Short- and Long-Term Outcomes of Indocyanine Green Fluorescence Navigation- Versus Conventional-Laparoscopic Hepatectomy for Hepatocellular Carcinoma: A Propensity Score-Matched, Retrospective, Cohort Study.

BACKGROUND: Indocyanine green (ICG) fluorescence imaging technology is increasingly widely used in laparoscopic hepatectomy. However, whether it can provide long-term survival benefits to patients with liver malignancies remains unclear. This study investigated the clinical effect of laparoscopic hepatectomy for hepatocellular carcinoma (HCC) using ICG imaging technology. METHODS: We retrospectively analyzed HCC patients who underwent laparoscopic hepatectomy at Zhongnan Hospital of Wuhan University from January 2016 to December 2020. Propensity score matching (PSM) was used to match patients undergoing ICG fluorescence navigation laparoscopic hepatectomy (ICG-FNLH) with those undergoing conventional laparoscopic hepatectomy (CLH) in a 1:1 ratio to minimize the influence of confounding factors. We compared perioperative status and long-term prognosis between the two groups and performed multivariate analysis to identify risk factors associated with overall survival and recurrence-free survival. RESULTS: The original cohort consisted of 141 patients, with 50 patients in each group (100 patients in total) after PSM. The anatomical liver resection rate, R0 resection rate, and resection margin distance in the ICG-FNLH group were higher than those in the CLH group. The intraoperative blood loss was lower than that in the CLH group. The recurrence-free survival and overall survival of the ICG-FNLH group were better than those of the CLH group. ICG-FNLH improved the recurrence-free survival of HCC patients (hazard ratio [HR] = 2.165, 95% confidence interval [CI]: 1.136-4.127, P = 0.024). CONCLUSIONS: Compared with CLH, ICG-FNLH can improve the recurrence-free survival rate of patients with hepatocellular carcinoma and may help to improve the long-term prognosis of patients.

Developing metabolic gene signatures to predict intrahepatic cholangiocarcinoma prognosis and mining a miRNA regulatory network.

BACKGROUND: Metabolic disturbance is closely correlated with intrahepatic cholangiocarcinoma (IHCC), and we aimed to identify metabolic gene marker for the prognosis of IHCC. METHODS: We obtained expression and clinical data from 141 patients with IHCC from public databases. Prognostic metabolic genes were selected using univariate Cox regression analysis. Unsupervised cluster analysis was applied to identify IHCC subtypes, and CIBERSORT was used for immune infiltration analysis of different subtypes. Then, the metabolic gene signature was screened using multivariate Cox regression analysis and the LASSO algorithm. The prognostic potential and regulatory network of the metabolic gene signature were further investigated. RESULTS: We screened 228 prognosis-related metabolic genes. Based on their expression levels, IHCC samples were divided into two subtypes, which showed significant differences in survival and immune cell infiltration. After LASSO analysis, eight metabolic genes including CYP19A1, SCD5, ACOT8, SRD5A3, MOGAT2, PFKFB3, PPARGC1B, and RPL17 were identified as the optimal genes for the prognosis signature. The prognostic model had excellent predictive abilities, with areas under the receiver-operating characteristic curves over 0.8. A nomogram model was also established based on two independent prognostic clinical factors (pathologic stage and prognostic model), and the generated calibration curves and c-indexes determined its excellent accuracy and discriminative ability to predict 1- and 5-year survival status (c-indexes>0.7). Finally, we found that miR-26a-5p, miR-27a-3p, and miR-27b-3p were the upstream regulators that mediate the involvement of gene signatures in metabolic pathways. CONCLUSION: We developed eight metabolic gene signatures to predict IHCC prognosis and proposed potential upstream regulatory axes of gene signatures.

Surgical Masks for Protection of Health Care Personnel Against Covid-19: Results from an Observational Study.

PURPOSE: The aim of the study was to describe the use of masks among health care personnel (HCP) exposed to index cases of coronavirus disease 2019 (COVID-19), and to evaluate any association with infection rate. METHODS: We did a retrospective, observational study of HCP at Zhongnan Hospital of Wuhan University for the management of COVID-19 (before person-to-person transmission was official confirmed, no additional protection was provided). A questionnaire was given to all staff listed on the roster in the clinical regions providing care for index patients with COVID-19. All participants were surveyed regarding hand-washing and use of surgical masks and gloves and were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data were analysed (Student's t test and Pearson χ2 test) for an association between infection and use of personal protective equipment. RESULTS: Exposure of a total of 299 non-infected and 30 infected staff was confirmed. None of the 149 staff who reported use of all three preventative measures (hand-washing and use of gloves and masks) became infect-ed. In contrast, all 30 of the staff who became infected had omitted at least one of the measures. Fewer staff who wore surgical masks (P=0.000003) became infected compared with those who did not. Infections rates were significantly lower in HCP from the internal medicine departments, as these personnel generally wore masks. CONCLUSION: An association was found between SARS-CoV-2 infection of HCP and the non-use of masks when working with index cases in clinical settings. We recommend that all HCP follow the strict instructions for prevention and treatment of nosocomial infection during intimate contact with COVID-19, especially staff from surgical departments.

Casein kinase 2 inhibitor CX-4945 elicits an anti-Warburg effects through the downregulation of TAp73 and inhibits gastric tumorigenesis.

Casein kinase 2 (CK2) has become a potential therapeutic target in gastric cancer; however, the underlying mechanism remains incompletely understood. TAp73, a structural homolog of the tumor suppressor p53, acts as a critical regulator of the Warburg effect. Recent study reveals that aberrant CK2 signaling is able to inhibit TAp73 function. Here we determine that TAp73 is overexpressed in AGS-1 but not in SNU-5 gastric cancer cell line as compared with normal gastric cells. In addition, we show that TAp73 expression is required for the maintenance of glucose uptake and lactate release in AGS-1 but not in SNU-5 gastric cancer cells. Importantly, the use of CX-4945, a selective inhibitor of protein kinase CK2, inhibits cell growth and invasion, and promotes cell apoptosis in AGS-1 with decreased TAp73 expression as well as downregulated glucose uptake and lactate release. Although TAp73 knockdown resulted in significant decreases in TAp73 expressions in SNU-5 cell line, no differences in glucose uptake and lactate release were observed between SNU-5 and normal gastric cells. Moreover, TAp73 gene overexpression promotes glucose uptake and lactate release and abolishes the anti-cancer effects of CX-4945 in gastric cancer cell line AGS-1. The impacts of CX-4945 on glycolysis and tumorigenesis were strongly limited in SNU-5 gastric cancer cell line. These findings suggest that CX-4945 elicits an anti-Warburg effects in gastric cancer overexpressing Tap73 and inhibits gastric tumorigenesis.

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells.

BACKGROUND: BMP9 induces both osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Nell1 is a secretory glycoprotein with osteoinductive and anti-adipogenic activities. We investigated the role of Nell1 in BMP9-induced osteogenesis and adipogenesis in MSCs. METHODS: Previously characterized MSCs iMEFs were used. Overexpression of BMP9 and NELL1 or silencing of mouse Nell1 was mediated by adenoviral vectors. Early and late osteogenic and adipogenic markers were assessed by staining techniques and qPCR analysis. In vivo activity was assessed in an ectopic bone formation model of athymic mice. RESULTS: We demonstrate that Nell1 expression was up-regulated by BMP9. Exogenous Nell1 potentiated BMP9-induced late stage osteogenic differentiation while inhibiting the early osteogenic marker. Forced Nell1 expression enhanced BMP9-induced osteogenic regulators/markers and inhibited BMP9-upregulated expression of adipogenic regulators/markers in MSCs. In vivo ectopic bone formation assay showed that exogenous Nell1 expression enhanced mineralization and maturity of BMP9-induced bone formation, while inhibiting BMP9-induced adipogenesis. Conversely, silencing Nell1 expression in BMP9-stimulated MSCs led to forming immature chondroid-like matrix. CONCLUSION: Our findings indicate that Nell1 can be up-regulated by BMP9, which in turn accelerates and augments BMP9-induced osteogenesis. Exogenous Nell1 may be exploited to enhance BMP9-induced bone formation while overcoming BMP9-induced adipogenesis in regenerative medicine.

A Novel Organ Culture Model of Mouse Intervertebral Disc Tissues.

The intervertebral disc (IVD) is a fibrocartilaginous joint between two vertebral bodies. An IVD unit consists of a gelatinous central nucleus pulposus, encased by the annulus fibrosus, which is sandwiched between cartilaginous endplates (EPs). The IVD homeostasis can be disrupted by injuries, ageing and/or genetic predispositions, leading to degenerative disc disorders and subsequent lower back pain. The complex structure and distinct characteristics of IVDs warrant the establishment of robust in vitro IVD organ culture for studying the etiology and treatment of disc degeneration. Here, we isolate mouse lumbar IVDs and culture the minimal IVD units in submersion or suspension medium supplemented with 2% bovine serum or 10% fetal bovine serum (FBS). We find the minimal IVD units remain healthy for up to 14 days when cultured in submersion culture supplemented with 10% FBS. New bone formation in the EPs of the cultured IVDs can be assessed with calcein labeling. Furthermore, the cultured IVDs can be effectively transduced by recombinant adenovirus, and transgene expression lasts for 2 weeks. Thus, our findings demonstrate that the optimized IVD organ culture system can be used to study IVD biology and screen for biological factors that may prevent, alleviate and/or treat disc degeneration.

The Prodomain-Containing BMP9 Produced from a Stable Line Effectively Regulates the Differentiation of Mesenchymal Stem Cells.

BACKGROUND: BMPs play important roles in regulating stem cell proliferation and differentiation. Using adenovirus-mediated expression of the 14 types of BMPs we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), which was undetected in the early studies using recombinant BMP9 proteins. Endogenous BMPs are expressed as a precursor protein that contains an N-terminal signal peptide, a prodomain and a C-terminal mature peptide. Most commercially available recombinant BMP9 proteins are purified from the cells expressing the mature peptide. It is unclear how effectively these recombinant BMP9 proteins functionally recapitulate endogenous BMP9. METHODS: A stable cell line expressing the full coding region of mouse BMP9 was established in HEK-293 cells by using the piggyBac transposon system. The biological activities and stability of the conditioned medium generated from the stable line were analyzed. RESULTS: The stable HEK-293 line expresses a high level of mouse BMP9. BMP9 conditioned medium (BMP9-cm) was shown to effectively induce osteogenic differentiation of MSCs, to activate BMP-R specific Smad signaling, and to up-regulate downstream target genes in MSCs. The biological activity of BMP9-cm is at least comparable with that induced by AdBMP9 in vitro. Furthermore, BMP9-cm exhibits an excellent stability profile as its biological activity is not affected by long-term storage at -80ºC, repeated thawing cycles, and extended storage at 4ºC. CONCLUSIONS: We have established a producer line that stably expresses a high level of active BMP9 protein. Such producer line should be a valuable resource for generating biologically active BMP9 protein for studying BMP9 signaling mechanism and functions.

The Calcium-Binding Protein S100A6 Accelerates Human Osteosarcoma Growth by Promoting Cell Proliferation and Inhibiting Osteogenic Differentiation.

BACKGROUND/AIMS: Although osteosarcoma (OS) is the most common primary malignancy of bone, its molecular pathogenesis remains to be fully understood. We previously found the calcium-binding protein S100A6 was expressed in ∼80% of the analyzed OS primary and/or metastatic tumor samples. Here, we investigate the role of S100A6 in OS growth and progression. METHODS: S100A6 expression was assessed by qPCR and Western blotting. Overexpression or knockdown of S100A6 was carried out to determine S100A6's effect on proliferation, cell cycle, apoptosis, tumor growth, and osteogenic differentiation. RESULTS: S100A6 expression was readily detected in human OS cell lines. Exogenous S100A6 expression promoted cell proliferation in vitro and tumor growth in an orthotopic xenograft model of human OS. S100A6 overexpression reduced the numbers of OS cells in G1 phase and increased viable cells under serum starvation condition. Conversely, silencing S100A6 expression induced the production of cleaved caspase 3, and increased early stage apoptosis. S100A6 knockdown increased osteogenic differentiation activity of mesenchymal stem cells, while S100A6 overexpression inhibited osteogenic differentiation. BMP9-induced bone formation was augmented by S100A6 knockdown. CONCLUSION: Our findings strongly suggest that S100A6 may promote OS cell proliferation and OS tumor growth at least in part by facilitating cell cycle progression, preventing apoptosis, and inhibiting osteogenic differentiation. Thus, it is conceivable that targeting S100A6 may be exploited as a novel anti-OS therapy.

A study on the differences in radon exhalation of different lithologies at various depths and the factors influencing its distribution in northern Shaanxi, China.

The inhalation of a high concentration of radon gas increases the risk of cancer. Therefore, it is of utmost necessity to pay due attention to the problem of environmental radon pollution. The high radioactivity above the coal slab causes serious radon radiation contamination on the mining grounds in coal mining areas such as the northern part of China and the western part of the United States. At present, there is a lack of research on radon exhalation in different lithologies. In this study, the differences in the radon exhalation of different lithologies at various depths and their controlling factors were studied by NMR and radon measurement. The results highlighted that the radon exhalation rates in different rocks varied from 0.3 to 0.6 Bq/m2·s. The average radon exhalation rate of the soil was 0.7 Bq/m2·s, and the radon exhalation rates of different lithologies followed the pattern red clay > loess > sandstone > mudstone > coal. The radon exhalation rate increased initially, followed by a decrease, and the radon exhalation rate was the highest at the boundary between the soil and rock layers. The radon exhalation rates of different lithologies have a strong correlation with the small pores (<0.1 µm), which govern the changes in the porous structure with depth. The results of this study are important from the perspective evaluation of environmental radon pollution.

Effect of heat treatment on the emission rate of radon from red sandstone.

High temperature and pore structure are important factors affecting the emission rate of radon in rocks. This study mainly focused on the correlation between radon emission rate and temperature in red sandstone. The results showed that in the temperature range of 25-400 °C, as the temperature increased, the connectivity of the internal pores of the sample became better, resulting in a significantly increased radon emission rate. The radon emission rate at 400 °C was 2.86 times the original. To explain the changes that occurred in the internal structure of the samples, the porosity characteristics of the samples after heat treatment were studied by nuclear magnetic resonance (NMR). It was found that the pore structure was also an important factor affecting the rate of radon emission. The smaller pore size of the micropores (r < 0.1 µm) inhibited the emission of radon in the sandstone. These results helped in understanding the mechanism of radon emission rate and provide an important basis for predicting rock fragmentation and coal fire.

[Simultaneous determination of nine typical anions in fire explosive residues by ion chromatography].

A novel method was developed for the simultaneous determination of nine typical anions (Cl-, NO2-, ClO3-, NO3-, CO32-, SO42-, S2O32-, SCN-and ClO4-) in explosive residues by ion chromatography (IC). A high capacity anion-exchange IonPac AS20 column (250 mm×4 mm) was used for the separation of the nine anions with gradient elution of KOH. The injection volume was 20 µL, the column temperature was 40℃, and the flow rate was 1.20 mL/min. Inhibitory conductivity detections were adopted in the determinations. Good linearities were found for all the nine anions in their respective ranges. The correlation coefficients were greater than 0.999. The method was applied to the determination of the inorganic ions in explosive residues. The average recovery of the nine ions ranged from 92.5% to 101.3%. The relative standard deviations of the nine anions ranged from 1.86% to 2.79% (n=6). The method is simple, quick, selective and sensitive and can meet the requirement of the analysis.

Energy Storage Analysis of a Mixed R161/MOF-5 Nanoparticle Nanofluid Based on Molecular Simulations.

The thermal properties of refrigerants can be modified by adding porous nanoparticles into them. Here, molecular simulations, including molecular dynamics and grand canonical Monte Carlo, were employed to study the thermal energy storage properties of an R161/MOF-5 nanofluid. The results show that the thermodynamic energy change of MOF-5 nanoparticles is linear to the temperature. The adsorption heat calculated by grand canonical Monte Carlo is close to that calculated by the Clausius⁻Clapeyron equation. Additionally, a negative enhancement of the thermal energy storage capacity of the R161/MOF-5 nanofluid is found near the phase transition area.

Monensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with <5% five-year survival rate due to late diagnosis, limited treatment options and chemoresistance. There is thus an urgent unmet clinical need to develop effective anticancer drugs to treat pancreatic cancer. Here, we study the potential of repurposing monensin as an anticancer drug for chemo-resistant pancreatic cancer. Using the two commonly-used chemo-resistant pancreatic cancer cell lines PANC-1 and MiaPaCa-2, we show that monensin suppresses cell proliferation and migration, and cell cycle progression, while solicits apoptosis in pancreatic cancer lines at a low micromole range. Moreover, monensin functions synergistically with gemcitabine or EGFR inhibitor erlotinib in suppressing cell growth and inducing cell death of pancreatic cancer cells. Mechanistically, monensin suppresses numerous cancer-associated pathways, such as E2F/DP1, STAT1/2, NFkB, AP-1, Elk-1/SRF, and represses EGFR expression in pancreatic cancer lines. Furthermore, the in vivo study shows that monensin blunts PDAC xenograft tumor growth by suppressing cell proliferation via targeting EGFR pathway. Therefore, our findings demonstrate that monensin can be repurposed as an effective anti-pancreatic cancer drug even though more investigations are needed to validate its safety and anticancer efficacy in pre-clinical and clinical models.

Effect of hydroxyapatite nanoparticles on the growth and p53/c-Myc protein expression of implanted hepatic VX2 tumor in rabbits by intravenous injection.

AIM: To evaluate the effect of hydroxyapatite nano-particles (Nano HAP) by intravenous injection on the inhibition of implanted hepatic VX(2) tumor growth in rabbits and cell p53/c-Myc protein expression. METHODS: 60 hepatic VX(2) tumor-bearing rabbits was randomly divided into five groups. Nano HAP collosol 20 mg/kg, 40 mg/kg, 5-FU solutions 20 mg/mL, mixed liquor of 5-FU solution 20 mg/mL and Nano HAP collosol 20 mg/kg were infused by vein, normal saline conducted as the control. The general state, weight, liver function and gross tumor volume were detected dynamically. The expression of p53 and c-Myc gene protein in tumor tissue was detected by immunohistochemistry methods. RESULTS: The growth of implanted hepatic VX(2) tumors was significantly inhibited in all therapy groups, 3 wk after the injection, the tumor control rates in Nano HAP collosol groups were 25.5% and 32.5% respectively, and the gross tumor volumes were obviously less than that of control group. (24.81 +/- 5.17 and 22.73 +/- 4.23 vs 33.32 +/- 5.26, P<0.05). The tumor control rate of 5-FU group was 43.7% (18.74 +/- 4.40 vs 33.32 +/- 5.26, P<0.05), but the general state of the animals after injection aggravated; and the adverse reaction in the drug combination group obviously decreased. Due to the effect of Nano HAP, the positive expression of tumor associated the mutated p53 and c-Myc in tumor tissue was decreased obviously compared with the control group. CONCLUSION: Nano HAP has evident inhibitory action on rabbit implanted hepatic VX(2) tumor in vivo, which may be the result of decreasing the expression of the mutated p53 and c-myc, and drug combination can obviously decrease the adverse reaction of 5-FU.

Predictive diagnosis of major depression using NMR-based metabolomics and least-squares support vector machine.

BACKGROUND: Major depressive (MD) disorder is a serious psychiatric disorder that can result in suicidal behavior if not treated. The MD diagnosis using a standardized instrument instead of a structured interview will be advantageous for treatment and management of the MD, but so far no such technique exists. We developed an integrated analytical method of NMR-based metabolomics and least squares-support vector machine (LS-SVM) for predictive diagnosis of the MD. METHODS: The metabolite profiles in clinical plasma samples obtained from 72 depressive patients and 54 healthy subjects were analyzed by NMR spectroscopy. Then, LS-SVM models with different kernels were trained and tested using 80% and 20% of samples, respectively. RESULTS: We found that the best performance for the MD prediction was achieved by LS-SVM equipped with RBF kernel. Moreover, the predictive performance of the MD using multi-biomarkers was largely improved as compared with that using a single biomarker. In this study, the LS-SVM-RBF using glucose-lipid signaling can achieve the MD prediction with the AUC values of 0.94 (0.89-0.99) in the training set and 0.96 (0.92-1.00) in the test set. CONCLUSION: The LS-SVM-RBF using glucose-lipid signaling obtained from NMR spectroscopy can be used as an auxiliary diagnostic tool for the MD.

Metabolic effects of basic fibroblast growth factor in streptozotocin-induced diabetic rats: A 1H NMR-based metabolomics investigation.

The fibroblast growth factors (FGFs) family shows a great potential in the treatment of diabetes, but little attention is paid to basic FGF (bFGF). In this study, to explore the metabolic effects of bFGF on diabetes, metabolic changes in serum and feces were analyzed in the normal rats, the streptozocin (STZ)-induced diabetic rats and the bFGF-treated diabetic rats using a 1H nuclear magnetic resonance (NMR)-based metabolomic approach. Interestingly, bFGF treatment significantly decreased glucose, lipid and low density lipoprotein/very low density lipoprotein (LDL/VLDL) levels in serum of diabetic rats. Moreover, bFGF treatment corrected diabetes-induced reductions in citrate, lactate, choline, glycine, creatine, histidine, phenylalanine, tyrosine and glutamine in serum. Fecal propionate was significantly increased after bFGF treatment. Correlation analysis shows that glucose, lipid and LDL/VLDL were significantly negatively correlated with energy metabolites (citrate, creatine and lactate) and amino acids (alanine, glycine, histidine, phenylalanine, tyrosine and glutamine). In addition, a weak but significant correlation was observed between fecal propionate and serum lipid (R = -0.35, P = 0.046). Based on metabolic correlation and pathway analysis, therefore, we suggest that the glucose and lipid lowering effects of bFGF in the STZ-induced diabetic rats may be achieved by activating microbial metabolism, increasing energy metabolism and correcting amino acid metabolism.

Transition to resistance: An unexpected role of the EMT in cancer chemoresistance.

Two recent studies provide intriguing evidence that challenges the role of the epithelial-mesenchymal transition (EMT) as a critical mediator of cancer metastasis, while revealing an unexpected role in cancer drug resistance.1,2 While these findings may not settle the EMT's role in metastasis, these studies suggest that targeting the EMT may inhibit both cancer metastasis and chemoresistance.

Wnt/ß-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance.

Wnt signaling transduces evolutionarily conserved pathways which play important roles in initiating and regulating a diverse range of cellular activities, including cell proliferation, calcium homeostasis, and cell polarity. The role of Wnt signaling in control of cell proliferation and stem cell self-renewal is primarily carried out through the canonical pathway, which is the best characterized among the multiple Wnt signaling branches. The past 10 years has seen a rapid expansion in our understanding of the complexity of this pathway, as many new components of Wnt signaling have been identified and linked to signaling regulation, stem cell functions, and adult tissue homeostasis. Additionally, a substantial body of evidence links Wnt signaling to tumorigenesis of many cancer types and implicates it in the development of cancer drug resistance. Thus, a better understanding of the mechanisms by which dysregulation of Wnt signaling precedes the development and progression of human cancer may hasten the development of pathway inhibitors to augment current therapy. This review summarizes and synthesizes our current knowledge of the canonical Wnt pathway in development and disease. We begin with an overview of the components of the canonical Wnt signaling pathway and delve into the role this pathway has been shown to play in stemness, tumorigenesis, and cancer drug resistance. Ultimately, we hope to present an organized collection of evidence implicating Wnt signaling in tumorigenesis and chemoresistance to facilitate the pursuit of Wnt pathway modulators that may improve outcomes of cancers in which Wnt signaling contributes to aggressive disease and/or treatment resistance.

A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells.

Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs + PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs + PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.