Sinomenine increases osteogenesis in mice with ovariectomyinduced bone loss by modulating autophagy

BACKGROUND A decreased autophagic capacity of bone marrow mesenchymal stromal cells(BMSCs)has been suggested to be an important cause of decreased osteogenic differentiation.A pharmacological increase in autophagy of BMSCs is a potential therapeutic option to increase osteoblast viability and ameliorate osteoporosis.AIM To explore the effects of sinomenine(SIN)on the osteogenic differentiation of BMSCs and the underlying mechanisms.METHODS For in vitro experiments,BMSCs were extracted from sham-treated mice and ovariectomized mice,and the levels of autophagy markers and osteogenic differentiation were examined after treatment with the appropriate concen-trations of SIN and the autophagy inhibitor 3-methyladenine.In vivo,the therapeutic effect of SIN was verified by establishing an ovariectomy-induced mouse model and by morphological and histological assays of the mouse femur.RESULTS SIN reduced the levels of AKT and mammalian target of the rapamycin(mTOR)phosphorylation in the phosphatidylinositol 3-kinase(PI3K)/AKT/mTOR signaling pathway,inhibited mTOR activity,and increased autophagy ability of BMSCs,thereby promoting the osteogenic differentiation of BMSCs and effectively alleviating bone loss in ovariectomized mice in vivo.CONCLUSION The Chinese medicine SIN has potential for the treatment of various types of osteoporosis,bone homeostasis disorders,and autophagy-related diseases.

Case Report and Clinical Management of a Case of Osteogenesis Imperfecta Detected in the Prenatal Period

Osteogenesis imperfecta is a hereditary disease characterized by bone fragility due to a defect in type I collagen synthesis. The diagnosis is typically suspected based on suggestive ultrasound findings and confirmed through genetic studies. We present a case of osteogenesis imperfecta suspected during obstetrical ultrasound at 19 weeks’ gestation, which was later confirmed radiographically through computed tomography. Due to the severity of the condition, therapeutic termination of pregnancy was indicated.

Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis

As the major cell precursors in osteogenesis, mesenchymal stem cells(MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers(SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene,ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis.Mechanistically, SEs recruited bromodomain containing 4(BRD4) at the site of ZBTB16, which then bound to RNA polymerase IIassociated protein 2(RPAP2) that transported RNA polymerase Ⅱ(POL Ⅱ) into the nucleus. The subsequent synergistic regulation of POL Ⅱ carboxyterminal domain(CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4^(fl/fl)Prx1-cre mice and osteoporosis(OP) models.Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway:Network Pharmacology Analysis and Experimental Validation

Objective Osteogenesis is vitally important for bone defect repair,and Zuo Gui Wan(ZGW)is a classic prescription in traditional Chinese medicine(TCM)for strengthening bones.However,the specific mechanism by which ZGW regulates osteogenesis is still unclear.The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis.Methods A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells(BMSCs).Results In total,487 no-repeat targets corresponding to the bioactive components of ZGW were screened,and 175 target genes in the intersection of ZGW and osteogenesis were obtained.And 28 core target genes were then obtained from a PPI network analysis.A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress,metal ions,and lipopolysaccharide.Additionally,KEGG pathway enrichment analysis revealed that multiple signaling pathways,including the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)signaling pathway,were associated with ZGW-promoted osteogensis.Further experimental validation showed that ZGW could increase alkaline phosphatase(ALP)activity as well as the mRNA and protein levels of ALP,osteocalcin(OCN),and runt related transcription factor 2(Runx 2).What’s more,Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT,and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002.Finally,the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002.Conclusion ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

Incorporation of Mg-phenolic networks as a protective coating for magnesium alloy to enhance corrosion resistance and osteogenesis in vivo

Magnesium(Mg) and its alloys have been intensively studied to develop the next generation of bone implants recently, but their clinical application is restricted by rapid degradation and unsatisfied osteogenic effect in vivo. A bioactive chemical conversion Mg-phenolic networks complex coating(e EGCG) was stepwise incorporated by epigallocatechin-3-gallate(EGCG) and exogenous Mg^(2+)on Mg-2Zn magnesium alloy. Simplex EGCG induced chemical conversion coating(c EGCG) was set as compare group. The in vitro corrosion behavior of Mg-2Zn alloy, c EGCG and e EGCG was evaluated in SBF using electrochemical(PDP, EIS) and immersion test. The cytocompatibility was investigated with rat bone marrow mesenchymal stem cells(r BMSCs). Furthermore, the in vivo tests using a rabbit model involved micro computed tomography(Micro-CT) analysis, histological observation, and interface analysis. The results showed that the e EGCG is Mgphenolic multilayer coating incorporated Mg-phenolic networks, which is rougher, more compact and much thicker than c EGCG. The e EGCG highly improved the corrosion resistance of Mg-2Zn alloy, combined with its lower average hemolytic ratios, continuous high scavenging effect ability and relatively moderate contact angle features, resulting in a stable and suitable biological environment, obviously promoted r BMSCs adhesion and proliferation. More importantly, Micro-CT, histological and interface elements distribution evaluations all revealed that the e EGCG effectively inhibited degradation and enhanced bone tissue formation of Mg alloy implants. This study puts forward a promising bioactive chemical conversion coating with Mg-phenolic networks for the application of biodegradable orthopedic implants.

Classification of osteogenesis imperfecta:Importance for prophylaxis and genetic counseling

Osteogenesis imperfecta(OI)is a genetically heterogeneous monogenic disease characterized by decreased bone mass,bone fragility,and recurrent fractures.The phenotypic spectrum varies considerably ranging from prenatal fractures with lethal outcomes to mild forms with few fractures and normal stature.The basic mechanism is a collagen-related defect,not only in synthesis but also in folding,processing,bone mineralization,or osteoblast function.In recent years,great progress has been made in identifying new genes and molecular mechanisms underlying OI.In this context,the classification of OI has been revised several times and different types are used.The Sillence classification,based on clinical and radiological characteristics,is currently used as a grading of clinical severity.Based on the metabolic pathway,the functional classification allows identifying regulatory elements and targeting specific therapeutic approaches.Genetic classification has the advantage of identifying the inheritance pattern,an essential element for genetic counseling and prophylaxis.Although genotype-phenotype correlations may sometimes be challenging,genetic diagnosis allows a personalized management strategy,accurate family planning,and pregnancy management decisions including options for mode of delivery,or early antenatal OI treatment.Future research on molecular pathways and pathogenic variants involved could lead to the development of genotype-based therapeutic approaches.This narrative review summarizes our current understanding of genes,molecular mechanisms involved in OI,classifications,and their utility in prophylaxis.

Osteogenesis Imperfecta: One Disease, Two or More Faces: A Case Report

Being such a rare condition in paediatrics, osteogenesis imperfecta (OI) is not a diagnosis which is made often. It is however, a diagnosis necessitating early diagnosis and timeous and effective management to improve morbidity and increase the quality of life for our patients. We report two cases of osteogenesis imperfecta in this case report to highlight the different phenotypic presentations. Both of these patients are unique in their presentations and each case highlights the importance of a high clinical index of suspicion by the practitioner in making the diagnosis of osteogenesis imperfecta. The first case is a patient who was diagnosed with osteogenesis imperfecta on day one of life. She had disproportionate short stature, blue sclera, a small chest and bowing of her lower limbs with swellings and tenderness over both of her femurs. A babygram radiograph revealed multiple fractures, with the presence of callus formation at some fracture sites suggesting intrauterine fractures. The second case is a patient who had normal anthropometry and was well at birth. She was subsequently diagnosed at two weeks of age when she presented to the Chris Hani Baragwanath Academic Hospital with an E. coli meningitis and she was suspected to have a right clavicular fracture and possibly rib fractures as she had pain on palpation over these areas. She was noted to have no blue sclera. Subsequent X-rays confirmed a right clavicular fracture as well as left and right rib fractures at different stages of healing. A lateral skull radiograph revealed Wormian bones. With no available genetic testing in South Africa, both diagnoses were made clinically. Both of our patients were started on zoledronic acid at three months of age and were followed up by the Metabolic Unit at the Chis Hani Baragwanath Academic Hospital. This case report of two patients highlights the characteristics important in diagnosing and treating this uncommon condition with varying phenotypical presentations, thus ensuring that the diagnosis is not missed or misdiagnosed: one disorder, two different faces.

壳聚糖/甘油磷酸钠/海藻酸钠/益母草碱水凝胶的抗炎与促成骨作用

背景:益母草碱具有改善微循环、抗氧化、抗细胞凋亡、清除自由基、抗炎、抗纤维化等多种生物活性作用,并且可促进骨髓间充质干细胞的成骨分化,具有应用于牙周炎治疗的潜能。目的:探讨益母草碱负载于壳聚糖/甘油磷酸钠/海藻酸钠水凝胶的抗炎与促成骨作用。方法:①分别制备壳聚糖/甘油磷酸钠/海藻酸钠水凝胶(空白水凝胶)与壳聚糖/甘油磷酸钠/海藻酸钠/益母草碱水凝胶(载药水凝胶),将两种水凝胶分别培养RAW 264.7细胞、MC3T3-E1细胞,利用CCK-8实验与活/死细胞染色检测水凝胶的细胞毒性。②将RAW 264.7细胞分5组培养:空白组常规培养24 h,脂多糖组加入脂多糖,单独水凝胶组加入脂多糖与空白水凝胶,载药水凝胶组加入脂多糖与载药水凝胶,抑制剂组加入脂多糖、载药水凝胶与PI3K抑制剂LY294002,处理24 h后,利用qRT-PCR法检测炎症相关因子mRNA表达,Western Blotting检测炎症相关因子与PI3K/AKT信号通路的蛋白表达。③将MC3T3-E1细胞分4组培养:空白组不加入任何材料,单独水凝胶组加入空白水凝胶,载药水凝胶组加入载药水凝胶,抑制剂组加入载药水凝胶与PI3K抑制剂LY294002,处理7 d后,进行碱性磷酸酶染色,利用qRT-PCR检测成骨相关因子mRNA表达水平,Western Blotting检测PI3K/AKT信号通路的蛋白表达。结果与结论:①CCK-8实验与活/死细胞染色结果显示,两种水凝胶无细胞毒性作用,具有良好的细胞相容性;②与空白组相比,脂多糖组白细胞介素6、肿瘤坏死因子α、白细胞介素1β的mRNA与蛋白表达升高(P<0.05),p-AKT、p-PI3K、p-p65、p-IκBα的蛋白表达升高(P<0.05);与脂多糖组比较,载药水凝胶组上述指标的mRNA与蛋白表达均降低(P<0.05);与载药水凝胶组相比,抑制剂组上述指标的mRNA与蛋白表达均降低(P<0.05);③载药水凝胶组碱性磷酸酶活性高于空白组、单独水凝胶组、抑制剂组(P<0.05);与空白组相比,单独水凝胶组碱性磷酸酶、Runx2、骨钙素与Ⅰ型胶原的mRNA表达升高(P<0.05),p-AKT、p-PI3K的蛋白表达升高(P<0.05);与单独水凝胶组相比,载药水凝胶组上述指标的mRNA与蛋白表达均升高(P<0.05);与载药水凝胶组相比,抑制剂组上述指标的mRNA与蛋白表达均降低(P<0.05);④结果表明,壳聚糖/甘油磷酸钠/海藻酸钠/益母草碱水凝胶具有抗炎与促成骨作用,该作用可能与调控PI3K/AKT信号通路相关。

3D打印导板在上颌骨前段牵引成骨中的应用及精度测量

目的·评估上颌骨前段牵引成骨(anterior maxillary segmental distraction osteogenesis,AMSDO)在治疗唇腭裂继发上颌骨发育不足中的作用以及3D打印导板在截骨中的精度。方法·收集12例接受了AMSDO治疗的唇腭裂患者的病例资料。手术前在患者的三维模型上进行虚拟手术,并通过3D打印将患者的截骨线制成牙支持式导板。术前(T0)、巩固期结束(T1)以及巩固期后6个月(T2)进行头影测量以评估AMSDO的效果及稳定性。导板的精度通过术后即刻CT与术前设计CT进行叠加,并通过计算位置和角度误差进行评估。结果·所有患者都顺利完成了牵引治疗,没有发生严重的并发症。SNA(S-N-A角)和覆盖从T0到T1以及T0到T2都发生了显著的改变。ANB(A-N-B角)、面部突度、硬腭长度都发生了变化,但是差异没有统计学意义。SNB(S-N-B角)基本没有发生变化。从T1到T2,所有的参数都没有发生明显变化。导板截骨精度在矢状向的线性均方根误差为0.90 mm,角度均方根误差为5.07°。结论·AMSDO是治疗唇腭裂继发上颌骨发育不足的一种有效方法。3D打印的截骨导板具有良好的精度,在减少手术并发症的同时降低了手术的难度。

使用数学模型对髁突颈骨折愈合过程中骨与软骨组织变化的研究

目的 使用数学模型模拟大鼠髁突颈骨折愈合过程中成骨与成软骨的动态变化过程,探索髁突颈骨折的愈合模式。方法 构建模拟大鼠髁突颈骨折愈合的数学模型,并统计该数学模型28 d内不同时间点生成的各参数(mb, mc, cb和cc)的数值,进而拟合骨、软骨、成骨细胞及成软骨细胞的密度云图和生长曲线并推算成骨方式。结果 数学模型模拟的骨面积比与大鼠骨折实验所测接近(P>0.05)。数学模型模拟的密度云图显示,在骨折后第3天至第7天成骨集中在骨膜周围,在第7天至第21天成骨集中在软骨所在区域并逐渐替代软骨。骨生长曲线与软骨生长曲线在骨折后第5至第8天与第21至第28天正相关,与第8至第14天呈负相关。成骨细胞生长曲线和成软骨细胞生长曲线均呈现先升后降的趋势,成软骨细胞在第6天达到最大密度,成骨细胞则在第13天达到最大密度。结论 数学模型能有效模拟大鼠髁突颈骨折愈合过程,可动态展示该过程中成骨与成软骨的动态变化,为研究髁突颈骨折的愈合方法提供了新思路。

龙血素B通过P38MAPK信号通路调控MC3T3-E1成骨分化和骨形成的机制

目的:探究龙血素B对小鼠胚胎成骨细胞前体细胞(MC3T3-E1细胞)成骨分化和骨形成的影响以及其通过P38MAPK信号通路调控成骨分化和骨形成的机制。方法:培养MC3T3-E1细胞,加入不同浓度(0、15、30、60、90、120μmol/L)龙血素B,采用CCK8法和流式细胞术(FCM)检测不同时间(24h、48h、72h)MC3T3-E1细胞增殖能力和凋亡情况,确定龙血素B促MC3T3-E1细胞成骨最佳的药物浓度及作用时间。培养MC3T3-E1细胞并分为空白组(不作干预),龙血素B组(加入龙血素B共培养),龙血素B+阻断剂组(加入龙血素B和P38抑制剂SB203580共培养),进行干预实验;采用碱性磷酸酶(ALP)活性检测试剂盒测定各组细胞ALP活性,qRT-PCR法检测各组细胞骨桥蛋白(OPN)基因、骨钙素(OCN)基因、骨唾液蛋白(BSP)基因表达水平,茜素红染色法观察各组细胞骨形成能力。结果:龙血素B可促进MC3T3-E1细胞增殖并抑制其凋亡,效果与浓度和干预时间相关,在90μmol/L浓度下干预48h促MC3T3-E1细胞生长作用最强;干预结束后第1、3、5天,龙血素B组细胞ALP活性较空白组升高,龙血素B+阻断剂组细胞ALP活性较龙血素B组降低(P<0.05);龙血素B组细胞OPN、OCN、BSP基因表达水平较空白组升高,龙血素B+阻断剂组细胞OPN、OCN、BSP基因表达水平较龙血素B组降低(P<0.05);干预结束后第21天,龙血素B组细胞钙化结节区域面积较空白组增大,龙血素B+阻断剂组细胞钙化结节区域面积较龙血素B组减小(P<0.05)。结论:龙血素B可提高MC3T3-E1细胞增殖活性,并通过激活P38MAPK信号通路促进MC3T3-E1细胞成骨分化和骨形成。

肌骨共生相关信号通路研究进展

骨质疏松症是一种以骨量降低、骨组织微结构损坏、骨脆性增加、易发生骨折为特征的全身代谢性骨病,而肌少症是以进行性全身肌量减少和功能减退为主要特征的综合征。基于二者共同的病理生理机制及密切相关性,逐渐衍生出“肌少-骨质疏松症”这一概念,以描述肌肉与骨骼同时发生衰减的现象。信号通路作为肌肉与骨骼之间重要的信号传递途径,如发生异常,则会导致肌少-骨质疏松症的发生。因此,本文就Hedgehog、Hippo、mTOR和MAPK等成骨与成肌相关信号通路进行综述,以期为肌少-骨质疏松症的靶向治疗提供新思路。

兼具显影与成骨功能多聚磷酸锶在磷酸钙骨水泥中的应用

背景:课题组前期研究发现,添加硫酸钡可以提高磷酸钙骨水泥的力学性能与显影性能,但伴随磷酸钙降解残存的显影剂难以降解,在植入部位产生的占位效应及破骨效应影响骨修复进程,因此需要开发一种可生物降解的新型显影材料。目的:探讨生物活性可降解材料多聚磷酸锶的显影能力及其对磷酸钙骨水泥理化性能与成骨效应的影响。方法:①分别制备磷酸钙骨水泥(CPC)、淀粉改性磷酸钙骨水泥(CPS)与含多聚磷酸锶(占骨水泥粉末的质量分数为20%)淀粉改性磷酸钙骨水泥(20%SrPP-CPN),表征3组骨水泥的理化性能。②将3组骨水泥浸提液分别与大鼠骨髓间充质干细胞共培养,检测细胞增殖、能量代谢与成骨分化能力。③在24只SD大鼠颅顶部两侧各制作直径5 mm的骨缺损,随机分为对照组(不进行任何干预)、CPC组、CPS组与20%SrPP-CPN组进行干预,每组6只。干预4,12周后取材进行相关检测。结果与结论:①与其他两组骨水泥比较,20%SrPP-CPN的显影能力增强、抗压强度与降解速率升高、固化时间延长,并且20%SrPP-CPN在降解过程中可稳定释放Sr^(2+)。②CCK-8实验显示20%SrPP-CPN不影响骨髓间充质干细胞的增殖;细胞饥饿实验(无血清培养)显示相较于其他两组骨水泥,20%SrPP-CPN可促进骨髓间充质干细胞的增殖;相较于其他两组骨水泥,20%SrPP-CPN可提升骨髓间充质干细胞内ATP浓度。碱性磷酸酶与茜素红染色显示,相较于其他两组骨水泥,20%SrPP-CPN可促进骨髓间充质干细胞的成骨分化。③在大鼠颅骨缺损实验中,Micro-CT扫描、组织学(苏木精-伊红、Masson染色)观察显示相较于CPC组、CPS组,20%SrPP-CPN组骨水泥降解明显,并且大量新生骨组织分散于降解的骨水泥中;免疫组化染色显示相较于CPC组、CPS组,20%SrPP-CPN组缺损处Runx2蛋白表达升高(P<0.01)。④结果表明,20%SrPP-CPN具有良好的显影性能与促成骨性能。

MXene纳米材料在生物医学应用中促成骨的机制研究

MXene纳米复合材料是一种新兴的生物活性材料,它具有良好的理化性能和生物相容性,已被用于各类组织工程和再生的研究。目前发现MXene纳米复合材料能够促进成骨相关细胞的黏附、增殖和分化,调控成骨相关基因和蛋白的表达和相关信号通路,促进缺损部位血管生成,从而促进骨缺损修复。这使其成为骨组织再生领域的热门研究材料。本文就MXene纳米复合材料促进骨再生作用的具体机制进行综述,为其基础及临床研究应用提供理论依据和新思路。

MiR-3609通过靶向调控CCND1抑制成骨细胞分化介导骨质疏松症发生

目的通过生物信息学分析和体外实验结合的方式验证miR-3609在骨质疏松症发病中的可能作用机制,为骨质疏松症的治疗提供新靶点。方法通过miRDB、miRWalk、TargetScan三大miRNA靶点分析数据库进行miR-3609下游靶点分析。双荧光素酶实验验证miR-3609和下游靶基因之间的靶向关系,RT-qPCR实验和WB实验验证miR-3609对下游靶基因表达的影响及WB实验检测miR-3609对抗凋亡基因Bcl2和成骨相关蛋白Runx2、OPG表达的影响。碱性磷酸酶实验及茜素红实验检测分析miR-3609对成骨细胞成骨分化、矿化的影响。结果首先,数据库靶点预测结果显示,CCND1可能是miR-3609导致骨质疏松发病的潜在靶点。其次,双荧光素酶实验验证了二者之间的靶向关系:miR3609的激活会下调成骨细胞中的CCND1表达。同时,碱性磷酸酶和茜素红实验结果表明miR3609的激活抑制成骨细胞向成骨分化及矿化。此外,miR3609的激活下调了成骨细胞成骨相关蛋白Runx2、OPG的表达,下调了抗凋亡蛋白Bcl2的表达。结论miR-3609可以通过靶向抑制CCND1表达抑制成骨细胞成骨分化、矿化,这可能是其导致骨质疏松发病的机制,靶向抑制miR-3609有望成为治疗骨质疏松症的新方向。

Magnesium-incorporated biocomposite scaffolds:A novel frontier in bone tissue engineering

Nonunion represents a crucial challenge in orthopedic medicine,demanding innovative solutions beyond the scope of traditional bone grafting methods.Among the various strategies available,magnesium(Mg)implants have been recognized for their biocompatibility and biodegradability.However,their susceptibility to rapid corrosion and degradation has garnered notable research interest in bone tissue engineering(BTE),particularly in the development of Mg-incorporated biocomposite scaffolds.These scaffolds gradually release Mg2+,which enhances immunomodulation,osteogenesis,and angiogenesis,thus facilitating effective bone regeneration.This review presents myriad fabrication techniques used to create Mg-incorporated biocomposite scaffolds,including electrospinning,three-dimensional printing,and sol-gel synthesis.Despite these advancements,the application of Mg-incorporated biocomposite scaffolds faces challenges such as controlling the degradation rate of Mg and ensuring mechanical stability.These limitations highlight the necessity for ongoing research aimed at refining fabrication techniques to better regulate the physicochemical and osteogenic properties of scaffolds.This review provides insights into the potential of Mg-incorporated biocomposite scaffolds for BTE and the challenges that need to be addressed for their successful translation into clinical applications.

Cinobufotalin prevents bone loss induced by ovariectomy in mice through the BMPs/SMAD and Wnt/β-catenin signaling pathways

Background:Osteoporosis is a chronic bone disease characterized by bone loss and decreased bone strength.However,current anti-resorptive drugs carry a risk of various complications.The deep learning-based efficacy prediction system(DLEPS)is a forecasting tool that can effectively compete in drug screening and prediction based on gene expression changes.This study aimed to explore the protective effect and potential mechanisms of cinobufotalin(CB),a traditional Chinese medicine(TCM),on bone loss.Methods:DLEPS was employed for screening anti-osteoporotic agents according to gene profile changes in primary osteoporosis.Micro-CT,histological and morphological analysis were applied for the bone protective detection of CB,and the osteogenic differentiation/function in human bone marrow mesenchymal stem cells(hBMMSCs)were also investigated.The underlying mechanism was verified using qRT-PCR,Western blot(WB),immunofluorescence(IF),etc.Results:A safe concentration(0.25mg/kg in vivo,0.05μM in vitro)of CB could effectively preserve bone mass in estrogen deficiency-induced bone loss and promote osteogenic differentiation/function of hBMMSCs.Both BMPs/SMAD and Wnt/β-catenin signaling pathways participated in CB-induced osteogenic differentiation,further regulating the expression of osteogenesis-associated factors,and ultimately promoting osteogenesis.Conclusion:Our study demonstrated that CB could significantly reverse estrogen deficiency-induced bone loss,further promoting osteogenic differentiation/function of hBMMSCs,with BMPs/SMAD and Wnt/β-catenin signaling pathways involved.

一个新发SERPINF1基因突变的Ⅵ型成骨发育不全病例家系分析

Ⅵ型成骨发育不全(OI)是由于SERPINF1基因突变致其编码的色素上皮衍生因子(PEDF)水平低下,从而导致骨矿化不足和矿化时间延长的一种罕见常染色体隐性遗传的单基因遗传病。本文对1例疑似Ⅵ型OI的女性患者及其家系进行了全外显子组基因测序和家系分析,结果显示患者SERPINF1基因NM_002615.5:c.786G>A(p.Lys262Lys)突变,该突变属于同义突变,符合常染色体隐性遗传模式。分析该致病基因的致病性和保守性后,最终通过辅助生殖技术帮助该患者生育了健康的后代。本研究报道了SERPINF1基因的新突变,丰富了OI的表型,补充了人类SERPINF1基因的突变数据库,为进一步研究Ⅵ型OI的基因型-表型相关性和未来对于此疾病的遗传咨询等提供依据。

万古霉素和妥布霉素局部给药对大鼠下颌骨再生的影响

目的建立大鼠颌骨缺损模型,观察局部应用万古霉素和妥布霉素粉剂对体内骨再生的影响。方法按照随机数字表法将24只SD雄性大鼠分为4组,每组各6只。对照组无需给予抗生素,万古霉素组给予万古霉素88μg/g,妥布霉素组给予妥布霉素176μg/g,联合组给予万古霉素88μg/g和妥布霉素176μg/g。每只大鼠制作5 mm全厚度标准颌骨缺损模型,并分别在骨缺损处放置抗生素粉末。术后12周收集标本,通过微型计算机断层扫描(micro-CT)分析缺损区骨体积/总骨体积比(BV/TV)、骨形成面积(BFA)和骨体积(BV)。再通过苏木精和伊红(HE)染色和马松染色进行组织学评估,分析骨再生情况。结果全部实验动物均无死亡,无万古霉素或妥布霉素相关毒性症状出现,伤口均愈合良好,无手术并发症及伤口感染。从micro-CT扫描分析,万古霉素组缺损区BV/TV、BFA、BV分别为(8.59±2.23)%、(10.33±2.01)%、(1.73±0.35)mm^(3),均小于对照组[(21.67±3.51)%、(27.55±2.60)%、(3.53±0.35)mm^(3)],而妥布霉素组BV/TV、BFA、BV分别为(45.03±3.20)%、(48.88±4.07)%、(9.06±0.56)mm^(3),均大于对照组,联合组缺损区BV/TV、BFA、BV分别为(22.67±4.04)%、(27.64±3.44)%、(3.41±0.33)mm^(3),均大于万古霉素组,差异均有统计学意义(P<0.05);对照组与联合组比较,差异均无统计学意义(P>0.05)。HE染色和马松染色支持micro-CT的扫描结果。万古霉素组的大鼠的每视野区成骨细胞数、骨组织百分比分别为(1.67±1.21)个、(3.83±1.47)%,均小于对照组[(6.33±1.03)个、(11.67±2.16)%],而妥布霉素组大鼠的每视野区成骨细胞数、骨组织百分比分别为(11.17±1.47)个、(29.50±2.81)%,均大于对照组,联合组大鼠的每视野区成骨细胞数、骨组织百分比分别为(6.33±0.82)个、(9.83±2.32)%,均大于万古霉素组,差异均有统计学意义(P<0.05);对照组与联合组比较,差异均无统计学意义(P>0.05)。结论对于骨缺损,推荐局部应用万古霉素联合妥布霉素,而非单用万古霉素。局部万古霉素给药可减少骨缺损区骨生成,而妥布霉素能促进骨再生。

体外冲击波治疗通过激活Wnt5a/Ca^(2+)信号通路改善大鼠股骨牵张成骨的分子机制

目的 探究体外冲击波治疗通过激活Wnt5a/Ca^(2+)信号改善大鼠股骨牵张成骨的机制。方法 60只8周龄Sprague Dawley雄性大鼠分为对照组、股骨牵张模型组和体外冲击波疗法(extracorporeal shock wave therapy,SWT)组(n=20)。使用Cat Walk XT系统测量大鼠步态参数。通过显微CT(Micro-CT)扫描分析大鼠股骨形态。组织病理学分级采用Mankin评分标准,并分析软骨下骨各区域Wnt5a阳性细胞百分比。实时聚合酶链反应分析大鼠股骨Ⅰ型胶原和骨桥蛋白mRNA表达。免疫组织化学染色分析骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)、血管内皮生长因子(vascular endothelial growth factor,VEGF)和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。蛋白印迹分析Wnt5a/Ca^(2+)信号通路蛋白表达。结果 模型组摆动速度、最大接触面积、单站姿、占空比和支架摆动时间低于对照组,ESWT组摆动速度、最大接触面积、单站姿、占空比和支架摆动时间高于模型组(P<0.05)。模型组骨小梁体积分数、小梁厚度和骨密度小于对照组,ESWT组骨小梁体积分数、小梁厚度和骨密度大于模型组(P<0.05)。模型组股关节病理组织学(Mankin)评分高于对照组,Wnt5a阳性细胞数量少于对照组,ESWT组股关节Mankin评分低于模型组,Wnt5a阳性细胞数量多于模型组(P<0.05)。与对照组相比,模型组大鼠软骨下骨板浅表区可见骨细胞陷窝,ESWT组股关节面损伤程度明显减轻(P<0.05)。ESWT诱导模型大鼠Wnt5a标记明显增加(P<0.05)。模型组Ⅰ型胶原和骨桥蛋白mRNA表达低于对照组,ESWT组Ⅰ型胶原和骨桥蛋白mRNA表达高于模型组(P<0.05)。模型组BMP2、VEGF和PCNA染色阳性细胞少于对照组,ESWT组BMP2、VEGF和PCNA染色阳性细胞多于模型组(P<0.05)。模型组CaMKⅡ、PLC、Wnt5a蛋白表达高于对照组,PKC蛋白表达高于对照组,ESWT组CaMKⅡ、PLC、Wnt5a蛋白表达低于模型组,PKC蛋白表达低于模型组(P<0.05)。结论 体外冲击波治疗通过激活Wnt5a/Ca^(2+)信号通路促进大鼠股骨牵张成骨新生血管形成、细胞增殖和成骨生长因子的表达,改善骨力学性能和骨矿化加速。