| ObjectiveBecause of Mesenchymal stem cells(MSCs) have high proliferative capacity, differentiation potential, low immunogenicity, does not involve ethical, is an important seed cells in Bone Tissue Engineering. In this study, we select bone marrow mesenchymal stem cells, To investigate the effect of BuShenHuoXue Decoction combined with bone marrow mesenchymal stem cells transplantation on fracture healing and its possible mechanism by animal experiment, under the guidance of traditional Chinese medicine theory,"The blood does not flow lead to stasis can not remove,The blood stasis does not remove lead to nonunion" and "kidney being in charge of bone, the marrow giving birth to bone".Analysis the rationality and feasibility of BuShengHuoXue Decoction for treatment of fracture, Clinical observation of the clinical curative effect of BuShenHuoXue Decoction for treatment of distal radius fractures, To provide a theoretical basis for TCM clinical application for treatment of fracture.Methods1.The experimental study(1)MSCs cell isolation, culture, identification and PKH26staining.The bone marrow adherent method and collagenase digested method was used to isolation MSCs, morphology of bone marrow origin and cortical bone derived mesenchymal stem cells was observed at different phase, compare their prolifer-ation capacity in vitro, cell cycle was analyzed,cell surface markers were assessed by Immunofluorescence, their differentiation ability induced into osteoblasts and adipocytes which were identified by alizarin red staining and oil red "0" staining, And with the PKH26fluorescent dye labeled BMSCs (2)The effect of BuShenHuoXue Decoction Combined with bone marrow mesenchymal stem cells vein transplantation on the healing of fractures in rats experimental study.The fracture rats were randomly divided into five groups:No modeling+BMSCs transplantation(A);modeling+BMSCs transplantation(B);BuShenHuoXue Decoction with high dose+BMSCs transplantation(C);BuShenHuoXue Decoction with middle dose+BMSCs transplantation (D);BuShenHuoXue Decoction with low dose+BMSCs transplantation(E).Analysis of the BMSCs Migration into the fracture site through the small animal in vivo fluorescence imaging system7days after modeling; Alkaline phosphatase and calcium content was detected by fundus vein blood in3、7、14days; The expression of Runx2protein in fracture site was detected by Western blot method in3、7、14days.2. Clinical study of BuShenHuoXue Decoction in the treatment of fractures of the distal radius.Simple random selected59distal radius fracture cases in Department of Orthopaedics and traumatology Hospital Hospital Affiliated to Guangzhou University of TCM. Divided into treatment group and control group:The treatment group of29cases treated by BuShenHuoXue Decoction, and30cases in control group, remaining the same routine therapy. Observation of bone metabolic indices:calcium serum and alkaline phosphatase serum, The improve-ment of clinical symptoms (Including visual analog scale score and The function of wrist joints Gartland&Werley score)Results1. The experimental study(1)Rat MSCs cell isolation, culture, identification and PKH26stainingThe collagenase digested method and bone marrow adherent method can effective separation of purified rat MSCs, the morphology, cell surface Immunofluorescence staining, osteogenic and adipogenic differentiation ability confirm that the cell is mesenchymal stem cells Complete BMSCs labeled with PKH26dye and The positive rate reached97%.(2)The effect of BuShenHuoXue Decoction Combined with bone marrow mesenchymal stem cells vein transplantation on the healing of fractures in rats experimental study.7days after modeling, analysis of small animal in vivo fluorescence imaging confirmed that PKH26labeled BMSCs migration into the fracture site. The mean fluorescence intensity of fracture site in high, medium and low dose group compared with the model group and blank group, there were statistical significance between the two groups. The3day, calcium and alkaline phosphatase levels of each group have no statistical significance (P>0.05); the7day, BuShenHuoXue Decoction in low dos Combined with MSCs transplantation group compare with model group(simple MSCs injection+fracture modeling)the serum calcium levels were statistically significant (P<0.05), the alkaline phosphatase have no statistically significant (P>0.05); the14day, Serum calcium content in high, medium and low dose group compared with the model group, there were statistical significance between the two groups (P<0.05), Serum alkaline phosphatase in high, middle dose group compared with the model group had statistical significance (P<0.05).In3、7、14days, the expression of runx2protein at fracture site in high, middle, low dose group compared with the model group and blank group had statistical significance (P<0.05)2. Clinical study of BuShenHuoXue Decoction in the treatment of fractures of the distal radius.Observation of bone metabolic indices:serum ALP levels of two groups before treatment, there were no statistical significance in the difference, After treatment, the treatment group and the control group have significant difference(P<0.05). serum calcium levels of two groups before treatment, there were no statistical significance in the difference, The serum calcium(mmol/L) in two groups before and after treatment there were statistical significance in the difference (P<0.05).After treatment, the treatment group and the control group have significant difference (P<0.05).Patients with VAS score after one month, VAS score of two groups before treatment, there were no statistical significance in the difference, The two groups before and after treatment there were statistical significant in the difference(P<0.05), after treatment, the treatment group and the control group have significant difference (P<0.05).The wrist joint function with Gartland&Werley score (Divided into4grades, Excellent:0-2score; good:3-8score; poor:9-20score; bad^21score)were assessed after two months:29cases in the treatment group, excellent in13cases, good in14cases, poor in1cases, bad in1cases, the excellent and good rate was93.1%;30cases in the control group:excellent in9cases, good in13cases, poor in8cases,bad in0cases, the excellent and good rate was73.3%. Compared with the excellent and good rate between the treatment group and the control,there were statistical significance in the difference.Conclusion1. Both the bone marrow and cortical bones can achieve isolation rat MSCs, and the bone marrow mesenchymal stem cells labeled with PKH26dye and the positive rate more than97%, provide seed cells for the next experiment.2. Strongger evidence confirmed that exogenous BMSCs can migration into the fracture site; BuShenHuoXue Decoction can promote the exogenous BMSCs migration into the fracture site. BuShenHuoXue Decoction combined with BMSCs transplantation can promote the expression of Runx2Protein in the fracture site and promote fracture healing, the mechanisms need to be further elucidated.3.BuShenHuoXue Decoction can increase the content of ALP in serum of patients with distal radius fractures and and improve the clinical symptoms, with no side effects. |
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