ObjectiveTo investigate the expression of p16INK4a in nucleus pulposus (NP) and to clarify its relationship with intervertebral disc degeneration so as to provide evidence for biological repair of intervertebral disc. MethodsThe NP specimens were obtained from 17 patients with intervertebral disc degeneration undergoing discectomy, who aged 40-50 years (mean, 45.4 years). Based on the preoperative MRI, there were 10 cases of grade Ⅲ degeneration, and 7 cases of grade IV degeneration. Cell senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-gal) activity. Senescence marker (p16INK4a) and disc degeneration markers [A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5), Aggrecan, and Sryrelated HMG box transcri ption factor 9 (Sox-9)] were determined in the NP specimens with immunohistochemistry and Western blot. The correlation between ADAMTS 5 and p16INK4a was analyzed. ResultsClustered distribution of green SA-β-gal-positive cells was seen in the NP with grade Ⅲ and IV degeneration. A few single round SA-β-gal-positive NP cells (NPCs) wrapped by the layered extracellular matrix were also seen in the NP with grade Ⅲ degeneration. It was difficult to see single distribution of NPCs in the NP with grade IV degeneration. The percentage of SA-β-gal-positive cells was 22.7%±5.4% and 37.1%±7.6% in the NP with grade Ⅲ and IV degeneration respectively, showing significant difference (t=-9.666, P=0.000). The percentages of p16INK4a-positive and ADAMTS 5-positive NPCs in the NP with grade IV degeneration were significantly higher than those with grade Ⅲ degeneration (P<0.05). The percentages of Aggrecan-positive and Sox-9-positive NPCs in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The protein expressions of Aggrecan and Sox-9 in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The NP with grade IV degeneration showed significantly higher protein expressions of p16INK4a and ADAMTS 5 (P<0.05). Importantly, there was a good correlation between p16INK4a and ADAMTS 5 protein expressions (r=0.908, P=0.000). ConclusionPremature senescent NPCs increase in the NP with the advancing disc degeneration. The expression of p16INK4a and its association with degeneration grades suggest that the p16INK4a may play a significant role in the pathogenesis of intervertebral disc degeneration.
ObjectiveTo isolate nucleus pulposus cells (NPCs) from the caudal and lumbar intervertebral disc of rat, and to identify the morphology and to compare the characteristics. MethodsThe whole spine was separated from 8-week-old Sprague Dawley rats under the sterile conditions. NPCs of different segments (lumbar group: L1,2-L6, S1; caudal group: C1,2-C17,18) were cultured by adherent cultivation approach. Cellular morphologic change was noted by HE staining and continuous observation under inverted phase contrast microscope. Besides, the aggrecan and collagen type Ⅱexpression were examined by toluidine blue and immunocytochemistry staining respectively. The total protein contents, senescence level, and the cell viability of passage 1-5 (P1-5) were detected. The growth curves of the P1 cells in lumbar and caudal groups were determined by cell counting kit 8. ResultsThe NPCs were isolated and identified successfully. The adherence time of the primary cells (the cell fusion reached 90%) in lumbar group was significantly longer than that in caudal group in primary generation (P<0.05). HE staining showed that cytoplasm was pink with the blue nucleus. Lumbar disc NPCs were spindle. The larger caudal disc NPCs were polygonal or irregular. Toluidine blue staining showed that the proteoglycan was stained as blue. In the cytoplasm of cells, collagen type Ⅱwas stained as brown surround the blue-black nucleus. The cell viability had no significant difference between lumbar and caudal groups and between different passages in the same group (P>0.05). The caudal disc NPCs reached their logarithmic growth phase after 3 days of culture, while the cells in lumbar segments did after 4-5 days of culture. The cell proliferation in caudal segments was more than that in lumbar segments at 3-9 days (P<0.05). The difference in the total protein contents was not significant between cells at P1-5 in 2 groups (P>0.05), and the caudal disc NPCs had higher protein contents than lumbar disc NPCs (P<0.05). There was no significant difference in cell senescence rate between cells at P1, P2, and P3 in 2 groups (P>0.05), but significant difference was shown in senescence rate between 2 groups in cells at P4 and P5 (P<0.05). ConclusionCaudal disc NPCs have a better status, which is more suitable for experiment as a seed cell than the lumbar disc NPCs in the same generation.
ObjectiveTo investigate the clinical efficiency of pelvic and sacrum tumor surgery using sizing balloon occlusion of the lower abdominal aorta. MethodsFrom January 2005 to June 2011, 156 patients were diagnosed to have sacrum or pelvic tumor and underwent surgery in our institution. Temporary balloon occlusion of abdominal aorta was used in 51 patients during the resection of sacrum and pelvic tumors (balloon group). Another 105 patients received the traditional surgery resection (control group). The results of the whole operation time, the volume of blood loss and transfusion, the complication and the total days of stay in hospital in the two groups were compared with each other. ResultsAfter the abdominal aorta was occluded, 92.2% of the patients in the balloon group had holistic resection or edge resection, while the number was 86.7% for the control group. In the balloon group, the average operation time was (171.96±65.16) minutes, the average intraoperative blood loss was (746.86±722.73) mL, and the blood transfusion was (411.76±613.73) mL. The postoperative lead flow was (294.50±146.09) mL, and the postoperative tube removal was within (2.98±1.07) days. Improvement of patients'condition was significantly better than the control group (P<0.05). No significant difference was found in the total days of stay in hospital and the postoperative complications between the two groups (P>0.05). ConclusionUsing abdominal aorta occlusion can effectively control intraoperative hemorrhage, and show the operation field clearly. It also can reduce operation time and control the blood transfusions. Appropriately extended balloon blocking time can obviously improve the tumor removal rate and the safety of the operation.
ObjectiveTo observe the ability of osteogenesis in vivo using the injected absorbable polyamine acid/calcium sulfate (PAA/CS) composites and assess their ability to repair bone defects. MethodWe selected 48 New Zealand white rabbits, and half of them were male with a weight between 2.0 and 2.5 kg. Bone defect models were made at the rabbit femoral condyle using electric drill, and the rabbits were divided into two groups. One group accepted implantation of the material at the defect, while nothing was done for the control group. After four, eight, twelve and sixteen weeks, the animals were killed. The line X-ray and hard tissue slices histological examination (HE, MASSON staining) were observed to assess the situation of degradation, absorption and bone formation of the material. ResultsFour weeks after operation, bone defect of the experimental group had no obvious callus growth on X-ray imaging. Histology showed that the material began to degrade and new immature trabecular bone grew. The bone defect of the experimental group had a small amount of callus growth on X-ray imaging after eight weeks. And histology showed that the material continued to degrade and new immature trabecular bone grew continually. There was an obvious callus growth after twelve weeks, and the bone defect area had smaller residual low-density shadow on X-ray imaging. Histology showed that most of the materials degraded and parts of woven bone grew into lamellar bone. After sixteen weeks, the composites were absorbed completely, replaced by new bone tissues, and the new bone was gradually changed from woven bone into mature plate of bone. There was no significant change in bone defect in the control group within twelve weeks, and part of bone defect hole became smaller, and partial edge repair could be detected. ConclusionsThe PAA/CS composites can be completely degraded and absorbed, with a certain activity of bone formation, expected to be used as bone repair materials.
ObjectiveTo investigate the value of computer assisted radiology and surgery solutions system (IQQA-Liver) in surgical planning for precise hepatectomy. MethodsThe clinical data of 95 cases performed precise hepatec-tomy from January 1, 2012 to June 30, 2013 in our hospital were retrospectively analyzed, and the computer assisted radiology and surgery solutions system was used for three dimensional quantitative analysis, volume measurement and designing for liver resection in all the cases before operation. ResultsThe intuitive and clear three dimensional images of all the 95 cases were obtained by using the computer assisted radiology and surgery solutions system, which could show the precise anatomical relationship of the liver, tumor, and main hepatic vascular.The three dimensional images could be observed at any angle and rotated freely, and could show the involved and needed to be resected vascular away from the tumor margin for 2 mm, 5 mm, 10 mm, and 20 mm and the liver volume dominated by this vascular.Of all the 95 cases, the total liver volume was (1 776.4±998.5) cm3, the proposed removed liver volume was (1 026.2±811.5) cm3, the functional residual liver volume was (795.3±522.6) cm3.The ratio of functional residual liver volume to the standard total liver volume was (58.2±25.1)%, which the ratio of patients without cirrhosis was > 30% and with cirrhosis was > 40%.All the 95 cases were implemented precise operation.The operation plan was designed based on a combination of factors such as the amount of functional residual liver volume and function.The surgery programs were changed for 13 cases, in which 9 cases were narrowed the scope of resection and 4 cases were expanded the scope of resection.No patients died perioperation. ConclusionBy using the computer assisted radiology and surgery solutions system (IQQA-Liver), we could precisely locate liver tumor, calculate the functional residual liver volume, identify the relationship between tumor and adjacent vascular, and ultimately help to design the optimal surgical plan.
ObjectiveTo investigate the regulation of human bone marrow mesenchymal stem cells (hBMSCs) osteogenic and adipogenic differentiations mediated by Wnt10b adenoviral vector in vitro. MethodsThe hBMSCs from ilial bone tissue in adults at passage 4 were infected by Wnt10b gene expression adenoviral vector (group A), Wnt10b-shRNA adenoviral vector (group B), and empty vector (group C), and non-transfected hBMSCs served as the blank control group. Then the cells were cultured separately in the circumstance of osteogenic induction, adipogenic induction, and non-induction. The alkaline phosphatase (ALP) staining, alizarin red staining, and oil red O staining were used to detect the osteogenic and adipogenic differentiations; real-time fluorescent quantitative PCR and Western blot were used to analyze the expressions of osteoblast and adipocyte genes and proteins. ResultsThe results of ALP staining were positive after osteogenic induction, group A showed strong staining, and group B showed the weakest staining. The results of alizarin red staining showed that there were a lot of patchy confluent brown mineralized nodules in group A; a few punctate brown mineralized nodules were seen in group B; and many punctuate brown mineralized nodules were found in groups C and D. The results of oil red O staining showed strong staining in groups B, C, and D after adipogenic induction, especially in group B; scattered or small clustered staining was observed in group A. The expressions of osteoblast genes and proteins were significantly higher in group A than groups B, C, and D, and in groups C and D than group B by real-time fluorescent quantitative PCR and Western blot test; however, the expressions of adipocyte genes and proteins showed a contrary tendency. ConclusionThe high level expression of Wnt10b can enhance osteogenic differentiation of hBMSCs, and the low level expression of Wnt10b can increase adipogenic differentiation of hBMSCs.
ObjectiveTo analyze the correlation between the trabecular microstructure and the clinical imaging parameters in the fracture region of osteoporotic hip so as to provide a simple method to evaluate the trabecular microstructure by a non-invasive way. MethodsBetween June 2012 and January 2013, 16 elderly patients with femoral neck fracture underwent hip arthroplasty were selected as the trial group; 5 young patients with pelvic fracture were selected as the control group. The hip CT examination was done, and cancellous bone volume/marrow cavity volume (CV/MV) was analyzed with Mimics 10.01 software in the control group. The CT scan and bone mineral density (BMD) measurement were performed on normal hips of the trial group, and cuboid specimens were gained from the femoral necks at the place of the tensional trabeculae to evaluate the trabecular microstructure parameters by Micro-CT, including bone volume fraction (BV/TV), trabecular number (Tb. N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th), connect density (Conn.D), and structure model index (SMI). The correlation between imaging parameters and microstructure parameters was analyzed. ResultsIn the trial group, the BMD value was 0.491-0.698 g/cm2 (mean, 0.601 g/cm2); according to World Health Organization (WHO) standard, 10 cases were diagnosed as having osteoporosis, and 6 cases as having osteopenia. The CV/MV of the trial group (0.670 1±0.102 0) was significantly lower than that of the control group (0.885 0±0.089 1) (t=-4.567, P=0.000). In the trial group, CV/MV had correlation with BV/TV, Tb.Th, and SMI (P<0.05); however, CV/MV had no correlation with Tb.N, Tb.Sp, or Conn.D (P>0.05). BV/TV had correlation with Tb.Th, Tb.N, Tb.Sp, and SMI (P<0.05), but it had no correlation with Conn.D (P=0.075). There was no correlation between BMD and microstructure parameters (P>0.05). ConclusionCV/MV obviously decreases in the osteoporotic hip, and there is a correlation between CV/MV and the microstructure parameters of BV/TV, Tb.Th, and SMI, to some extent, which can reflect the variety of the microstructure of the trabeculae. There is no correlation between BMD of femoral neck and microstructure parameters.
Systems-based integrated course is a advanced and mainstream educational mode in western medical education, and also it is a hotspot of current medical education reform in China. In this article, we introduce our practice and the students' feedback of the orthopedic module of 8-year clinical medicine systems-based integrated course. During practice, we follow the integral curriculum design of Sichuan University, establish a team of teachers, integrate the curriculum of relevant disciplines, and improve teaching methods, ameliorate evaluation forms, etc. Through the implementation of this course, we recognize that this new course system aims to cultivate excellent clinical doctors, which focuses on the bilateral permeation and reconstruction among disciplines, especially between basic curriculums and clinical curriculums, changes traditional teaching methods, and strengthens the ability of practice and lifelong learning.