[Surgical management of sacral neurogenic tumors].

OBJECTIVE: To observe the efficacy and complications of one-stage tumor resection to treat primary sacral neurogenic tumors and to discuss some details in the clinically relevant anatomy. METHODS: A retrospective analysis of 26 patients with neurogenic turors of the sacral spine who were surgically treated from January 2001 to January 2018, including 16 males and 10 females, aged from 21 to 69 years old with an average age of (39.3±10.9) years old. The courses of diseases ranged from 3 to 56 months with an average of (17.9±10.1) months. The diameters of presacral components ranged from 3.3 to 19.6 cm with an average of (8.7±4.1) cm. The proximal margin of presacral lesions was above the L5S1 level in 6 cases, and lower than L5S1 in 20 cases. A posterior incision approach for one-stage complete resection of the tumor was used firstly, and an anterior approach was combined when necessary. Spinal-pelvic reconstruction with the modified Galveston technique was also carried out in relevant cases. Whether to preserve the tumor-involved nerve roots depended on the situation during the operation. The operation time, intraoperative blood loss, pain relief, and complications were recorded. The lumbosacral spine stability and sacral plexus neurological function were evaluated during postoperative follow-up, and local recurrence and distant metastasis were examined as well. RESULTS: Total excision was achieved in all 26 patients, with an operation time of (160.4±35.3) mins and an intraoperative blood loss of (1 092.3±568.8) ml. Tumors have been removed via a posterior-only approach in 21 cases and via combined anterior/posterior approaches in 5 cases. The diameter of presacral masses components ranged from 11.3 to 19.6 cm with an average of (15.1±3.2) cm in patients with combined anterior/posterior approaches, and ranged from 3.3 to 10.9 cm with an average of (7.2±2.4) cm in patients with a posterior-only approach. Five of the six patients whose proximal margin of presacral masses was above the L5S1 level adopted combined anterior/posterior approaches, and 20 patients lower than the L5S1 level adopted the posterior-only approach. All the patients were followed up for 6 to 82 months with an average of(45.4±18.2)months. Postoperative lumbosacral pain and lower extremity radicular pain were significantly relieved, and sensation, muscle strength and bowel and bladder function were also improved to varying degrees. The postoperative early complications included superficial wound infection in 1 case and cerebrospinal fluid leakage in 2 cases. Pathology confirmed 17 cases of schwannoma, 7 cases of neurofibroma and 2 cases of malignant schwannoma. Local recurrence was observed in two cases of benign neurogenic tumors. One patient with a malignant nerve sheath tumor had lung metastasis, who died 20 months after the operation. In 17 cases of upper sacral neurogenic tumors, 4 cases did not undergo spinal-pelvic reconstruction with internal fixation, of which 2 cases suffered from postoperative segmental instability. Tumor-involved nerve roots were resected during surgery in 7 cases. One of these patients who had S2 and S3 nerve roots sacrificed simultaneously had an impaired bladder and bowel function postoperatively, and did not recover completely. In the other 6 cases, the neurological function was not damaged obviously or recovered well. CONCLUSION: The posterior approach can directly expose the lesions, and it is also convenient to deal with nerve roots and blood vessels. The operation time, intraoperative blood loss, degree of symptom relief, complication rate, and recurrence and metastasis rate can be controlled at an appropriate level. It is a safe and effective surgical approach. When the upper edge of the presacral mass is higher than the L5S1 level or the diameter of the presacral mass exceeds 10 cm, an additional anterior approach should be considered. The stress between the spine and pelvis is high, and internal fixation should be used to restore the mechanical continuity of the spine and pelvis during resection of neurogenic tumors of the high sacral spine. Most of the parent nerve roots have lost their function. Resection of a single parent nerve root is unlikely to cause severe neurological dysfunction, while the adjacent nerve roots have compensatory functions and should be preserved as much as possible during surgery.

Sensitive osteosarcoma diagnosis through five-base telomerase product-triggered CRISPR-Cas12a enhanced rolling circle amplification.

Osteosarcoma is the most frequent primary malignant bone tumor, composed of mesenchymal cells producing osteoid and immature bone. The sensitive detection of telomerase plays a pivotal role in the early diagnosis and therapeutic treatment of osteosarcoma. We report here an in vitro strategy for sensitive telomerase activity detection through the integration of rolling circle amplification (RCA) and a clustered regularly spaced short palindrome repeats (CRISPR)-Cas12a system. In the proposed strategy, telomerase substrate (TS) primers are easily controlled to extend five bases (GGGTT) to give short telomerase extension products (TEP) with definite lengths without adding dATP. The resulting short TEPs can then cyclize the padlock through hybridizing with its two terminals and thus initiate the following RCA. To obtain an improved sensitivity, the CRISPR-Cas12a system is attached to collaterally cut surrounding DNA reporter probes after recognizing the target single strand DNA sequence in the RCA products. The highlights of this strategy are as follows: (i) the short TEP triggered strategy is excellent at detecting low telomerase activity and thus contributes to the early diagnosis of malignant tumors; (ii) highly sensitive telomerase activity detection which is easy to operate from RCA initiated CRISPR-Cas12a; (iii) opening up of a new avenue for telomerase activity detection with a CRISPR-Cas12a system. Finally, the proposed strategy exhibited sensitive telomerase activity detection under optimized experimental parameters and has great application potential for the clinical diagnosis of malignant tumors and the development of anti-cancer drugs.

Suppressing CHD1L reduces the proliferation and chemoresistance in osteosarcoma.

Osteosarcoma (OS) is the most common bone malignant tumor. However, the genetic basis of OS pathogenesis is still not understood, and occurrence of chemo-resistance is a major reason for the high morbidity of OS patients. Recently, chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) has been identified as a gene related to malignant tumor progression. Unfortunately, its effects on OS development and drug resistance are still not understood. In the study, we attempted to investigate the effects of CHD1L on tumorigenesis and chemoresistance in OS. We found that CHD1L expression was markedly up-regulated in OS samples, especially in cisplatin (cDDP)-resistant patients. We also showed that OS cells with CHD1L knockdown were more sensitive to cDDP treatment with lower IC50 values. In addition, we found that CHD1L deletion markedly reduced cell proliferation and induced apoptosis in OS cells with cDDP resistance. Moreover, the properties of cancer stem cells were highly suppressed in cDDP-resistant OS cells following CHD1L knockdown. Furthermore, multidrug resistance protein 1 (MDR-1) expression levels were dramatically decreased in OS cells with cDDP resistance when CHD1L was suppressed. Functional analysis indicated that CHD1L knockdown clearly restrained the activation of ERK1/2, protein kinase B (AKT) and NF-κB signaling pathways in cDDP-resistant OS cells. Consistently, animal experiments suggested that CHD1L suppression mitigated cDDP resistance in the generated in vivo xenografts. Collectively, CHD1L could modulate chemoresistance of OS cells to cDDP, and thus may be inspiring findings for overcoming drug resistance in OS.

Functions of Exogenous RUNX2 in Giant Cell Tumor of Bone In Vitro.

OBJECTIVES: This research aimed to investigate the relative level of Runt-related transcription factor 2 (RUNX2) in giant cell tumor of bone (GCTB). Through the histopathological similarities between osteoporosis and GCTB, the biological functions of exogenous RUNXS were demonstrated in GCTB cell lines. This generated awareness of the molecular mechanism of the biogenesis and metastasis of GCTB, as well as showing the pathways and processes involved in this study. This research also expected to provide hints for the clinical treatment of patients with GCTB, to release the tumor burden and reduce the recurrence rate and metastasis of patients with this condition. METHODS: The expression of RUNX2 in the tumors was verified by Western Blot, qRT-PCR and immunohistochemistry, compared with the normal tissues' adjacent tumors. Subsequently, the plasmids expressing RUNX2 were constructed, amplified and transfected into the 0404 cell line through transfection kits (0.4, 0.8, 1.6, 2.4 ng/µl). After that, the proliferation, migration, invasion, cellular viability and apoptosis of 0404 cell lines were examined by EDU assay, wound healing assay, transwell assay, annexin v staining, and CCK8 assay, respectively. RESULTS: The messenger RNA (mRNA) level of RUNX2 in tumors was over 100 folds more than the normal tissues. The protein level of tumors upregulated 8.32(±4.41) folds relatively. After the transfection of RUNX2 overexpressed plasmids into the 0404 cell line, the mRNA level of RUNX2 increased approximately 530.11(±24.87), 1117.96(±77.68), 2835.09(±45.22) and 4781.51(±79.37) folds respectively, and the protein level was upregulated about 4.12(±1.15), 16.73(±1.63), 21.53(±2.41) and 23.39(±0.85) folds respectively. The proliferation of 0404 cells was inhibited by 2.13(±1.02)% of 1.6 ng/µl group and 3.03(±1.76)% of 2.4 ng/µl group. And the migration was inhibited about 45.56(±6.13)%, 50.79(±5.27)%, 63.15(±8.62)% and 93.90(±3.65)% respectively. The invasion was decreased about 14.49(±5.4)%, 37.02(±6.52)%, 42.24(±2.59)% and 48.97(±10.61)% respectively. Meanwhile, FITC Annexin V/PI apoptosis assay demonstrated that RUNX2 plasmids could promote apoptosis rate around 4.15(±0.27)%, 5.07(±0.27)%, 7.61(±0.45)% and 11.32(±1.02)% respectively, and CCK8 proved these plasmids could weaken cellular viability in a concentration-dependent manner with the time passing. CONCLUSIONS: RUNX2 is highly expressed in giant cell tumors of bone. The RUNX2 overexpressed plasmids we constructed could be successfully transfected into 0404 cell line. Far more importantly, the exogenous RUNX2 can seriously block the biological functions of 0404 cell line in a concentration-dependent manner, including proliferation, translocation, invasion, cellular viability, and apoptosis. Meanwhile, the mechanism was hypothesized and discussed in the article.

Mechanical and Biological Properties of a Biodegradable Mg-Zn-Ca Porous Alloy.

OBJECTIVES: As promising alternative to current metallic biomaterials, the porous Mg scaffold with a 3-D open-pore framework has drawn much attention in recent years due to its suitable biodegradation, biocompatibility, and mechanical properties for human bones. This experiment's aim is to study the mechanical properties, biosafety, and osteogenesis of porous Mg-Zn alloy. METHODS: A porous Mg-2Zn-0.3Ca (wt%) alloy was successfully prepared by infiltration casting, and the size of NaCl particles was detected by a laser particle size analyzer. The microstructure of the Mg-2Zn-0.3Ca alloy was characterized by the stereoscopic microscope and Sirion Field emission scanning electron microscope. X-ray computerized tomography scanning (x-CT) was used to create the 3-D image. The degradation rate was measured using the mass loss method and the pH values were determined together. The engineering stress-strain curve, compressive modulus, and yield strength were tested next. The bone marrow stromal cells (BMSC) were cultured in vitro. The CCK-8 method was used to detect the proliferation of the BMSC. Alkaline phosphatase (ALP) and alizarin red staining were used to reflect the differentiation effects. After co-culturing, cell growth on the material's surface was observed by scanning electron microscope (SEM). The cell adhesion was tested by confocal microscopy. RESULTS: The obtained results showed that by using near-spherical NaCl filling particles, the porous Mg alloy formed complete open-cell foam with a very uniform size of pores in the range of 500-600 µm. Benefitting from the small size and uniform distribution of pores, the present porous alloy exhibited a very high porosity, up to 80%, and compressive yield strength up to 6.5 MPa. The degradation test showed that both the pH and the mass loss rate had similar change tendency, with a rapid rise in the early stage for 1-2 day's immersion and subsequently remaining smooth after 3 days. In vitro cytocompatibility trials demonstrated that in comparison with Ti, the porous alloy accelerated proliferation in 1, 3, 5, and 7 days (P < 0.001), and the osteogenic differentiation test showed that the ALP activity in the experimental group was significantly higher (P = 0.017) and has more osteogenesis nodules. Cell adhesion testing showed good osteoconductivity by more BMSC adhesion around the holes. The confocal microscopy results showed that cells in porous Mg-based alloy had better cytoskeletal morphology and were larger in number than in titanium. CONCLUSIONS: These results indicated that this porous Mg-based alloy fabricated by infiltration casting shows great mechanical properties and biocompatibilities, and it has potential as an ideal bone tissue engineering scaffold material for bone regeneration.

Expression of Matrix Metalloproteinase-9 and CD34 in Giant Cell Tumor of Bone.

OBJECTIVE: Giant cell tumor of bone (GCTB) invades extensively and metastasizes, however, the pathological grade and imaging findings are not accurate predictors of its prognosis. Thus, the aim of this study was to explore the relationships between expression of cluster of differentiation (CD)34 and matrix metalloproteinase-9 (MMP-9) and the biological behavior of GCTB with the hope of identifying predictors of prognosis. METHODS: Sixty-eight patients with GCTBs attending our institution from September 2008 to August 2013 were enrolled in this prospective study and grouped according to tumor location. Relevant patient characteristics were assessed. Additionally, the expression of CD34 and MMP-9 in these patients was assayed by an immunohistochemistry staining procedure and the relationships between CD34/MMP-9 and microvessel density (MVD) analyzed by Spearman correlation analysis. RESULTS: It was found that CD34 factor localizes in the cytoplasm of the endothelial cells of small blood vessels in the tumor stroma and is strongly expressed in GCTBs. In addition, radiological grading showed that there was significantly more CD34 antibody-labeled MVD in invasive than in non-invasive tumors (P < 0.05) and significantly more CD34 antibody-labeled MVD in patients who developed recurrences than in those who did not (P < 0.05). Expression of MMP-9 was localized in the cytoplasm of tumor cells and the rate of MMP-9 positivity in GCTBs was significantly higher in active and invasive tumors than in non-invasive tumors (P < 0.01). Moreover, there were significantly more MVDs in MMP-9-positive than in MMP-9 negative tumors (P < 0.01). CD34 and MMP-9 are positively correlated with MVD values in GCTBs and closely correlated with their grade of malignancy. CONCLUSION: Expression of CD34 and MMP-9 accurately predicts clinical behavior detection and prognosis of GCTBs.