Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
Objective To investigate the diagnosis and effectiveness of improved percutaneous kyphoplasty (PKP) for patients with thoracolumbar metastatic tumors, who could not tolerate anesthesia and open operation. Methods Between September 2009 and September 2010, 16 patients with thoracolumbar metastatic tumors underwent improved PKP. Of 16 patients, 7 were male and 9 were female with an average age of 64.5 years (range, 60-73 years). All patients had vertebralmetastasis tumor. The disease duration was 3-6 months with an average of 4 months. The visual analogue scale (VAS) score was 8.9 ± 0.8. No spinal cord compression and nerve root compression was observed. The involved vertebrae included T7 in 1 case, T8 in 1, T12 in 1, L2 in 2, L3 in 2, L4 in 3, T1, 2 in 1, T3, 4 in 1, T7, 8 in 1, T11, 12 in 1, T7-L1 in 1, and T12-L4 in 1. Nine patients had vertebral compression fracture with a vertebral compression rate below 75%. Results All patients were successfully performed PKP. There was no serious adverse reactions in cardiopulmonary and brain vascular systems and no perioperative death. The biopsy results showed that all were metastatic adenocarcinoma. All patients were followed up 9-18 months mean, 14 months). Complete pain rel ief was achieved in 14 cases and partial rel ief in 2 cases 6 months after operation according to World Health Organization criterion, with a pain-rel ief rate of 87.5%. The VAS score was 1.8 ± 0.6 at 6 months postoperatively, showing significant difference when compared with the preoperative score (P lt; 0.05). Two patients had cement leakages in 3 vertebrae with no symptoms at 6 months postoperatively. During follow-up, 12 patients died and the others survived with tumor. Conclusion For patients with thoracolumbar metastatic tumors who can not tolerate anesthesia and open operation, improved PKP has the advantages such as minimal invasion, high diagnostic rate, and early improvement of pain in the biopsy and treatment. It can improve patient’s qual ity of l ife in the combination of radiotherapy or chemotherapy.
Objective To transplant intravenously human brain-derived neurotrophic factor (hBDNF) genemodified bone marrow mesenchymal stem cells (BMSCs) marked with enhanced green fluorescent protein (EGFP) to injured spinal cord of adult rats, then to observe the viabil ity of the cells and the expressions of the gene in spinal cord, as well as theinfluence of neurological morphological repairing and functional reconstruction. Methods Ninety-six male SD rats weighing (250 ± 20) g were randomly divided into 4 groups: hBDNF-EGFP-BMSCs transplantation group (group A, n=24), Ad5-EGFPBMSCs transplantation group (group B, n=24), control group (group C, n=24), and sham operation group (group D, n=24). In groups A, B, and C, the spinal cord injury models were prepared according to the modified Allen method at the level of T10 segment, and after 3 days, 1 mL hBDNF-EGFP-BMSCs suspension, 1 mL Ad5-EGFP-BMSCs suspension and 1 mL 0.1 mol/L phosphate buffered sal ine (PBS) were injected into tail vein, respectively; in group D, the spinal cord was exposed without injury and injection. At 24 hours after injury and 1, 3, 5 weeks after intravenous transplantation, the structure and neurological function of rats were evaluated by the Basso-Beattie-Bresnahan (BBB) score, cortical somatosensory evoked potential (CSEP) and transmission electron microscope. The viabil ity and distribution of BMSCs in the spinal cord were observed by fluorescent inverted phase contrast microscope and the level of hBDNF protein expression in the spinal cord was observed and analyzed with Western blot. Meanwhile, the expressions of neurofilament 200 (NF-200) and synaptophysin I was analyzed with immunohi stochemistry. Results After intravenous transplantation, the neurological function was significantly improved in group A. The BBB scores and CSEP in group A were significantly higher than those in groups B and C (P lt; 0.05) at 3 and 5 weeks. The green fluorescence expressions were observed at the site of injured spinal cord in groups A and B at 1, 3, and 5 weeks. The hBDNF proteinexpression was detected after 1, 3, and 5 weeks of intravenous transplantation in group A, while it could not be detected in groups B, C, and D by Western blot. The expressions of NF-200 and synaptophysin I were ber and ber with transplanting time in groups A, B, and C. The expressions of NF-200 and synaptophysin I were best at 5 weeks, and the expressions in group A were ber than those in groups B and C (P lt; 0.05). And the expressions of NF-200 in groups A, B, and C were significantly ber than those in group D (P lt; 0.05), whereas the expressions of synaptophysin I in groups A, B, and C were significantly weaker than those in group D (P lt; 0.05). Ultramicrostructure of spinal cords in group A was almost normal. Conclusion Transplanted hBDNF-EGFP-BMSCs can survive and assemble at the injured area of spinal cord, and express hBDNF. Intravenous implantation of hBDNF-EGFP-BMSCs could promote the restoration of injured spinal cord and improve neurological functions.
Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism．Methods Different generations(P1, P2, P3 and P4) of the chondrocytes were detected with the methods of histochemistry for β-galactosidase (β-gal)， electronmicroscope for ultromicrostructure， immunocytochemistry for proliferating cell nuclear antigen (PCNA)，alcian blue stain for content and structure of sulfatglycosaminoglycan (GAG) of extracellular matrix (ECM)，reverse transcriptionpolymerase chain reaction (RTPCR) for content of collagen Ⅱ，flow cytometry for cell life cycle and proliferative index(PI) to observe senescence of chondrocytes．Results In the 4th passage，the chondrocytes emerging quantitively positive express of β-gal，cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electronmicroscope ，cell life cycle being detented on G1 phase(83．8％)，while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PI decreased(16．2％)，while in P1, P2, P3, it was 20.9%, 20.8%, 19.2%. The positive percentage of PCNA，the content of GAG(long chain molecule) and the positive expression of collagen Ⅱ diminished,all detections above were significantly different (Plt;0.01) when compared the 4th passage with the preceding passages．Conclusion Chondrocytes show the onset of senescence in the 4th passage．
Objective To observe the effect of pilose antler polypeptides(PAP)on the apoptosis of rabbit marrow mesenchymal stem cells (MSCs) differentiated into chondrogenic phenotype by interleukin 1β (IL-1β) so as to optimize the seeding cells in cartilage tissue engineering. Methods The MSCs were separated from the nucleated cells fraction of autologus bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into chondrogenic phenotype by transforming growth factor β1(TGF-β1) and basic fibroblast growth factor(bFGF). According to different medias, the MSCs were randomly divided into four groups: group A as black control group, group B(100 ng IL-1β)，group C(10 μg/ml PAP+100 ng IL-1β) and group D(100 ng/ml TGF-β1 +100 ng IL-1β). The samples were harvested and observed by morphology, flow cytometry analysis, RT-PCR and ELISA at 24, 48 and 72 hours. Results The intranuclear chromatin agglutinated into lump and located under nulear membranes which changed into irregular shapeat 24 hours. The intranuclear chromatin agglutinated intensifily at 48 hours. Then the nucear fragments agglutinated into apoptosic corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosic rate of cells, the mRNA expression of Caspase-3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A,C and D(Plt;0.01). Conclusion Caspase-3 is involved in aoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3.
OBJECTIVE To investigate the possibility of repairing the cartilage cartilage defect with homogeneous chondrocytes combined with Pluronic. METHODS: Homogeneous cartilage chondrocytes of adult New Zealand rabbits were harvested and cultured in vitro, which were marked by 3H-TdR and mixed with Pluronic. The medial or lateral condyle defects were made (phi 4 mm, extending down to the calcified zone) in 20 rabbits. In the experimental group, the right defects were repaired by homogeneous chondrocytes combined with Pluronic; in the control group, the left defects were repaired by Pluronic only or were left un-repaired. The animals were sacrificed in the 4th, 8th and 16th weeks after operation respectively. The repair results were observed and the cell source of repair tissue was distinguished. RESULTS: In the experimental group, the cartilage defects were repaired by the cartilage-like tissue after 8 weeks of operation; the defects were completely filled with mature cartilage tissue, which integrated smoothly with articular cartilage 16 weeks later. In the control group, only a small amount fibrous tissues were seen on the surface of defects. Autoradiographic assessment showed that the repair cells came from the implants, but not from self-chondrocytes. CONCLUSION: It is a good way to repair articular cartilage defects with homograft of tissue engineering cartilage. It is a convenient method to mark with 3H-TdR to discriminate the resource of the repair cells.
Objective To prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.
ObjectiveTo analyse the effectiveness of unicompartmental knee arthroplasty (UKA) for the patients with spontaneous osteonecrosis of the knee (SONK). MethodsBetween January 2012 and December 2016, 31 patients with SONK was admitted and treated with medial UKA. All patients were examined by both plain radiography and magnetic resonance images. The patients were composed of 5 men and 26 women with an average age of 64.3 years (range, 48-79 years), and with 16 left joints and 15 right joints. The average disease duration was 14.7 months (range, 6-26 months). Preoperative visual analogue scale (VAS) was 6.00±1.15, Hospital for Special Surgery (HSS) score was 55.77±11.03, and knee range of motion (ROM) was (114.68±10.40)°. The imaging examinations showed that all the lesions were located in the medial compartment of the knee joint and there were 19 patients with Aglietti stage Ⅳ and 12 patients with Aglietti stage Ⅴ. Preoperative femorotibial angle (FTA) was (177.39±1.63)° and posterior tibial slope (PTS) was (84.05±1.39)°. ResultsAll the incisions healed by first intention. All patients were followed up 14-46 months (mean, 25 months). At last follow-up, VAS score was 2.06±0.72 and HSS score was 86.45±3.67, which both improved significantly when compared with preoperative scores (t=22.73, P=0.00; t=–14.72, P=0.00). ROM was (118.06±3.80)° with no significant difference when compared with preoperative ROM (t=–1.78, P=0.08). The X-ray films showed there was no severe adverse events, such as periprosthetic infection, aseptic loosening, bearing dislocation, and so on. At last follow- up, PTS was (85.30±1.19)° with significant difference compared with preoperative one (t=–4.07, P=0.00); while FTA was (177.51±1.98)° with no significant difference when compared with preoperative FTA (t=–0.38, P=0.71). ConclusionUKA may be an optional management for SONK with minimally invasive, bone-preserving, and rapid recovery.
ObjectiveTo investigate the effect of granulocyte colony-stimulating factor (G-CSF) mobilizing the bone marrow mesenchymal stem cells (BMSCs) homing to the spinal cord injury sites in rats, and to evaluate the feasibility of G-CSF mobilizing the BMSCs home to the injured spinal cord. MethodsTwenty-four healthy adult female Sprague Dawley rats were injected with 1 mL green fluorescence protein labeled BMSCs (GFP-BMSCs, 1×106 cells/mL) into tail vein at 12 hours before operation. They were randomly divided into sham operation group (group A), sham operation+G-CSF group (group B), spinal cord injury group (group C), and spinal cord injury+G-CSF group (group D), with 6 rats in each group. In groups C and D, spinal cord injury model was established by T10 level spinal cord hemisection. In groups A and B, only laminectomy was performed without injury to the spinal cord. Groups B and D were injected with G-CSF (10 μg/kg·d) at 1 hour after operation for 3 consecutive days, and groups A and C were injected with the same amount of saline. The Basso-Beattie-Bresnahan (BBB) score was used to estimate the neurological function of rats and the expressions of tumor necrosis factor α (TNF-α) and stromal-derived factor 1 (SDF-1) were detected by ELISA method at 1, 3, 7, 14, 21, and 28 days after operation. The spinal cord samples of rats were sacrificed at 28 days after operation for immunohistochemical staining to observe the expression of cytokines, including SDF-1, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and TNF-α, and immunofluorescence staining to observe GFP-BMSCs positive cells, double-stained fluorescent yellow GFP/neuronal nuclear antigen (NeuN) positive neurons, and GFP/glial fibrillary acidic protein (GFAP) positive neurons. The number of glial cells and apoptosis were detected by TUNEL method. ResultsThe BBB score of groups A and B had no significant change at each time point after operation. At 1 day after operation, the BBB score of groups C and D decreased to the lowest level, and then gradually increased. The BBB score of group D was significantly higher than that of group C at all time points except 1 day after operation (P<0.05). At 3, 7, 14, 21, 28 days after operation, the levels of TNF-α and SDF-1 in groups C and D were significantly higher than those in groups A and B (P<0.05), but the levels of TNF-α in group D were significantly lower than those in group C at each time point, and the levels of SDF-1 were significantly higher than those in group C (P<0.05). Immunohistochemical staining showed that the expressions of SDF-1, BDNF, VEGF, and TNF-α in groups C and D were significantly higher than those in groups A and B (P<0.05); the expressions of SDF-1, BDNF, and VEGF in group D were significantly higher than those in group C, and the expression of TNF-α was significantly lower than that in group C (P<0.05). Immunofluorescence staining showed that the number of GFP-BMSCs, GFP/NeuN, and GFP/GFAP positive cells in groups C and D were significantly higher than those in groups A and B, and in group D than in group C (P<0.05). TUNEL assay showed that the number of apoptotic cells in groups C and D was significantly lower than that in groups A and B, and in group D than in group C (P<0.05). ConclusionG-CSF can mobilize BMSCs to the spinal cord injury site and promote repair effect by down-regulating TNF-α to promote the anti-apoptosis function and up-regulating SDF-1, BDNF, VEGF to promote BMSCs migration.