The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.

Bone fracture is one of the most common injuries. Despite the high regenerative capacity of bones, failure of healing still occurs to near 10% of the patients. Herein, we aim to investigate the modulatory role of neurofibromatosis type I gene (NF1) to osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and new bone formation after fracture in a rat model. We studied the NF1 gene expression in normal and non-union bone fracture models. Then, we evaluated how NF1 overexpression modulated osteogenic differentiation of BMSCs, autophagy activity, mTORC1 signalling and osteoclastic bone resorption by qRT-PCR, Western blot and immunostaining assays. Finally, we injected lentivirus-NF1 (Lv-NF1) to rat non-union bone fracture model and analysed the bone formation process. The NF1 gene expression was significantly down-regulated in non-union bone fracture group, indicating NF1 is critical in bone healing process. In the NF1 overexpressing BMSCs, autophagy activity and osteogenic differentiation were significantly enhanced. Meanwhile, the NF1 overexpression inhibited mTORC1 signalling and osteoclastic bone resorption. In rat non-union bone fracture model, the NF1 overexpression significantly promoted bone formation during fracture healing. In summary, we proved the NF1 gene is critical in non-union bone healing, and NF1 overexpression promoted new bone formation after fracture by enhancing autophagy and inhibiting mTORC1 signalling. Our results may provide a novel therapeutic clue of promoting bone fracture healing.

LncRNA ENST00000563492 promoting the osteogenesis-angiogenesis coupling process in bone mesenchymal stem cells (BMSCs) by functions as a ceRNA for miR-205-5p.

Pain, physical dysfunction, and mental disorders caused by bone nonunion bring great burden to patients. Bone mesenchymal stem cells (BMSCs) isolated from bone nonunion patients with poor proliferation and osteogenic ability are compared with that from normal bone-healing patients. Long noncoding RNAs (lncRNAs) are a class of RNAs that are more than 200 nucleotides in length, lack an open-reading frame encoding a protein, and have little or no protein-coding function, and could regulate gene expression, which is involved in the regulation of important life activities, such as growth, development, aging, and death at epigenetic, transcriptional, and post-transcriptional levels. In this study, we intended to investigate the difference of lncRNA expression between patients with nonunion and normal fracture healing. Our result found that lncRNA ENST00000563492 was downregulated in bone nonunion tissues. LncRNA ENST00000563492 promotes osteogenic differentiation of BMSCs through upregulating the expression of CDH11. On the other hand, LncRNA ENST0000563492 could improve the osteogenesis-angiogenesis coupling process through enhancing the expression of VEGF during osteogenic differentiation of BMSCs. LncRNA ENST00000563492 functions as a ceRNA for miR-205-5p that was targeting CDH11 and VEGF. LncRNA ENST00000563492 could promote the osteogenesis of BMSCs in vivo. Our result indicated that lncRNA ENST00000563492 may be a new target for bone nonunion.

NSM00158 Specifically Disrupts the CtBP2-p300 Interaction to Reverse CtBP2-Mediated Transrepression and Prevent the Occurrence of Nonunion.

Carboxyl-terminal binding proteins (CtBPs) are transcription regulators that control gene expression in multiple cellular processes. Our recent findings indicated that overexpression of CtBP2 caused the repression of multiple bone development and differentiation genes, resulting in atrophic nonunion. Therefore, disrupting the CtBP2-associated transcriptional complex with small molecules may be an effective strategy to prevent nonunion. In the present study, we developed an in vitro screening system in yeast cells to identify small molecules capable of disrupting the CtBP2-p300 interaction. Herein, we focus our studies on revealing the in vitro and in vivo effects of a small molecule NSM00158, which showed the strongest inhibition of the CtBP2-p300 interaction in vitro. Our results indicated that NSM00158 could specifically disrupt CtBP2 function and cause the disassociation of the CtBP2-p300-Runx2 complex. The impairment of this complex led to failed binding of Runx2 to its downstream targets, causing their upregulation. Using a mouse fracture model, we evaluated the in vivo effect of NSM00158 on preventing nonunion. Consistent with the in vitro results, the NSM00158 treatment resulted in the upregulation of Runx2 downstream targets. Importantly, we found that the administration of NSM00158 could prevent the occurrence of nonunion. Our results suggest that NSM00158 represents a new potential compound to prevent the occurrence of nonunion by disrupting CtBP2 function and impairing the assembly of the CtBP2-p300-Runx2 transcriptional complex.

Defective Proliferation and Osteogenic Potential with Altered Immunoregulatory phenotype of Native Bone marrow-Multipotential Stromal Cells in Atrophic Fracture Non-Union.

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing.

MiR-26a promotes fracture healing of nonunion rats possibly by targeting SOSTDC1 and further activating Wnt/ß-catenin signaling pathway.

Nonunion is a serious complication after fracture due to its difficulty of self-healing. MicroRNA-26a (miR-26a) has been known to play a crucial role in bone metabolism. In this study, we established a rat nonunion model by removing periosteum, and found that miR-26a was significantly upregulated. Osteogenic differentiation of mesenchymal stem cells (MSCs) isolated from bone marrow transfected with miR-26a mimics was significantly enhanced, evidenced by increased calcium deposition and expression levels of alkaline phosphatase (ALP) and osteocalcin. Bioinformatics analysis suggested that sclerostin domain-containing 1 (SOSTDC1) may be a target of miR-26a, which was confirmed by dual-luciferase assay and western blot. Besides, miR-26a was used for nonunion rats. Delightfully, radiographs of nonunion rats with miR-26a mimics administration showed obvious new bone formation compared with nonhealing control. Hematoxylin-eosin and Masson staining assays revealed that osteogenesis capacity was greatly enhanced by miR-26a mimics' administration. In addition, miR-26a mimics could promote osteogenic differentiation in nonunion rats, evidenced by increased protein levels of ALP and osteocalcin, while SOSTDC1 was suppressed. The injection of miR-26a mimics also gave rise to phosphorylation of GSK3ß and nuclear accumulation of ß-catenin, which indicated the activation of canonical Wnt/ß-catenin signaling. In conclusion, we demonstrated that miR-26a promoted fracture healing of rats with nonunion in vivo and osteogenic differentiation of MSCs in vitro, possibly by targeting SOSTDC1, and that Wnt/ß-catenin signaling pathway was involved in this process.

miR-381 modulates human bone mesenchymal stromal cells (BMSCs) osteogenesis via suppressing Wnt signaling pathway during atrophic nonunion development.

The osteogenic differentiation of human bone mesenchymal stromal cells (BMSCs) has been considered as a central issue in fracture healing. Wnt signaling could promote BMSC osteogenic differentiation through inhibiting PPARγ. During atrophic nonunion, Wnt signaling-related factors, WNT5A and FZD3 proteins, were significantly reduced, along with downregulation of Runx2, ALP, and Collagen I and upregulation of PPARγ. Here, we performed a microarray analysis to identify differentially expressed miRNAs in atrophic nonunion tissues that were associated with Wnt signaling through targeting related factors. Of upregulated miRNAs, miR-381 overexpression could significantly inhibit the osteogenic differentiation in primary human BMSCs while increase in PPARγ protein level. Through binding to the 3'UTR of WNT5A and FZD3, miR-381 modulated the osteogenic differentiation via regulating ß-catenin nucleus translocation. Moreover, PPARγ, an essential transcription factor inhibiting osteogenic differentiation, could bind to the promoter region of miR-381 to activate its expression. Taken together, PPARγ-induced miR-381 upregulation inhibits the osteogenic differentiation in human BMSCs through miR-381 downstream targets, WNT5A and FZD3, and ß-catenin nucleus translocation in Wnt signaling. The in vivo study also proved that inhibition of miR-381 promoted the fracture healing. Our finding may provide a novel direction for atrophic nonunion treatment.

Knockdown of MiR-140-5 promotes osteogenesis of adipose-derived mesenchymal stem cells by targeting TLR4 and BMP2 and promoting fracture healing in the atrophic nonunion rat model.

OBJECTIVE: This study aims to assess the effect and mechanism of genetically modified adipose-derived mesenchymal stem cells (ASCs) with recombinant lentiviruses mediated knockdown of miR-140-5p in ASCs' osteogenesis in vitro and atrophic nonunion rat model. MATERIALS AND METHODS: This study included 36 male adult Sprague-Dawley (SD) rats weighing 400 g to 450 g from the experimental animal facility of our university. Approval was obtained from the University Animal Care Committee before the study. Rats' ASCs were prepared and genetically modified with lentivirus (Lv)-empty (NC) or Lv-miR-140-5p-TuD (inhibitors). After that, the expressions of RUNX2 and osteocalcin (OCN) were detected in the ASCs. To confirm the mechanisms of miR-140-5p in ASCs, we predicted the target genes by bioinformatics analysis and then the target genes were verified by luciferase reporting assay. The artificial atrophic nonunion was created in the rat's femoral bone. Animals were randomly divided into three groups according to the material implanted into bone defects space: AT scaffolds (AT group, n=12), AT scaffold with Lv-NC modified (AT+ASCs+Lv-NC group, n=12), AT scaffold with the Lv-miR-140-5p-TuD modified ASCs (AT+ASCs+Lv-miR-140-5p-TuD group, n=12). After four weeks, the rats were euthanized for the following radiographic examination, histologic study and biomechanical testing. RESULTS: MiR-140-5p was down-regulated during osteogenic differentiation of ASCs, and inhibition of MiR-140-5p promoted osteogenesis of ASCs in vitro. Inhibition of MiR-140-5p promoted osteogenesis of ASCs and enhanced fracture in the atrophic nonunion rat model: AT+ASCs+Lv-NC group, AT+ASCs+Lv-miR-140-5p-TuD group resulted in a better bone formation and higher BMD and BMC than AT group, while excellent bone formation and the highest BMD and BMC were observed in AT+ASCs+Lv-miR-140-5p-TuD group. Both AT+ASCs+Lv-NC group and AT+ASCs+Lv-miR-140-5p-TuD group presented more mature characteristics in the micro-architecture than AT group, whereas AT+ASCs+Lv-miR-140-5p-TuD group presented the highest BV/TV, Tb.Th and Tb.N as well as the lowest Tb.Sp. The peak load of the operated femur increased by 94.43% AT+ASCs+Lv-miR-140-5p-TuD group, 50.68% in AT+ASCs+Lv-NC group compared to the control AT group, respectively. The result of luciferase reporting assay showed that miR-140-5p could directly target TLR4 and BMP2. CONCLUSIONS: This study demonstrates that lentiviruses-mediated knockdown of miR-140-5p can significantly promote osteogenesis of ACSs by directly regulating its' target genes, TLR4 and BMP2, and that combined adipose scaffold with genetically modified ASCs can significantly enhance fracture-healing and bone formation in the atrophic nonunion rat model.

Periosteal Mesenchymal Progenitor Dysfunction and Extraskeletally-Derived Fibrosis Contribute to Atrophic Fracture Nonunion.

Atrophic nonunion represents an extremely challenging clinical dilemma for both physicians and fracture patients alike, but its underlying mechanisms are still largely unknown. Here, we established a mouse model that recapitulates clinical atrophic nonunion through the administration of focal radiation to the long bone midshaft 2 weeks before a closed, semistabilized, transverse fracture. Strikingly, fractures in previously irradiated bone showed no bony bridging with a 100% nonunion rate. Radiation triggered distinct repair responses, separated by the fracture line: a less robust callus formation at the proximal side (close to the knee) and bony atrophy at the distal side (close to the ankle) characterized by sustained fibrotic cells and type I collagen-rich matrix. These fibrotic cells, similar to human nonunion samples, lacked osteogenic and chondrogenic differentiation and exhibited impaired blood vessel infiltration. Mechanistically, focal radiation reduced the numbers of periosteal mesenchymal progenitors and blood vessels and blunted injury-induced proliferation of mesenchymal progenitors shortly after fracture, with greater damage particularly at the distal side. In culture, radiation drastically suppressed proliferation of periosteal mesenchymal progenitors. Radiation did not affect hypoxia-induced periosteal cell chondrogenesis but greatly reduced osteogenic differentiation. Lineage tracing using multiple reporter mouse models revealed that mesenchymal progenitors within the bone marrow or along the periosteal bone surface did not contribute to nonunion fibrosis. Therefore, we conclude that atrophic nonunion fractures are caused by severe damage to the periosteal mesenchymal progenitors and are accompanied by an extraskeletal, fibro-cellular response. In addition, we present this radiation-induced periosteal damage model as a new, clinically relevant tool to study the biologic basis of therapies for atrophic nonunion. © 2018 American Society for Bone and Mineral Research.

MiR-133a inhibits fracture healing via targeting RUNX2/BMP2.

OBJECTIVE: To analyze the mechanism of miR-133a in inhibiting fracture healing through regulating runt-related transcription factor 2 (RUNX2) signaling pathway. PATIENTS AND METHODS: A total of 80 patients with fracture admitted to our hospital from January 2016 to January 2017 were divided into 2 groups according to nonunion fracture or healing fracture: nonunion fracture group (n = 40) and control group (n= 40). After admission, patients underwent the surgery, respectively, and the bone tissues were taken for stand-by application. The expression level of bone morphogenetic protein 2 (BMP2) was detected using the immunohistochemical method, the expression level of RUNX2 protein was detected by Western blotting, and the expression level of micro ribonucleic acid (miR)-133a was detected by quantitative polymerase chain reaction (qPCR). Moreover, the bioinformatics method was used to predict the target gene of miR-133a, and the luciferase reporter gene was used to detect the binding of miR-133a to RUNX2. RESULTS: Compared with those in control group, the expression of BMP2 and the relative expressions of RUNX2 protein and miR-133a were significantly decreased; the differences were statistically significant (p < 0.05). Pearson correlation analysis showed that miR-133a was negatively correlated with RUNX2. After overexpression of miR-133a, the expression level of RUNX2 was decreased, but it was increased significantly after interference in miR-133a. Besides, it was found in dual-luciferase reporter assay that miR-133a bound to RUNX2. CONCLUSIONS: MiR-133a inhibits the bone formation through inhibiting the RUNX2/BMP2 signaling pathway, thereby negatively regulating the fracture healing.

Genetic predisposition to fracture non-union: a case control study of a preliminary single nucleotide polymorphisms analysis of the BMP pathway.

BACKGROUND: Despite the known multi-factorial nature of atrophic fracture non-unions, a possible genetic predisposition for the development of this complication after long bone fractures remains unknown. This pilot study aimed to address this issue by performing a preliminary SNP analysis of specific genes known to regulate fracture healing. METHODS: A total of fifteen SNPs within four genes of the Bone Morphogenetic Protein (BMP) pathway (BMP-2, BMP-7, NOGGIN and SMAD6) were examined, in 109 randomly selected patients with long bone fractures as a result of motor vehicle accident, fall or direct blow. There were sixty-two patients with atrophic non-union and forty-seven patients (54 fractures) with uneventful fracture union. Overall SNPs frequencies were computed with respect to patient's age, gender, smoking habits, fracture-associated parameters and the use of nonsteroidal anti-inflammatory drugs (NSAIDs), and tested for their association to the impaired bone healing process, using binary logistic regression (STATA 11.1; StataCorp, Texas USA). RESULTS: Statistical analysis revealed age to be an important covariate in the development of atrophic non-union (p = 0.01, OR 1.05 [per year]), and two specific genotypes (G/G genotype of the rs1372857 SNP, located on NOGGIN and T/T genotype of the rs2053423 SNP, located on SMAD6) to be associated with a greater risk of fracture non-union (p = 0.02, OR 4.56 and p = 0.04, OR 10.27, respectively, after adjustment for age). CONCLUSIONS: This is the first clinical study to investigate the potential existence of genetic susceptibility to fracture non-union. Even though no concrete conclusions can be obtained from this pilot study, our results indicate the existence of a potential genetically predetermined impairment within the BMP signalling cascade, initiated after a fracture and when combined with other risk factors could synergistically increase the susceptibility of a patient to develop non-union. Further research is desirable in order to clarify the genetic component and its role and interaction with other risk factors in the development of atrophic long bone non-union, as simple genetic testing may contribute to the early identification of patients at risk in the future and the on-time intervention at the biologic aspects of bone healing.

Genetic factors responsible for long bone fractures non-union.

INTRODUCTION: Approximately 10-15% of all fractures of long bones heal with delay, prolonged immobilization and repetitive operative interventions. Despite intense investigations, the pathomechanism of impaired healing of skeletal tissue remains unclear. An important role in the pathomechanism of mal-union of close fractures plays subclinically proceeding infections. AIM: The question arises whether colonization and proliferation of bacteria in the fracture gap could be related to the mutation of genes for factors regulating local antimicrobial response, such as pathogen recognizing receptors (PRR), cytokines and chemokines. METHODS: We carried out studies in patients with delayed long bone fractures estimating the frequency of mutation of genes crucial for pathogen recognition (TLR2, TLR4 and CD14), and elimination (CRP, IL-6, IL-1ra), as well as wound healing (TGF-ß). The molecular milieu regulating healing process (IGF-1, COLL1a, TGF-ß, BMP-2, and PDGF) was validated by Western blot analysis of the gap tissue. RESULTS: Microbiological investigations showed the presence of viable bacterial strains in 34 out of 108 gaps in patients with non-healing fractures (31.5%) and in 20 out of 122 patients with uneventful healing (16.4%) (P < 0.05). The occurrence of mutated TLR4 1/W but not 2/W gene was significantly higher (P < 0.05) in the non-healing infected than sterile group. In the non-healing infected group 1/W mutated gene frequency was also higher than in healing infected. In the TGF-ß codon 10 a significantly higher frequency of mutated homozygote T and heterozygote C/T in the non-healing infected versus non-healing sterile subgroup was observed (P < 0.05). Similar difference was observed in the non-healing infected versus healing infected subgroup (P < 0.05). The CRP (G1059C), IL1ra (genotype 2/2), IL-6 (G176C), CD14 (G-159T), TLR2 (G2259A) and TLR4/2 (Thr399Ile) polymorphisms did not play evident role in the delay of fracture healing. CONCLUSIONS: Individuals bearing the mutant TLR 4 gene 1/W (Asp299Gly) and TGF-ß gene codon 10 mutant T and T/C allele may predispose to impaired pathogen recognition and elimination, leading to prolonged pathogen existence in the fracture gaps and healing delays.