Preclinical Evaluation of Bioactive Small Intestinal Submucosa-PMMA Bone Cement for Vertebral Augmentation.

In vertebroplasty and kyphoplasty, bioinert poly(methyl methacrylate) (PMMA) bone cement is a conventional filler employed for quick stabilization of osteoporotic vertebral compression fractures (OVCFs). However, because of the poor osteointegration, excessive stiffness, and high curing temperature of PMMA, the implant loosens, the adjacent vertebrae refracture, and thermal necrosis of the surrounding tissue occurs frequently. This investigation addressed these issues by incorporating the small intestinal submucosa (SIS) into PMMA (SIS-PMMA). In vitro analyses revealed that this new SIS-PMMA bone cement had improved porous structure, as well as reduced compressive modulus and polymerization temperature compared with the original PMMA. Furthermore, the handling properties of SIS-PMMA bone cement were not significantly different from PMMA. The in vitro effect of PMMA and SIS-PMMA was investigated on MC3T3-E1 cells via the Transwell insert model to mimic the clinical condition or directly by culturing cells on the bone cement samples. The results indicated that SIS addition substantially enhanced the proliferation and osteogenic differentiation of MC3T3-E1 cells. Additionally, the bone cement's biomechanical properties were also assessed in a decalcified goat vertebrae model with a compression fracture, which indicated the SIS-PMMA had markedly increased compressive strength than PMMA. Furthermore, it was proved that the novel bone cement had good biosafety and efficacy based on the International Standards and guidelines. After 12 weeks of implantation, SIS-PMMA indicated significantly more osteointegration and new bone formation ability than PMMA. In addition, vertebral bodies with cement were also extracted for the uniaxial compression test, and it was revealed that compared with the PMMA-implanted vertebrae, the SIS-PMMA-implanted vertebrae had greatly enhanced maximum strength. Overall, these findings indicate the potential of SIS to induce efficient fixation between the modified cement surface and the host bone, thereby providing evidence that the SIS-PMMA bone cement is a promising filler for clinical vertebral augmentation.

Generation of a human iPSC line (CHCMUi002-A) from peripheral blood mononuclear cells from a chronic granulomatous disease patient.

Chronic granulomatous disease (CGD) is a rare X-linked recessive primary immunodeficiency disease (PID). Herein, a human induced pluripotent stem cell (iPSC) line was generated from the peripheral blood mononuclear cells (PBMCs) of a CGD patient with a mutation (c.785_786delTT) in the CYBB gene. These iPSCs showed the expression of pluripotency markers, the ability to differentiate into three germ layers. They offer a promising technique for studying the pathogenesis and conducting drug screening for CGD patients.

Neutralization against Omicron subvariants after BA.5/BF.7 breakthrough infection weakened as virus evolution and aging despite repeated prototype-based vaccination1.

BACKGROUND: Omicron had swept the mainland China between December 2022 and January 2023, while SARS-CoV-2 still continued to evolve. To fully prepare for the next wave, it's urgent to evaluate the humoral immune response post BA.5/BF.7 breakthrough infection against predominant sub-lineages among existing vaccination strategies and the elders. METHOD: This study enrolled a longitudinal young-adult cohort from 2/3-dose vaccination to 1 month after breakthrough infection, and an elder cohort at 1 month after breakthrough infection. Seral samples were collected and tested for humoral immune response to SARS-CoV-2 subvariants including WT, BA.2, BA.5, BF.7, BQ.1.1, CH.1.1, XBB.1.5. RESULTS: BA.5/BF.7 breakthrough infection induced higher neutralization activity than solely vaccination in all SARS-CoV-2 strains, while the latest Omicron subvariants, BQ.1.1, CH.1.1, XBB.1.5, exhibited the strongest neutralization evasion ability. There was a negative correlation between age and humoral immune response in WT, BA.5, BQ.1.1, and XBB.1.5. Compared to non-vaccination groups, breakthrough infection in two-dose vaccination groups had significantly higher neutralizing antibody against WT, BA.2, BA.5, BF.7 but not to BQ.1.1, CH.1.1, XBB.1.5 while booster dose against the prototype prior-breakthrough would not further significantly enhance individual's humoral responses against the latest Omicron subvariants. CONCLUSIONS: Newer variants manifest increasing immune evasion from neutralization and repeated prototype-based booster vaccines may not further enhance neutralizing antibody against emerging new variants. Older adults have lower levels of neutralizing antibody. Future vaccination strategies should aim to enhance effective neutralization to contemporary variants.

An early-onset SLE patient with a novel paternal inherited BACH2 mutation.

BACH2-related immunodeficiency and autoimmunity (BRIDA) is an inborn error of immunity, newly reported in 2017, presenting with symptoms of immunoglobulin deficiency and ongoing colitis. Studies using a mouse model have demonstrated that BACH2 deficiency predisposes individuals to systemic lupus erythematosus (SLE); however, no BACH2 deficiency has been reported in SLE patients. Here we describe a patient with BRIDA presenting with early-onset SLE, juvenile dermatomyositis, and IgA deficiency. Whole exome sequencing analysis of the patient and her parents revealed a novel heterozygous point mutation in BACH2, c.G1727T, resulting in substitution of a highly conserved arginine with leucine (R576L), which is predicted to be deleterious, in the patient and her father. Reduced BACH2 expression and deficient transcriptional repression of the BACH2 target, BLIMP1, were detected in PBMCs or lymphoblastoid cell lines of our patient. Notably, extreme reduction of memory B cells was detected in the patient's father, although he had no obvious symptoms. SLE symptoms and recurrent fever were relieved by treatment with prednisone combined with tofacitinib. Thus, we present the second report of BRIDA and demonstrate that BACH2 may be a monogenic cause of SLE.

Clinical and immunological characteristics of five patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in China-expanding the atypical phenotypes.

Background: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare disorder of the immune regulatory system caused by forkhead box P3 (FOXP3) mutations. Abnormal numbers or functions of regulatory T (Treg) cells account for the various autoimmune symptoms. We aimed to explore the molecular genetics and phenotypic spectra of patients with atypical IPEX syndrome in China. Methods: We analyzed the molecular, clinical and immune phenotype characteristics of five Chinese patients with FOXP3 mutations. Results: We summarized the molecular and phenotypic features of five patients with FOXP3 mutations, including two novel mutations. Four of the five patients displayed atypical phenotypes, and one developed immune-related peripheral neuropathy. Three of the five patients showed normal frequencies of Treg cells, but the proportions of subsets of Treg cells, CD4+ T cells and B cells were out of balance. Conclusions: Our report broadens the understanding of the clinical features of atypical IPEX syndrome. Our detailed analyses of the immunological characteristics of these patients enhance the understanding of the possible mechanisms underlying the clinical manifestations.

Generation of human induced pluripotent stem cell line from peripheral blood mononuclear cells from an activated phosphoinositide 3-kinase δ syndrome patient.

Activated phosphoinositide 3-kinase δ syndrome (APDS) is a rare autosomal dominant primary immunodeficiency disease (PID) which was caused by the acquired mutation of PIK3CD gene. In this study, we generated a human induced pluripotent stem cell (hiPSC) line CHCMUi001-A from the peripheral blood mononuclear cells (PBMCs) of a APDS patient, who has a heterozygous mutation (c.3061 G > A) in the PIK3CD gene. This iPSC line presented a normal karyotype and exhibited characteristics of pluripotent stem cells. This iPSC line can be very useful for not only studying disease mechanisms but also developing new potential clinical treatments for APDS patients.

Circular gap forming device and two-dimensional area calculation for in vitro cell migration study.

Cell wound healing assay is an important experimental technique for the detection of cell migration in vitro. At present, scratch on monolayer cells using a pipette tip is commonly used. However, it is difficult to guarantee the scratch with same width, and only the migration distance of a certain part is calculated. Therefore, the experimental method needs to be optimized. ATRA was used to treat hepa1-6 mouse hepatoma cells. Circular wound with diameter of 0.2 cm were formed by a circular gap forming device. The whole cell wound region could be captured under the microscope to observe cell migration. There are almost no crawling cells in the wound region. The migration capacity of hepa1-6 cells was evaluated by calculating the healing area. ATRA could significantly inhibit the migration of hepa1-6 cells. Compared with linear wound, the standard deviation of wound healing rate in the circular cell wound method is smaller. The circular cell wound method can ensure the dynamic observation of the same wound region, and calculate the healing area at the two-dimensional level with small error and high repetition rate. It is reliable and easy to operate, can be widely used in laboratory.

A retrospective analysis of eleven gene mutations, PD-L1 expression and clinicopathological characteristics in non-small cell lung cancer patients.

OBJECTIVES: To investigate the associations among expression of programmed cell death ligand 1 (PD-L1), eleven mutated genes, and clinicopathological characteristics in 273 patients with non-small cell lung cancer (NSCLC). METHODS: We retrospectively examined tumor PD-L1 expression in 247 surgically resected primary and 26 advanced NSCLC patients by immunohistochemistry using SP263 antibody assay. Gene mutations of EGFR, TP53, KRAS, PIK3CA, ERBB2, MET, RET, ALK, BRAF, ROS1, and APC were examined by NGS sequence. Data analysis was carried out using SPSS 22.0. The associations among PD-L1 expression, eleven mutated genes and clinicopathological characteristics were assessed by univariate and multivariate analysis. RESULTS: Among the total 273 patients, 68 (24.9%) patients were positive for PD-L1 expression. Data showed that mutated rate of EGFR gene was the highest with 63.0% (172/273), followed by TP53 (11.7%, 32/273) and KRAS (5.5%, 15/273). The female, non-smoker, and patients with adenocarcinoma (ADC) were more likely to have EGFR mutations. Multivariate logistic regression showed that PD-L1 expression was significantly associated with Non-ADC, lymphatic invasion, EGFR wild type and TP53 mutation (p = 0.041, <0.001, 0.004 and 0.014, respectively). Moreover, PD-L1 expression in adenocarcinoma was associated with lymphatic invasion, mutation of TP53 and KRAS gene (p = 0.012, <0.025 and 0.041, respectively). CONCLUSIONS: Mutations of EGFR, KRAS and TP53 should be routinely detected in clinical practice to better guide the immunotherapy for NSCLC patients. Future investigations are warranted to illustrate the potential mechanisms between driver mutations and PD-L1 expression for guiding immunotherapy in patients with NSCLC.

[Gene editing for the treatment of primary immunodeficiency disease].

Gene editing is an advanced technique based on artificial nucleases and can precisely modify genome sequences. It has shown great application prospects in the field of medicine and has provided a new precision therapy for diseases. Primary immunodeficiency disease is a group of diseases caused by single gene mutation and characterized by recurrent and refractory infections, with an extremely high mortality rate. The application of gene editing has brought hope for curing these diseases. This article reviews the development of gene editing technology and briefly introduces the research and application of gene editing technology in primary immunodeficiency disease.

All-trans retinoic acid reverses malignant biological behavior of hepatocarcinoma cells by regulating miR-200 family members.

As a potential chemo-therapeutic agent, all-trans retinoic acid (ATRA) can significantly reverse epithelial-mesenchymal transition (EMT) of hepal-6 hepatocarcinoma cell line in vitro, but the mechanism is unclear. The expression profile of microRNA-200 (miR-200) families is different in hepatocellular carcinoma. In this study, we found that ATRA treatment could up-regulate the expression of miR-200a-3p, 200c-3p, and 141-3p, which were involved in ATRA regulated proliferation and apoptosis of hepal-6 cell, but not colony formation. Meanwhile, miR-200a-3p, 200c-3p, and 141-3p could recovery ATRA inhibited migration and invasion abilities of hepal-6 cells at various levels. miR-200a-3p and 200c-3p prevented ATRA from inducing the differentiation and hepatic functions of hepal-6 cells. Antagomir specific for miR-200a-3p and 200c-3p down-regulated the expression of CK18, but only miR-200a-3p antagomir played prominent role in regulating the expression of these mesenchymal markers, N-Cadherin, Snail and Twist. The transcriptional activities of 8 transcription factors were up-regulated and 35 transcription factors were down-regulated by ATRA. Compared with ATRA group, inhibition of miR-200a-3p, 200c-3p, and 141-3p significantly strengthened the expression of Fra1/Jun (AP1), Ets1/PEA3, Brn3, and Zeb1/AREB6 at varying degrees. Therefore, this result suggested that ATRA may suppress EMT through down-regulating miR-200a-3p, 200c-3p and 141-3p related transcription factors. miR-200 and their downstream genes might be the potentially specific targets for the treatment of hepatocarcinoma.

Erratum: Urine-derived stem cells accelerate the recovery of injured mouse hepatic tissue.

[This corrects the article on p. 5131 in vol. 12, PMID: 33042410.].

Indocyanine Green Uptake and Periodic Acid-Schiff Staining Method for Function Detection of Liver Cells are Affected by Different Cell Confluence.

Hepatic stem cell transplantation has been demonstrated as an effective alternative therapy for the end-stage liver failure patients. Therefore, the functional detection of hepatic stem cell is essentially required. The present study confirmed that adenovirus BMP9 (Ad-BMP9) could increase the ALB-Gluc activity of HP14-19 hepatic progenitor cells, the expression of specific hepatic markers ALB, TAT, UGT1A were up-regulated while the hepatic stem cell markers DLK, AFP were down-regulated, and the number of positive Periodic acid-Schiff (PAS) stained cells were significantly higher than those in control group. However, the indocyanine green (ICG) uptake failed to be detectable in induced hepatocytes, which was inconsistent. By using another cell line LC14d, we found out that positive ICG uptake cells were located in the area of low cell density, while positive PAS stained cells were mainly concentrated in the area where cells were overlapped, indicating that different cell confluence might affect the outcomes of ICG uptake and PAS staining. A manual wound healing of Ad-BMP9 induced HP14-19 cells was made, the crawling cells were stained positive for ICG but not for PAS. Therefore, our finding may provide evidence for better application of PAS staining and ICG uptake assay in functional detection of mature hepatocytes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00453-8.

Hypoxia Preconditioning Promotes the Proliferation and Migration of Human Urine-Derived Stem Cells in Chronically Injured Liver of Mice by Upregulating CXCR4.

Our previous studies reported that urine-derived stem cells (USCs) possess a strong self-renewal ability and multidirectional differentiation potential and thus are an ideal candidate cell source for hepatocellular transplantation. USC transplantation may repair the pathological changes of chronic liver injury to a certain extent, and hypoxia pretreatment may improve the recovery efficiency of USCs. Therefore, the present study aimed to investigate the possible mechanism of the improved recovery efficiency of hypoxia-pretreated USCs. A chronic liver injury model was established by intraperitoneal injection of carbon tetrachloride into nude mice. USCs were transplanted via caudal vein injection. Hematoxylin and eosin staining and Masson's staining were performed to determine the pathology of the liver. Immunofluorescence and frozen section biopsy were performed to determine differentiation and cell fusion in vivo. Cell coculture was used to detect cell fusion in vitro. The proliferative ability of USCs was evaluated using cell viability and colony formation assays, and the migratory functions of USCs were evaluated using wound healing and transwell assays. The degeneration of hepatocytes and the level of fibrosis in the hypoxia transplantation group were improved compared with the normoxia transplantation group. It was found that exogenous USCs may be differentiated into functional hepatocytes or fused with hepatocytes in vivo. C-X-C motif chemokine (CXC) ligand 12 (CXCL12) expression levels in liver tissue of the chronic liver injury model were upregulated compared with those in the control group. The expression of CXC receptor 4 (CXCR4) in hypoxia-pretreated USCs was also significantly upregulated. The results suggested that USCs fused with different types of liver cells and that hypoxia treatment promoted the fusion rate in vitro by upregulating CXCR4 signaling. Furthermore, hypoxia pretreatment promoted cell proliferation, migration, and cell fusion by inducing CXCR4 signaling, leading to USC-elicited liver tissue recovery following injury in vivo.

All-trans-retinoic acid inhibits the malignant behaviors of hepatocarcinoma cells by regulating autophagy.

Hepatocellular carcinoma is the fourth leading cause of cancer-related deaths due to its high rate of recurrence and metastasis. All-trans-retinoic acid (ATRA) can inhibit the malignant behaviors of hepatocarcinoma cells. Autophagy is reportedly involved in the migration and metastasis of various cancer cells. This study aimed to investigate the effect of autophagy on the function of ATRA on hepatocarcinoma cells, and to explore its possible underlying mechanism. Hepatocarcinoma cell lines, Hepa1-6 and HepG2, were treated with ATRA and autophagy inhibitors, including 3-methyladenine (3-MA) and Bafilomycin (Baf). Transmission electron microscopy, laser scanning, western blot, and real-time PCR demonstrated that ATRA induces autophagy in hepatocarcinoma cells. Trypan blue staining, a wound healing assay, and a transwell assay showed that 3-MA and Baf reverses the inhibitory functions of ATRA on the proliferation, migration, and invasion of hepatocarcinoma cells. Flow cytometry, Hoechst staining, periodic acid-Schiff staining, and indocyanine green uptake validated that 3-MA and Baf reverses the function of ATRA on apoptosis and the differentiation of hepatocarcinoma cells. Real-time PCR, western blot, and an immunofluorescence assay demonstrated that the reversal of the epithelial-mesenchymal transition (EMT) process by ATRA is weakened when autophagy is inhibited. Additionally, we confirmed that Bcl-2 is associated with the induction of ATRA-induced autophagy instead of the PI3K/Akt/mTOR pathway. These findings suggest that ATRA induces autophagy and autophagic cell death through the Bcl-2/Beclin1 pathway. Furthermore, ATRA-induced autophagy is involved in the inhibitory effect of ATRA on the malignant behaviors of hepatocarcinoma cells by reversing the EMT process.

Urine-derived stem cells accelerate the recovery of injured mouse hepatic tissue.

Urine-derived stem cells (USCs) are autologous stem cells that exhibit self-renewal ability and multi-lineage differentiation potential. These characteristics make USCs an ideal cell source for hepatocellular transplantation. Here, we investigated the biological characteristics of USCs and their potential use for the treatment of chronic liver injury. We characterized the cell-surface marker profile of USCs by flow cytometry and determined the osteogenic, adipogenic, and hepatic differentiation capacities of USCs using histology. We established a chronic liver-injury model by intraperitoneally injecting carbon tetrachloride into nude mice. USCs were then transplanted via tail vein injection. To determine liver function and histopathology following chronic liver injury, we calculated the liver index, measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and performed histological staining. USCs were small, adherent cells expressing mesenchymal but not hematopoietic stem-cell markers. Some induced USCs underwent osteogenic and adipogenic differentiation. When co-cultured with hepatic progenitor cells, about 10% of USCs underwent hepatic differentiation. The ALT and AST levels of the USC-transplanted group were lower than that of the chronic liver-injury model group, and there were no significant differences between the two USC-transplanted groups. However, hepatocyte degeneration and liver fibrosis substantially improved in the hypoxia-pretreated USC-transplanted group compared with the normoxia USC-transplanted group. Taken together, USCs display desirable proliferation and differentiation characteristics, and USC transplantation partially improves abnormal liver function and pathology associated with chronic liver injury. Furthermore, hypoxia pretreatment promotes cell proliferation, migration, and colony formation by inducing autophagy, leading to USC-elicited liver tissue recovery following injury in vivo.

ATRA induces the differentiation of hepatic progenitor cells by upregulating microRNA-200a.

Hepatic progenitor cells (HPCs) are potential seed cells for hepatocyte transplantation treatment of liver diseases. ATRA can induce the differentiation and mature function of hepatic progenitor cells, but the mechanism is still poorly understood. Here, by using microRNA array to analyze the expression profiles of microRNA (miR), we found that miR-200 family molecules in HPCs were upregulated after ATRA treatment, especially miR-200a-3p, 200c-3p, and 141-3p. ATRA induction could downregulate the expression of hepatic stem markers Oct4 and AFP, and improve the expression of hepatic markers ALB, CK18, and TAT, and the activity of ALB-GLuc, as well as indocyanine green uptake and glycogen storage function of HPCs. These above effects of ATRA on HPC differentiation were almost inhibited by blocking of miR-200a-3p, but not miR-200c-3p and 141-3p using antagomir. Cell autophagy is associated with ATRA regulation in HPCs, compared with control group, the expression of LC3 and Beclin1 increased in ATRA-treated HPCs, and orange and red fluorescent spot, which represents autophagy flow, also enhanced after ATRA treatment. However, ATRA-induced cell autophagy level was inhibited in antagomir-200a-3p+ATRA-treated cells. Therefore, the present study indicates that antagomir-200a-3p is related to ATRA-induced hepatic differentiation of HPCs through regulating cell autophagy, supporting the possible use of ATRA as a key inducer in HPC-based therapy of liver diseases.

Reversibly immortalized hepatic progenitor cell line containing double suicide genes.

A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 T­antigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these long­term immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSV­tk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxP­mediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSV­tk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSV­tk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5­fluorocytosine (5­FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days post­implantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.

[Changes in autophagy during maturation and differentiation of Hepa1-6 cells induced by all-trans retinoic acid].

OBJECTIVE: To investigate the effects of different concentrations of all-trans retinoic acid (ATRA) on the maturation, differentiation and autophagy of Hepa1-6 cells. MONTHOD: Hepa1-6 cells were treated with 0.1, 1, and 10 µmol/L ATRA, and the changes in the expressions of hepatic specific markers were detected using real-time PCR and Western blotting. Indocyanine green (ICG) and periodic acid-schiff (PAS) staining was used to assess the functional maturation of Hepa1-6 cells, and the cell-cell junction and autophagy were observed under transmission electron microscopy to determine the optimal concentration of ATRA for treatment. The expressions of autophagy-related markers in the cells were detected using Western blotting, and confocal microscopy was used to observe the autophagic flow in the cells transfected with ptfLC3 plasmid. RESULTS: Compared with the control cells, the hepatocytes treated with ATRA showed a concentration-dependent decrease in AFP expression and increase in the expressions of ALB, CK18, TAT and ApoB. ICG and PAS staining revealed significantly increased number of positive cells after ATRA treatment. Following ATRA treatment, the cells exhibited obviously increased tight junctions, cytoskeleton and number of autophagosomes under transmission electron microscopy. ATRA treatment resulted in significantly increased the expressions of autophagy-related markers LC3-II, Beclin-1, RAB7 and P62 and also an increased ratio of LC3-II/LC3-I(P<0.05). Confocal microscopy revealed obviously increased green and red spots in the cells after ATRA treatment. CONCLUSION: ATRA can induce the maturation and differentiation and enhance the level of autophagy in Hepa1-6 cells.

[Twist regulates proliferation, migration and invasion of osteosarcoma cells in vitro].

OBJECTIVE: To investigate the role of Twist in regulating the proliferation, migration, and invasion of osteosarcoma cells with different levels of malignancy. METHODS: The baseline expressions of Twist in 3 different osteosarcoma cell lines (143B, MG63 and TE85) were detected using real-time PCR and Western blotting. The cells were infected with the recombinant adenoviruses Ad-Twist or Ad-siTwist for Twist overexpression or knockdown, respectively, and the cell growth curves were drawn to assess the cell proliferation. The migration abilities and invasiveness of the cells were evaluated using wound healing assay and Transwell assay. Luc-labeled 143B cells infected with Ad-Twist or Ad-siTwist were intrathecally injected to establish nude mouse models bearing osteosarcoma xenografts, in which the tumor formation was monitored using living body imaging technique. RESULTS: The baseline expressions of Twist in the 3 osteosarcoma cells were significantly higher than that in C3H10 cells (P<0.05). Twist expression was the highest in 143B cells followed by MG63 cells, and was the lowest in TE85 cells, indicating its positive correlation with the level of malignancy of the osteosarcoma cells. Ad-Twist or Ad-siTwist infection efficiently enhanced or lowered Twist expressions at both mRNA and protein levels in osteosarcoma cells (P<0.05). Twist overexpression resulted in enhanced proliferation, migration and invasion abilities of osteosarcoma cells, and Twist knockdown obviously inhibited the cell proliferation, migration and invasion. In nude mice, 143B cells with Twist overexpression showed accelerated tumor formation compared with the control cells, while Twist knockdown significantly inhibited the tumor formation ability of the cells. CONCLUSION: Twist overexpression can promote the proliferation, migration, invasion and tumorigenicity of osteosarcoma cells.