The Effect Of Needle Knife Combined With Stem Cell Intervention On The Proliferation Of KOA Rabbit Chondrocytes

ObjectiveArthritis is the most common aging-related skeletal muscle disease.The number of patients worldwide accounts for 26.6%of the total population.It ranks in the top 4 of the top ten high-risk diseases for women and men for the top 8.Knee joints account for the largest proportion of affected joints in Asians.The incidence of knee osteoarthritis(Knee Osteoarthritis,KOA)in people over 60 years of age in China has reached 80%.With the increasing aging of the global population,research on prevention and treatment of KOA has become a public health issue related to national economy and people's livelihood.Biomechanical factors and lack of stem cells after aging are considered to be the main causes of KOA.In recent years,stem cells have carried out many researches on interventions for arthritis due to their anti-inflammatory and regenerative properties.The regulation of systemic infusion and targeted injection of affected areas have also become one of the emerging effective intervention methods for arthritis.Needle-knife therapy is guided by the traditional theory of traditional Chinese medicine and tendons,combined with modern anatomical and biomechanical intervention methods.The clinical effect of KOA is definite.The basic research has also made some progress.Previous studies have found that the needle-knife releases muscles and tendons to restore the knee joint Mechanical imbalance and benign stress stimulation promote cartilage anabolic metabolism and ease cartilage matrix degradation.The ability of chondrogenic differentiation and paracrine effects of stem cells to regulate the microenvironment has been proven;can stem cells play a synergistic role after the needle knife regulates the mechanical properties of KOA to improve the cartilage stress environment?This study started with the study of chondrocyte proliferation-related factors p-AK T,GSK3?,CyclinD1,and CDK4,combined with the biological characteristics of stem cells,and studied the intervention methods of needle knife,stem cells,needle knife and stem cell intervention on KOA rabbit behavior,knee joint imaging,Morphology of cartilage,and molecular biology of chondrocytes.The aim is to verify that needle-knife combined with stem cells interfere with KOA,strengthen the molecular biological mechanism of needle-knife for repair of cartilage damage,and also make stem cell therapy play a better regeneration role,and provide experimental basis for needle-knife combined with stem cell therapy for KOA.MethodsThirty clean and healthy New Zealand male rabbits aged 6 months were divided into 5 groups of 6 animals each.The blank group was left without any treatment.The other group was Videman method.Intervention began in the week.The model group did not intervene;in the acupuncture group,one week after modeling,the rabbit left knee had medial femoral muscle,lateral femoral muscle,rectus femoris,biceps femoris tendon attachment point,and peri-knee muscle abdominal cord nodules.Needle-knife operation was performed twice a week for 4 weeks.In the first week of the intervention,the stem cell group injected 1 million stem cells in 2 ml of 0.9%sodium chloride solution and injected it into the ear margin vein.On the same day of the first intervention in the Zhou Needle Knife group,100,000 stem cells were dissolved in 2 ml of 0.9%sodium chloride solution and injected into the rabbit joint cavity.The needle-knife combined with the stem cell group,based on the same operation of the needle-knife group,is the same time and the same dose of stem cells as the stem cell group.One week after the intervention,rabbits in each group were modified with Lequesne MG knee joint behavior score,knee joint PROM comparison,knee MRI imaging observation,knee cartilage scanning electron microscope observation,and RT-PCR to detect rabbit cartilage p-AKT,GSK3?,CyclinDl,and CDK4 mRNA expressions.Immunohistochemistry and Western Blot were used to detect p-AKT,GSK3?,CyclinD1,and CDK4 protein expressions in rabbit cartilage.The data was statistically analyzed using SPSS 20.0 statistical software,and the sample data of each group was expressed as mean±standard deviation(±s);if the data conformed to the normal distribution and the variances were uniform,the comparison between multiple groups was performed by One-way ANOVA(One-way ANOVA));,Followed by pairwise comparison by LSD test;if the variance is not uniform,the Welch method is used.If the data is not normally distributed,a non-parametric test is used.P<0.05 was considered statistically significant,and P<0.01 was considered statistically significant.Results1 Rabbit knee joint behavior score results of each group6equesne MG score:Compared with the blank group,the Lequesne MG score of the model group,needle knife group,stem cell group,and needle knife plus stem cell group was significantly increased after modeling(P<0.01)and there was no significant difference between each group(P>0.05).Compared with the model group,the Lequesne MG score of the stem cell group decreased after the intervention(P<0.05),and there was no statistical difference in the Lequesne MG score of the needle knife group and the needle knife plus stem cell group(P>0.05).Compared with before intervention,the Lequesne MG score of the model group was not statistically different after the intervention(P>0.05);the Lequesne MG score of the needle-knife group and the stem cell group was significantly reduced after the intervention(P<0.01);the needle-knife plus stem cell group The Lequesne MG score decreased after the intervention(P<0.05).PROM value:Compared with the blank group,the PROM values of the model group,the needle knife group,the stem cell group,and the needle knife plus stem cell group were significantly reduced after modeling(P<0.01),and there was no significant difference between the groups(P>0.05).Compared with the model group,the PROM value of the needle-knife group and the needle-knife-plus-stem cell group increased significantly after modeling(P<0.01),and the PROM value of the stem cell group was not statistically different(P>0.05).Compared with the pre-intervention,the PROM value of the model group was not statistically different after the intervention(P>0.05);the PROM value of the needle-knife group and the needle-knife plus stem cell group were increased after the intervention(P<0.05);There was no significant difference in the PROM value after the operation(P>0.05).2 MRI imaging results of rabbit knee joints in each groupIn the normal group,the cartilage surface was smooth and intact,bilateral joint spaces were equally wide,and there was a small amount of fluid in the joint cavity.In the model group,the cartilage was thinner,the surface was frizzy,the joint space was narrowed,the joint cavity effusion increased,the bone marrow was edema,the soft tissue was significantly swollen,and the fascial fluid was intact.Soft tissue swelling and fascial edema can still be seen in a small amount of fluid;individual cells can see frizz on the cartilage surface,and a large amount of joint effusion can clearly see swelling of soft tissue and fascia;Cavity fluid,occasionally soft tissue swelling,fascial edema.3 Scanning electron microscope observation results of rabbit cartilage in each groupIn the blank group,the cartilage surface was smooth,the surface amorphous material was uniformly covered and the bulge was shallow,and the superficial structure was basically complete;in the model group,the cartilage surface was found to be thin or even disappeared,the bright layer was completely disappeared,and the underlying collagen fibers and amorphous material were exposed.The superficial flat chondrocytes below;in the needle-knife group,the surface of the cartilage is rough,and the smooth surface is seen.The ridges are arranged neatly,and the surface is covered with amorphous material.The exfoliation and collagen fibers are not seen,and the shallow pits are obvious.The stem cell group It can be seen that the surface of the cartilage is rough,and the amorphous material appears to be lumpy and cracked.Partially,even exfoliation,collagen is lifted,the shallow pit structure disappears,the ditch deepens and increases;the needle cartilage plus stem cell group shows that the cartilage surface is smooth,and the amorphous material is covered relatively uniformly.The ditch became deeper and arranged regularly,but no obvious parallel arrangement was seen.Small fissures were common,and the cartilage surface was full of pits with regular size and morphological structure.The gaps were uniform and the collagen fibers were bare.4 Rabbit cartilage p-AKT,GSK3?,CyclinDl,CDK4 PCR test results in each groupCompared with the blank group,the expression of p-AKT and GSK3? mRNA in the model group was significantly increased(P<0.01),and the expression of CyclinDl and CDK4 mRNA was decreased(P<0.05).Compared with the model group,the expression of p-AKT and GSK3? mRNA in the needle-knife group was significantly reduced(P<0.01),and the expression of CyclinDl and CDK4 mRNA was increased(P<0.05,P<0.01);the expression of p-AKT and GSK3? mRNA in the stem cell group was significantly reduced(P<0.01),CDK4 mRNA expression was significantly increased(P<0.01);p-AKT,GSK3? mRNA expression was significantly reduced in the needle knife plus stem cell group(P<0.01),and CyclinDl,CDK4 mRNA expression was increased(P<0.05,P<0.01).Compared with the stem cell group,the GSK3? mRNA expression in the needle-knife group and the needle-knife plus stem cell group was significantly reduced(P<0.01),and the CDK4 mRNA expression was increased(P<0.05)5 Immunohistochemical results of p-AKT,GSK3?,CyclinDl,and CDK4 in rabbit cartilage in each groupCompared with the blank group,the expression of p-AKT and GSK3? protein was significantly increased in the model group(P<0.01),and the expressions of CyclinDl and CDK4 protein were significantly decreased(P<0.01).Compared with the model group,the expression of p-AKT protein in the needle-knife group was decreased(P<0.05),and the expression of CyclinDl protein was increased(P<0.05);the expression of p-AKT and GSK3?protein in the needle-knife plus stem cell group was reduced(P<0.05).CyclinD1,CDK4 protein expression was increased(P<0.01).There were no significant differences in the expression of p-AKT,GSK3?,CyclinDl,and CDK4 between the three groups of the needle knife group,the stem cell group,and the needle knife plus stem cell group(P>0.05)6 Rabbit cartilage p-AKT,GSK3?,CyclinDl,CDK4 Western Blot test resultsCompared with the blank group,the expression of p-AKT and GSK3? protein in the model group was significantly increased(P<0.01),and the expressions of CyclinDl and CDK4 protein were significantly reduced(P<0.01).Compared with the model group,CyclinDl protein expression in the needle knife group was significantly increased(P<0.01);p-AKT,GSK3? protein expression was reduced in the needle knife plus stem cell group(P<0.05,P<0.01),and the expressions of CyclinDl and CDK4 protein were significantly increased.High(P<0.01).There was no significant difference in the expression of p-AKT and GSK3? protein between the three groups of the needle knife group,the stem cell group,and the needle knife plus stem cell group(P>0.05).Compared with the stem cell group,the expression of CyclinDl protein in the needle knife group increased.High(P<0.05),CyclinD1,CDK4 protein expression increased in the needle knife plus stem cell group(P<0.05);compared with the needle knife group,CDK4 protein expression in the needle knife plus stem cell group increased(P<0.05)Conclusion1 Needle knife and stem cell intervention can inhibit the knee arthritis reaction in KOA rabbits,relieve cartilage degeneration,and improve joint mobility.However,needle-knife intervention is more advantageous in improving knee joint activity,and stem cell intervention is more advantageous in reducing KOA level;2 Needle-knife and stem cell intervention can regulate the expression of KOA chondrocyte proliferation-related factors p-AKT,GSK3?,CyclinD1,and CDK4 mRNA and protein,while needle-knife combined with stem cells can synergistic ally reduce p-AKT and GSK3? mRNA in KOA chondrocyte And protein expression,synergistic ally promote the expression of CyclinDl,CDK4 mRNA and protein,confirming that the combination of needle knife and stem cells is more conducive to repair of KOA cartilage damage.