| ObjectiveKnee osteoarthritis is a common disease in the elderly, connects with rheumatism involving bone in Chinese medicine. The main TCM pathogenes are renal deficidncy and blood stasis. This disease is the syndrome of the root (Ben) deficiency and the branch(Biao) sthenia. In this case, the root is deficiency of kidneyenergy while the branch is blood stasis and phlegm stasis. Therefore, invigorating kidney and promoting blood flow treatment is the prevailing therapy. Kidney Jing encompasses both kidney yin and kidney yang. The root cause is kidney-Yang deficiency. Treatment focuses on warming and recuperating kidney Yang. This study was designed to observe the clinical curative effect of warming kidney and activating blood circulation to Treat Knee OA.Recently, research has found that articular cartilage degeneration is the root cause in OA. Once articular cartilage is injured or degenerated, it has very limited capacities for self-repair and regeneration. the regeneration of articular cartilage is a continuing challenge in orthopaedic practice. Under the circumstances, tissue engineering approaches have been investigated with the aim to repair cartilage. Human stromal mesenchymalstem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e. g. osteoblasts and chondroblast and thus they are being introduced into clinical trials for cartilage regeneration.Chinese medicine considers the kidney generating marrow and dominating bone, and the growth and repair of bone and cartilage depends on the Kidney Jing. Experimental kidney-Supplementing medicine might promote the body to produce growth factors and promote proliferation and differentiation of osteogenic, chondrogenic cells which was studied mainly on the metabolism and endocrine of bone and cartilage and the differentiation of mesenchymal stem cells by modern medical research. In this study, from bone marrow have been isolated, cultured in vitro in order to observe proliferation of hMSCs and differentiation of chondroblast induced by Epimedium icariin which is the active monomer components of Kidney-Warming Chinese Medicine Longspur Epimedium.MethodClinical research:All the cases were selected from orthopedic outpatient department of Guangzhou Hospital of Integrated Chinese and Western Medicine diagnosed with knee OA, a total of80cases were divided into Wenshen Huoxue group, Wenshen group,Huoxue group and the control group by random table. Patients in the Wenshen Huoxue group were treated with the combination of Xianlinggubao capsule and Huayuzengtong capsule, Wenshen group were treated with Xianlinggubao capsule, Huoxue group were treated with Huayuzengtong capsule, while the control group were treated with glucosamine capsule for4weeks. According to the Phase II clinical trial program with knee osteo-arthritis syndrome semi-quantitative scoring criteria, the changes of clinical symptoms, signs, TCM syndromes were observed to evaluate therapeu-tic efficiency.Experimental study:1.The hMSCs obtained from bone marrow were cultured for2-3generations in vitro, and the cell morphology of hMSCs were observed, and cell phenotype were identifide by flow cytometry with fluorescent staining of anti-CD29mAb-PE, anti-CD44mAb-FITC, anti-CD45mAb-PE, anti-CD105mAb-FITC, anti-CD34mAb-PE, anti-CD90mAb-FITC.2. The3rd generation cells were inoculated to the cover glass slices as the experimental cells, which was divided into five groups, a blank group, the induced group, low-dose Icariin group, middle dose and high dose Icariin group. After hBMSCs were adherent to the surface of the culture dish, replaced the medium and add new medium. The medium of the blank group contained the original. The medium of the induced group was replaced with induction medium. Each dose Icariin group was replaced the corresponding dose Icariin+10%FBS H-DMEM medium. Afere3days the medium of each group was changed1times. MTT assay was used to measure the OD value of from the second day to the eighth day, and take the average to draw the cell proliferation curve. 3.The3rd generation cells were divided into five groups:non-induced group, the induced group, low-dose Icariin group, middle dose and high dose Icariin group, cultured for21days. To observe the effect of Epimedium icariin on cartilage differentiation of hMSCs, the cell morphology was observed under a fluorescence microscope, and toluidine blue staining, type II collagen SABC immunohistochemical method, PCR of collagen mRNA were performed.ResultClinical research:1. An evaluation of clinical efficacy:the pairwise comparison was performed with LSD method. It is indicated that, in general, the curative effect of Wenshen Huoxue group was better than the other three groups, while there was no significant difference among three groups, Wenshen group, Huoxue group and the control group (P>0.05).2. Pain scores before and after treatment:Wenshen Huoxue group had significant pain relief effect (P<0.01), superior to all other groups, while it is showed that similar efficacy in the other three groups.3. Knee swelling scores before and after treatment:No significant difference was found among groups(P>0.05).4. Scores of the knee complete squat or semi squat before and after treatment:the function of the complete squat or semi squat in Wenshen Huoxue group was improve significantly better than the other three groups (P<0.01). No significant difference was found among the three groups:Wenshen group, Huoxue group and control group(P>0.05).5. Scores of up and down stairs before and after treatment:there was no significant difference among the groups(P>0.05).6. The knee anchylosis Points before and after treatment:The knee anchylosis was improved significantly in Wenshen Huoxue group and Wenshen group better than the other two groups(P<0.01),No significant difference was found between the Huoxue group and control group(P>0.05).7. Range of motion of before and after treatment:there was no significant difference among the groups(P>0.05).8. Soreness and weakness of waist and knees before and after treatment: the symptom was improved significantly in Wenshen Huoxue group and Wenshen group better than the other two groups (P<0.01). No significant difference was found between the Huoxue group and control group(P>0.05). 9.Aversion to cold before and after the treatment:the treatment effect in Wenshen blood group and Wenshen group was superior to the other two groups(P<0.01). No significant difference was found between the Huoxue group and control group(P>0.05).Experimental study:1.Human bone marrow mesenchymal stem cells in vitro were isolated, cultured and displays the phenotype characteristics of MSCs indentified by flow cytometry.2. The growth curve of hMSCs is S-shaped. For the first two days in subculture, there was a slight increase in the number of cells. From the third day on, cell number increased significantly, especially in the5-6th days. The cell growth come into plateau period on the7-8th days. The proliferation were increased in high dose Icariin group and the induced group than the other two groups.3. Morphology observation in the fluorescence microscope:we have observed that cells emitted blue light in high-dose Icariin group and the induced group by the combination of mouse monoclonal antibody against human type II collagen and mouse IgG-PE, indicating the high expression of type II collagen of differentiation of chondrocytes in hMSCs. Cells emitted weak blue light in the middle-dose and low-dose group indicating that the weak expression of type â…¡Icollagen in hMSCs. In non-induced group blue light can not be observed in the fluorescence microscopy, which means no differentiation of chondrocytes.Toluidine blue staining:the staining in high-dose and induced group showed that cytoplasm was purple red, which was rich Glycosaminoglycan. In the low and middle dose icariine group and non-induced group no purple red staining was founded.Identification of type â…¡ collagen by SABC immunohistochemistry method:the brown particles in the high-dose Icariin group and induced group were observed, indicating that hMSCs differentiated to chondrocytes.There was no significantly differentiation of chondrocytes in low and medium dose Icariin group and the non-induced group.Quantitative PCR test results:the expression of type I and typeâ…¡collagen showed significantly increased in the high doses icariine group and the induced group. No expression of type I and type â…¡ collagen was observed in non-induced group. ConelusionFirst, Kidney-Warming Blood-Quickening is a good and effective method to treat osteoarthritis of the knee, which played a important role in the Traditional Chinese Medicine.Second, human mesenchymal stem cells from bone marrow can be cultured and amplified in vitro.Third,Icariine, Kidney-Warming Chinese Medicine, can promote a prolifer-ation of hMSCs, and induce the differentiation of chondrocytes.
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