical vertebrae,lumbar vertebrae and so on.The clinical onset is relatively slow.The main symptoms are joint pain,limited mobility,dysfunction,etc.The most significant general changes are articular cartilage damage,bone loss and changes in the shape of the articular surface.The repair of articular cartilage is very slow and difficult because it has no blood supply and no distribution of nerves and lymphoid tissues.There is no clinically effective treatment for osteoarthritis.Mesenchymal stem cells(MSCs)belong to pluripotent stem cells,which are derived from the early mesoderm.They have low immunogenicity,good proliferative capacity and multipotential differentiation potential,and have been widely studied and used by scholars.At present,MSCs have been injected into the knee joint cavity to treat osteoarthritis,mainly by dividing MSCs into chondrocytes and then repairing damaged articular cartilage.It may be more effective in repairing articular cartilage,if it can promote chondrogenic differentiation.Umbilical cord mesenchymal stem cells(UC-MSCs)have stronger proliferative capacity and differentiation potential than bone marrow mesenchymal stem cells(BMSCs).Growth factors are indispensable components of MSCs during chondrogenic differentiation.Transforming growth factor p(TGF-p)is a classical inducing factor to induce chondrogenic differentiation of mesenchymal stem cells.Hyaluronic acid(HA),one of the main components of the cartilage matrix,can be used as a regulatory factor to induce cartilage differentiation.Parathyroid hormone related protein(PTHrP)can inhibit chondrocyte hypertrophy within a certain range.So,transforming growth factor β3(TGF-β3)was used as a control,which combined with HA and PTHrP,to observe its effect on chondrogenic differentiation.Furthermore,it was applied to a mouse osteoarthritis model to observe the effect on articular cartilage repair in vivo.ObjectiveTo investigate the effects of TGF-β3,HA and PTHrP on chondrogenic differentiation of HUC-MSCs in vitro,and to optimize the combination of culture conditions.A model of knee osteoarthritis in mice was constructed to observe the effect of repairing articular cartilage in vivo.Methods1.Effects of TGF-β3,HA,PTHrP on chondrogenic differentiation of HUC-MSCs and;Isolation and culture of C57 mouse adipose-derived mesenchymal stem cells:Umbilical cord was isolated in vitro,and HUC-MSCs were cultured in an adherent manner for morphological examination,surface antibody detection,adipogenic and osteogenic differentiation.P3 generation cells were added with different combinations of cytokines for chondrogenic differentiation,which were divided into 1-21d TGF-β3(G1),1-28d TGF-β3(G2),and 1-21d TGF-β3/1-21d HA(G3),1-28d TGF-β3/1-28d HA(G4),1-21d TGF-β3/1-21d HA/14-21d PTHrP(G5),1-28d TGF-β3/1-28d HA/14-28d PTHrP(G6),1-28d TGF-β3/1-28d HA/21-28d PTHrP(G7).The C57 mouse fat was isolated in vitro,and Mouse adipose-derived mesenchymal stem cells(MAD-MSCs)were cultured in an adherent manner for morphological examination,surface antibody detection,adipogenic,osteogenic and chondrogenic differentiation.2.C57 mouse knee osteoarthritis model:Medial meniscectomy was used.The medial incision of the knee joint was taken,and the switch capsule was cut along the upper edge of the patellar ligament,the intact medial meniscus was removed,and then the incision was sutured.Mice were euthanized at 4 weeks and 8 weeks post-operatively.Lower limbs were fixed in formalin for 72 hours and whole knee joint were decalcified for 20 minutes.hematoxylin-eosin staining(HE),Alcian Blue-Periodic acid Schiff staining(AB-PAS)and Immunohistochemical staining of type Ⅱ collagen were performed to observe the expression of cartilage matrix and cartilage matrix protein in knee joint of mice.The MANIN score assessed the extent of osteoarthritis.3.Repair of osteoarthritic cartilage in mice by different MSCs and HUC-MSCs combined with TGF-β3,HA and PTHrP:After successful modeling of osteoarthritis in C57 mice,the bilateral knee joints were injected with 10 ul of normal saline,HUC-MSCs,MAD-MSCs,and HA TGF-β3 PTHrP HUC MSCs.Eight mice were taken in each group after 4 weeks and 8 weeks.The knee joint was removed to formalin for 72 hours and then decalcified.HE staining,safranin-fixed green staining were performed to observe the expression of cartilage matrix and cartilage matrix protein in knee joint of mice.The MANIN score assessed the extent of osteoarthritis.Results1、Cultured for 28 days Compared with the 21 days,the expression levels of COL2α1,COL10α1,SOX9 and ACAN increased in group TGF-β3 and the group TGFβ3/HA;in the group TGFβ3/HA/PTHrP,the expression levels of COL2al,SOX9 and ACAN increased,the COL10α1 decreased.When cultured with the same time,compared with the TGF-β3 group,the expressions of COL2α1,SOX9 and ACAN was increased in the TGFP3/HA group;compared with the TGF-β3/HA group,the expression levels of COL2α1,ACAN and SOX9 increased,the expression of COL10α1 decreased in the TGFβ3/HA/PTHrP group,and the effect of PTHrP addition for 2 weeks was more obvious than that of 1 week.The difference was statistically significant(p<0.05).The results of HE and Alcian blue staining were consistent with the results of real-time qPCR.2、After 4 weeks of modeling,the cartilage tissue structure was disordered but the layers could be divided,the joint surface was slightly worn and not smooth,the chondrocytes were mildly moderately hyperplasia,some areas were mild to moderate fibrosis,and the AB-PAS staining was mildly moderately reduced.The line is complete or multi-level.After 8 weeks of modeling,the cartilage tissue structure was severely disordered,the layers were inseparable,cracks appeared,the chondrocytes were too small,the AB-PAS staining was severely reduced or uncolored,the tidal line was blurred,the synovial tissue was proliferated and villi-like extended into the joint cavity.The expression of type II collagen was decreased with time to varying degrees,the MANKIN score was statistically different at 4 weeks and 8 weeks.those indicated that the degree of arthritis was progressive and the modeling was successful.3.After 4 weeks and 8 weeks of treatment,it was found that the articular cartilage surface had different degrees of repair.In the group of saline,the surface of the articular cartilage of the knee joint was not smooth,the structure of the cartilage was disordered,the safranin-fixed green staining were very light or uncolored.Compared with the saline group,the repaired cartilage layer of HUC-MSCs and MAD-MSCs group was thinner,the cartilage matrix was less,the positive staining area was slightly enlarged and the staining was deeper,and the MANKIN score was lower(P<0.05).However,the difference between the HUC-MSCs and MAD-MSCs group was not statistically significant(P>0.05).The HA+TGF-β3+PTHrP+HUC-MSCs group had a slightly thicker cartilage layer and more cartilage matrix,the lower MANKIN scores(P<0.05),as against the single cell group.Conclusion1.By flow cytometry,adipogenic differentiation,osteogenic differentiation,chondrogenic differentiation,they are identified as HUC-MSCs and MAD-MSCs,and have the potential of multi-directional differentiation which consistent with the definition of mesenchymal stem cells.TGF-β3,HA,and PTHrP can promote cartilage differentiation to different extents.TGF-β3 and HA are combined for 28 days,PTHrP is added on the 14th day.This combination promotes the best chondrogenic differentiation of HUC-MSCs,and inhibited cell hypertrophy.2.Medial meniscus resection can successfully construct a mouse knee osteoarthritis model,and the degree of lesions is progressive.So,medial meniscus resection can be used for the study of osteoarthritis.3.Intra-articular injection of HUC-MSCs and MAD-MSCs can promote the repair of articular cartilage in mice with osteoarthritis.There is no immunological rejection in the injection of HUC-MSCs into the knee joint.There is no difference in the repair effect.HUC-MSCs combined with HA,TGF-β3,and PTHrP can promote the repair of knee joint articular cartilage in mice and inhibit the hypertrophy of chondrocytes.