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Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.

Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFβ/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood. Keywords: BMP9/GDF2, Bone morphogenetic proteins (BMPs), Hepatic metabolism, Mesenchymal stem cells, Neurogenesis, Osteogenic differentiation, Pulmonary arterial hypertension, Tumorigenesis

Extracellular vesicles (EVs) such as microvesicles (MIVs) play an important role in intercellular communications. MIVs are small membrane vesicles sized 100–1000 nm in diameter that are released by many types of cells, such as mesenchymal stem cells (MSCs), tumor cells and adipose-derived stem cells (ADSC). As EVs can carry out autocrine and paracrine functions by controlling multiple cell processes, it is conceivable that EVs can be used as delivery vehicles for treating several clinical conditions, such as to improve cardiac angiogenesis after myocardial infarction (MI). Here, we seek to investigate whether ADSC-derived MIVs contain microRNAs that regulate angiogenesis and affect cell migration of endothelial cells. We first characterized the ADSC-derived MIVs and found that the MIVs had a size range of 100–300 nm, and expressed the MIV marker protein Alix. We then analyzed the microRNAs in ADSCs and ADSC-derived MIVs and demonstrated that ADSC-derived MIVs selectively released a panel of microRNAs, several of which were related to angiogenesis, including two members of the let-7 family. Furthermore, we demonstrated that ADSC-derived MIVs promoted the cell migration and invasion of the HUVEC endothelial cells. The PKH26-labeled ADSC-derived MIVs were effectively uptaken into the cytoplasm of HUVEC cells. Collectively, our results demonstrate that the ADSC-derived MIVs can promote migration and invasion abilities of endothelial cells, suggesting pro-angiogenetic potential. Future studies should focus on investigating the roles and mechanisms through which ADSC-derived MIVs regulate angiogenesis. Keywords: Adipose-derived stem cells (ADSCs), Angiogenesis, Let-7, microRNA, Microvesicle (MIV)

With the significant financial burden of chronic cutaneous wounds on the healthcare system, not to the personal burden mention on those individuals afflicted, it has become increasingly essential to improve our clinical treatments. This requires the translation of the most recent benchtop approaches to clinical wound repair as our current treatment modalities have proven insufficient. The most promising potential treatment options rely on stem cell-based therapies. Stem cell proliferation and signaling play crucial roles in every phase of the wound healing process and chronic wounds are often associated with impaired stem cell function. Clinical approaches involving stem cells could thus be utilized in some cases to improve a body's inhibited healing capacity. We aim to present the laboratory research behind the mechanisms and effects of this technology as well as current clinical trials which showcase their therapeutic potential. Given the current problems and complications presented by chronic wounds, we hope to show that developing the clinical applications of stem cell therapies is the rational next step in improving wound care. Keywords: Chronic inflammation, Chronic wounds, Growth factors, Personalized medicine, Skin, Stem cells, Wound healing

Mesenchymal stem cells (MSCs) are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes, chondrocytes, adipocytes, tenocytes and myocytes. MSCs represent one of the most commonly-used adult progenitors and serve as excellent progenitor cell models for investigating lineage-specific differentiation regulated by various cellular signaling pathways, such as bone morphogenetic proteins (BMPs). As members of TGFβ superfamily, BMPs play diverse and important roles in development and adult tissues. At least 14 BMPs have been identified in mammals. Different BMPs exert distinct but overlapping biological functions. Through a comprehensive analysis of 14 BMPs in MSCs, we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs. Nonetheless, a global mechanistic view of BMP signaling in regulating the proliferation and differentiation of MSCs remains to be fully elucidated. Here, we conducted a comprehensive transcriptomic profiling in the MSCs stimulated by 14 types of BMPs. Hierarchical clustering analysis classifies 14 BMPs into three subclusters: an osteo/chondrogenic/adipogenic cluster, a tenogenic cluster, and BMP3 cluster. We also demonstrate that six BMPs (e.g., BMP2, BMP3, BMP4, BMP7, BMP8, and BMP9) can induce I-Smads effectively, while BMP2, BMP3, BMP4, BMP7, and BMP11 up-regulate Smad-independent MAP kinase pathway. Furthermore, we show that many BMPs can upregulate the expression of the signal mediators of Wnt, Notch and PI3K/AKT/mTOR pathways. While the reported transcriptomic changes need to be further validated, our expression profiling represents the first-of-its-kind to interrogate a comprehensive transcriptomic landscape regulated by the 14 types of BMPs in MSCs.

Effective bone tissue engineering can restore bone and skeletal functions that are impaired by traumas and/or certain medical conditions. Bone is a complex tissue and functions through orchestrated interactions between cells, biomechanical forces, and biofactors. To identify ideal scaffold materials for effective mesenchymal stem cell (MSC)-based bone tissue regeneration, here we develop and characterize a composite nanoparticle hydrogel by combining carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP) (designated as CMCh-ACP hydrogel). We demonstrate that the CMCh-ACP hydrogel is readily prepared by incorporating glucono δ-lactone (GDL) into an aqueous dispersion or rehydrating the acidic freeze-dried nanoparticles in a pH-triggered controlled-assembly fashion. The CMCh-ACP hydrogel exhibits excellent biocompatibility and effectively supports MSC proliferation and cell adhesion. Moreover, while augmenting BMP9-induced osteogenic differentiation, the CMCh-ACP hydrogel itself is osteoinductive and induces the expression of osteoblastic regulators and bone markers in MSCs in vitro. The CMCh-ACP scaffold markedly enhances the efficiency and maturity of BMP9-induced bone formation in vivo, while suppressing bone resorption occurred in long-term ectopic osteogenesis. Thus, these results suggest that the pH-responsive self-assembled CMCh-ACP injectable and bioprintable hydrogel may be further exploited as a novel scaffold for osteoprogenitor-cell-based bone tissue regeneration.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with 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.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small regulatory noncoding RNAs, binding to complementary target mRNAs and resulting in mRNA translational inhibition or degradation, and they play an important role in regulating many aspects of physiologic and pathologic processes in mammalian cells. Thus, efficient manipulations of miRNA functions may be exploited as promising therapeutics for human diseases. Two commonly used strategies to inhibit miRNA functions include direct transfection of chemically synthesized miRNA inhibitors and delivery of a gene vector that instructs intracellular transcription of miRNA inhibitors. While most miRNA inhibitors are based on antisense molecules to bind and sequester miRNAs from their natural targets, it is challenging to achieve effective and stable miRNA inhibition. Here we develop a user-friendly system to express circular inhibitors of miRNA (CimiRs) by exploiting the noncanonical head-to-tail backsplicing mechanism for generating endogenous circular RNA sponges. In our proof-of-principle experiments, we demonstrate that the circular forms of the hsa-miR223-binding site of human β-arrestin1 (ARRB1) 3′ UTR sponge RNA (BUTR), the bulged anti-miR223 (cirBulg223) and bulged anti-miR21 (cirBulg21), exhibit more potent suppression of miRNA functions than their linear counterparts. Therefore, the engineered CimiR expression system should be a valuable tool to target miRNAs for basic and translational research. Keywords: microRNA, miRNA, miRNA sponge, circular RNA, miRNA inhibitor, competing endogenous RNA, miRNA decoy, noncoding RNA, oncomiR

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing β-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced β-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of β-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.

Understanding the bone and musculoskeletal system is essential to maintain the health and quality of life of our aging society. Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiate into multiple tissue types including bone. We demonstrated that BMP9 is the most potent osteogenic factors although molecular mechanism underlying BMP9 action is not fully understood. Long noncoding RNAs (lncRNAs) play important regulatory roles in many physiological and/or pathologic processes. Here, we investigated the role of lncRNA Rmst in BMP9-induced osteogenic differentiation of MSCs. We found that Rmst was induced by BMP9 through Smad signaling in MSCs. Rmst knockdown diminished BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro, and attenuated BMP9-induced ectopic bone formation. Silencing Rmst decreased the expression of Notch receptors and ligands. Bioinformatic analysis predicted Rmst could directly bind to eight Notch-targeting miRNAs, six of which were downregulated by BMP9. Silencing Rmst restored the expression of four microRNAs (miRNAs). Furthermore, an activating Notch mutant NICD1 effectively rescued the decreased ALP activity caused by Rmst silencing. Collectively, our results strongly suggest that the Rmst-miRNA-Notch regulatory axis may play an important role in mediating BMP9-induced osteogenic differentiation of MSCs.

MicroRNAs (miRNAs) are ~22 nucleotide noncoding RNAs that are involved in virtually all aspects of cellular process as their deregulations are associated with many pathological conditions. Mature miRNAs is generated through a series of tightly-regulated nuclear and cytoplasmic processing events of the transcribed primary, precursor and mature miRNAs. Effective manipulations of miRNA expression enable us to gain insights into miRNA functions and to explore potential therapeutic applications. Currently, overexpression of miRNAs is achieved by using chemically-synthesized miRNA mimics, or shRNA-like stem-loop vectors to express primary or precursor miRNAs, which are limited by low transfection efficacy or rate-limiting miRNA processing. To overcome rate-limiting miRNA processing, we developed a novel strategy to express mature miRNAs (mMIRs) which are driven by converging U6/H1 dual promoters. As a proof-of-concept study, we constructed mMIR expression vectors for hsa-miR-223 and hsa-Let-7a-1, and demonstrated that the expressed mature miRNAs effectively silenced target gene expression, specifically suppressed miRNA reporter activity, and significantly affected cell proliferation, similar to respective primary and precursor miRNAs. Furthermore, these mature miRNA expression vectors can be easily converted into retroviral and adenoviral vectors. Collectively, our simplified mature miRNA expression system should be a valuable tool to study miRNA functions and/or to deliver miRNA-based therapeutics.

OBJECTIVE: To determine the feasibility, safety, and efficacy of common pedicle screw placement under direct vision combined with dome shaped decompression via small incision for double segment thoracolumbar fracture with nerve injury. METHODS: A retrospective analysis was performed on the clinical data of 32 patients with double segment thoracolumbar fracture with nerve injury undergoing common pedicle screw placement under direct vision combined with dome shaped decompression via small incision between November 2011 and November 2015 (combined surgery group), and another 32 patients undergoing traditional open pedicle screw fixation surgery (traditional surgery group). There was no significant difference in gender, age, cause of injury, time of injury-to-surgery, injury segments and Frankel classification of neurological function between two groups (P>0.05). The length of soft tissue dissection, the operative time, the blood loss during surgery, the postoperative drainage, the visual analogue scale (VAS) of incision after surgery, and recovery of neurological function after surgery were evaluated. RESULTS: All cases were followed up 9 to 12 months (mean, 10.5 months) in combined surgery group, and 8 to 12 months (mean, 9.8 months) in traditional surgery group. The length of soft tissue dissection, the operative time, the blood loss during surgery, the postoperative drainage, and the postoperative VAS score in the combined surgery group were significantly better than those in the traditional surgery group (P

Percutaneous kyphoplasty (PKP) surgery is generally accepted as a minimally invasive treatment for osteoporotic vertebral compression fractures (OVCFs). However, hidden blood loss (HBL) caused by this procedure is usually disregarded. This study aimed to investigate the amount of HBL and its influencing factors after PKP surgery. A total of 160 patients were retrospectively examined from January 2014 to January 2016, and their clinical and radiological data were recorded and analyzed. Preoperative and postoperative hematocrit (Hct) and hemoglobin (Hb) levels were also documented. HBL was calculated using Gross formula. Different factors, including gender, age, bone mineral density (BMD), number of fracture levels, hypertension, diabetes mellitus, operative time, percentage of vertebral height loss, percentage of vertebral height restoration, and cement leakage, were examined. Multivariate linear regression analysis was performed to elucidate the related clinical or radiological factors of HBL. A total of 122 patients with 169 levels were eligible for inclusion in the study. The mean HBL was 279 ± 120 mL, and the postoperative Hb loss was 8.2 ± 3.9 g/L. Multivariate linear regression analysis revealed that HBL was positively associated with operative time (P = .000), percentage of vertebral height loss (P = .037), and percentage of vertebral height restoration (P = .000). By contrast, HBL was not associated with gender (P = .874), age (P = .148), BMD (P = .134), number of fracture levels (P = .079), hypertension (P = .259), diabetes mellitus (P = .495), and cement leakage (P = .975). The postoperative incidence of anemia significantly increased by 39.3% compared with that of the preoperative incidence (χ2 = 21.432, P = .000). For patients with OVCFs, the amount of HBL after PKP is much larger than that observed perioperatively. Operative time, percentage of vertebral height loss, and percentage of vertebral height restoration are influencing factors of HBL.

The aim of this review was to compare the clinical outcomes between operative and conservative management for primary patella dislocation (PPD).PubMed Medline, EMBASE, Google scholar, and the Cochrane Library were systematically searched for randomized controlled trials that compared operative technique versus conservative technique for PPD. The results of eligible studies were independently extracted and analyzed according to the following: patient's satisfaction, Kujala score, Tegner score, and redislocation rate. Random-effect and fixed-effect models were adopted to calculate the weight mean difference and the odds ratio for continuous and dichotomous variables with 95 % confidence interval.Seven studies met the inclusion criteria, resulting in 402 (216 surgery and 186 conservation) patients available for the present study. A meta-analysis showed no significant differences between the two treatment groups in terms of patient's satisfaction and Kujala score. However, significant differences in Tegner score (P0.00001) and redislocation rate (P = 0.002) were observed in favor of surgical treatment. In the subgroup analysis, surgical intervention achieved higher (P = 0.002) Kujala score in short term (5 years), while conservation gained advantage (P = 0.003) in long term (5 years). There was no significant difference in dislocation rate in long term.Surgical treatment might provide better clinical results in short term. More persuasive evidence is still needed to proof the effect of surgical management in long time.

Laminectomy is a widely accepted treatment for lumbar disorders, and epidural fibrosis (EF) is a common complication. EF is thought to cause post-operative pain recurrence after laminectomy or discectomy. All-trans retinoic acid (ATRA) has shown anti-fibrotic, anti-inflammatory, and anti-proliferative functions. The object of this study was to investigate the effects of ATRA on the prevention of EF in post-laminectomy rats. In vitro, the anti-fibrotic effect of ATRA was demonstrated with cultured fibroblasts count, which comprised of those that were cultured with/without ATRA. In vivo, rats underwent laminectomy at the L1-L2 levels. We first demonstrated the beneficial effects using 0.05% ATRA compared to vehicle (control group). We found that a higher concentration of ATRA (0.1%) achieved dose-dependent results. Hydroxyproline content, Rydell score, vimentin-positive cell density, fibroblast density, inflammatory cell density and inflammatory factor expression levels all suggested better outcomes in the 0.1% ATRA rats compared to the other three groups. Presumably, these effects involved ATRA's ability to suppress transforming growth factor (TGF-β1) and interleukin (IL)-6 which was confirmed with reverse-transcriptase polymerase chain reaction (RT-PCR). Finally we demonstrated that ATRA down-regulated nuclear factor (NF)-κB by immunohistochemistry and western blotting for p65 and inhibition of κB (IκBα), respectively. Our findings indicate that topical application of ATRA can inhibit fibroblast proliferation, decrease TGF-β1 and IL-6 expression level, and prevent epidural scar adhesion in rats. The highest concentration employed in this study (0.1%) was the most effective. ATRA suppressed EF through down-regulating NF-κB signaling, whose specific mechanism is suppression of IκB phosphorylation and proteolytic degradation.

Skeletal cryptococcosis, an aspect of disseminated cryptococcal disease or isolated skeletal cryptococcal infection, is a rare but treatable disease. However, limited information is available regarding its clinical features, treatment, and prognosis. This systematic review examined all cases published between April 1977 and May 2013 with regard to the factors associated with this disease, including patient sex, age, and epidemiological history; affected sites; clinical symptoms; underlying diseases; laboratory tests; radiological manifestations; and delays in diagnosis, treatment, follow-up assessments, and outcomes. We found that immune abnormality is a risk factor but does not predict mortality; these observations are due to recent Cryptococcus neoformans var gattii (CNVG) outbreaks (Chaturvedi and Chaturvedi, 2011). Dissemination was irrespective of immune status and required combination therapy, and dissemination carried a worse prognosis. Therefore, a database of skeletal cryptococcosis cases should be created.

To compare the related clinical outcomes of total disc replacement (TDR) versus fusion in management of lumbar degenerative disc disease (LDDD)and provide available basis for choice of surgical procedure.Computer systematically researched PubMed,EMBase, COCHRANE Library, CBMWin, CNKI, VIP databases for randomized controlled trials comparing TDR and fusion for LDDD. Data were searched until October 2013. The available statistical data was extracted after methodological assessment. The statistical soft RevMan 5.1 was used to analyze the results.Total 1 658 cases of patients in 6 studies were conducted, including 543 cases of fusion and 1 115 cases of TDR. The results of Meta-analysis showed that TDR was superior to fusion in term of visual analogue scale (VAS) (OR = -3.33, 95%CI:-5.94--0.71, P = 0.01) , Oswestry disability index (ODI) (OR = -5.21, 95%CI:-7.51--2.92, P = 0.00) , complication (OR = 0.45, 95%CI:0.21-0.95, P = 0.04) . There were no statistically difference regarding operating time, blood loss and reoperation (P0.05). However, there was no difference in term of complication in two-year and five-year sub-analysis.Regardless TDR may be more effective comparable to lumbar fusion at the immediate postoperative time, vigorous evidence is still requisite to certify the result in long-term follow-up.