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Background: This study aimed to investigate the clinical outcome and related risk factors of fetal lateral ventriculomegaly (VM). Methods: A retrospective analysis was performed on 255 cases diagnosed as fetal VM. Prenatal imaging examination was carried out. The pregnancy outcomes were investigated through follow-up. According to the prognosis of children, they were divided into case group and control group. Multivariate logistic regression was used to analyze the factors influencing the prognosis of hydrocephalus. Results: After excluding the cases with either loss of follow-up or incomplete information, 102 cases were followed up. Twelve cases with poor prognosis were set as the case group. According to the maternal age, gestational age, gender of children, and follow-up time, 3 cases were selected from the other 90 cases for each child in the case group, respectively, and selected as the control group. Paired comparative analysis was performed on 48 cases. Using prognosis as a dependent variable, multivariate logistic regression analysis of the statistically significant factors indicated that the change speed of width ratio (CSWR) and maximum lateral ventricular width (MW) were associated with fetal prognosis. Conclusions: Our results suggested that CSWR and MW may have the value of predicting fetal prognosis.

Osteosarcoma (OS) is the most common malignant tumor of bone. It has recently been demonstrated that galectin-3, a multifunctional β-galactoside-binding, is significantly upregulated in OS tissues, and is correlated with its progression and metastasis. However, the detailed role of galectin‑3 in the regulation of cellular biological processes in OS cells has remained to be elucidated. The present study reported that the mRNA and protein levels of galectin‑3 were significantly increased in OS tissues compared to those in their matched normal adjacent tissues. Furthermore, galectin‑3 was upregulated in three OS cell lines, Saos‑2, MG63 and U2OS, when compared with that in the human osteoblast cell line hFOB1.19. Knockdown of galectin‑3 by galectin‑3‑specific small interfering RNA markedly inhibited OS‑cell proliferation and induced cell apoptosis. Furthermore, silencing of galectin‑3 expression significantly inhibited OS cell migration and invasion, accompanied with a marked decrease in the protein expression of matrix metalloproteinase 2 and ‑9. Mechanistic investigation suggested that the mitogen‑activated protein kinase kinase/extracellular signal‑regulated protein kinase signaling pathway may be involved in the galectin‑3‑mediated OS cell invasion. In conclusion, the present study was the first to report that silencing of galectin‑3 inhibited the malignant phenotypes of osteosarcoma in vitro. Therefore, galectin-3 may serve as a potential therapeutic target for OS.

[This corrects the article DOI: 10.1371/journal.pone.0093608.].

Background: The supercapsularpercutaneously-assisted total hip (SuperPATH) approach is a micro-invasive approach that was developed to minimize surgical disruption of soft-tissue during routine total hip arthroplasty (THA). This study aimed to assess early outcomes and learning curves of the SuperPATHapproachin one Chinese hospital’s experience.Methods: Early outcomes of the first consecutive 78SuperPATH cases (80 hips) performed by same surgeon were evaluated. The patients were divided into 4 groups according to the surgical order. The incision, intraoperative blood loss, hospital stay, Harris hip score and complications occurrence in each group were evaluated. Learning curves were assessed using operative time and intraoperative blood loss as surrogates.Results:The operation time and intraoperative blood loss of group A and B was more than that of group C and D, and the difference was statistically significant (PConclusion:In summary, for surgeons who familiar with the standard posterolateral approach, they could achieve more familiar with SuperPATH after 40 case of surgery.

The supercapsular percutaneously assisted total hip (SuperPATH) approach is a microinvasive approach that was developed to minimize surgical disruption of soft tissue during routine total hip arthroplasty (THA). This study was aimed at assessing early outcomes and learning curves of the SuperPATH approach in one Chinese hospital’s experience. Early outcomes of the first consecutive 78 SuperPATH cases (80 hips) performed by the same surgeon were evaluated. The patients were divided into 4 groups according to the surgical order. The incision, intraoperative blood loss, hospital stay, Harris hip score, and complication occurrence in each group were evaluated. Learning curves were assessed using operative time and intraoperative blood loss as surrogates. The operation time and intraoperative blood loss of groups A and B were more than those of groups C and D, and the difference was statistically significant (P<0.05); however, there was no statistically significant difference between the two groups (group A vs. group B, P=0.426; group A vs. group B, P=0.426). There was no statistically significant difference in terms of incision length and hospital stay, and Harris hip score at the last follow-up was increased with statistically significant difference when compared with that preoperatively among the 4 groups. One case of periprosthetic fracture occurred in group A. No other complication, such as joint dislocation, sciatic nerve injury, prosthesis loosening, periprosthetic infection, and deep vein thromboembolism, occurred in the 4 groups. In summary, for surgeons who are familiar with the standard posterolateral approach, they could achieve more familiarity with SuperPATH after 40 cases of surgery.

ObjectiveThe purpose of this study is to retrospectively analyze the clinical efficacy of free fibula autograft and wrist arthroplasty in the treatment of giant cell tumors (GCT) of distal radius.MethodsWe retrospectively reviewed 26 patients with GCT of distal radius who underwent free autogenous fibula graft and wrist arthroplasty for repairing residual defect after en-block resection. The length of the fibula graft was 8.2 cm (6–10 cm). Postoperative follow-up regularly for an mean of 66.9 months. Bone healing was assessed by radiographs, pain was assessed by Visual Analog Scale (VAS) score and limb function was evaluated by Musculoskeletal Tumor Society (MSTS) score and disabilites of the arm, shoulder and hand (DASH) score. The range of motion (ROM) of wrist and grip strength were also evaluated.ResultThere were four males and 22 females with an mean age of 36.7 years (19–60 years); the mean length of lesions was 4.8 cm (2.3–6.6 cm); 21 primary cases and five recurrent cases; eight cases of Campanacci Grade II, 18 cases of Grade III. We had no postoperative lung metastasis and only one case had a local recurrence, three cases (11%) with subluxation of lower ulnoradial joints and five cases (19%) showed narrowing of wrist joint space. The mean postoperative VAS pain score was 0.7 ± 0.7 and grip strength retained 71% of the normal hand, MSTS score was 27.7 ± 1.1 and DASH score was 9.0 ± 3.7. The ROM of the involved wrist only slightly restricted and no donor complications. Postoperative wrist joint function was significantly improved.ConclusionStrict surgical resection boundary and solid reconstruction of wrist joint capsule are the key to achieving excellent oncological prognosis and function of distal radius GCT.

The morphology and histology changes in the medial meniscus after posterior cruciate ligament (PCL) rupture are poorly understood. Forty-eight rabbits were divided into matched mode pairs; each rabbit had an experimental side, in which the PCL was transacted, and a control side. At the 4, 8, 16 and 24 weeks after the PCL transection, each of the 12 rabbits was killed. Histology was performed to detect the expression of the tissue inhibitors of metalloproteinases-1 (TIMP-1), matrix metalloproteinase (MMP)-1 and MMP-13 in the medial meniscus. We found that medial meniscus displayed significant degenerative characteristics in morphology. The histological evaluation of the degeneration found that the expression levels of TIMP-1, MMP-1 and MMP-13 in the medial meniscus were higher in the experiment side than those in the control side (P

// Hongbo He 1 , Qing Liu 1 , Can Zhang 1 , Zhan Liao 1 , Yupeng Liu 1 , Jun Wan 1 , Jian Tian 1 , Xiaopeng Tong 1 , Lichun Sun 1,2,3 and Wei Luo 1 1 Department of Orthopedics, Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmacy & Pharmacology, University of South China, Hengyang, China 3 Department of Medicine, School of Medicine, Tulane Health Sciences Center, New Orleans, LA, USA Correspondence to: Wei Luo, email: luowei0928@126.com Keywords : fibrous dysplasia; whole-exome sequencing (WES); transcriptome sequencing (RNAseq); single gene sequencing; SNV/InDel Received: May 07, 2017 Accepted: November 13, 2017 Epub: January 03, 2018 Abstract Fibrous dysplasia (FD) of bone is a rare and non-inheritable bone disease. Its precise molecular mechanisms remain unclear. We collected FD patient samples and applied the innovative technology of whole-exome sequencing (WES) and transcriptome sequencing (RNAseq) to analyze the gene mutations and expression profiles. By comparing gene mutations with both WES and RNAseq, and further filtering these mutations with gene expressional significance (average > 1TPM), we eventually identified 20 genes with non-synonymous single nucleotide variations (SNVs) occurring in the exonic regions of all FD samples. About 40 genes with SNVs were mutated in 50% of the FD samples, including GNAS (Guanine Nucleotide Binding Protein Alpha) and RTN4 (Reticulon 4). Particularly, in three of these patients (Patient 1, 3 and 4), GNAS were identified as having SNVs, with the nucleotide changed in exon 8 and the amino acid residue changed in position 201 (GNAS: NM_000516: exon8: c.G602A: p.R201H; or c.C601T: p.R201C). We only observed 2 genes, FAM21B (mutation type: deletion) and GAGE2A (mutation type: amplification), with copy number variations (CNVs) occurred in FD patients. When analyzing signaling pathways and biological functions, we predicted some key genes, such as GPCRs, G proteins, chemokine and ANK-associated genes, and their associated signaling networks might play critical roles in FD development. We further confirmed these observations via single gene sequencing and Q-PCR. Thus, the mutations of these genes may be connected with fibroblast and bone development disorders. These findings may provide a clue for our next investigation of FD pathogenesis and its molecular mechanisms.

One of the greatest obstacles to current cancer treatment efforts is the development of drug resistance by tumors. Despite recent advances in diagnostic practices and surgical interventions, many neoplasms demonstrate poor response to adjuvant or neoadjuvant radiation and chemotherapy. As a result, the prognosis for many patients afflicted with these aggressive cancers remains bleak. The insulin-like growth factor (IGF) signaling axis has been shown to play critical role in the development and progression of various tumors. Many basic science and translational studies have shown that IGF pathway modulators can have promising effects when used to treat various malignancies. There also exists a substantial body of recent evidence implicating IGF signaling dysregulation in the dwindling response of tumors to current standard-of-care therapy. By better understanding both the IGF-dependent and -independent mechanisms by which pathway members can influence drug sensitivity, we can eventually aim to use modulators of IGF signaling to augment the effects of current therapy. This review summarizes and synthesizes numerous recent investigations looking at the role of the IGF pathway in drug resistance. We offer a brief overview of IGF signaling and its general role in neoplasia, and then delve into detail about the many types of human cancer that have been shown to have IGF pathway involvement in resistance and/or sensitization to therapy. Ultimately, our hope is that such a compilation of evidence will compel investigators to carry out much needed studies looking at combination treatment with IGF signaling modulators to overcome current therapy resistance.

Background To explore the association between the rupture of posterior cruciate ligament (PCL) and the radial displacement of medial meniscus under the conditions of different flexion and various axial loads. Methods The radial displacement value of medial meniscus was measured for the specimens of normal adult knee joints, including 12 intact PCLs, 6 ruptures of the anterolateral bundle (ALB), 6 ruptures of the postmedial bundle (PMB), and 12 complete ruptures. The measurement was conducted at 0°, 30°, 60°, and 90° of knee flexion angles under 200 N, 400 N, 600 N, 800 N and 1000 N of axial loads respectively. Results The displacement values of medial meniscus of the ALB rupture group increased at 0° flexion under 800 N and 1000 N, and at 30°, 60° and 90° flexion under all loads in comparison with the PCL intact group. The displacement values of the PMB rupture group was higher at 0° and 90° flexion under all loads, and at 30° and 60° flexion under 800 N and 1000 N loads. The displacement of the PCL complete rupture group increased at all flexion angles under all loads. Conclusions Either partial or complete rupture of the PCL can increase in the radial displacement of the medial meniscus, which may explain the degenerative changes that occuring in the medial meniscus due to PCL injury. Therefore, early reestablishment of the PCL is necessarily required in order to maintain stability of the knee joint after PCL injury.

Objectives Chordoma is a rare malignant bone tumour arising from notochordal remnants. Long non-coding RNA LOC554202, as the host gene of miR-31, contributes to various cancer developments. However, little is known about the biological function of LOC554202 in chordoma. Here, the relationship between LncRNA LOC554202, miR-31 and EZH2 was elucidated in chordoma. Materials and methods The levels of LOC554402, miR-31, EZH2, RNF144B, and epithelial-mesenchymal transition (EMT) markers were measured in chordoma tissues and the chordoma cell lines via quantitative real-time PCR (qRT-PCR) or Western blot. FISH assay demonstrated the LOC554402 expression in chordoma tissues. The chordoma cell lines, U-CH1 and JHC7, were transfected with siRNA or miRNA mimics and analysed for cell proliferation ability, apoptosis, cell migration, and invasion. RNA pull down, RIP assay, and Luciferase Reporter Assay were used to analyze the interaction between LOC554202 and EZH2. Animal tumour xenografts were generated, and qRT-PCR was performed to investigate EZH2, miR-31, and RNB144B expression on tumour growth in vivo. Results We found elevated expression of LOC554202 was associated with a decreased level of miR-31 in cancer tissues. Knockdown of LOC554202 or overexpression of miR-31 suppressed the proliferation, migration, and invasion of chordoma cells. Unexpectedly, EZH2 as a binding protein of LOC554202, and it was positively regulated by LOC554202, leading to the reduced expression of miR-31. Furthermore, the impaired function of miR-31 restored expression of the oncogene RNF144B and maintained the metastasis-promoting activity in vitro. The results in vivo confirmed the anti-tumour effects of knockdown of LOC554202, which inhibited EZH2/miR-31 to activate the oncogene RNF144B. Conclusion Our results suggest that LOC554202 may play an important role in the progression of chordoma by the direct upregulation of EZH2 and indirect promotion of RNF144B via miR-31.

Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/β-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells.

Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

Aberrant activation of β-catenin signaling plays an important role in human tumorigenesis. However, molecular mechanisms behind the β-catenin signaling deregulation are mostly unknown because genetic alterations in this pathway only account for a small fraction of tumors. Here, we investigator if other major pathways can regulate β-catenin signaling activity. By employing a panel of chemical activators and/or inhibitors of several cellular signaling pathways, we assess these modulators' effects on luciferase reporter driven by β-catenin/TCF4-responsive elements. We find that lithium-stimulated β-catenin activity is synergistically enhanced by protein kinase C activator PMA. However, β-catenin-regulated transcriptional (CRT) activity is significantly inhibited by casein kinase II inhibitor DRB, MEK inhibitor PD98059, G-proteins and their receptor uncoupling agent suramin, protein tyrosine kinase inhibitor genistein, and PI-3 kinase inhibitor wortmannin, suggesting that these cellular pathways may participate in regulating β-catenin signaling. Interestingly, the Ca⁺⁺/calmodulin kinase II inhibitor HDBA is shown to activate β-catenin activity at low doses. Furthermore, Wnt3A-stimulated and constitutively activated CRT activities, as well as the intracellular accumulation of β-catenin protein in human colon cancer cells, are effectively suppressed by PD98059, genistein, and wortmannin. We further demonstrate that EGF can activate TCF4/β-catenin activity and induce the tyrosine phosphorylation of β-catenin protein. Thus, our results should provide important insights into the molecular mechanisms underlying Wnt/β-catenin activation. This knowledge should facilitate our efforts to develop efficacious and novel therapeutics by targeting these pathways.

Introduction: Whether the expression level of MMP-1, MMP-13 and TIMP-1 has association with the degeneration of lateral meniscus after posterior cruciate ligament (PCL) fracture is poorly understood. The aim of this study was to investigate the influence of PCL fracture on lateral meniscus, including morphological changes, histological changes and roles of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression level in the secondary injury. Materials and Methods: Sixty male rabbits were used as PCL transection models and randomized into the PCL-transection side, which underwent PCL transection surgery, and the control side, which underwent PCL exposure without transection. On 4, 8, 12, 16 and 24 weeks after PCL-transection, 12 rabbits were randomly killed for H&E staining to determine the histological changes of lateral meniscus. Immunohistochemical staining was undertaken to evaluate the expression level of MMP-1, MMP-13 and TIMP-1 in lateral meniscus. The results were statistically analyzed using SPSS 15.0. Results: The lateral meniscus of PCL-transection side presented abnormal morphology. Histological evaluation score of meniscal degeneration in PCL-transection group was higher than that in the control group with statistical difference (P < 0.05). The expression levels of MMP-1, MMP-13 and TIMP-1 were significantly elevated in meniscus of the PCL-transection group with statistical difference (P < 0.05). MMP-1 expression displayed an increasing trend firstly then kept stable after PCL transection; MMP-13 and TIMP-1 expression displayed high level firstly then decreased in advanced stage after PCL transection. Conclusions: PCL transaction may induce a coordinated response of degeneration of lateral meniscus in a time-dependent manner. The high expression level of MMP-1, MMP-13 and TIMP-1 would contribute to the degeneration of lateral meniscus after PCL transection.

Cartilage tissue engineering holds great promise for treating cartilaginous pathologies including degenerative disorders and traumatic injuries. Effective cartilage regeneration requires an optimal combination of biomaterial scaffolds, chondrogenic seed cells, and biofactors. Obtaining sufficient chondrocytes remains a major challenge due to the limited proliferative capability of primary chondrocytes. Here we investigate if reversibly immortalized mouse articular chondrocytes (iMACs) acquire long-term proliferative capability while retaining the chondrogenic phenotype. Primary mouse articular chondrocytes (MACs) can be efficiently immortalized with a retroviral vector-expressing SV40 large T antigen flanked with Cre/loxP sites. iMACs exhibit long-term proliferation in culture, although the immortalization phenotype can be reversed by Cre recombinase. iMACs express the chondrocyte markers Col2a1 and aggrecan and produce chondroid matrix in micromass culture. iMACs form subcutaneous cartilaginous masses in athymic mice. Histologic analysis and chondroid matrix staining demonstrate that iMACs can survive, proliferate, and produce chondroid matrix. The chondrogenic growth factor BMP2 promotes iMACs to produce more mature chondroid matrix resembling mature articular cartilage. Taken together, our results demonstrate that iMACs acquire long-term proliferative capability without losing the intrinsic chondrogenic features of MACs. Thus, iMACs provide a valuable cellular platform to optimize biomaterial scaffolds for cartilage regeneration, to identify biofactors that promote the proliferation and differentiation of chondrogenic progenitors, and to elucidate the molecular mechanisms underlying chondrogenesis.

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.

Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.

Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

Background/Aims: Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. Molecular mechanisms underlying the pathogenesis of OS remain to be fully understood. Several members of the E-F hand calcium-binding S100 protein family are differentially expressed in human cancers. We previously showed that S100A6 is highly expressed in OS tumors. In this study, we investigated the role of S100A4 in regulating OS proliferation and osteogenic differentiation. Methods/Results: Endogenous S100 expression was examined by semi-quantitative PCR in human OS lines. Adenoviral vector-mediated overexpression and RNAi knockdown of S100A4 were used to assess S100A4's effects on cell proliferation, migration and invasion and osteogenic differentiation. Apoptosis was assessed by using anti-caspase-3 immunostaining and flow cytometry with annexin V staining. Early osteogenic marker alkaline phosphatase (ALP) and late markers osteocalcin (OCN) and osteopontin (OPN) were assessed to determine the status of osteogenic differentiation. We found that S100A4 was elevated in metastatic MG63.2 cells. S100A4 knockdown inhibited cell proliferation, prolonged cell doubling time, and induced significant apoptosis. Silencing S100A4 expression in OS cells delayed cell wounding closure and diminished the numbers of migrated OS cells in transwell invasion assay. Furthermore, silencing S100A4 expression stimulated ALP activity, as well as late markers OPN and OCN, in both OS cells and mesenchymal stem cells. Conclusion: Our results strongly suggest that S100A4 may promote OS tumor growth by regulating the cell cycle, reducing apoptosis, and inhibiting osteogenic differentiation. Thus, S100A4 may serve as a marker for tumorigenic potential, as well as a therapeutic target.

Background/Aims: We have demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent BMPs in regulating osteoblast differentiation of mesenchymal stem cells (MSCs) although the molecular mechanism underlying BMP9-induced osteogenesis remains to be fully elucidated. It is known that epigenetic regulations play an important role in regulating the stem cell potency and lineage commitment. Here, we investigate if the inhibition of histone deacetylases (Hdacs) affects BMP9-induced osteogenic differentiation of MSCs. Methods: Using the Hdac inhibitor trichostatin A (TSA), we assess that TSA enhances BMP9-mediated osteogenic markers and matrix mineralization in MSCs, and bone formation in mouse embryonic limb explants. Results: We find that the endogenous expression of most of the 11 Hdacs is readily detectable in MSCs. BMP9 is shown to induce most Hdacs in MSCs. We demonstrate that TSA potentiates BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs, as well as late osteogenic markers osteopontin (OPN) and osteocalcin (OCN) and matrix mineralization. Fetal limb explant culture studies reveal that TSA potentiates BMP9-induced endochondral bone formation, possibly by expanding hypertrophic chondrocyte zone of growth plate. Conclusion: Our findings strongly suggest histone deacetylases may play an important role in fine-tuning BMP9-mediated osteogenic signaling through a negative feedback network in MSCs. Thus, Hdac inhibitors may be used as novel therapeutics for bone fracture healing.

Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.

Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.

Background: The relationship between medial meniscus tear and posterior cruciate ligament (PCL) injury has not been exactly explained. We studied to investigate the biomechanical effect of partial and complete PCL transection on different parts of medial meniscus at different flexion angles under static loading conditions. Materials and Methods: Twelve fresh human cadaveric knee specimens were divided into four groups: PCL intact (PCL-I), anterolateral bundle transection (ALB-T), posteromedial bundle transection (PMB-T) and PCL complete transection (PCL-T) group. Strain on the anterior horn, body part and posterior horn of medial meniscus were measured under different axial compressive tibial loads (200-800 N) at 0°, 30°, 60° and 90° knee flexion in each groups respectively. Results: Compared with the PCL-I group, the PCL-T group had a higher strain on whole medial meniscus at 30°, 60° and 90° flexion in all loading conditions and at 0° flexion with 400, 600 and 800 N loads. In ALB-T group, strain on whole meniscus increased at 30°, 60° and 90° flexion under all loading conditions and at 0° flexion with 800 N only. PMB-T exihibited higher strain at 0° flexion with 400 N, 600 N and 800 N, while at 30° and 60° flexion with 800 N and at 90° flexion under all loading conditions. Conclusions: Partial PCL transection triggers strain concentration on medial meniscus and the effect is more pronounced with higher loading conditions at higher flexion angles.