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Objective@#To study the effect of growth differentiation factor (GDF) 11-silenced bone marrow stromal cells (BMSCs) on bone regeneration in early steroid-induced osteonecrosis of the femoral head in rats.@*Methods@#After GDF11 expression in BMSCs was inhibited by siRNA, the knockdown efficiency and transfection cytotoxicity were detected. The further experiments both in vitro (n=3) and in vivo (n=8) were divided into 4 groups respectively: blank control group (without any intervention), model group (glucocorticoid treatment), experimental group (siRNA transfection and glucocorticoid treatment) and negative control group (negative control transfection and glucocorticoid treatment). The BMSCs were induced into osteogenic differentiation. Alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the osteogenic differentiation ability of the cells while reverse transcription polymerase chain reaction (qRT-PCR) was employed to detect the relative expression levels of osteogenic markers. The osteogenesis in the necrotic femoral head was evaluated by microCT, H&E staining, immunohistochemistry staining and biomechanical test.@*Results@#No transfection cytotoxicity was found (P>0.05). The ALP staining and alizarin red staining showed that the osteogenic differentiation of BMSCs in the experimental group was better than that in the model group. At the level of mRNA, the relative expression of ALP, runt-related transcription factor (Runx) 2, osteocalcin (OCN) and type Ⅰ collagen (α1) (COL1A1) in the blank control group (1.00±0.09, 1.02±0.23, 1.03±0.30 and 1.02±0.25, respectively) were significantly higher than those in the model group (0.46±0.11, 0.50±0.11, 0.35±0.01 and 0.57±0.02, respectively) but significantly lower than those in the experimental group (1.97±0.30, 0.94±0.19, 1.50±0.18 and 1.28±0.37) (all P<0.05). MicroCT images and quantitative analysis showed that the bone mass in the experimental group was significantly increased compared with the model group (P<0.05). Histological examination showed better bone regeneration and higher expression of Runx2 and COL1 in the necrotic femoral head in the experimental group than in the model group. Improved biomechanical properties were shown in the experimental group compared with the model group (P<0.05).@*Conclusions@#Silence of GDF11 expression may alleviate the inhibitory effect of glucocorticoid on osteogenic differentiation of BMSCs. Early transplantation of GDF11-silenced BMSCs may promote osteogenesis in the necrotic femoral head in rats.
ABSTRACT
Objective To study the effect of growth differentiation factor (GDF) 11-silenced bone marrow stromal cells (BMSCs) on bone regeneration in early steroid-induced osteonecrosis of the femoral head in rats.Methods After GDF11 expression in BMSCs was inhibited by siRNA,the knockdown efficiency and transfection cytotoxicity were detected.The further experiments both in vitro (n =3) and in vivo (n =8)were divided into 4 groups respectively:blank control group (without any intervention),model group (glucocorticoid treatment),experimental group (siRNA transfection and glucocorticoid treatment) and negative control group (negative control transfection and glucocorticoid treatment).The BMSCs were induced into osteogenic differentiation.Alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the osteogenic differentiation ability of the cells while reverse transcription polymerase chain reaction (qRT-PCR) was employed to detect the relative expression levels of osteogenic markers.The osteogenesis in the necrotic femoral head was evaluated by microCT,H& E staining,immunohistochemistry staining and biomechanical test.Results No transfection cytotoxicity was found (P ＞ 0.05).The ALP staining and alizarin red staining showed that the osteogenic differentiation of BMSCs in the experimental group was better than that in the model group.At the level of mRNA,the relative expression of ALP,runt-related transcription factor (Runx) 2,osteocalcin (OCN) and type Ⅰ collagen (α1) (COL1A1) in the blank control group (1.00 ± 0.09,1.02 ± 0.23,1.03 ± 0.30 and 1.02 ± 0.25,respectively) were significantly higher than those in the model group (0.46±0.11,0.50±0.11,0.35±0.01 and0.57±0.02,respectively) but significantly lower than those in the experimental group (1.97±0.30,0.94±0.19,1.50±0.18 and 1.28 ±0.37) (all P ＜ 0.05).MicroCT images and quantitative analysis showed that the bone mass in the experimental group was significantly increased compared with the model group (P ＜ 0.05).Histological examination showed better bone regeneration and higher expression of Runx2 and COL1 in the necrotic femoral head in the experimental group than in the model group.Improved biomechanical properties were shown in the experimental group compared with the model group (P ＜ 0.05).Conclusions Silence of GDF11 expression may alleviate the inhibitory effect of glucocorticoid on osteogenic differentiation of BMSCs.Early transplantation of GDF11-silenced BMSCs may promote osteogenesis in the necrotic femoral head in rats.
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BACKGROUND:A unified standard for cervical pedicle screw placement does not currently exist;therefore, it is difficult to quantitatively evaluate the clinical effects of the technique. Digital navigation can provide a reference for accurate and safe location, orientation, and placement of cervical pedicle screws. OBJECTIVE:To investigate whether digital navigation can greatly increase the accuracy and safety of cervical pedicle screw placement. METHODS:This was a prospective, single-center, randomized control ed, open-label trial. Seventy-six patients with cervical spine fracture scheduled to receive treatment in the Department of Orthopedics, Affiliated Hospital of Nantong University, China were randomly divided into three groups to undergo cervical pedicle screw internal fixation. Patients in the cervical lamina partial excision group (n=26, 160 screws) underwent partial cervical lamina excision and cervical pedicle screw internal fixation;those in the pipeline-dredge discharge group (n=27, 156 screws) underwent pipeline-dredge discharge and cervical pedicle screw internal fixation;and those in the digital navigation group (n=23, 162 screws) underwent digital navigation-assisted cervical pedicle placement. Al patients were evaluated at 12 and 36 months. The primary outcome was the percentage of screws graded I when evaluating the penetration degree of the cervical pedicle screws, which evaluates the accuracy of screw placement, 12 months after internal fixation. Secondary outcomes included:(1) the percentage of screws graded I when evaluating the penetration degree of cervical pedicle screws 36 months after internal fixation;(2) bony fusion rate of the atlantoaxial joint, used to evaluate fracture healing, 12 and 36 months after internal fixation;(3) Visual Analogue Scale spine score, used to evaluate cervical neck pain, prior to and 12 and 36 months after internal fixation;(4) American Spinal Injury Association Classification, used to evaluate improvement in neurological function, prior to and 12 and 36 months after internal fixation;and (5) adverse events, used to evaluate the safety of each pedicle screw implantation method, 12 and 36 months after internal fixation. This trial protocol was approved by Medical Ethics Committee, Affiliated Hospital of Nantong University, China, and was performed in accordance with the guidelines of the Declaration of Helsinki, formulated by the World Medical Association. Signed informed consent regarding trial procedure and treatment was obtained from each patient. DISCUSSION:This trial protocol compared the effects of three cervical pedicle screw internal fixation methods for the treatment of cervical spine fracture, and investigated and compared the accuracy and safety of digital navigation-assisted cervical pedicle screw placement with partial cervical lamina excision and pipeline-dredge discharge. We hoped to provide quantitative evidence for the clinical use of digital navigation in orthopedics, especial y in cervical pedicle screw placement. TRIAL REGISTRATION:This trial was registered at ClinicalTrials.gov identifier:NCT02880839 on 19 August 2016.
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<p><b>OBJECTIVE</b>To establish a simple method to detect four ATP7B gene mutations in Wilson disease using allele-specific PCR(AS-PCR) with whole blood polymerase chain reaction.</p><p><b>METHODS</b>Four allele-specific PCR primers specific for the mutations(G2333T, C2850T, G2855A, G2975T) were designed, and PCR was optimized to screen the whole blood samples. The amplified gene products with mutation were separated with agarose gel electrophoresis to detect the pattern of point mutation and allele types. Exons 8, 12 and 13 of the ATP7B gene were amplified with PCR, and the amplification products were sequenced to confirm the mutation.</p><p><b>RESULTS</b>The detection of four ATP7B gene mutations by AS-PCR with whole blood was accomplished with 100% accuracy. In the 27 healthy subjects, the mutation rate of G2855A was 51.8%. No mutation was detected for G2333T, C2850T and G2975T. Among the 22 patients, 11 were mutated for G2333T, C2850T or G2975T. The mutation rate was therefore 50%.</p><p><b>CONCLUSION</b>Our experiment has established an AS-PCR based method for detecting four ATP7B gene mutations using whole blood samples, which has provided a simple and effective means for the early diagnosis of Wilson disease. This method is rapid, convenient, accurate and economical for detecting point mutations of the ATP7B gene.</p>