MicroRNA 98-5p Overexpression Contributes to Delayed Fracture Healing via Targeting BMP-2.

MicroRNAs (miRNAs) are related to the regulation of bone metabolism. Delayed fracture healing (DFH) is a common complication after fracture surgery. The study attempted to examine the role of miR-98-5p and bone morphogenetic protein (BMP)-2 with the onset of DFH. A total of 140 patients with femoral neck fracture were recruited, including 80 cases with normal fracture healing (NFH) and 60 cases with DFH. MC3T3-E1 cells were induced cell differentiation for cell function experiments. Real-time quantitative polymerase chain reaction (RT-qPCR) was carried out to test mRNA levels. Cell proliferation and apoptosis were determined via CCK-8 and flow cytometry assay. Luciferase reporter assay was done to verify the targeted regulatory relationship of miR-98-5p with BMP-2. In comparison with NFH cases, DFH patients owned high levels of serum miR-98-5p and low concentration of BMP-2, and the levels of the two indexes are significantly negatively correlated. Both miR-98-5p and BMP-2 had the ability to predict DFH, while their combined diagnostic value is the highest. BMP-2 was demonstrated to be the target gene of miR-98-5p. Overexpression of BMP-2 reversed the role of miR-98-5p in MC3T3-E1 cell proliferation, apoptosis and differentiation. Increased miR-98-5p and decreased BMP-2 serve as potential biomarkers for the diagnosis of DFH. MiR-98-5p overexpression inhibits osteoblast proliferation and differentiation via targeting BMP-2.

Cell-associated type I collagen in nondegenerate and degenerate human articular cartilage.

Chondrocytes with abnormal morphology are present in nondegenerate human cartilage suggesting dedifferentiation to a fibroblastic phenotype and production of a mechanically-weakened matrix of unknown composition. We determined the relationship between in situ chondrocyte morphology, chondrocyte clusters, and levels of cell-associated collagen type I. Chondrocyte morphology in fresh femoral head cartilage from 19 patients with femoral neck fracture and collagen type I labelling was identified with Cell TrackerTM fluorescence and immunofluorescence, respectively, in axial/coronal orientations using confocal microscopy with images analysed by ImarisTM . In axial images of grade 0 cartilage, 87 ± 8% were normal chondrocytes with a small (10 ± 6%) abnormal population possessing ≥1 cytoplasmic process. More normal chondrocytes (78 ± 11%) were collagen type I negative than those labelling positively (p < 0.001). For abnormal chondrocytes, 81 ± 14% labelled negatively for collagen type I compared to those labelling positively (19 ± 3%; p = 0.007; N(n)=11(3)). Overall, approximately 9% of the cells in normal cartilage labelled for collagen type I. With degeneration, the percentage of normal chondrocytes decreased (p < 0.001) but increased for abnormal cells (p = 0.036) and clusters (p = 0.003). A larger percentage of normal, abnormal and clustered chondrocytes now demonstrated collagen type I labelling (p = 0.004; p = 0.009; p = 0.001 respectively). Coronal images exhibited increased (p = 0.001) collagen type I labelling in the superficial zone of mildly degenerate cartilage with none in the mid or deep zones. These results show that collagen type I was identified around normal and abnormal chondrocytes in nondegenerate cartilage, which increased with degeneration. This suggested the presence of mechanically weak fibro-cartilaginous repair tissue in otherwise macroscopically nondegenerate human cartilage which progressed with degeneration as occurs in osteoarthritis.

Effect of monthly intravenous ibandronate injections on vertebral or non-vertebral fracture risk in Japanese patients with high-risk osteoporosis in the MOVER study.

We examined the efficacy of intravenous (IV) ibandronate 1 mg/month in patient subgroups in the phase III MOVER study. Here we present results of analyses on the incidence of fractures in patients with prevalent vertebral fractures (1 or ≥2, and ≥3) at screening and femoral neck (FN) bone mineral density (BMD) T scores ≥-2.5 or <-2.5, and <-3.0 at baseline. The per-protocol set comprised 1134 patients (ibandronate 0.5 mg/month n = 376; ibandronate 1 mg/month n = 382; risedronate oral 2.5 mg/day n = 376). The incidence of vertebral fractures in patients with 1 or ≥2 prevalent vertebral fractures was 11.2 and 20.4 %, respectively, with ibandronate 1 mg/month, and 12.6 and 22.1 %, respectively, with risedronate. In patients with FN BMD T scores ≥-2.5 or <-2.5, the vertebral fracture incidence was 13.7 and 16.4 %, respectively, with ibandronate 1 mg/month, and 17.3 and 19.1 %, respectively, with risedronate. The incidence of non-vertebral fractures in patients with ≥2 prevalent vertebral fractures or FN BMD T score <-2.5 was 7.6 and 7.6 %, respectively, with ibandronate 1 mg/month, and 9.5 and 9.4 %, respectively, with risedronate. Fracture incidence was consistently lower, but not significant, with ibandronate 1 mg/month than with risedronate in patients with ≥2 prevalent vertebral fractures and FN BMD T score <-2.5. The efficacy of the fracture reduction of monthly IV ibandronate appears consistent and seemingly independent of the number of prevalent vertebral fractures or baseline BMD values.

Hypoxia-Induced MicroRNA-429 Promotes Differentiation of MC3T3-E1 Osteoblastic Cells by Mediating ZFPM2 Expression.

BACKGROUND/AIMS: Hypoxia has been reported to regulate osteoblastic differentiation of bone cells and cartilage development. However, information concerning the molecular mechanisms remains largely unknown. METHODS: The expression of miR-429 was evaluated by quantitative real-time PCR analysis. To test whether miR-429 directly regulate the expression level of ZFPM2 at transcription level, dual-luciferase reporter gene assay was performed. Western blotting was performed to detect osteogenesis related protein expression. The cell proliferation, apoptosis, alkaline phosphatase activity and matrix mineralization were performed to assess the functions of miR-429 in vitro and in vivo the effects of miR-429 on fracture healing. RESULTS: Expression of miR-429 was increased in MC3T3-E1 cells treated with 200 µM CoCl2 by qRT-PCR, and overexpression of miR-429 promoted cell differentiation, and enhanced alkaline phosphatase activity and matrix mineralization. Luciferase reporter assays suggested that miR-429 directly targets the 3'UTR of ZFPM2. In addition, knockdown of ZFPM2 could phenocopy the effects of miR-429 expression. Furthermore, overexpression of ZFPM2 in miR-429-expressing MC3T3-E1 cells suppressed cell differentiation. CONCLUSIONS: Our results provide valuable insight into the potential role of hypoxia in regulation of osteoblastic cell differentiation.

Fibroblast Growth Factor-23, Sclerostin, and Bone Microarchitecture in Patients With Osteoporotic Fractures of the Proximal Femur: A Cross-sectional Study.

This cross-sectional observational cohort study was designed to simultaneously investigate bone microarchitecture and serum markers of bone metabolism in elderly osteoporotic patients experiencing a trochanteric or femoral neck fracture. Special emphasis was put on renal function, sclerostin and fibroblast growth factor-23 (FGF-23). Eighty-two patients (median age: 84 years; 49 trochanteric fractures) scheduled for emergency surgery due to an osteoporotic fracture participated. Bone specimens for ex vivo microcomputed X-ray tomography were sampled during surgery. Blood samples for laboratory workup were collected before surgery (t0) and 1 day afterward (t1). Fifty-eight patients consented to dual-energy X-ray absorptiometry scanning of the lumbar spine and/or contralateral femoral neck after recovery during the in-patient stay. Samples were grouped according to the site of fracture. Regression coefficients were controlled for age and/or estimated glomerular filtration rate (eGFR), if appropriate. Patients experiencing a femoral neck fracture presented with better preserved renal function (eGFR) and lower C-terminal fragment of fibroblast growth factor-23 (cFGF-23) concentrations compared to those with trochanteric fractures. By contrast, serum sclerostin was similar at both time points and did not differ between groups. Age-adjusted correlation analysis revealed negative associations between eGFR and cFGF-23 determined at t1 (R=-0.34; p<0.05) as well as between eGFR and sclerostin levels at t0 (R=-0.45; p<0.05) in patients with trochanteric and femoral neck fractures, respectively. Our study provides evidence that not only an age-related decline of renal function but also the type of skeletal injury may contribute to the circulating concentrations of cFGF-23.

Profiling microRNA expression during fracture healing.

BACKGROUND: The discovery of microRNA (miRNA) has revealed a novel type of regulatory control for gene expression. Increasing evidence suggests that miRNA regulates chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. We hypothesized that the functions of certain miRNAs and changes to their expression pattern may play crucial roles during the process of fracture healing. METHODS: Standard healing fractures and unhealing fractures produced by periosteal cauterization at the fracture site were created in femurs of seventy rats, with half assigned to the standard healing fracture group and half assigned to the nonunion group. At post-fracture days 3, 7, 10, 14, 21, and 28, total RNA including miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture day 14. For further analysis, we selected highly up-regulated five miRNAs in the standard healing fracture group from the microarray data. Real-time PCR was performed with miRNA samples at each time point above mentioned to compare the expression levels of the selected miRNAs between standard healing fractures and unhealing fractures and investigate their time-course changes. RESULTS: Microarray and real-time polymerase chain reaction (PCR) analyses on day 14 revealed that five miRNAs, miR-140-3p, miR-140-5p, miR-181a-5p, miR-181d-5p, and miR-451a, were significantly highly expressed in standard healing fractures compared with unhealing fractures. Real-time PCR analysis further revealed that in standard healing fractures, the expression of all five of these miRNAs peaked on day 14 and declined thereafter. CONCLUSION: Our results suggest that the five miRNAs identified using microarray and real-time PCR analyses may play important roles during fracture healing. These findings provide valuable information to further understand the molecular mechanism of fracture healing and may lead to the development of miRNA-based tissue engineering strategies to promote fracture healing.

Matrix metalloproteinase 13 expression in response to double-stranded RNA in human chondrocytes.

OBJECTIVE: To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA). METHODS: Differential expression of PRRs was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively. RESULTS: The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid-inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain-like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation-associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C). CONCLUSION: Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.

Bone marrow levels of 25 hydroxy vitamin D are not depressed in cases of hip fracture compared with controls.

There is little information on tissue as distinct from plasma levels of vitamin D metabolites in cases of hip fracture compared with controls. Femoral neck fractures in the elderly are associated with increased cortical remodelling and endosteal resorption, leading to regional increases in porosity and reduced cortical thickness. Vitamin D metabolites play a central role in the maintenance of normal serum calcium levels and may, through interactions with parathyroid hormone, exert an important influence on bone structure. To investigate whether hip fracture might be associated with tissue vitamin D deficiency, we have measured by radioimmunoassay the levels of 25 hydroxy vitamin D (25 (OH)D) in bone marrow samples extracted from the proximal femurs of 16 female subjects who had suffered fracture (mean age = 82.1 years, standard error (se) 1.9) and nine sex matched post mortem controls (mean age = 83.8 years, se 2.5). Twenty five (OH)D concentrations were significantly greater in the fracture cases (median = 3.7, IQR = 2.5-3.9 ng/g) than in the control group (median = 1.5, IQR = 0.9-2.3 ng/g; P = 0.0007, non-parametric Wilcoxon/Kruskal-Wallis test). It was suggested in the 1970s that bone loss and hip fracture risk in the UK were driven by vitamin D deficiency. Our results suggest that the alterations in femoral neck bone microstructure and remodelling in hip fracture cannot be assigned to the single cause of relative deficiency of vitamin D. Vitamin D deficiency or insufficiency may nevertheless increase remodelling and loss of bone tissue and contribute causally to a minority of hip fractures.

Integrated proteomics and metabolomics analysis of sclerosis-related proteins and femoral head necrosis following internal fixation of femoral neck fractures.

Femoral head necrosis (FHN) is a serious complication after femoral neck fractures (FNF), often linked to sclerosis around screw paths. Our study aimed to uncover the proteomic and metabolomic underpinnings of FHN and sclerosis using integrated proteomics and metabolomics analyses. We identified differentially expressed proteins (DEPs) and metabolites (DEMs) among three groups: patients with FNF (Group A), sclerosis (Group B), and FHN (Group C). Using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses, we examined the roles of these proteins and metabolites. Our findings highlight the significant differences across the groups, with 218 DEPs and 44 DEMs identified between the sclerosis and FNF groups, 247 DEPs and 31 DEMs between the FHN and sclerosis groups, and a stark 682 DEPs and 94 DEMs between the FHN and FNF groups. Activities related to carbonate dehydratase and hydrolase were similar in the FHN and sclerosis groups, whereas extracellular region and lysosome were prevalent in the FHN and FNF groups. Our study also emphasized the involvement of the PI3K-Akt pathway in sclerosis and FHN. Moreover, the key metabolic pathways were implicated in glycerophospholipid metabolism and retrograde endocannabinoid signaling. Using western blotting, we confirmed the pivotal role of specific genes/proteins such as ITGB5, TNXB, CA II, and CA III in sclerosis and acid phosphatase 5 and cathepsin K in FHN. This comprehensive analyses elucidates the molecular mechanisms behind sclerosis and FHN and suggests potential biomarkers and therapeutic targets, paving the way for improved treatment strategies. Further validation of the findings is necessary to strengthen the robustness and reliability of the results.

Is cartilage sGAG content related to early changes in cartilage disease? Implications for interpretation of dGEMRIC.

OBJECTIVE: This study investigates sulphated glycosaminoglycans (sGAG) content changes in early osteoarthritis (OA), and whether contrast-enhanced magnetic resonance imaging (MRI) of cartilage in vitro may identify early event of OA pathology. METHOD: Osteochondral plugs from patients with hip OA or femoral neck fracture (reference group) were collected and analysed by 1.5 T MRI with ΔR1 as a measure of cartilage contrast concentration. Cartilage hydration, contents of sGAG, cartilage oligomeric matrix protein (COMP), hydroxyproline, denatured collagen, and aggrecan TEGE(392) neoepitope were determined and histological grading was performed. RESULTS: sGAG content correlated to ΔR1, although no difference in either of these parameters was detectable between OA and reference cartilage at 4 h of contrast equilibration. In contrast, biochemical analysis of other cartilage matrix constituents showed distinct alterations typical for early cartilage degradation in OA cartilage and with clear evidence for increased aggrecan turnover. CONCLUSION: In the present in vitro study, cartilage sGAG content could not distinguish between early OA cartilage and reference cartilage. Given, that delayed gadolinium enhanced MRI of cartilage (dGEMRIC) indicates early events in the pathogenesis of OA in vivo, our results from the in vitro studies imply other, additional factors than cartilage sGAG content, e.g., alterations in diffusion or increased supply of contrast agent in the diseased joint. Alternatively, an altered dGEMRIC reflects later stages of OA, when sGAG content decreases. Further investigations are warranted, to understand variations in sGAG content in pathology, an essential background for interpreting dGEMRIC measurements.

Human bone material characterization: integrated imaging surface investigation of male fragility fractures.

UNLABELLED: The interrelation of calcium and phosphorus was evaluated as a function of bone material quality in femoral heads from male fragility fracture patients via surface analytical imaging as well as scanning microscopy techniques. A link between fragility fractures and increased calcium to phosphorus ratio was observed despite normal mineralization density distribution. INTRODUCTION: Bone fragility in men has been recently recognized as a public health issue, but little attention has been devoted to bone material quality and the possible efficacy in fracture risk prevention. Clinical routine fracture risk estimations do not consider the quality of the mineralized matrix and the critical role played by the different chemical components that are present. This study uses a combination of different imaging and analytical techniques to gain insights into both the spatial distribution and the relationship of phosphorus and calcium in bone. METHODS: X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry imaging techniques were used to investigate the relationship between calcium and phosphorus in un-embedded human femoral head specimens from fragility fracture patients and non-fracture age-matched controls. The inclusion of the bone mineral density distribution via backscattered scanning electron microscopy provides information about the mineralization status between the groups. RESULTS: A link between fragility fracture and increased calcium and decreased phosphorus in the femoral head was observed despite normal mineralization density distribution. Results exhibited significantly increased calcium to phosphorus ratio in the fragility fracture group, whereas the non-fracture control group ratio was in agreement with the literature value of 1.66 M ratio in mature bone. CONCLUSIONS: Our results highlight the potential importance of the relationship between calcium and phosphorus, especially in areas of new bone formation, when estimating fracture risk of the femoral head. The determination of calcium and phosphorus fractions in bone mineral density measurements may hold the key to better fracture risk assessment as well as more targeted therapies.

Low-level chronic exposure to cadmium enhances the risk of long bone fractures: a study on a female rat model of human lifetime exposure.

In the present paper, the hypothesis that low chronic exposure to cadmium (Cd) enhances the risk of long bone fractures was investigated in a female rat model simulating human lifetime exposure in non-Cd-polluted areas. For this purpose, the femur and both tibias of control female rats and those exposed to Cd (1 mg Cd I(-1) in drinking water for 24 months since weaning) were assigned to geometric, densitometric (bone mineral content, BMC, and density, BMD), radiographic and biomechanical studies as well as assessing their chemical composition. The exposure to Cd disturbed mineralization (decreased BMD and minerals content, including calcium, magnesium, zinc, copper and iron) and weakened the biomechanical strength of the femur and tibia, enhancing their fragility. The Z-score values for the BMD revealed osteopenia of the femur and tibia in 20 and 30% of the Cd-exposed female rats, respectively, and osteoporosis in 80 and 70%, respectively. In 30% of the Cd-exposed animals, femoral neck fracture was evident in the radiographic picture. The findings seem to confirm the hypothesis that a low exposure to Cd during the lifetime may be an important risk factor for osteoporosis and fractures of long bones, and especially for femoral neck fracture in elderly women. The results indicate that greater attention should be paid to Cd as an environmental risk factor for the increasing rate of osteoporosis and bone fractures in old population.

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis.

OBJECTIVE: Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). METHODS: Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. RESULTS: Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. CONCLUSION: Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

Comparison of femoral morphology and bone mineral density between femoral neck fractures and trochanteric fractures.

BACKGROUND: Many studies that analyzed bone mineral density (BMD) and skeletal factors of hip fractures were based on uncalibrated radiographs or dual-energy xray absorptiometry (DXA). QUESTIONS/PURPOSES: Spatial accuracy in measuring BMD and morphologic features of the femur with DXA is limited. This study investigated differences in BMD and morphologic features of the femur between two types of hip fractures using quantitative computed tomography (QCT). PATIENTS AND METHODS: Forty patients with hip fractures with normal contralateral hips were selected for this study between 2003 and 2007 (trochanteric fracture, n=18; femoral neck fracture, n=22). Each patient underwent QCT of the bilateral femora using a calibration phantom. Using images of the intact contralateral femur, BMD measurements were made at the point of minimum femoral-neck cross-sectional area, middle of the intertrochanteric region, and center of the femoral head. QCT images also were used to measure morphologic features of the hip, including hip axis length, femoral neck axis length, neck-shaft angle, neck width, head offset, anteversion of the femoral neck, and cortical index at the femoral isthmus. RESULTS: No significant differences were found in trabecular BMD between groups in those three regions. Patients with trochanteric fractures showed a smaller neck shaft angle and smaller cortical index at the femoral canal isthmus compared with patients with femoral neck fractures. CONCLUSIONS: We conclude that severe osteoporosis with thinner cortical bone of the femoral diaphysis is seen more often in patients with trochanteric fracture than in patients with femoral neck fracture. LEVELS OF EVIDENCE: Level IV, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.

The natural trends of C-reactive protein after hip arthroplasty for femoral neck fracture without infection.

ABSTRACT: The aim of this study was to estimate the degree of normalization of C-reactive protein (CRP) at 2-weeks and 4-weeks after hip arthroplasty after femoral neck fracture. We also wished to determine whether the degree of CRP normalization differs after total hip arthroplasty (THA) compared to bipolar hemiarthroplasty (BH). We also wanted to analyze the patient factors that may influence CRP normalization.We conducted a retrospective study of 135 patients who had undergone THA (32 cases) or BH (103 cases) for femoral neck fracture by single surgeon from January 2015 to December 2019. We analyzed CRP levels during the preoperative period, the early postoperative period, the 2-week postoperative period, and the 4-week postoperative period.In THA, CRP was normalized in 4 patients (12.5%) and in 15 patients (46.9%) within 2-weeks and 4-weeks after surgery, respectively. In BH, CRP was normalized in 16 patients (15.5%) and in 52 patients (50.5%) within 2-weeks and 4-weeks after surgery, respectively. There were no statistical differences between THA and BH. Compared to women, men were 3.78 (95% confidence interval, 1.05-13.63) times less likely to have normalized CRP at 2-weeks after surgery (P = .042). Compared to women, men were 3.01 (95% confidence interval, 1.44-6.27) times less likely to have normalized CRP at 4-weeks after surgery (P = .003).Only 50% of patient's CRP level was normalized during 4-week postoperative period. In men, CRP levels were significantly higher than women in whole period. In the case of THA, the CRP level was higher only in early postoperative period compared to BH, and there was no difference since then.

MALAT1 knockdown promoted cell viability and migration of LPS-treated MG-63 cells via sponging miR-212.

BACKGROUND: Most fractures could heal after treatment, around 5-10 % of patients still develop delayed union and nonunion. Evidence has increasingly shown that abnormal expression of long noncoding RNAs is closely related to the occurrence and development of various diseases including fracture healing. However, evidence regarding the effect of MALAT1 on fracture healing remains limited. OBJECTIVES: In this study, we attempt to explore the role of MALAT1 during the process of femoral neck fracture healing and elucidate the underlying mechanism of this disease. METHODS: We first detect the expression of lncRNAs in serums from 3 pairs of patients with delayed femoral neck fracture healing and healthy volunteers using lncRNA microarray. And the expression of long noncoding RNA MALAT1 in serums and LPS-treated MG-63 cells was measured using qRT-PCR. CCK-8 assay, cell migration and qRT-PCR were applied to the role of MALAT1 knockdown in LPS-treated MG-63 cells. ELISA was used for the measurement of inflammatory cytokines in serums of patients and healthy volunteers. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. RESULTS: MALAT1 expression was up-regulated in serum of patients with delayed union of femoral neck fracture. MALAT1 knockdown promoted cell viability and migration, reduced inflammation in LPS-treated MG-63 cells. The bioinformatics analysis showed MALAT1 acts as a molecular sponge for miR-212. And SOX6 was a target of miR-212. Besides, MALAT1 knockdown suppressed SOX6 expression via targeting miR-212 in LPS-treated MG-63 cells. CONCLUSIONS: These data suggest MALAT1 knockdown promoted the biological behavior of LPS-treated MG-63 cells via sponging miR-212, which may provide a new therapeutic avenue for delayed union of femoral neck fracture.

Bone screws of porous silicon carbide coated with tantalum improve osseointegration and osteogenesis in goat femoral neck fractures.

Traditional metal materials, such as stainless steel and titanium (Ti) alloys, are still the gold standards for fracture fixation. However, the elastic moduli of these materials differ from that of human cortical bone, and the stress shielding effect affects fracture healing, leading to secondary fractures. Herein, a new porous Ta coated SiC (pTa-SiC) scaffold using in internal fixation devices with good mechanical and biological properties was prepared based on porous silicon carbide (SiC) scaffold and tantalum (Ta) metal. The osteogenic and osseointegration properties of the pTa-SiC scaffold were investigated by bothin vitroandin vivotests. The results showed that compared with porous titanium (pTi), the pTa-SiC promoted the proliferation, migration, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. Moreover, the internal fixation tests were carried out in a goat load-bearing femoral neck fracture model. Histological results showed good osseointegration around the pTa-SiC screws. And the acid etching results showed that bone cells grew tightly on the pTa-SiC throughout bone canaliculi, and the growth mode was contact osteogenesis, which indicated good biological fixation effects. Therefore, it is reasonable to be expected that the new pTa-SiC scaffold with excellent mechanical and biological properties could be a promising candidate for bone implant field.

HIF/Ca2+/NO/ROS is critical in roxadustat treating bone fracture by stimulating the proliferation and migration of BMSCs.

AIMS: Fracture site is regionally hypoxic resulting from vasculature disruption. HIF-1αplays an essential role in fracture repair. This study aims to investigate the influence of FG4592 on the femur fracture of SD rats and the proliferation, migration of BMSCs. MATERIALS AND METHODS: After the femoral fracture model was established, computed tomography imaging and histological analyses were used to quantify bone healing and the expression of CD90, HIF-1α, VEGF were observed by means of immunohistochemistry method on Day 10 and Day 20. In addition, CCK-8 assay, transwell, flow cytometric analysis, laser confocal microscopy assay, western blot and rT-PCR were performed to text the proliferation and migration of BMSCs using FG4592. KEY FINDINGS: In vivo, FG4592 facilitated the repair of bone fracture by increasing the number of BMSCs and cartilage formation. In vitro, FG4592 markedly improved the proliferation, migration of BMSCs via upregulation of intracellular Ca2+, NO and concomitant decrease of ROS. Gene silencing of HIF-1α resulted in the opposite phenomenon in BMSCs with the treatment of FG4592. SIGNIFICANCE: The transplantation of BMSCs is the most promising candidate for the treatment of fracture non-union. We illustrated that FG4592 promoted the proliferation, migration of BMSCs via the HIF/Ca2+/NO/ROS pathway and further accelerated fracture healing. These results provide a deeper understanding for the mechanism of HIF in promoting fracture healing.

Subchondral insufficiency fracture of the femoral head may be associated with hip dysplasia: a pilot study.

BACKGROUND: Subchondral insufficiency fracture of the femoral head occurs mainly in elderly patients with osteoporosis. Spontaneous resolution is observed after nonoperative treatment in some patients whereas other show progressive joint destruction requiring THA. Several studies report the occurrence of subchondral insufficiency fracture of the femoral head in dysplastic hips. QUESTIONS/PURPOSES: We asked whether the extent of hip dysplasia or osteoporosis was greater in patients with subchondral insufficiency fracture of the femoral head than in normal control subjects. PATIENTS AND METHODS: We compared the clinical and imaging findings of 13 patients with subchondral insufficiency fractures of the femoral head and 12 patients scheduled for TKA with asymptomatic hips. Age, gender, and body mass index were comparable in the two groups. RESULTS: Higher mean Sharp angles, lower acetabular head indices, lower center-edge angles, and higher acetabular roof angles in patients with subchondral insufficiency fracture of the femoral head than in those with asymptomatic hips suggested a greater degree of hip dysplasia. Bone mineral density and serum levels of Type I collagen cross-linked N-telopeptide and bone alkaline phosphatase were similar in the two groups. CONCLUSIONS: We speculate an excessive amount of stress on the acetabular edge from dysplasia may be associated with the occurrence of subchondral insufficiency fracture of the femoral head.

LncRNA HAGLR accelerates femoral neck fracture healing through negatively regulating miRNA-19a-3p.

OBJECTIVE: This study aims to uncover the function of long non-coding RNA (lncRNA) HAGLR in the healing process of femoral neck fracture and the underlying mechanism. PATIENTS AND METHODS: Expression levels of HAGLR, microRNA-19a-3p (miRNA-19a-3p) and TGFBR2 in fractured femoral neck tissues and adjacent normal tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Regulatory effects of HAGLR on viability, apoptosis, migration, and protein levels of BALP and Osteocalcin in MC3T3-E1 cells were determined. Dual-Luciferase reporter gene assay was conducted to assess the binding in HAGLR/miRNA-19a-3p/TGFBR2. In addition, relative levels of TGFBR2, p-smad2, p-smad3, and RUNX2 in MSCs influenced by HAGLR were detected. RESULTS: HAGLR was downregulated in fractured femoral neck tissues. Knockdown of HAGLR reduced viability and migration, enhanced apoptotic rate, as well as downregulated BALP and Osteocalcin in MC3T3-E1 cells. HAGLR served as miRNA-19a-3p sponge, and miRNA-19a-3p directly targeted 3'-untranslated region (3'-UTR) of TGFBR2. Knockdown of HAGLR downregulated expressions of TGFBR2, p-smad2, p-smad3, and RUNX2 in MC3T3-E1 cells, indicating the inhibited TGF-ß pathway. CONCLUSIONS: LncRNA HAGLR/miRNA-19a-3p/TGFBR2 regulatory loop accelerates the healing process of femoral neck fracture by inhibiting the TGF-ß pathway.