Time course changes to structural,mechanical and material properties of bone in rats after complete spinal cord injury

Objective:Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats.Methods:8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy.Results:The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone.Conclusion:Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.     

兰州市1907例不同年龄正常人群骨密度测定综合分析

目的：了解兰州地区正常人群骨密度的变化特点,分析其变化规律,为预防和治疗骨质疏松症提供科学依据。方法：使用天津圣鸿公司SHY-I数字式骨密度测定仪对兰州地区1907人进行检测,其中男1381例,女526例,分别做左前臂尺、桡骨测量。年龄20～85岁,每10岁为一年龄组进行统计分析。结果：男、女组骨密度峰值均在30-39岁,峰值后随年龄增加而骨密度下降,女性下降较男性显著。骨量减少及骨质疏松患病率在40岁后随年龄增长而增高,女性高于男性。老年人骨量减少及骨质疏松患病率高于中青年人,老年女性骨质疏松患病率与老年男性比较有明显差异（P〈0．05）。结论：兰州地区健康人群骨密度随年龄变化,并与性别有关。骨密度的检测在骨质疏松症的早期预防和治疗中具有重要意义。     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

熊果酸对酒精性骨质疏松大鼠骨形成、骨矿化的影响

目的：探讨熊果酸对酒精所致骨质疏松大鼠骨形成、骨矿化的影响。方法：雄性Wistar大鼠60只,按体重随机分为空白对照 组、熊果酸对照组、模型组、熊果酸低、中、高剂量组,同时分别给予生理盐水、150 mg/kg 熊果酸、50%酒精,50 mg/kg 熊果酸,100 mg/kg 熊果酸,150 mg/kg 熊果酸灌胃。熊果酸对照组生理盐水剂量同空白组,熊果酸低、中、高剂量组酒精剂量同模型组。灌胃共 持续8 周。磷钼酸法检测血清磷(P)含量,比色法检测血清钙(Ca)含量,酶联免疫吸附（ELISA）法检测血清骨钙素(BGP)、骨形成蛋 白-2(BMP-2)浓度;HE 染色法观察股骨结构的病理学变化。结果：与空白对照组相比较,模型组血清BGP、BMP-2 和Ca、P 均明显 降低,且有统计学差异(P < 0.05),但熊果酸对照与空白对照组各项指标结果相近。熊果酸中、高剂量组大鼠血清BGP、Ca 和P 水 平均较模型组有显著升高,差异具有统计学意义(P < 0.05),但仅熊果酸高剂量组血清BMP-2 显著升高(P < 0.05)。股骨组织HE 染色结果显示,空白对照组骨小梁致密、规则且较粗,粗细均匀;模型组骨小梁稀松、不规则、粗细不均匀,甚至可见骨小梁断裂; 熊果酸中、高剂量组骨小梁致密、规则、较厚、粗细均匀,未见骨小梁断裂。结论：熊果酸能够促进酒精性骨质疏松大鼠的骨形成, 抑制骨矿物质的流失,在改善酒精致骨质疏松方面有一定的保护作用。     

Study of selected genes of Wnt signaling pathway in relation to the parameters in the bone tissue of the laying hens

The Wnt signaling pathway plays a critical role in almost all aspects of skeletal development and homeostasis. Many studies suggest the importance of this signaling pathway in connection with bone metabolism through many skeletal disorders caused by mutations in Wnt signaling genes. The knowledge gained through targeting this pathway is of great value for skeletal health and diseases, for example of increased bone mass in the case of osteoporosis. Our objective was to focus on the detection of single nucleotide polymorphisms and investigate the associations between possible polymorphisms in selected genes that are part of those signaling pathways and parameters of bones in hens of ISA Brown hybrids (bone breaking strength, length, width, and bone mass). Different regions of the GPR177, ESR1 and RUNX2 genes were studied, using PCR and sequencing, in a total of forty-eight samples for each marker. Thirteen polymorphisms have been discovered in selected regions of studied genes, whereas these polymorphisms were only within the GPR177 gene. Eight of these polymorphisms were synonymous and five were in the intron. The tested regions of the ESR1 and RUNX2 genes were monomorphic. The only statistically significant difference was found within the GPR177 gene (exon 2) and the bone length parameter, in the c.443 + 86G > A polymorphism. However, this polymorphism was found in the intron, and no other one was found within the selected regions to show associations with the observed bone parameters.     

血清Irisin、SOST、H2S与膝骨关节炎合并骨质疏松症患者骨密度、骨代谢标志物的相关性及其预测价值

摘要 目的：探讨血清鸢尾素（Irisin）、骨硬化蛋白（SOST）、硫化氢（H₂S）与膝骨关节炎（KOA）合并骨质疏松症（OP）患者骨密度、骨代谢标志物的相关性,分析Irisin、SOST、H₂S预测KOA合并OP的价值。方法：选取2020年4月至2023年4月我院收治的179例KOA患者,根据是否合并OP将其分为OP组（68例）和非OP组（111例）。检测血清Irisin、SOST、H₂S和骨代谢标志物骨钙素（OC）、骨碱性磷酸酶（BALP）、Ⅰ型原胶原N-端前肽（PINP）、抗酒石酸酸性磷酸酶异体（TRACP5b）水平,股骨颈、腰椎L1～4骨密度。Pearson分析血清Irisin、SOST、H₂S与股骨颈、腰椎L1～4骨密度和血清骨代谢标志物的相关性,受试者工作特征（ROC）曲线分析血清Irisin、SOST、H₂S对KOA合并OP的预测价值。结果：OP组血清Irisin、H₂S、OC、BALP、PINP水平,股骨颈、腰椎L1～4骨密度低于非OP组（P＜0.05）,TRACP、SOST水平高于非OP组（P＜0.05）。OP组血清Irisin、H₂S水平与股骨颈、腰椎L1～4骨密度,血清OC、BALP、PINP水平呈正相关（P＜0.05）,与血清TRACP水平呈负相关（P＜0.05）;SOST水平与股骨颈、腰椎L1～4骨密度,血清OC、BALP、PINP水平呈负相关（P＜0.05）,与血清TRACP水平呈正相关（P＜0.05）。Irisin、SOST、H₂S预测KOA合并OP的曲线下面积为0.784、0.773、0.755,联合预测KOA合并OP的曲线下面积为0.908,高于单独预测。结论：KOA合并OP患者血清Irisin、H₂S水平降低、SOST水平增高,低水平Irisin、H₂S和高水平SOST与骨密度降低、骨代谢异常有关,可用于预测KOA合并OP患者骨代谢异常状态和骨质流失风险。     

Wnt通路中蓬松蛋白DNA甲基化对骨质疏松患者骨髓间充质干细胞成骨分化影响

摘要 目的：探讨蓬松蛋白（DVL）DNA甲基化对骨质疏松（OP）患者骨髓间充质干细胞（BMSCs）成骨分化及Wnt通路的影响。方法：分离、培养OP患者的BMSCs,成骨诱导培养BMSCs 0、7、14、21天,观察BMSCs细胞形态变化,检测碱性磷酸酶（ALP）染色及活性,检测茜素红染色及钙结节形成,荧光定量聚合酶链式反应（RT-PCR）及免疫印迹检测远端缺失同源盒5（Dlx5）、核心结合蛋白因子2（Runx2）、成骨细胞特异性转录因子（OSX）、Ⅰ型胶原蛋白（Colla Ⅰ）表达。检测DVL1、Wnt、糖原合成酶激酶3（GSK3）、β连环蛋白（β-catenin）表达及DVL DNA甲基化水平。在成骨诱导培养基中加入甲基转移酶抑制剂5-Aza,将BMSCs分为对照组（Control组）、甲基转移酶抑制剂组（5-Aza组）、甲基转移酶抑制剂+si-NC组（5-Aza+si-NC组）、甲基转移酶抑制剂+si-Wnt组（5-Aza+si-Wnt组）,依次进行ALP活性测定,茜素红染色及钙结节形成测定。RT-PCR检测Dlx5、Runx2、OSX、Colla 1水平,免疫印迹检测Dlx5、Runx2、OSX、Colla Ⅰ、DVL1、Wnt、GSK3、β-catenin的蛋白表达量,并检测DVL DNA甲基化水平。结果：成骨诱导后BMSCs具有强的ALP活性和矿化结节生成能力,且随着培养时间的增长,BMSCs细胞ALP活性和矿化结节生成能力增强,Dlx5、Runx2、OSX、Colla Ⅰ mRNA水平和Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza组较Control组ALP染色加深,活性增强,钙结节形成增多（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza+si-Wnt组较5-Aza+si-NC组ALP染色变浅,活性降低,钙结节形成减少（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达降低,DVL DNA甲基化水平升高（P＜0.05）。结论：DVL DNA甲基化可以通过抑制Wnt/β-catenin信号通路抑制OP患者BMSCs成骨分化。     

2型糖尿病合并骨质疏松症患者外周血炎性因子表达与骨代谢、骨密度丢失的关系

摘要 目的：探讨2型糖尿病合并骨质疏松症患者外周血中炎性因子NLR、IL-1β、TNF-α与骨代谢指标骨密度、活性维生素D、PTH、ALP及生化指标FPG、2hPG、HbA1c、空腹C肽、BMI、血脂、血尿酸之间的关系,为该类患者的诊断和治疗提供参考。方法：选取2020年9月至2021年9月在本院内分泌科就诊的2型糖尿病合并骨质疏松症患者80例作为观察组,同时选取同期健康体检者80例作为对照组。比较两组患者的一般资料,采用ELISA法检测两组患者外周血中NLR、IL-1β、TNF-α的水平,采用双能X线吸收法（DEXA）检测两组患者的骨密度,采用放射免疫法检测两组患者外周血中活性维生素D、PTH的水平,采用生化分析仪检测两组患者外周血中ALP、FPG、2hPG、HbA1c、空腹C肽、BMI、血脂、血尿酸的水平。结果：观察组患者外周血中NLR、IL-1β、TNF-α的水平均高于对照组（P<0.05）,观察组患者骨密度低于对照组（P<0.05）,观察组患者外周血中活性维生素D水平低于对照组（P<0.05）,观察组患者外周血中PTH水平高于对照组（P<0.05）,观察组患者外周血中ALP水平高于对照组（P<0.05）,观察组患者外周血中FPG、2hPG、HbA1c水平高于对照组（P<0.05）,观察组患者空腹C肽水平低于对照组（P<0.05）,观察组患者BMI高于对照组（P<0.05）,观察组患者血脂异常率高于对照组（P<0.05）,观察组患者血尿酸水平高于对照组（P<0.05）。观察组患者外周血中NLR、IL-1β、TNF-α与骨密度呈负相关（P<0.05）,与活性维生素D呈负相关（P<0.05）,与PTH、ALP呈正相关（P<0.05）,与FPG、2hPG、HbA1c、空腹C肽、BMI、血脂、血尿酸无显著相关性（P>0.05）。结论：2型糖尿病合并骨质疏松症患者外周血中炎性因子NLR、IL-1β、TNF-α水平升高,可能与骨代谢紊乱和骨密度丢失有关,提示该类患者存在慢性低度炎症状态,应加强对其骨代谢和骨密度的监测和干预,以降低骨折风险和改善生活质量。     

Pharmacological studies of the large-scaled purified genistein from Huaijiao (Sophora japonica – Leguminosae) on anti-osteoporosis

In this report, we used genistein that was extracted from a Chinese herbal medicine Huaijiao (Sophora japonica – Leguminosae) to evulate its pharmacological function on anti-osteoporosis. This genistein is purified in a large-scale production from Huaijiao by a state-of-art method as described by Tian et al. [2004. The preparation of genistein and LC-MS/MS on-line analysis. Drug Devel. Res. 61, 6–12]. Chemical structure of the isolated genistein was examined by using various techniques including nuclear magnetic resonant spectrum, infrared absorption spectrum, ultraviolet absorption spectrum and mass spectrum, and was proved to be identical to those purified from soybean in a small scale as previously reported. We randomly divided female SD rats into 6 groups, including control, ovariectomized model, Nilestriol-treated, and three level of dosages of genistein-treated. We evaluated the pharmacological effects of genistein against osteoporosis by measuring the bone density of femur and bone mineral group including calcium, phosphorous, and magnesium. The consequences of genistein treatment on bone histology and morphology were also determined by measuring the trabcular area, thickness and number. Our results indicated that treatment with a 4.5 or 9 mg/kg dosage of genistein could also prevent osteoporosis significantly at the 4th week after treatment. In comparison with the anti-osteoporosis effects of soybean genistein, the genistein extracted from Huaijiao has the same beneficial effect on anti-osteoporosis.