BACKGROUND:Preliminary experiments have demonstrated that the expression of Notch-1 in the degenerated intervertebral disc was increased. However, the role of Notch-1 in the nucleus pulposus-like cel differentiation of mesenchymal stem cel s remains unclear.
OBJECTIVE:To investigate the bone marrow mesenchymal stem cel s in suppressing the degeneration of intervertebral discs after Notch1 gene knockout.
METHODS:(1) Bone marrow mesenchymal stem cel s were isolated from the femur bone of four New Zealand rabbits weighing 0.4-0.5 kg, under deep anesthesia, and then purified with discontinuous gradient density centrifugation method. (2) Notch1 shRNAs and blank plasmid shRNA were designed, synthesized, and transiently transfected into these mesenchymal stem cel s, and the differentiation of mesenchymal stem cel s was induced with transforming growth factor beta-1. (3) Ten New Zealand rabbits weighing 1.0-1.5 kg were involved in this study. The rabbits’ intervertebral discs in L3-4, L4-5 and L5-6 were stabbed by a needle, and nucleus pulposus tissue was harvested for modeling. The cel s were divided into blank plasmid transfected with transforming growth factor beta-1 group, shRNA-Notch-1 plasmid transfected with transforming growth factor beta-1 group, and non-transfected treated with transforming growth factor beta-1 group. Two weeks later the treated cel s were transplanted into L3-4, L4-5 and L5-6, respectively.
RESULTS AND CONCLUSION:(1) Four weeks after the cel transplantation, the signal intensity of T2 weighted images in the L3-4 (non-transfection group) and L5-6 (blank plasmid group) discs was increased by magnetic resonance imaging, and the significant difference was found in L4-5 discs (shRNA-Notch-1 plasmid group) in comparison with other two groups (P<0.05). (2) Toluidine blue staining showed that, the expression of proteoglycan in the L4-5 discs (shRNA-Notch-1 plasmid group) was significantly higher than that in L3-4 (non-transfection group) and L5-6 (blank plasmid group) discs. (3) Reverse transcription-polymerase chain reaction and western blot assay showed that, the expression of col agen II and proteoglycan mRNA and protein in the L4-5 discs (shRNA-Notch-1 plasmid group) were significantly increased compared with L3-4 (non-transfection group) and L5-6 (blank plasmid group) discs. Transplantation of bone marrow mesenchymal stem cel s of Notch-1 gene knockout rabbits can effective restore the degenerated intervertebral discs.

[Objective] To study the effect of histone deacetylase inhibitor-Trichostatin A(TSA) on proliferation and differentiation of human osteogenic sarcoma cell. [Methods]Human osteogenic sarcoma cell line MG-63 was treated in vitro with various concentrations of TSA, a potent and specific histone deacetylase inhibitor. Proliferation suppression was observed by MTT method and inverted microcopy before and after TSA treatment, and the cell growth curve was obtained. The cell growth and invasion ability were measured with the colony-formation rate in soft agar test. Flow cytometry was used to investigate the cell cycle.[Results]TSA significantly inhibited the proliferation of the human osteogenic sarcoma cell in a dose-dependent and time-dependent manner. The cell experienced benigh morphological differentiation. The colony-formation rate in semi-solid agar was significantly decreased(P

Intervertebral disc (IVD) degeneration is considered to be the main cause of low back pain (LBP),however,there are lack of long-lasting and effective methods of clinical treatment.Tissue engineering technique based on stem cells becomes an essential research direction on repair of IVD degeneration at present,and its effectiveness and feasibility have been confirmed,but it is difficult to maintain the sufficiency and vitality of stem cells in IVD.Previous studies showed that stem cells existed naturally in IVD,and stem cells from stem cell niche could migrate to IVD physiologically to maintain the IVD environment balance under the adverse microenvironment.Unfortunately,these behaviors cannot preclude IVD degeneration.Therefore,theoretical basis for the regeneration of nucleus pulposus (NP) in situ can be obtained from studying the mechanism that the endogenous repair failure during IVD degeneration,the cell death and the migration of stem cells in IVD,and the key regulatory targets to sustain the quantity and quality of the stem cells.Although there have been few researches to study the mechanism of the cell death and the migration of stem cells in IVD so far,studies demonstrated that the major inducing factors of IVD degeneration (pressure and hypoxia) could decrease the number of NP cells by autophagy,apoptosis and necroptosis,and chemokines and their receptors played a critical role in the migration of mesenchymal stem cells.These researches provide a clue for studying the mechanism of endogenous repair failure during IVD degeneration.We reviewed the current research situation and progress of the mechanism that endogenous repair failure during IVD degeneration in the following articles.First,we exhibited the potential of IVD stem cells in IVD degeneration repair.Second,the effect of the adverse microenvironment (pressure,hypoxia,etc) on the migration of IVD stem cells was discussed.Third,the mechanism of the stem cell death,autophagy,apoptosis and necroptosis under the adverse (pressure,hypoxia,etc.) microenvironment,and the correlation between the IVD stem cells migration and autophagy,apoptosis and necroptosis was studied.And then tissue engineering of NP was also discussed to achieve the endogenous repair of IVD degeneration.These studies will provide an innovative research direction on endogenous repair and a new strategy of early therapy for IVD degeneration.

[Objective]To explore the effect of external fixator combined with vaccum sealing drainage(VSD) for the treatment of infection after internal fixation in tibial and fibular fracture.[Method]Thirty-six patients with infection after internal fixation in tibial and fibular fracture were treated by external fixator combined with VSD from June 2004 to June 2008.[Result]During the following-up period from 8-30 months in thirty-two patients,the infection in all cases were controlled effectively,no case of recurrence was found in all patients,and satisfactory healing were found in 32 cases.The average time of fracture union were 4.5 months;the removal time of external fixation were 5 months on average.[Conclusion]The combined operative procedure is simple,reliable and less traumatic and allow to early functional exercises.Therefore,it is a satisfactory measure for the infection after internal fixation in tibial and fibular fracture.

BACKGROUND: Interverbral disc degeneration (IDD) is the major cause of low back pain, which is considered as the pathological basis of intervertebral disc herniation and other degenerative diseases that severely affect the patients' quality of life. At present, neither conservative treatment nor operative treatment can reverse or inhibit the pathological development of IDD.OBJECTIVE: To summarize the progress of stem cell therapy for IDD, and to further analyze its challenge and potential solutions.METHODS: Relevant studies in PubMed database published before September 2016 were retrieved. Studies about stem cell therapy in IDD in vitro and vivo as well clinical trials were enrolled to analyze its existing problems.RESULTS AND CONCLUSION: (1) Stem cell therapy provides a new strategy for IDD treatment. So far, a large number of studies in vitro and vivo as well as clinical trials have reported the remarkable effect of stem cell therapy. (2) However, problems such as how to deal with microenvironment of interverbral disc, choose proper stem cells and master indications will be a challenge in the future.

BACKGROUND: Previous researches indicate that ADp14ARF transfecting positive tumor cell line of p53 can inhibit the proliferation; in addition, the inhibitory effect is superior to transfection negative tumor cell line of p53. Whether simultaneous transfection of p14ARF and p53 genes can increase expression and accumulation of p53 and accelerate apoptosis of tumor cells needs further studies.OBJECTIVE: To construct double plasmid expression vector plRES-p14ARF-p53 by using gene engineering so as to observe the inhibitory effect on proliferation of osteogenic sarcoma cells.DESIGN: Randomized controlled observation.SETTING: Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the Public Laboratory Platform, Immune Researching Room, Basic Medical College, Tongji Medical College, Huazhong University of Science and Technology from January 2005 to October 2006. Human osteogenic sarcoma MG-63 cells were provided by Cell Laboratory, Immune Researching Room, Tongji Medical College, Huazhong University of Science and Technology. plRES-p53 plasmid and plRES vector containing p53total-length gene order were provided by Wuhan Jingsai Biology Company.METHODS: Based on gene engineering, p14DNA (0.5 kb) was amplified from cultured L02 cells of normal human hepatic cells into plRES vector. Recombinant plasmid plRES-p14ARF-p53 was determined with polymerase chain reaction (PCR) and restriction enzyme and transfected into human osteogenic sarcoma MG-63 cells through mediation of liposome to screen positive clones. Otherwise, cells were divided into three groups, including blank control group (MG-63cells), blank vector control group (stably transfecting plRES-neo cells) and p14ARF-p53 group (stably transfecting plRES-p14ARF-p53 cells). ① DNA content and cycle of tumor cells were measured by using flow cytometry before and after transfection. ② Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to detect quantitative and semi-quantitative expression of p53 and p14ARF protein in tumor cells after stable transfection. ③Thiazole blue chromatometry and growth curve were used to observe proliferation.MAIN OUTCOME MEASURES: ① DNA content and cycle of osteogenic sarcoma cells; ② expressions of p53 and p14ARF protein in tumor cells; ③ proliferation.RESULTS: Double plasmid expression vector plRES-p14ARF-p53 was constructed successfully. ① DNA content and cycle of osteogenic sarcoma cells: Flow cytometry demonstrated that tumor cells mainly stayed in G1 phase after transfection. ② Protein expression: RT-PCR and Western blot indicated that p14ARF and p53 gene independently expressed in target cell mRNA and protein, respectively. ③ Cell growth: At 24, 48, 72 and 96 hours after MG-63 transfection, inhibitory rates of tumor cells were 33.43%, 69.37%, 66.19% and 75.26%, respectively, which was significant difference as compared with blank vector control group (P ＜ 0.01).CONCLUSION: Wild p53 and p14ARF can synergistically inhibit the proliferation and accelerate the apoptosis of osteogenic sarcoma cells.

BACKGROUND: How to deal with bone defect is a big problem to surgeons. In recent years, the development in the technology of molecular biology and tissue engineering provides broad prospect for the clinical treatment of bone defect, which is one of the important study directions in department of orthopedics. The transforming growth factor-beta 1(TGF-β1),one of the important factors in bone formation, plays an important role in bone metabolism and recovery.OBJECTIVE: To observe the therapeutic effects of bone defects with osteoblasts transfected . By TGF-β1 combining with biomimetic biodegradable polymer scaffolds.DESIGN: Randomized controlled trial.SETTING: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Siderophilin, trypsin, 3H-proline and sirius red, etc.MATERIALS: The experiment was completed in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from March to August in 2003. Twenty healthy adult Sprague-Dawley(SD) rats of SPF grade, weighing 200-250 g, were provided by the Experimental Animal Center of Tongji Medical College.METHODS: The osteoblasts transfected by TGF-β1 gene, combining with poly-DL-lactic acid scaffolds modified with poly-L-lysine, were transplanted into rat tibia defect. Radiographs and histological analysis were performed to evaluate the repair effects.MAIN OUTCOME MEASURES: The X-ray evaluation and histology observation were performed at the 4th and 8th weeks after the operation.RESULTS: Totally 20 SD rats were included in result analysis without one rat missing. ①In the experiment group, X-ray image indicated callus formation, while histology observation showed osteoid tissue and new bone formation, and osteoblasts attached to the surface of the materials after 4 weeks. Eight weeks later, the defect was essentially repaired, and the bone density of new bone was similar to that of the autogenous bone. ②In the control group, there was no formation of callus and osteoid tissue, and few osteoblasts attached to the surface of the materials, and a lot of lymphocytes infiltrated and blood capillary grew in the lacune of materials after 4 weeks. Eight weeks later, the imbedded materials were substituted mostly by fibrous tissue.CONCLUSION: The ideal repair effect of bone defect can be obtained through the combination of molecular biology with tissue engineering.

Objective To study the age peak of incidence,pathologic sites,therapeutic effect and complications of different treatment for hip fractures in the elderly.Methods Hip fractures,therapy,elderly were used as key words to search articles both in English and Chinese from 1979 to 2017.The articles were filtrated by title,abstract and full text and 32 of them were left.All the patients were objects of this study,but the repeated cases were excluded.The patients' data such as age of onset,gender,fracture type,operation method,follow-up time,and complications were collected and analyzed by systematic analysis.Results Data of 2 758 patients in 32 papers had been collected for analysis.There were 1791 femoral neck fractures and 967 intertrochanteric fractures,with a ratio of 1.85:1.The patients' age of onset was ranging from 54 to 92 years old,with an average of 76.7 years old,and the age peak of incidence was from 70 to 79 years old.The sex ratio was 1:1.66 (1 037 males:1721 females).All the patients had been followed up for 0.5 to 18 years,(average,7.8 years).Among 927 femoral neck fractures who were treated by 3 lag screws,there were 278 cases of non-union (29.9％),139 femoral head necrosis(15.1％) and 19 internal fixation loosening (2.0％);but for 183 femoral neck fractures who were treated by dynamic hip screws (DHS),there were 51 cases of non-union (28.1％),31 femoral head necrosis(17.0％),3 internal fixation losening(1.8％),and 27 cutting effect (15％).Then for 400 femoral neck fractures who were treated by hemiarthroplasty,there were 6 cases of dislocation(1.5％),14 shaft fracture (3.5％),8 deep infection (2.0％);while for 281 femoral neck fractures who were treated by total hip arthroplasty,there were 14 cases of dislocation (5.0％),11 shaft fracture (4.0％),and 10 deep infection (3.5％).On the other hand,for 354 intertrochanteric fractures who were treated by proximal femoral nail or proximal femoral nail antirotation internal fixation (PFN or PFNA),there were 18 cases of trochanter fracture (5.0％),28 hip varus (8.1％),32 displacement (9.0％);and for 210 intertrochanteric fractures who were treated by Gamma nail,there were 17 cases of trochanter fracture (7.9％),11 cutting effect (5.0％),6 displacement (3.0％);and for 135 intertrochanteric fractures who were treated by DHS,there were 8 cases of cutting effect (6.1％),7 displacement (5.2％);and for 101 intertrochanteric who were treated by proximal femoral locking plate,there were 5 cases of displacement (5.0％);Also for 89 intertrochanteric fractures who were treated by hemiarthroplasty,there were 2 cases of dislocation(1.8％),3 shaft fracture (3.0％),1 deep infection(1.5％);and for 78 intertrochanteric fractures who were treated by total hip arthroplasty,there were 4 cases of dislocation (4.8％),3 shaft fracture (4.0％),2 deep infection (2.5％).Conclusion The elderly hip fractures' age peak of incidence is from 70 to 79 years old,and femoral neck fracture takes up a majority of it.Female patients are more than male patients.The femoral neck fractures are mainly treated by 3 lag screws internal fixation;non-union and femoral head necrosis are the common complications postoperation.While the intertrochanteric fractures are mainly treated by proximal femoral nails;displacement,hip varus and trochanter fractures are the common complications postoperation.The artificial hip replacement is the effective remedy measure for secondary femoral head necrosis after hip fractures.

BACKGROUND: Osteoblasts have become a kind of important seed cells in bone tissue engineering. However, it is difficult to harvest osteoblasts, and the purity and calcification ability of osteoblasts isolated by different methods are inconsistent. OBJECTIVE: To compare the purity and calcification ability of osteoblasts induced from mouse bone marrow mesenchymal stem cells, MC3T3 cell lines, and cultured primarily from the neonatal mouse cranium. METHODS: Mouse bone marrow mesenchymal stem cells were isolated by differential adhesion method, and after passaing, passage 3 cells were cultured in osteogenic induction medium for 21 days. MC3T3 cell lines were cultured in osteogenic induction media 1 and 2 for 21 days. Osteoblasts were cultured primarily from neonatal mouse cranium by type Ⅰ coll agenase digestion method. Calcium nodules of osteoblasts obtained by three methods were observed by Alizarin red staining to detect osteogenic activity of cells. RESULTS AND CONCLUSION: (1) There were average 16.3 calcium nodules per low-power field after osteogenic induction of bone marrow mesenchymal stem cells. (2) There were sparsely distributed calcium nodules in MC3T3 cells after induction with osteogenic induction medium 1, accounting for 1.7 calcium nodules per low-power field, while there were dense calcium nodules in MC3T3 cells after induction with osteogenic induction medium 2, accounting for 44.6 calcium nodules per low-power field. There was a significant difference in the calcium nodule formation ability between the two groups (P < 0.01). (3) After primary culture, there was only 0.6 calcium nodule per low-power field. (4) Except for the insignificant difference between osteogenic induction medium 1 and primary culture groups, there were significant differences in pair-wise comparison of any other two groups. Except the insignificant difference between group I of MC3T3 inducing conditional media and primary culture osteoblasts, there were significant differences in the osteogenic ability between groups (P < 0.01). In conclusion, it is a better method to culture MC3T3 cells in osteogenic induction medium 2 containing dexamethasone, because many uncontrol able factors are involved in the isolation and culture of bone marrow mesenchymal stem cells.

BACKGROUND: Recent studies have demonstrated that energy metabolism-related genes play important roles in intervertebral disc (IVD) cell adaptation to negative environmental factors, such as hypoxia and insufficiency of nutrient. But the effects of these genes in pressure-induced intervertebral disc degeneration remain uncertain.OBJECTTVE: To investigate continuous pressure-induced expression of energy metabolism genes: hypoxia-inducible factor 1α(HIF-lo), glucose transporter-1 (GLUT-1) and vascular endothelial growth factor (VEGF) in rabbit annulus fibrosus (AF).DESIGN: A randomized controlled experiment.SETTING: The Central Laboratory and the Laboratory of Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the Central Laboratory and the Laboratory of Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from May to October 2005. Twenty-five Japanese white rabbits(about 4 months old, weighting 2.5-3.0 kg, provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology) were selected to establish the animal model.METHODS: A controllable pressure-induced rabbit intervertebral disc degeneration model was adopted to impose various pressured on rabbit IVDs in vivo. The survived animals whose IVDs were compressed successfully were divided randomly into 4 groups. The IVDs were treated with no pressure as control (control group), with 15 kg axial load for 24 hours (24hours group), 72 hours (72 hours group), and 24 hours with 48 hours free for self-reparation (reparation group). Reverse transcriptase-polymerase chain reaction was used to detect the expression of HIF-1α and GLUT-1. Western Blot and immunohistochemical test were carried out for the content and distribution of VEGF.MAIN OUTCOME MEASURES: Expression of HIF-1α and GLUT-1, content and distribution of VEGF.RESULTS: Twenty of 25 rabbits entered result analysis. Two rabbits were missed because of vertebral fracture, while death of 3 rabbits within 1-3 days postoperatively caused another loss. ①HIF-1α. A very low expression was detected in the control group, while the expression in the 24 hours group was raised over 20 times than that in the control group (t=25.022, P＜0.01). The expression in the 72 hours group and reparation group decreased as compared with the 24hours group. ②GLUT-1 expressed weakly in the control group. The expression in the 24 hours group rose a lot as compared with the control group (t=18.314, P＜0.01) and the expression in the 72 hours group rose slightly than that in the 24 hours group (t =2.819, P＜0.05). The expression in the reparation group is close to that in the 24 hours group. ③Littie VEGF content was detected in the control group, while the content rose significantly in the other 3 groups. Immunohistochemical staining showed more VEGF positive stained cells in outer AF than in inner AF.CONCLUSION: Continuous pressure can strongly up-regulate the expression of energy metabolism gene: HIF-1α,GLUT-1 and VEGF in vivo. These genes play important roles in AF adaptation and reparation in over load-caused damage.