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1.
Journal of Southern Medical University ; (12): 409-414, 2019.
Article in Chinese | WPRIM | ID: wpr-772086

ABSTRACT

OBJECTIVE@#To assess the geometrical matching of a new anatomical adaptive titanium mesh cage (AA-TMC) with the endplate and its effect on cervical segmental alignment reconstruction in single- and two-level anterior cervical corpectomy and fusion (ACCF) and compare the compressive load at the endplate between the AA-TMC and the conventional titanium mesh cage (TMC).@*METHODS@#Twelve cervical cadaveric specimens were used to perform single- and two-level ACCF. The interbody angle (IBA), interbody height (IBH) and the interval between the AA-TMC and the endplate were evaluated by comparison of the pre- and postoperative X-ray images. The maximum load at the endplate was compared between the AA-TMC and TMC based on American Society for Testing and Materials (ASTM) F2267 standard.@*RESULTS@#No significant differences were found between the preoperative and postoperative IBA and IBH in either single-level ACCF (11.62°±2.67° 12.13°±0.69° and 23.90±2.18 mm 24.23±1.13 mm, respectively; > 0.05) or two-level ACCF (15.63°±5.06° 16.16°±1.05°and 42.93±3.51 mm 43.04±1.70 mm, respectively; > 0.05). The mean interval between the AA-TMC and the endplate was 0.37 ± 0.3 mm. Compared to the conventional TMC, the use of AA-TMC significantly increased the maximum load at the endplate in both single-level ACCF (719.7±5.5 N 875.8±5.2 N, < 0.05) and two-level ACCF (634.3±5.9 N 873±6.1 N, < 0.05).@*CONCLUSIONS@#The use of AA-TMC in single-level and two-level ACCF can significantly increase the maximum load at the endplate to lower the possibility of implant subsidence and allows effective reconstruction of the cervical alignment.


Subject(s)
Humans , Biomechanical Phenomena , Cervical Vertebrae , Prostheses and Implants , Spinal Fusion , Surgical Mesh , Titanium , Treatment Outcome
2.
Chinese Journal of Trauma ; (12): 686-692, 2019.
Article in Chinese | WPRIM | ID: wpr-754700

ABSTRACT

Objective To evaluate the preliminary clinical effect of anterior anatomical reduction plate fixation on the treatment of atlantoaxial dislocation. Methods A retrospective case series study was conducted to analyze the 13 patients with atlantoaxial dislocation admitted to the second affiliated hospital of Xi'an Jiaotong University from January 2016 to December 2017. There were eight males and five females, aged 20-57 years, with an average age of 42 years. All patients received transoropharyngeal reconstruction and atlantoaxial anterior anatomical reduction plate fixation, 12 of which underwent the surgery for the first time but one had the revision surgery. The operation time and intraoperative bleeding were recorded. The angle of the clivus axis was measured, and the reduction of the atlantoaxial spine and the fusion of bone graft were observed. The neurological function was evaluated by Japanese Orthopedic Association ( JOA ) score and the improvement rate of spinal cord function was calculated. The complications were also recorded. Results All patients were followed up for 10-30 months [(14. 2 ± 5. 0)months]. The operation time was 150-285 minutes [(216. 8 ± 36. 7)minutes]. The intraoperative blood loss was 50-130 ml [(80. 5 ± 19. 7)ml]. The slope axis angle was (113. 2 ± 9. 1)° before operation and (145. 8 ± 6. 7)° after operation, with an average increase of 32. 6° (P<0. 01). Anatomical reduction was obtained in nine patients, and partial reduction in four patients. At the last follow-up, the atlantoaxial fusion was obtained in all patients, and the healing time was ( 4. 6 ± 1. 1 ) months. Postoperative neurological symptoms were improved compared with those before operation. The JOA score was improved from preoperative (8. 7 ± 1. 7) points to postoperative (14. 3 ± 1. 2) points, with an average increase of 5. 6 points (P<0. 01). The average improvement rate of spinal cord function was 69%. Except for one patient with cerebrospinal fluid leakage, there were no complications such as spinal cord, nerve, blood vessel injury or wound infection after operation. Conclusions Anterior atlantoaxial anatomical reduction plate fixation can effectively restore the dislocated atlantoaxial joint, restore slope axis angle, improve bone fusion rate, and improve nerve function. It can be used as an alternative or supplement to posterior fixation.

3.
Progress in Modern Biomedicine ; (24): 5369-5372, 2017.
Article in Chinese | WPRIM | ID: wpr-614990

ABSTRACT

Objective:To discuss the clinical efficacy of minimally invasive locking plate and the traditional open reduction with internal fixation in the treatment of proximal humerus fractures.Methods:89 cases of patients with proximal humeral fractures were selected and divided into two groups according to different surgical methods.The observation group (45 cases) was given minimally invasive locking plate,while the control group (44 cases) was treated with the traditional open reduction and internal fixation.The operation time,hospitalization time,fracture healing time,intraoperative blood loss,Neer score and Constant-Murley score at 1 month after postoperatiion were compared between two groups.Results:The operation time,hospitalization,fracture healing time of observation group were significantly shorter than those of the control group,and the bleeding amount of observation group was less than that of the control group (P<0.05).At 1 months after operation,the Constant-Murley scores of observation group were significantly better than those of the control group (P<0.05).The For Neer scores,excellent rate of observation group (91.1%) were significantly higher than those of the control group (68.1%,P<0.05).Conclusion:Compared with the traditional open reduction with internal fixation,minimally invasive locking plate was better,safer,faster and more effectively for promoting the recovery of shoulder function of patient with proximal humerus fractures.

4.
Chinese Journal of Tissue Engineering Research ; (53): 5642-5649, 2016.
Article in Chinese | WPRIM | ID: wpr-504847

ABSTRACT

BACKGROUND:We have designed and manufactured a novel artificial cervical vertebra and intervertebral complex (ACVC) which combines the cervical titanium cage with the artificial cervical disc, and also developed the ACVC with a hydroxyapatite biocoating (ACVC-HA). OBJECTIVE:To evaluate biomechanical properties of the joint system, and the role of HA coating in promoting osseointegration and long-term stability. METHODS:Twenty-four goats were randomly divided into three groups and underwent the anterior C2/3 and C3/4 discectomy, and C3 subtotal corpectomy, fol owed by ACVC implantation (group 1) and ACVC-HA implantation (group 2), and given no intervention (black control group), respectively. group. At 12 weeks after surgery, C1-5 samples were col ected to undergo biomechanical tests and histological staining. RESULTS AND CONCLUSION:Prior to the fatigue test, compared with the blank control group, the range of motion and neural zone of groups 1 and 2 in the directions of flexion-extension and lateral bending showed no significant differences, but the above indicators were significantly increased in the direction of rotation (P<0.05). Additional y, the stiffness in al three directions was significantly lower than that in the blank control group (P<0.05). There were no significant differences in the range of motion and neural zone in al directions between groups 1 and 2. Similar results were found after the fatigue test. The histological staining showed that both two implants had good biocompatibility and abradability, but more new bone formed on the ACVC-HA. These results suggest that ACVC can effectively reconstruct the motor function of the cervical spine after decompression. Furthermore, HA coating can markedly improve bone-implant interface to promote osseointegration.

5.
Chinese Journal of Tissue Engineering Research ; (53): 671-676, 2016.
Article in Chinese | WPRIM | ID: wpr-485801

ABSTRACT

BACKGROUND:Cauda equina syndrome often induces skin hypoesthesia in the perineal area, poor urine-stool control, and impairs male function. After peripheral nerve fiber injury, apoptosis of neurons appeared. This is associated with the nature of the injury, the types of neurons, the species of animals, the age, and the distance between neurons. OBJECTIVE:To explore the motor neuron apoptosis and expression of apoptosis-associated protein in the anterior horn of the spinal cord after acute cauda equina compression. METHODS:A total of 27 canines were randomly divided into three groups. In the compression and control groups, models of cauda equina compression were established. In the normal group, no models were established. Compression group received water sac compression for 4, 8, 12, 24, 48, 72 and 168 hours, with three models in each group. In the control group, only water sac was implanted, but water was not injected. Terminal deoxynucleotidyl transferase TdT-mediated biotin dUTP nick end-labeling assay was used to detect the apoptosis of neurons in the anterior horn of the spinal cord. Bcl-2, Bax and Caspase-3 protein expressions were measured by immunohistochemical staining (strept avidin-biotin complex). Gray values of positive cels of Bax, Bcl-2 and Caspase-3 protein expressions were detected using Qwin550Cw image colection and analysis system. RESULTS AND CONCLUSION:The apoptosis of motor neuron occurred in the compression groups. At 12 hours of compression, positive cels were detected, and the number of positive cels reached a peak at 72 hours. Bax and Bcl-2 protein expression was smal in the normal group. Caspase-3 protein expression was not detected in the normal and control groups. Bax and Bcl-2 protein expression was significantly increased at 8 hours, peaked at 72 hours and reduced to a normal level at 168 hours. The increased range of Bax protein expression was bigger than that of Bcl-2. Caspase-3 protein began to express at 12 hours, peaked at 72 hours and reduced to a low level at 168 hours. Bax and Caspase-3 protein expression peaked at 72 hours, and Bcl-2 protein expression was not obviously increased. These findings verified that after acute cauda equina compression, the apoptosis of neurons occurred in the anterior horn of the spinal cord. Bax and Bcl-2 protein expression showed an antagonistic action. In the Bax/Bcl-2 complex, Bax protein in a high expression promoted apoptosis, induced Caspase-3 protein expression, and neuronal apoptosis.

6.
Chinese Journal of Tissue Engineering Research ; (53): 5973-5978, 2016.
Article in Chinese | WPRIM | ID: wpr-503594

ABSTRACT

BACKGROUND:Animal studies have shown that cauda equina compression can induce apoptosis of lumbosacral spinal cord anterior horn motor neurons. OBJECTIVE:To explore the pathological change in lumbosacral spinal cord after acute cauda equina compression in dogs. METHODS:A total of 27 dogs were randomly divided into nine groups, with three dogs in each group. There were one normal control group, seven experimental groups and one sham surgery group. In the experimental group, an empty water sac was implanted above epidural fat below L6 vertebral plate. Compression was given by injecting water at 4, 8, 12, 24, 48, 72 and 168 hours. In the sham surgery group, an empty water sac was implanted, but compression was not given. At the time of compression, the spinal cord sent out by cauda equina nerve and adjacent to the head end was subjected to histopathological examination. RESULTS AND CONCLUSION:(1) Results of light microscope:at 4-48 hours of compression, spinal cord anterior horn motor neurons did not alter. At 72 hours, motor neurons became smal , cel membrane shrank and separated from surrounding tissues. Cel s were homogenous and darkly stained. At 168 hours, motor neurons disappeared, but spinal cord sections of the adjacent head end did not shown abnormal motor neurons in the spinal cord anterior horn. (2) Results of electron microscope:at 12 hours, spinal cord tissue began to swel , and the swel ing aggravated with prolonged time of compression. The swel ing of glial cel s was apparent. At 168 hours, myelin sheath structure dissolved;axons showed vacuolization;axoplasm spil ed, and exhibited inflammatory injury-like changes. (3) Apoptotic results of spinal cord anterior horn motor neurons:apoptosis appeared at 12 hours of compression, became increased, and showed an increased trend at 168 hours.

7.
Chinese Journal of Tissue Engineering Research ; (53): 6296-6301, 2016.
Article in Chinese | WPRIM | ID: wpr-503413

ABSTRACT

BACKGROUND:Previous study has found that hsa-miR-182 is probably related to the apoptosis-related genes such as cytochrome C (Cycs C) and calcineurin subunit CnB (PPP3R1) in nucleus pulposus cells. OBJECTIVE:To determine whether miR-182 plays a regulatory role in nucleus pulposus cel apoptosis by detecting the relative gene expression levels after transfecting miR-182 with Cycs C and PPP3R1 into nucleus pulposus cel s via plasmid delivery. METHODS:After a bioinformatics prediction about miR-182, miR-182 and target genes were transfected into the nucleus pulposus cel s, and at the same time, blank control group was established. Then the expression levels of the target genes were detected through cel lysis. RESULTS AND CONCLUSION:miR-182 significantly inhibited the expression of Cycs C in nucleus pulposus cel s compared with the blank control group (P<0.05). Compared with the blank control group, miR-182 made no inhibitory effect on the expression of PPP3R1. These findings suggest that miR-182 may play a regulatory part in nucleus pulposus cel apoptosis by inhibiting the expression of Cycs C.

8.
Chinese Journal of Tissue Engineering Research ; (53): 4860-4864, 2015.
Article in Chinese | WPRIM | ID: wpr-476308

ABSTRACT

BACKGROUND:Titanium aloy and stainless steel are two common internal fixation materials, but there are some difference in their therapeutic effects and biocompatibility. OBJECTIVE:To explore the therapeutic effects and biocompatibility of titanium aloy and stainless steel internal fixation materials for the treatment of spinal tuberculosis. METHODS:Seventy-one spinal tuberculosis patients, 35 males and 36 females, aged 17-81 were enroled. Among them, 35 patients received titanium aloy internal fixation, and the 36 patients underwent stainless steel internal fixation. At the end of 12-month folow-up, Cobb angle changes, therapeutic effect and Frankel grade were analyzed in the two groups. RESULTS AND CONCLUSION:Before treatment, there was no difference in the spinal kyphosis angle and Frankel grade between the two groups. At the last folow-up, the Frankel grade and Cobb angle were both improved in the two groups (P < 0.05), but there was stil no difference between the two groups. The cure rate was 97% (n=34) in the titanium aloy group and 92% in the stainless steel group (n=33), and no significant difference was found between the two groups. These two kinds of internal fixation materials exhibited good biocompatibility, and no infection and other adverse reactions occurred. These findings indicate that both titanium aloy and stainless steel as internal fixation materials have good biocompatibility and therapeutic effects.

9.
Chinese Journal of Postgraduates of Medicine ; (36): 4-7, 2014.
Article in Chinese | WPRIM | ID: wpr-474708

ABSTRACT

Objective To investigate the clinical results and related key points of surgical treatment for Hawkins Ⅲ talus neck fractures.Methods Forty-one patients with Hawkins Ⅲ talus neck fracture were treated.The fractures occurred on the left side in 21 patients and on the right side in 20 patients.All patients were performed internal fixation by internal and lateral approaches.The weight bearing should be adjusted with follow-up.The functional results were evaluated by American Orthopaedic Foot and Ankle Society (AOFAS).Results The average duration of follow-up was (37.7 ± 8.2) months.All fractures gained union and the average union time was (4.1 ±0.5) months.The average AOFAS score after treatment was (79.3 ± 2.6) scores which was higher than that before treatment [(35.1 ± 8.0) scores],and there was significant difference (P =0.026).There were 11 cases in excellent,17 cases in good,11 cases in fair and 2 cases in poor.The excellent and good rate was 68.3% (28/41).Traumatic arthritis occurred in 18 cases (43.9%,18/41),involved tibial astragaloid joint in 4 cases,involved subtalar joint in 6 cases,involved tibial astragaloid joint and subtalar joint in 8 cases.Avascular necrosis occurred in 7 cases (17.1%,7/41).Conclusions The effect of surgical treatment for Hawkins Ⅲ talus neck fracture via a bilateral approaches is satisfactory.Pay more attention to protect blood circulation intraoperative and perform anatomic reduction according to the characteristic of body of talus displacement.After operation,functional rehabilitation should be directed by the principle of early movement,later weighted.

10.
Journal of Pharmaceutical Analysis ; (6): 120-122, 2010.
Article in Chinese | WPRIM | ID: wpr-621617

ABSTRACT

Objective To explore the law of apoptosis of lumbar spinal cord neurons in cauda equina syndrome (CES). Methods Cauda equina of rats was compressed by a piece of silica gel stick. From day 1 to day 28, the lumbar spinal cord specimens were harvested and assessed by Nissl's staining and TUNEL staining. Results Compression of cauda equina caused lesion and apoptosis of neurons in lumbar spinal cord, and the extent of apoptosis reached the peak on 7th day after compression. Conclusion Apoptosis of neurons in lumbar spinal cord might be one of the reasons why patients with CES get poor prognosis.

11.
Journal of Pharmaceutical Analysis ; (6): 175-178, 2010.
Article in Chinese | WPRIM | ID: wpr-621590

ABSTRACT

Objective To analyze proliferation and differentiation of glial fibrillary acid protein (GFAP)- and nestin-positive (GFAP+/nestin+) cells isolated from the subventricular zone following fluid percussion brain injury to determine whether GFAP+/nestin+ cells exhibit characteristics of neural stem cells. Methods Male Sprague-Dawley rats, aged 12 weeks and weighing 200-250 g, were randomly and evenly assigned to normal control group and model group. In the model group, a rat model of fluid percussion brain injury was established. Five days later, subventricular zone tissue was resected from each group and made into single cell suspension. After serum-free neural stem cell medium culture and subsequent serum-induced differentiation, cell type, proliferation and differentiation capacities were determined by immunofluorescence staining and flow cytometry. Results At 3-7 days after fluid percussion brain injury, nestin+/GFAP+ cells in the single cell suspension from the model group significantly outnumbered those from the normal control group (P<0.01). In the model group, an increased number of small neurospheres with smooth cell edge and bulged center formed after primary culture, and were clearly visible with the increase of culture time and medium replacement. After several passages, many clonal spheres were obtained, suggesting strong self-proliferatiing capacity. Neurospheres from the model group differentiated into astrocytes, neurons and oligodendrocytes. Conclusion GFAP+/nestin+ cells isolated from the adult rat subventricular zone after fluid percussion brain injury are thought to be neural stem cells because of their self-renewal and multi-differentiation capacities.

12.
Journal of Xi'an Jiaotong University(Medical Sciences) ; (6): 221-223,238, 2010.
Article in Chinese | WPRIM | ID: wpr-597474

ABSTRACT

Objective To explore the expression and effect of transforming growth factors-β1 (TGF-β1) and heat shock protein (HSP47) on gluteal muscle contracture (GMC). Methods We collected contraction band and adjacent muscle from GMC patients and explored the expression of TGF-β1 and HSP47 using immunohistohemistry, reverse transcription and polymerase chain reaction (RT-PCR), and Western blot analysis. Results TGF-β1 and HSP47 were intensely expressed in fibroblast cells and vascular endothelial cells. The expression of them increased 8.1-fold and 3.6-fold at the mRNA level, respectively (P<0.05). The same changes were found at the protein level, which increased 11.2-fold and 7.6-fold, respectively (P<0.05). Conclusion Up-regulation of TGF-β1 and HSP47 may initiate fibrotic cascade in the gluteal muscles of GMC patients.

13.
Chinese Journal of Tissue Engineering Research ; (53): 1053-1057, 2010.
Article in Chinese | WPRIM | ID: wpr-403528

ABSTRACT

BACKGROUND: There are no effective treatments for spinal cord injury. Transplantation of olfactory ensheathing cells (OECs) has achieved great progress in repairing spinal cord injury. OBJECTIVE: To observe the effect of OECs transplantation on pathological and ultrastructural alterations of spinal cord, and the role in spinal cord injury developing.METHODS: A total of 60 SD rats were randomly divided into blank, model, transplantation and DF12 groups, with 15 animals in each group. The entire vertebral plate of T_(10), and partial vertebral plate of T_9 and T_(11) of blank group were cut open, and gelatin sponge was used for hemostasis. In the model group, the spinal cord was excised. In the transplantation and DF12 groups, OECs and DF12 culture solution were injected following spinal cord excision. The incision was sutured. Two rats from each group were anesthetized 1, 3, 7, 14, 28, 42, and 56 days following injury, and injured areas were observed by light microscopy and electron microscopy. RESULTS AND CONCLUSION: Following spinal cord injury, pathological and ultrastructural changes occurred, such as hemorrhage, edema, degeneration, necrosis, cavitation, gliacyte proliferation and nerve fiber regeneration. OECs transplantation attenuated neuronal and nerve fiber necrosis, relieved degree of pathological reaction, protected injured neurons, prevented gliacyte proliferation and increased nerve fiber regeneration. Results show that OECs transplantation ameliorated pathological reactions and promoted spinal cord injury repair.

14.
Journal of Pharmaceutical Analysis ; (6): 212-216, 2007.
Article in Chinese | WPRIM | ID: wpr-621707

ABSTRACT

Objecttive To observe the repaired effect of distinct source olfactory ensheathing cells (OECs) on spinal cord injury (SCI) rats. Methods These OECs were dissociated from olfactory bulb and olfactory mucosa of SD rats and transplanted to the injuried region of spinal cord injury rats. The function of nerve, motor evoked potential of hind legs and the histopathlogical diversities of injuried spinal cord were observed. Results The OECs grafts into the SCI area could survive longer time. The BBB scale, incubation stage of EP and histopathologic manifestations showed that the group with transplanted OECs regained more improvement in hindlimb than the control group. Conclusion The OECs of two sources have the same ability to regain and improve the axonal function which can promote axons regeneration of SCI.

15.
Journal of Pharmaceutical Analysis ; (6): 83-85,89, 2006.
Article in Chinese | WPRIM | ID: wpr-625019

ABSTRACT

Objective To explore the influence of the acute cauda equina compression on the lumbosacral spinal cord; To clarify the pathologic change of the motor neuron after acute cauda equina compression. Methods 27 canis familiaris were randomly divided into 9 groups (3 in each) : one for normal group, one for control group, and seven for compression groups. The control group and compressed groups was given operation and the sac made of silica gel was placed under the lamina of L5-6. Water was injected into the sac until their posterior legs paralysis in compressed groups, the animals had been compressed for 4, 8, 12, 24, 48, 72, 168 hours. The control group were not injected water. Cells apoptosis was investigated with the technology of TdT-mediated biotin dUTP nick end-labeling (TUNEL)staining. The Bcl-2 Bax and Caspase-3 protein was investigated by immunohistochemical method. Results TUNEL staining cells in anterior horn presented after compressed 8-12 hours, and at 72 hours the number of positive cells got to maximum, it decreased subsequently after 168 hours. The protein of Bax, Bcl-2 expressed a little in normal motor neuron. The caspase-3 protein didn't express in normal cell. They all reached the peak at 72 hours after compression.Conclusion The apoptosis of motor neuron occurred earlier after cauda equina acute compression. Bax protein restrained Bcl-2 protein then active caspase-3 and conduced apoptosis of motor neuron.

16.
Journal of Pharmaceutical Analysis ; (6): 138-141, 2001.
Article in Chinese | WPRIM | ID: wpr-621808

ABSTRACT

Objective To find out an effective therapeutic method for and observe whether there is any synergistic action or not between fetal spinal cord transplantation (FST) and methylprednisolone (MP).Methods Fifty male adult SD rats were randomly divided into group A,B,C,D and E,10 in each group.Group A was treated with both large dosage of MP and FST,group B with MP only, grop C with FST only and group D without any treatment.Group E served as blank control.Fetal spinal cord was obtained from 14-day pregnant rats .Spinal cord Somatosensory evoked potential (SSEP) examination and behavior observation were performed in 24 hours and in 8 months after treatment By the way of reduced silver staining, the condition of nerve plerosis and regeneration could be observed.Results There were significant differences in the latent period and amplitude of N1 wave in SSEP between group A and group B,C and D (P<0.05).No obvious behavior changes were found except partial sensory recovery in the left lower limbs in Group A.Histologically,more nerve fibers contacting with branches at injury area could be found in Group A than in Group B,C and D.Conclusion The combination of large dosage of MP and FST can produce synergistic effect in the recovery of the injured spinal cord.

17.
Chinese Journal of Orthopaedics ; (12)2000.
Article in Chinese | WPRIM | ID: wpr-537718

ABSTRACT

Objective To explore the indications of physiotherapy and operative treatment for gluteal muscles contracture, and discuss the principles and necessity of classification in order to facilitate clinical management. Methods The standards of classification of gluteal muscles contracture were based on the signs and pathological examination, It consisted of three stages upon our standards. 210 patients could be divided into stage Ⅰof 23 cases, stage Ⅱand Ⅲ of 187 cases. For the patients of stageⅠ, physiotherapy was performed; However, all cases of stageⅡand Ⅲ, operative treatment was adopted by small superomedial incision above the greater trochanter. Furthermore, in the cases of stage Ⅱ, tight fibrotic gluteal muscles and thick contracted fascia lata should be released; For stage Ⅲ lesion, the release should be extended to the capsule of hip, and incision of the posterior capsule was necessary. Results In the group of stageⅠ, The symptoms and signs were relieved by maneuver , the rate of excellent and good results was 39%; while in the group of stageⅡand Ⅲ, it was 97% by operative treatment. Conclusion The staging system of gluteal muscles contracture could demonstrate exactly the severity degree of the disease, and is useful in choosing therapeutic method. Non-operatiive method can obtain satisfactory results in about 1/3 cases of stage Ⅰof gluteal muscles contracture; operative method is indicated for the patients of stage Ⅱand Ⅲ.

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