Downregulation of miR-221-3p by LncRNA TUG1 Promoting the Healing of Closed Tibial Fractures in Mice.

Objective: To probe into the effect of LncRNA TUG1 on the healing of closed tibial fracture in mice. Methods: The closed tibial fracture model of mice was established, selecting the mouse osteoblast line MC3T3-E1, with the cells separated into four groups. The expression levels of TUG1 and miR-221-3p were determined by RT-qPCR analysis, with the targeting relationship between TUG1 and miR-221-3p authenticated by dual luciferase reporter (DLR) assay, detection of cell migration (CM) ability based on Transwell cell migration (TCM) assay, and cell proliferation (CP) acquired by cell counting kit-8 (CCK-8). Results: Prediction results of the target gene by bioinformatics software showed that miR-221-3p had binding sites with the 3'-UTR of TUG1, and DLR assay authenticated the targeting relationship between LncRNA TUG1 and miR-221-3p. Downregulation of TUG1 inhibited osteoblast CP and CM and promoted osteoblast cell apoptosis (CA). Cell cycle analysis indicated that miR-221-3p provoked cell cycle arrest in G1 stage of MC3T3-E1 cells. The siLncRNA-NC group had higher anticyclin D1 and D3 levels than the siLncRNA TUG1 group, with a lower CA rate in the former, implying that miR-221-3p overexpression inhibited osteoblast CP and CM and LncRNA TUG1 inhibited CA. Downregulation of miR-221-3p partly reversed the retardation out of downregulating TUG1 on osteoblast CP and CM. Bcl-2 level was higher in the LncRNA TUG1 group compared to the siLncRNA TUG1 and miR-221-3p overexpression groups, with remarkably lower SDF-1 level in the miR-221-3p overexpression group than those in the control, miRNA-NC, and LncRNA TUG1 groups. Conclusion: The downregulation of miR-221-3p by LncRNA TUG1 can promote the healing of closed tibial fractures in mice.

LncRNA SNHG1 Delayed Fracture Healing via Modulating miR-181a-5p/PTEN Axis.

Background:The purpose of this study was to investigate the role of Long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in delayed healing of tibial fractures and its potential regulatory mechanisms.Methods: 51 patients with normal healing of tibial fractures and 46 patients with delayed healing of tibial fractures were enrolled. RT-qPCR was performed to analyze SNHG1, microRNA (miRNA)-181a-5p, and PTEN levels. ROC curves were used to detect the predictive value of SNHG1 for delayed healing in fracture patients. Subsequently, the regulation of osteogenic markers by SNHG1 and miR-181a-5p was analyzed in MC3T3-E1. Cell proliferation and apoptosis were quantified by CCK-8 and flow cytometry. The binding between SNHG1/miR-181a-5p/PTEN was detected by dual-luciferase reporter gene assay.Results: Serum SNHG1 and PTEN expression were upregulated and miR-181a-5p expression was downregulated in patients with delayed fracture healing. SNHG1 decreased the level of osteogenic markers in MC3T3-E1, inhibited the proliferation, and stimulated apoptosis of MC3T3-E1 (P < 0.05). SNHG1 acted as a sponge for miR-181a-5p, and elevation of miR-181a-5p abolished the inhibition of osteogenic differentiation and promotion of apoptosis by SNHG1 (P < 0.05). PTEN was identified as a target of miR-181a-5p and involved in this regulatory process. Finally, elevated SNHG1 was a feasible predictive biomarker in patients with delayed fracture healing.Conclusion: The current study revealed that SNHG1/miR-181a-5p/PTEN axis inhibited osteoblast differentiation and proliferation and promoted apoptosis, thus leading to the inhibition of tibial fracture healing. Our findings contribute to the understanding of the mechanisms underlying delayed tibial fracture healing.

Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

Automated reanalysis, a novel way to diagnose an ultra-rare condition: Fibronectin-1-related spondylometaphyseal dysplasia (SMD-FN1).

We report a further case of spondylometaphyseal dysplasia - corner fracture type due to the fibronectin-1 gene (SMD-FN1) in a child originally thought to have metaphyseal chondrodysplasia-Brussels type (MCD Brussels). We highlight phenotypic differences with the SMD-FN1 published reports. This case is unique in terms of the method of molecular confirmation. Findings from the 100 000 Genomes Project were originally negative (in both tier 1 and 2); however, subsequent reanalysis, initiated by an automated search for new gene-disease associations in PanelApp, highlighted a candidate diagnostic variant. Our child had short stature, facial dysmorphism, spondylometaphyseal dysplasia and corner fractures and a heterozygous de novo missense variant in FN1 (c.675C>G p.(Cys225Trp), which was likely pathogenic. The variant matched the clinical and radiological features and a diagnosis of SMD-FN1 was confirmed. We explore the diagnostic journey of this patient, compare her findings with the previous 15 patients reported with SMD-FN1 and discuss the diagnostic utility of automated reanalysis. We consider differences and similarities between MCD Brussels and SMD-FN1, by reviewing literature on both conditions and assess whether they are in fact the same disorder.

Expression of miR-182 in patients with fracture of tibial plateau and its regulative effects on the fracture healing.

OBJECTIVE: To investigate the expression of miR-182 in patients with fracture of tibial plateau (FTP) and its effects on osteoblasts and fracture healing. PATIENTS AND METHODS: The patients with fracture of tibial plateau treated in our hospital and healthy subjects who received physical examination from January 2017 to January 2018 were collected. The expression of miR-182 in the serum was detected. The osteoblasts from SD rats were cultured and transfected with miR-182, anti-miR-182, miR-NC or anti-miR-NC using transfection reagent LipofectamineTM 2000. The proliferation, alkaline phosphatase (ALP) activity, calcification and osteogenic gene expression of osteoblasts were detected. The rat models with fracture of tibial plateau were divided into control group, fracture group, fracture+miR-182 group, and fracture+anti-miR-182 group. The levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor ß (TGFß) in serum were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the controls, the expression of miR-182 in serum was significantly elevated in patients with fracture of tibial plateau. Overexpression of miR-182 inhibited the proliferation of osteoblasts, while the knockdown of miR-182 increased the proliferation. MiR-182 could decrease the ALP activity of osteoblasts, while anti-miR-182 increased the ALP activity. Osteoblast calcification ability was significantly decreased by overexpression of miR-182. Knockdown of miR-182 increased the calcification ability of osteoblasts and the expression of osteogenic genes. MiR-182 could inhibit the expression of osteogenic genes. The levels of VEGF, EGF and TGFß in the fracture group were higher than those in the control group, while the levels in the fracture+miR-182 group were higher than those in the fracture group. The levels of VEGF, EGF and TGFß in the anti-miR-182 group were lower than those in the fracture group. CONCLUSIONS: MiR-182 is elevated in patients with fracture of tibial plateau, which can inhibit the proliferation and differentiation of osteoblasts and affect the fracture healing. The knockdown of miR-182 might be a new method for treating fracture healing.

Experimental study of expression profile and specific role of human microRNAs in regulating atrophic bone nonunion.

The expression profile and specific roles of microRNAs (miRNAs) in regulation of atrophic bone nonunion are not fully understood. Here, we present evidence that miRNAs are involved in regulation of several osteogenic genes and may contribute to the development of atrophic bone nonunion.The miRNA expression profile of repairing tissues in atrophic bone nonunion patients (group A) and in callus tissues from patients with healed fractures (group B) were quantitatively measured. microRNA microarrays were used to identify differentially expressed miRNAs, and the bioinformatics methods were used to predict the potential target genes. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase reporter assay were performed in human bone marrow stromal cells (hBMSCs) to validate the microarray results.Nine miRNAs in group A were up-regulated 1.5 times compared to group B, while the other 9 miRNAs in group A were down-regulated 1.5 times. Several target regions of these miRNAs were identified in the osteogenic genes, as well as in the other genes in their families or related regulatory factors. Four miRNAs (hsa-miR-149, hsa-miR-221, hsa-miR-628-3p, and hsa-miR-654-5p) could play important roles in regulating bone nonunion development. hBMSCs transfected with these miRNAs significantly decreased mRNA levels of alkaline phosphatase, liver/bone/kidney (ALPL), platelet derived growth factor subunit A (PDGFA), and bone morphogenetic protein 2 (BMP2). Lower protein expression levels were observed using western blotting, confirming that ALPL, PDGFA, and BMP2 were directly targeted by hsa-miR-149, hsa-miR-221, and hsa-miR-654-5p, respectively.In summary, hsa-miR-149, hsa-miR-221, and hsa-miR-654-5p may play important biological roles by repressing osteogenic target genes ALPL, PDGFA, and BMP2, and, therefore, contributing to progression of atrophic bone nonunion.

Ablation of Ephrin B2 in Col2 Expressing Cells Delays Fracture Repair.

Ephrin B2 is critical for endochondral bone development. In this study, we investigated its role in fracture repair by deleting ephrin B2 in type II collagen (Col.2) expressing cells. We used a nonstable tibia fracture model to evaluate fracture repair at 3 sites: intramembranous bone formation, endochondral bone formation, and intramedullary bone formation. We observed that during fracture repair, deletion of ephrin B2 impaired periosteal stem cell activation, inhibited their proliferation, decreased their survival, and blocked their differentiation into osteoblasts and chondrocytes. In addition, deletion of ephrin B2 decreased vascular endothelial growth factor production as well as vascular invasion into the fracture site. These changes led to reduced cartilage to bone conversion in the callus with decreased new bone formation, resulting in impaired fracture repair. Our data indicate that ephrin B2 in Col2-expressing cells is a critical regulator of fracture repair, pointing to a new and potentially targetable mechanism to enhance fracture repair.

PTH promotes rabbit tibial fracture healing via the Notch signaling pathway.

OBJECTIVE: To explore the effect of parathyroid hormone (PTH) on the expression of Jagged1 in the rabbit tibial fracture healing, and its function and mechanism in this process via the Notch signaling pathway. MATERIALS AND METHODS: A total of 60 New Zealand white rabbits were randomly divided into control group (n=30) and experimental group (n=30). Then, a rabbit tibial fracture model was established. After surgery, the rabbits in experimental group were given 10 µg/kg PTH (1-34) once a day for 5 days a week, while those in control group were given an equal volume of normal saline. Six rabbits were randomly selected from each group at 1, 2, 3, 4, and 6 weeks after surgery to collect right tibia specimens. Next, X-ray examination, bone mineral density (BMD) test, histological detection, and serum biochemical test were performed. Additionally, the messenger ribonucleic acid (mRNA) expression levels of Notch1 and Jagged1 in the Notch signaling pathway were measured via polymerase chain reaction (PCR) assay. Their protein levels were detected through Western blotting analysis. RESULTS: The healing and BMD in experimental group were better than those in control group since cortical and medullary bridging was observed in the rabbits of experimental group at the 6th week after surgery. Plasma level of alkaline phosphatase (ALP), P content, and the product of Ca and P significantly increased (p<0.05) in experimental group. The pathological morphology of the calluses stained with hematoxylin-eosin (HE) in experimental group was overtly superior to that in control group. The PCR results revealed that both mRNA and protein levels of Notch1 and Jagged1 were lower in control group than those in experimental group (p<0.05). CONCLUSIONS: PTH (1-34) promotes the rabbit tibial fracture healing by regulating Jagged1 ligand molecules in the Notch signaling pathway.

Low-expression of lncRNA-ANCR promotes tibial fracture healing via targeting RUNX2.

OBJECTIVE: To explore the role of long non-coding ribonucleic acid-anti-differentiation non-coding ribonucleic acid (lncRNA-ANCR) in tibial fracture healing in rabbits by regulating the runt-related transcription factor 2 (RUNX2) expression. MATERIALS AND METHODS: A total of 60 healthy adult rabbits were evenly divided into Control group (n=20), Fracture group (n=20), and Lnc group (n=20). Then, RUNX2 transfection, Real Time Polymerase Chain Reaction (PCR) assay and relevant instruments were carried out and used to determine the differences in dry weight, bone mineral density, bone mechanical strength, and RUNX2 expression in tibiae among three groups of rabbits. RESULTS: Comparison of the bone mineral density in rabbit tibiae among the three groups showed that the bone mineral density was significantly lower in Fracture group than that in Control group (p<0.05), and it was slightly higher in Lnc group than in Fracture group (p<0.05). The dry weight of the full-length tibiae in Fracture group was significantly decreased compared with that in Control group (p<0.05), and Lnc group had an increased dry weight of tibiae in comparison with Fracture group (p<0.05). The maximum load, flexural strength, elastic stress, elastic strain, elastic modulus, maximum stress, and maximum strain in Fracture group were lower than those in both Control group and Lnc group (p<0.05). Compared with those in Fracture group, the amount of new collagen was overtly increased in Lnc group, and that of mature collagen was decreased (p<0.05). The relative expression level of RUNX2 in tibial bone tissues was evidently lower in Fracture group than that in Control group (p<0.05), and it was markedly higher in Lnc group than that in Fracture group (p<0.05). CONCLUSIONS: Down-regulating lncRNA-ANCR activates and triggers the expression of RUNX2 that facilitates the growth and metabolism of bone tissues to play an important role in the repair of bone tissues and promote the healing of the tibial fracture.

MiR-140-3p overexpression activates the Wnt signaling pathway to promote fracture healing.

OBJECTIVE: To study the mechanism of micro ribonucleic acid (miR)-140-3p participating in the regulation of fracture healing in rats. MATERIALS AND METHODS: A total of 50 male Sprague-Dawley (SD) rats were randomly divided into five groups, namely, group A [phosphate-buffered saline (PBS)] (n=10), group B (miR-140-3p mimics) (n=10), group C [ mimics negative control (NC)] (n=10), group D [antisense oligonucleotide (ASO)-miR-140-3p] (n=10), and group E (ASO NC) (n=10). A rat model of fracture was established on all the rats through the operation. From the successful establishment of the model, the rats in group A were intraperitoneally injected with 50 µL PBS (2 nmol) once a week for 6 weeks, and those in group B, C, D, and E were injected with equivalent volume of miR-140-3p mimics, mimics NC, ASO-miR-140-3p, and ASO NC, respectively, once a week since the successful establishment of model for 6 weeks. The fracture healing in the rats was evaluated via imaging. Meanwhile, Real Time-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression of miR-140-3p in the five groups. Wnt and ß-catenin expressions in the five groups were detected by means of Western blotting (WB). Alkaline phosphatase (ALP) and its quantized statistical value in the five groups were detected through immunohistochemical staining. RESULTS: The expression of miR-140-3p was stimulated in miR-140-3p mimics group and inhibited in ASO-miR-140-3p group. The detection of the miR-140-3p expression level in the five groups via RT-PCR showed that miR-140-3p mimics group had a remarkably higher miR-140-3p expression than the other four groups. The differences were statistically significant (p<0.05). The WB assay verified that the Wnt and ß-catenin expressions in miR-140-3p mimics group were notably higher than those in control groups, and there were statistically significant differences (p<0.05). Compared with those in the groups injected with PBS, ASO miR-140-3p, mimics NC, and ASO NC, there were evidently more callus tissues, better healed and more blurred fracture lines, as well as no translocation and looseness of internal fixation, in the group injected with miR-140-3p mimics, suggesting that the stimulation of the miR-140-3p expression promotes the fracture healing in the rats. The results of immunohistochemical staining indicated that the number of ALP-positive osteoblasts in the rats in miR-140-3p mimics group was increased markedly in comparison with that in the remaining groups (p<0.05), implying that the differentiation of osteoblasts in the rats was affected in miR-140-3p mimics group. CONCLUSIONS: The overexpressed miR-140-3p in the rats with fracture can promote fracture healing by activating the Wnt signaling pathway.

LncRNA KCNQ1OT1 delayed fracture healing through the Wnt/ß-catenin pathway.

OBJECTIVE: The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) on fracture healing and its possible mechanism. PATIENTS AND METHODS: Abnormal lncRNAs were compared between patients with delayed fracture healing and those with normal fracture healing using gene expression profiling method. LncRNA expression in patients was verified by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Subsequently, the model of tibial fracture was successfully established in rabbits. The effect of lncRNA KCNQ1OT1 expression on tibial fracture healing in rabbits was explored. Meanwhile, the effects of lncRNA KCNQ1OT1 on cell proliferation and apoptosis were investigated by knockdown and overexpression experiments with HC-a as a cell model. Furthermore, Western blotting was used to explore the expressions of proteins in signaling pathway affected by lncRNA KCNQ1OT1. RESULTS: Gene expression profiling and qRT-PCR revealed that lncRNA KCNQ1OT1 was significantly down-regulated in bone tissues of patients with delayed fracture healing. Compared with the control group, knocking down lncRNA KCNQ1OT1 remarkably reduced the serum levels of alkaline phosphatase (ALP) and osteoprotegerin (OPG) in rabbits, and markedly decreased bone trabecular growth index (p<0.05). In HC-a cells, overexpression of lncRNA KCNQ1OT1 activated the Wnt/ß-catenin signaling pathway, which could be suppressed by knocking down lncRNA KCNQ1OT1. Cell Counting Kit-8 (CCK-8) assay and 5(6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) results manifested that lncRNA KCNQ1OT1 remarkably promoted the proliferation and inhibited apoptosis of HC-a cells by activating the Wnt/ß-catenin signaling pathway. CONCLUSIONS: LncRNA KCNQ1OT1 plays a vital role in delayed fracture healing. Moreover, it can induce cell proliferation and inhibit cell apoptosis by activating the Wnt/ß-catenin signaling pathway. Therefore, KCNQ1OT1 may be used as a biomarker to predict the occurrence of delayed fracture healing.

HMGB1 promotes bone fracture healing through activation of ERK signaling pathway in a rat tibial fracture model.

This work was to investigate potential roles of HMGB1-mediated ERK pathway in the healing process of bone fracture. Rat tibial fracture models were established and divided into control (rats with normal saline), HMGB1 (rats with HMGB1), and HMGB1+ PD98059 groups (rats with HMGB1 and 1 mg/kg of ERK1/2 inhibitor PD98059) with 30 rats per each. The healing of rats' fracture was observed by X-ray films, the morphological changes of bone fractures by HE staining, the callus formation by micro-CT and biomechanical test, and the expression of osteogenesis-related genes, HMGB1 and ERK-related proteins by qRT-PCR and Western blot. Rats in the HMGB1 group was increased in X-ray scores, peak torque, torsional stiffness, and the bone volume fraction (bone volume/total volume, BV/TV); meanwhile, those rats presented elevations in osteogenesis-related genes and HMGB1 expressions, as well as p-ERK/ERK ratio. However, rats in the HMGB1+ PD98059 group was significantly reduced in X-ray score, peak torque, torsional stiffness, and BV/TV, as well as the expression of osteogenesis-related genes and the ratio of p-ERK/ERK, as compared to those from HMGB1 group. HMGB1 could promote the expressions of osteogenesis-related genes and accelerate the healing process of fracture via activation of the ERK signaling pathway.

Silencing long non-coding RNA MEG3 accelerates tibia fraction healing by regulating the Wnt/ß-catenin signalling pathway.

As fracture healing is related to gene expression, fracture healing is prospected to be implicated in long non-coding RNAs (lncRNAs). This study focuses on the effects of epigenetic silencing of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) on fracture healing by regulating the Wnt/ß-catenin signalling pathway. Genes expressed in fracture were screened using bioinformatics and the subcellular location of MEG3 was determined using FISH. Next, we successfully established tibia fracture (TF) models of C57BL/6J and Col2a1-ICAT mice and the effect of silencing lncRNA MEG3 on fracture healing was detected after TF mice were treated with phosphate buffer saline (PBS), MEG3 siRNA and scramble siRNA. X-ray imaging, Safranin-O/fast green and haematoxylin-eosin (HE) staining and histomorphometrical and biomechanical analysis were adopted to observe and to detect the fracture healing conditions. Additionally, the positive expression of collagen II and osteocalcin was examined using immunohistochemistry. At last, in the in vitro experiment, the relationship of MEG3 and the Wnt/ß-catenin signalling pathway in fraction healing was investigated. MEG3 was located in the cell nucleus. In addition, it was found that MEG3 and the Wnt/ß-catenin signalling pathway were associated with fraction healing. Moreover, silencing MEG3 was proved to elevate callus area and maximum bending load and to furthermore enhance the recanalization of bone marrow cavity. Finally, MEG3 knockdown elevated levels of Col10a1, Runx2, Osterix, Osteocalcin, Wnt10b and ß-catenin/ß-catenin whereas it reduced p-GSK-3ß/GSK-3ß levels. Taken together, our data supported that epigenetic silencing of lncRNA MEG3 could promote the tibia fracture healing by activating the Wnt/ß-catenin signalling pathway.

Effector memory regulatory T cells were most effective at suppressing RANKL but their frequency was downregulated in tibial fracture patients with delayed union.

Delayed union and nonunion occur in a minor subject of bone fractures, presenting ongoing challenges to treatment. RANKL, which promotes the differentiation of bone-resorbing osteoclasts, is thought to negatively impact bone healing. In this study, we recruited patients with isolated closed tibial fracture, who were later categorized into normal healing and delayed healing groups based on their healing progression. The regulatory T cell (Treg) compartment was then investigated in each patient. Based on CD45RA and CD62L expression, we distinguished circulating Treg cells into CD45RA+CD62L+ naive (N), CD45RA-CD62L+ central memory (CM), and CD45RA-CD62L- effector memory (EM) subsets. Compared to normal patients, delayed patients presented significantly lower EM Treg proportion and significantly higher N Treg proportion. Among the N, EM, and CM Treg cells, the EM Treg cells were the most potent at suppressing RANKL expression in T conventional (Tconv) cells. This functionality of EM Treg cells was present in both normal healing patients and delayed healing patients, and was dependent on IL-10, as neutralization of IL-10 resulted in significantly elevated RANKL expression. EM Treg cells presented the highest IL-10 and TGF-ß expression directly ex vivo, as well as after anti-CD3/anti-CD28/IL-2 stimulation. CM Treg cells did not present high expression of inhibitory cytokines ex vivo, but was capable of upregulating cytokine expression upon stimulation. N Treg cells, on the other hand, presented limited capacity to upregulate inhibitory cytokines. In summary, our study identified that, while the EM Treg cells were the most effective at suppressing RANKL, they in delayed union patients were present at lower frequencies with functional impairment, resulting in decreased RANKL suppression. Hence, bone-resorbing osteoclast formation may be favored in these patients thus suggesting a possible mechanism for delayed bone healing.

Jagged1 promotes mineralization in human bone-derived cells.

OBJECTIVES: The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. METHODS: Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. RESULTS: From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. CONCLUSION: Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

STEEL participates in fracture healing through upregulating angiogenesis-related genes by recruiting PARP 1.

OBJECTIVE: To explore the effect of STEEL on fracture healing and its underlying mechanism. PATIENTS AND METHODS: A total of 31 patients with long bone fracture and who received reoperation because of bone nonunion, delayed union or healing disorder in the Wuxi Nine Hospital Affiliated to Soochow University from July 2016 to February 2018 were selected. The bone callus at the fracture site was collected from each patient during the reoperation. QRT-PCR (Quantitative Real-Time Polymerase Chain Reaction) was used to detect STEEL expression in the callus tissues of the treatment group (bone nonunion or delayed union) and the control group. In addition, we measured the number of blood vessels in the fracture tissues by immunohistochemistry. After the construction of tibial fracture model in mice, STEEL expression and the total number of blood vessels in the treatment group (sawing treatment) and the control group (sham operation) were detected, respectively. For in vitro experiments, CCK-8 (cell counting kit-8) assay was performed to detect cell proliferation after knockdown or overexpression of STEEL in the vascular endothelial cells. The binding condition of STEEL and its interacting proteins were detected by RIP (RNA binding protein immunoprecipitation), and the binding of PARP 1 [poly (ADP-ribose) polymerase 1] with gene promoter was observed by ChIP (chromatin immunoprecipitation assay). Western blot was used to detect the expression level of VEGF (vascular endothelial growth factor). RESULTS: STEEL expression and the vascular density in the callus tissues of the treatment group were significantly lower than those of the control group. Downregulated STEEL remarkably decreased the proliferation ability of HUVEC cells. Meanwhile, the vascular density was also significantly decreased in mice with a tibial fracture. Overexpressed STEEL obtained the opposite results. STEEL could interact with PARP 1 to regulate expressions of downstream genes. Moreover, STEEL could also promote angiogenesis by elevating VEGF expression. CONCLUSIONS: We showed that STEEL expression could partly represent the angiogenesis of fracture sites. Moreover, it promoted angiogenesis by elevating VEGF expression.

FGFR3 deficient mice have accelerated fracture repair.

Bone fracture healing is processed through multiple biological stages that partly recapitulates the skeletal development process. FGFR3 is a negative regulator of chondrogenesis during embryonic stage and plays an important role in both chondrogenesis and osteogenesis. We have investigated the role of FGFR3 in fracture healing using unstabilized fracture model and found that gain-of-function mutation of FGFR3 inhibits the initiation of chondrogenesis during cartilage callus formation. Here, we created closed, stabilized proximal tibia fractures with an intramedullary pin in Fgfr3-/-mice and their littermate wild-type mice. Fracture healing was evaluated by radiography, micro-CT, histology, and real-time polymerase chain reaction (RT-PCR) analysis. The fractured Fgfr3-/- mice had increased formation of cartilaginous callus, more fracture callus, and more rapid endochondral ossification in fracture sites with up-regulated expressions of chondrogenesis related gene. The fractures of Fgfr3-/- mice healed faster with accelerated fracture callus mineralization and up-regulated expression of osteoblastogenic genes. The healing of fractures in Fgfr3-/- mice was accelerated in the stage of formation of cartilage and endochondral ossification. Downregulation of FGFR3 activity can be considered as a potential bio-therapeutic strategy for fracture treatment.

Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies.

Spondylometaphyseal dysplasia (SMD) corner fracture type (also known as SMD "Sutcliffe" type, MIM 184255) is a rare skeletal dysplasia that presents with mild to moderate short stature, developmental coxa vara, mild platyspondyly, corner fracture-like lesions, and metaphyseal abnormalities with sparing of the epiphyses. The molecular basis for this disorder has yet to be clarified. We describe two patients with SMD corner fracture type and heterozygous pathogenic variants in COL2A1. These two cases together with a third case of SMD corner fracture type with a heterozygous COL2A1 pathogenic variant previously described suggest that this disorder overlaps with type II collagenopathies. The finding of one of the pathogenic variants in a previously reported case of spondyloepimetaphyseal dysplasia (SEMD) Strudwick type and the significant clinical similarity suggest an overlap between SMD corner fracture and SEMD Strudwick types. © 2016 Wiley Periodicals, Inc.

Orthopedic surgery modulates neuropeptides and BDNF expression at the spinal and hippocampal levels.

Pain is a critical component hindering recovery and regaining of function after surgery, particularly in the elderly. Understanding the role of pain signaling after surgery may lead to novel interventions for common complications such as delirium and postoperative cognitive dysfunction. Using a model of tibial fracture with intramedullary pinning in male mice, associated with cognitive deficits, we characterized the effects on the primary somatosensory system. Here we show that tibial fracture with pinning triggers cold allodynia and up-regulates nerve injury and inflammatory markers in dorsal root ganglia (DRGs) and spinal cord up to 2 wk after intervention. At 72 h after surgery, there is an increase in activating transcription factor 3 (ATF3), the neuropeptides galanin and neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), as well as neuroinflammatory markers including ionized calcium-binding adaptor molecule 1 (Iba1), glial fibrillary acidic protein (GFAP), and the fractalkine receptor CX3CR1 in DRGs. Using an established model of complete transection of the sciatic nerve for comparison, we observed similar but more pronounced changes in these markers. However, protein levels of BDNF remained elevated for a longer period after fracture. In the hippocampus, BDNF protein levels were increased, yet there were no changes in Bdnf mRNA in the parent granule cell bodies. Further, c-Fos was down-regulated in the hippocampus, together with a reduction in neurogenesis in the subgranular zone. Taken together, our results suggest that attenuated BDNF release and signaling in the dentate gyrus may account for cognitive and mental deficits sometimes observed after surgery.