"Sclerotic Band" type of classification system and measurement of necrotic area for osteonecrosis of the femoral head.

Osteonecrosis of the femoral head is a common orthopedic disease. Based on years of clinical experience and significant imaging data, this study aimed to elucidate a new type of it, to help improve prognosis in young adults and provide a basis for hip preservation treatment.From January 2014 to December 2016, a total of 211 patients undergoing hip preservation surgery for femoral head necrosis at our hospital were enrolled in this study. Coronal plane classification and cross-sectional area analysis were performed by nuclear magnetic resonance imaging (computed tomography optional) in cases meeting the inclusion criteria. Meanwhile, a new method of classification and calculating the necrotic area was proposed. The application simulation was conducted using sample cases. Additionally, treatment methods were recommended. We used our method to compare the outcome of the selected patients with the JIC classification so as to judge the advantages and disadvantages.The " pressure bone trabecular angle " of the femoral head was measured, and the "sclerotic band" (Zhang Ying) type of classification system and the "quartile" (Zhang Ying) method of measurement were used in 2 sample cases. After analysis, it is more accurate than JIC.The "Sclerotic band" type of classification system and 'quartile' methods are new methods to evaluate the stability of femoral head necrosis. They are convenient for clinical application and easily adopted.

Cinobufagin Suppresses The Characteristics Of Osteosarcoma Cancer Cells By Inhibiting The IL-6-OPN-STAT3 Pathway.

BACKGROUND: Current clinical treatments for osteosarcoma are limited by disease recurrence and primary or secondary chemoresistance. Cancer stem-like cells have been proposed to facilitate the initiation, progression, recurrence and chemoresistance of osteosarcoma. Furthermore, previous studies have reported that IL-6-STAT3 pathway is overexpressed in various types of cancer and contributes to cell proliferation, apoptosis, invasion/migration, chemoresistance and modulation of stemness features. AIM: To examined the effect of cinobufagin on cancer progression and modulation of stemness features in osteosarcoma, and investigated the molecular mechanisms underlying such effects. METHODS: Human osteosarcoma cell lines U2OS/MG-63 were recruited in this study. Cell proliferation, migration, and invasion were determined by MTT assay, colony formation assay,wound healing assay, and cell invasion assay respectively. Its effect on stemness was assessed by flow cytometry and mammosphere formation. The protein expression levels of related proteins were detected by Western blot. The xenograft model, immunofluorescence staining and immunohistochemistry were used to determine the effect of cinobufagin on tumorigenicity in vivo experiment. RESULTS: We found that cinobufagin suppressed the viability of U2OS/MG-63 spheroids/parent cells in a time-and dose-dependent manner. Notably, cinobufagin had no effect on the viability of hFOB 1.19 cells. Moreover, cinobufagin induced apoptosis, increased the width of wounds, reduced invasive osteosarcoma spheroids/parent cell numbers and reduced EMT phenotype and OPN levels in U2OS/MG-63 spheroids as well as U2OS/MG-63 parent cells lines. Noticeablely, we found that OPN levels were higher in spheroids group than that in parent cells. In addition, cinobufagin ameliorated the proportion of CD133-positive cells, the size of spheroids and Nanog, Sox-2 and Oct3/4 protein levels. Our in vivo experiments showed that cinobufagin consistently reduced tumor volume,the expressions of OPN, Sox-2, Oct3/4, Nanog and p-STAT3 by the immuno histochemistry staining as well as CD133 expression in tumor tissues by immunofluorescence analysis. From a mechanistic point of view, cinobufagin was shown to inhibit IL-6-OPN-STAT3 signaling pathway. Exogenous IL-6/OE-OPN/overexpression STAT3 attenuated the induction of cinobufagin-mediated apoptosis and the suppression of stemness properties respectively. CONCLUSION: Collectively, our data demonstrated that cinobufagin inhibited the viability and tumorigenesis capability of osteosarcoma cells by blocking IL-6- OPN-STAT3 signaling pathway. Cinobufagin may therefore represent a promising therapeutic agent for osteosarcoma management.

IL-6 Promotes Cancer Stemness and Oncogenicity in U2OS and MG-63 Osteosarcoma Cells by Upregulating the OPN-STAT3 Pathway.

Background: Cancer stem cells (CSCs) are associated with tumor development, chemoresistance, recurrence, metastasis, and even prognosis. Interleukin (IL)-6 overexpression has been implicated in the development of various cancers, including osteosarcoma. This study aimed to investigate the role of IL-6 in modulating clinicopathological features, malignant traits, and stemness in osteosarcoma, and to determine the mechanisms underlying IL-6-mediated osteosarcoma progression. Methods: Patients with osteosarcoma (n = 54) and healthy controls (n = 50) were selected. No patients received any pre-operative cancer treatment. Serum levels of IL-6 were determined in patients with osteosarcoma by ELISA and their relationship with pathological features and prognosis analyzed. The 3-(4,5-dimethyl -2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) and colony formation assays were used to evaluate cell proliferation, transwell assays were used to assess the invasive potential of cells, and cell migration rates were analyzed using a wound healing assay. Tumor self-renewal was detected using a spheroid formation assay and CD133 and CD44 expression assessed by flow cytometry. Protein levels of NANOG, SOX2, OCT3/4, OPN, and epithelial-to-mesenchymal transition (EMT)-related markers, and the phosphorylation status of STAT3, were determined by western blotting. Finally, cell viability was determined with or without cisplatin (cis-dichlorodiammineplatinum [DDP])/adriamycin (ADR) treatment. Xenograft tumor models were established by subcutaneous injection of osteosarcoma spheroids, with or without IL-6. Results: Serum IL-6 levels were higher in osteosarcoma patients than controls. There was no significant association of serum IL-6 level with age, sex and tumor size; however, it was associated with TNM stage, and lung metastasis (P < 0. 05). IL-6 significantly increased proliferation and colony formation of osteosarcoma cells, and enhanced their invasion and migratory potential, thus promoting an EMT-like phenotype and elevated chemoresistance of to DDP/ADR. Spheroid size/proportion of CD133+CD44+ cells and SOX2, OCT3/4, and NANOG protein levels were elevated by IL-6 treatment in a time-dependent manner; however, IL-6 did not substantially influence any of these features in hFOB 1.19 and T98G cells. Knockdown of IL-6 reduced cell viability, colony formation, and invasion/migration ability, and reversed EMT, whereas it increased chemosensitivity to DDP/ADR. Blocking IL-6 expression with siRNA also caused loss of stemness, including reducing self-renewal ability, and reduced the proportion of CD133/CD44-positive cells, and expression of stemness-related genes. Pretreatment with the STAT3 inhibitor, S3I-201, decreased sphere size, and downregulated NANOG, SOX2, and OCT3/4 protein levels, compared with IL-6 treatment alone. Furthermore, OPN levels were elevated in response to IL-6 and an anti-OPN antibody effectively blocked IL-6-induced spheroid formation and STAT3 phosphorylation. In vivo, tumor size and weight were higher in IL-6 treated mice than controls. Conclusions: IL-6 mediates promotion of osteosarcoma spheroid stemness by activating OPN/STAT3 signaling.

Cinobufagin induces autophagy-mediated cell death in human osteosarcoma U2OS cells through the ROS/JNK/p38 signaling pathway.

The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin â…¤/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis.

[Subtype classification of Garden III femoral neck fractures in the elderly based on frontal Garden index and its clinical significance].

OBJECTIVE: To retrospectively study postoperative Garden III femoral neck fractures in the elderly so as to explore the different degree of displacement of Garden III femoral neck fracture, and discuss the basis and clinical significance of the subtype classification. METHODS: A total of 492 patients with complete clinical data out of the 1397 patients with femoral neck fractures treated by closed reduction and internal fixation with cannulated compression screws from September 2005 to September 2010 were included in the study. Each patient's frontal Garden Index was measured. On the basis of the frontal Garden Index, these cases were divided into three types:type A, which frontal Garden Index was more than or equal to 140°, included 53 males and 84 females with an average age of(65.3±7.2) years old ranging from 60 to 75 years old; type B, more than 120°and less than 140°, included 79 males and 172 females with an average age of (67.5±3.6) years old;and type C, less than or equal to 120°, included 38 males and 66 with an average age of(68.6±5.7) years old. Aspects were followed up including complications, consequences and hip joint function. The fracture healing and femoral head necrosis were compared among three types. RESULTS: Operative incision of 492 cases was primary healing, and no infection and other complications occurred. All patients were followed up from 2 to 10 years with an average of 6.3 years, the healing of femoral neck fracture occurred in 432 cases, and the total union rate was 87.8%. Femoral head necrosis occurred in 83 cases, and the total necrosis rate of femoral head was 16.9%. The nonunion rate of type A was 6.6%, type B was 13.5%, and type C was 16.3%, there were significant differences among three types(χ²2AB=4.377, P=0.036;χ²2AC=5.872, P=0.015;χ²2BC=0.469, PBC=0.494). The necrosis rate of femoral head of group A was 8.8%, group B was 16.7%, and group C was 27.9%, there were significant differences among three groups(χ²2AB=4.704, P=0.030;χ²2AC=15.317, P=0.000;χ²2BC=5.715, P=0.017). CONCLUSIONS: It is different for the degree of displacement of Garden III femoral neck fracture in the elderly. Based on frontal Garden Index to differentiate degree of fracture displacement, Garden III femoral neck fracture would be divided into A, B and C subtypes. The prognosis of Garden III femoral neck fracture in the elderly is negatively related to its degree of displacement, which has clinical significance to make treatment plan for Garden III femoral neck fracture in the elderly.

[Radiographic analysis of the osseous fixation zone for the iliac crest external fixation with Schanz screws].

OBJECTIVE: To radiographically analyze the osseous fixation zone for the iliac crest external fixation with Schanz screws and in order to guide their placement. METHODS: Nine adults with 2.0-mm-slice continuous pelvic axial CT scans were selected as research subjects. Each CT scan data was imported into MIMICS 10.0. The osseous fixation zone the upper portion of the anterior column of the acetabulum which is located between the anterior superior iliac spine and the gluteal medius pillar and between the iliac crest and the acetabulum-for the iliac crest external fixation with Schanz screws was reconstructed into true sagittal and true coronal planes by using the software. Then the measurements were taken on the reconstructed planes with measuring tools. Finally, the measured data was analyzed. RESULTS: The palpable iliac crest segment, which was of 49.6 mm width and located 16.5 mm posterior to the anterior superior iliac spine could be used to locate the start points of the Schanz screws. Under the above-mentioned iliac crest segment, the osseous zone was deep, got ample bony materials and could intraosseously contain Schanz screws with 5.0 mm diameter. The screws could be safely inserted to a minimal depth of 71.7 mm towards the acetabular dome and to a maximal depth of 143.5 mm posterior to the acetabulum. CONCLUSION: The study can guide the effective insertion of the iliac crest Schanz screws. By setting a suitable start point in the above-mentioned iliac crest region and angling correctly relative to the acetabulum,the Schanz screw can be inserted into the relative strong cancellous bone above or posterior to the acetabulum with a considerable depth, to getting more bone engagement.

[Radiographic anatomical analysis of the pelvic Teepee view].

OBJECTIVES: To research radiographic anatomy of the main structure of the pelvic Teepee view, including its azimuth direction and view anatomy structure. METHODS: From June 2013 to June 2014 adult pelvic CT examination results were filtered, excluding skeletal deformities and pelvic osseous destruction caused by tumors, trauma, etc. The data of 2.0 mm contiguous CT scan of 9 adults' intact pelves was,selected and input into Mimics 10.01 involving 7 males and 2 females with an average age of (41.2±10.3) years old. Utilizing the software, the 3D CT reconstructions of the pelves were completed. Setting the transparency being high,the pelvic 3D reconstructions were manipulated from the pelvic anteroposterior view to the combined obturator oblique outlet view and fine-tuned till the regular Teepee-or teardrop-shaped appearance emerges. Cutting tools of the software were at the moment applied to separate the "Teepee" from the main pelvis for each reconstruction. Then the "Teepee" and the rest (main) part of the pelvis were displayed in different color to facilitate the analysis on the Teepee, iliac-oblique, and anteroposterior views. RESULTS: The "Teepee" started from the posterolateral aspect of the anterior inferior iliac spine and finished at the cortex between the posterior superior iliac spine and the posterior inferior iliac spine in a direction of being from caudal-anterior-lateral to cranial-posterior-medial. The radiographic anatomical composition of the "Teepee" contained one tip, one base,and two aspects. With the inner and outer iliac tables being the inner and outer aspects of the "Teepee", the tip is consequently formed by their intersection. The base is imaged from the cortex of the greater sciatic notch. The medial-inferior-posterior portion of the "Teepee" contains a small part of sacroiliac joint and its corresponding side of bone of the sacrum. CONCLUSIONS: The "Teepee" is a zone of ample osseous structures of the pelvis, aside from a small medial-inferior-posterior portion, the main zone of which can be accepted as a safe osseous zone for the anchor of implants stabilizing certain pelvic and acetabular fracture patterns. The Teepee view can be utilized as guidance for the safe percutaneous insertion of such implants.

[S2 iliosacral screw insertion technique].

OBJECTIVE: To introduce a technique pertaining to S2 iliosacral screw insertion. METHODS: The screw pathway was first measured on the preoperative pelvic CT scan or the standard sacral lateral radiograph to make sure the existence of the "safe zone" in the S2 segment for screw insertion. Under general anesthesia, patients were positioned supine or prone, depending on the injury pattern of pelvic ring or associated injuries requiring concomitant operation. The operation field was routinely sterilized using iodine and subsequent alcohol solution and draped. The tip of a guide wire was inserted through a stab wound to the posterior outer iliac table, manipulated in the "safe zone" being enclosed by the anterior aspect of the S2 nerve root tunnel, the anterior aspect of the sacral vertebrae, and the inferior aspect of the S1 foramen under the guidance of the standard sacral lateral fluoroscopy, and then the tip was hammered one to two millimeters into the iliac cortex. The guide wire progressed along the trajectory between the inferior aspect of the S1 foramen and the superior aspect of the S2 foramen on the pelvic outlet fluoroscopic view, and then along the posterior to the anterior aspect of the S2 sacral vertebrae and alae on the pelvic inlet fluoroscopic view with a predetermined length. At that moment, in order to ensure the safety, another standard sacral lateral view was imaged to detect the guide wire's tip which should locate posterior to the anterior aspect of the sacral vertebrae and anterior to the anterior aspect of the S2 nerve root tunnel. Subsequently, the depth was measured, the trajectory was drilled and tapped, and the screw was inserted. Following the removal of the guide wire, the wound was irrigated and sutured. RESULTS: Utilizing this insertion technique, there were 30 S2 iliosacral screws in total being placed to stabilize the injured and unstable posterior pelvic ring in 27 patients. Each S2 screw was accompanied by an ipsilateral S1 screw. The S2 screw location was completely intraosseous in all patients, which was verified by postoperative pelvic outlet and inlet radiographs and CT scans. The insertion accuracy was 100 percent in the present series. CONCLUSION: The S2 iliosacral screw insertion technique is safe and reproducible to guide the placement of the S2 screw, enhancing the stability for the compromised posterior pelvic ring.

[Effect of the anterior aspect of sacral nerve root tunnel on iliosacral screw placement on the standard lateral image of sacrum].

OBJECTIVE: To introduce the location and course of S1, S2 sacral nerve root tunnel and to clarify the significance of the anterior aspect of sacral nerve root tunnel on placement of iliosacral screw on the standard lateral sacral view. METHODS: Firstly the data of 2.0 mm slice pelvic axial CT images were imported into Mimics 10.0, and the sacrum, innominate bones, and sacral nerve root tunnels were reconstructed into 3D views respectively, which were rotated to the standard lateral sacral views, pelvic outlet and inlet views. Then the location and course of the S1, S2 sacral nerve root tunnel on each view were observed. RESULTS: The sacral nerve root tunnel started from the cranial end and anterior aspect of the vertebral canal of the same segment and ended up to the anterior sacral foramen with a direction from cranial-posterior-medial to caudal-anterior-lateral. The tunnel had a lower density than the iliac cortex and greater sciatic notch on the pelvic X-rays,especially on the standard sacral lateral view, on which it showed up as a disrupted are line and required more careful recognition. CONCLUSION: It can prevent the iliosacral screw from penetrating the sacral nerve root tunnel and vertebral canal when recognizing the anterior aspect of sacral nerve root tunnel and choosing it as the caudal-posterior boundary of the "safe zone" on the standard lateral sacral view.