生育三烯酚通过激活Wnt/β-catenin信号减少激素诱导骨质疏松大鼠骨量流失

目的观察生育三烯酚对糖皮质激素性骨质疏松(glucocorticoid-induced osteoporosis,GIOP)大鼠骨量影响并探索可能的机制。方法将30只12周龄雄性SD大鼠随机分为3组:对照组(n=10)、地塞米松组(DEX,n=10)及地塞米松+生育三烯酚组(DEX+TTS,n=10)对应给予DEX及TTS干预。12周后取双侧股骨进行微型计算机断层扫描(Micro-CT)和骨生物力学检测。同时检测抗酒石酸酸性磷酸酶(TRACP)、Ⅰ型胶原交联羧基末端肽(CTX-I)、碱性磷酸酶(ALP)和骨钙素(OC)。蛋白印迹检测各组BMP-2、OPG及RANKL蛋白表达改变。结果 DEX组大鼠的骨密度(bone mineral density,BMD)、骨显微结构和骨生物力学指标均明显低于对照组(P0.05)。骨吸收指标(TRACP和CTX-I)升高,骨形成指标(ALP和OC)降低。TTS治疗后骨密度、骨显微结构、骨生物力学指标均有明显改善。与DEX相比,DEX+TTS组的TRACP和CTX-I显著降低,ALP和OC显著升高。蛋白印迹结果表明DEX组的BMP-2和OPG明显降低,而RANKL水平显著上升; TTS治疗后BMP-2和OPG明显上升,而RANKL水平显著降低。结论 TTS可以通过降低骨吸收和促进骨形成及激活Wnt/β-catenin信号来提高GIOP大鼠骨密度、骨微观结构和骨强度。     

黄芪多糖通过PI3K/AKT/mTOR促进激素性骨质疏松症大鼠成骨细胞增殖

目的 探讨黄芪多糖在体内外通过PI3K/AKT/mTOR信号通路对糖皮质激素诱导的骨质疏松症（glucocorticoid-induced osteoporosis,GIOP）的保护作用。方法 从分化的骨髓间充质干细胞（bone marrow mesenchymal stem cells,BM-MSCs）中培养成骨细胞,分为PBS组、模型组、LY294002组、黄芪多糖组和LY294002+黄芪多糖干预组。通过CCK-8法和碱性磷酸酶（alkaline phosphatase,ALP）染色检测细胞增殖和分化。MDC染色观察自噬体的形成。Western blot检测Beclin-1、p62等信号通路及自噬相关因子的蛋白表达。大鼠分为对照组、模型组、LY294002组、黄芪多糖组和LY294002+黄芪多糖组。比较各组大鼠的骨密度、骨组织形态学参数、组织中通路和自噬相关因子的表达。结果 黄芪多糖促进成骨细胞的增殖和分化能力（P＜0.05）。与模型组相比,黄芪多糖组PI3K/AKT/mTOR通路相关磷酸化蛋白的表达、成骨细胞的增殖分化能力、自噬体及自噬相关因子的表达均升高,但在LY294002组中发现了相反的结果（P＜0.05）。在体内实验中,与模型组相比,GIOP大鼠通过黄芪多糖干预改善了骨密度和骨形态参数,并提高了软骨组织中自噬相关因子的表达,而LY294002干预则表现出相反的结果（P＜0.05）。LY294002部分逆转了黄芪多糖对GIOP中成骨分化和骨形态参数的影响。结论 黄芪多糖通过PI3K/AKT/mTOR通路对GIOP发挥保护作用,可能与诱导自噬和促进成骨细胞增殖有关。     

益生菌制剂通过PI3K/Akt信号通路改善骨质疏松大鼠的骨代谢和骨量

摘要：目的 从磷脂酰肌醇3激酶/丝氨酸-苏氨酸激酶（PI3K/AKT）通路,观察肠道菌群调节剂-益生菌制剂对骨代谢及骨量的影响,为益生菌治疗骨质疏松症（osteoporosis,OP）提供可靠资料。方法 建立肠道菌群失调性OP大鼠模型,并设置为正常对照组、模型组、益生菌制剂组、PI3K/AKT通路激活剂组、益生菌制剂+通路激活剂组,每组15只。给药干预治疗14 d后,检测粪便含水量及菌群分布;通过ELISA法检测血清炎症因子及骨生物学指标变化、骨密度仪检测骨密度、灰化法检测骨灰重、HE及抗酒石酸酸性磷酸酶（TRACP）染色观察骨病理损伤及破骨细胞骨吸收数目、Western blot法检测PI3K/Akt通路相关蛋白表达。结果 与正常对照组相比,模型组大鼠肠道菌群失衡,血清炎症因子升高,骨代谢异常（促骨形成因子减少,β-CTX及RANKL等破骨活性降低）,PI3K/Akt通路活性升高（P<0.05）。益生菌制剂可纠正肠道菌群失衡,抑制血清炎症因子释放、改善OP大鼠骨代谢失衡,并抑制PI3K/Akt通路活性（P<0.05）。PI3K/Akt通路激活剂可削弱益生菌制剂的上述作用,加重骨代谢失衡及肠道菌群失调（P<0.05）。结论 益生菌制剂干预治疗,可减弱菌群失调并纠正OP大鼠的骨代谢异常,且其作用可能与抑制PI3K/Akt通路激活有关。     

GLP-1受体激动剂利拉鲁肽对糖皮质激素性骨质疏松大鼠的疗效和作用机制研究

目的讨论GLP-1受体激动剂利拉鲁肽对糖皮质激素性骨质疏松(GIOP)大鼠模型骨代谢指标的影响,包括对骨量、骨组织微结构、骨生物力学、骨转换标志物的作用。方法选取30只8周龄雄性SD大鼠,随机分为对照组、地塞米松组、利拉鲁肽干预组,每组各10只。除对照组外,各组均予以地塞米松1 mg/kg肌肉注射(2次/周)诱导糖皮质激素性骨质疏松。利拉鲁肽组同时给予利拉鲁肽200μg/(kg·d)皮下注射干预。对照组给予同等体积0. 9%氯化钠肌肉注射。干预12周后收集两侧股骨和第五腰椎行Micro-CT和骨生物力学检测。同时收集外周血清检测抗酒石酸酸性磷酸酶(Str ACP)、Ⅰ型胶原交联羧基端肽(CTX-1)、碱性磷酸酶(ALP)、和骨钙素(OC)。结果地塞米松组股骨远端干骺端和第五腰椎的骨密度(BMD)、组织骨密度(TMD)、骨体积分数(BV/TV)、骨小梁分离度(Tb.Sp)、骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N)、连接密度(Conn.D)、最大载荷和弹性模量均较对照组明显降低,骨吸收指标Tr ACP、CTX-1显著升高,骨形成指标ALP、OC显著降低(均P0. 05)。利拉鲁肽干预组大鼠的骨密度、骨微结构、骨生物力学指标得到明显改善,且Tr ACP、CTX-1明显降低,ALP、OC明显升高(均P0. 05)。结论利拉鲁肽能对抗糖皮质激素的致骨质疏松作用,改善糖皮质激素造成的骨质疏松状态,机制可能与减少骨吸收以及促进骨形成有关。     

异补骨脂素对去卵巢大鼠骨代谢的影响研究

目的研究异补骨脂素对去卵巢大鼠骨代谢的影响。方法取3月龄雌性SD大鼠40只,随机分为4组,假手术对照组,模型组,雌二醇组(苯甲酸雌二醇0.2 mg/kg,皮下注射,每周1次),异补骨脂素组。手术6 w后,异补骨脂素组用25 mg/kg(此浓度经过前期浓度筛选得到)异补骨脂素灌胃,每天1次,1周停止1次,模型组灌胃与用药组等体积生理盐水,假手术组正常饲养。12 w后双能X骨密度仪检测全身骨密度后处死,分离骨组织,于AG-X系列台式电子万能试验机检测左侧股骨和椎骨生物力学指标,血清中骨钙素(osteocalcin,OC)和抗酒石酸性磷酸酶5b(tartrate resistant acid phosphatase 5b,TRACP5b)检测,右侧股骨护骨素(osteoprotegerin,OPG),RANKL免疫组织化学染色,剥离心、肝、脾、肺、肾、肾上腺和子宫称重,计算器官指数,并做常规病理学检测。结果与假手术组比较,模型组大鼠体质量、全身骨密度、股骨骨密度、腰椎骨密度及血清OC、TRACP 5b,胫骨微组织结构和股骨生物力学性能差异极显著,有统计学意义(P0.01),说明骨质疏松造模良好;与模型组比较,异补骨脂素组大鼠体质量、全身骨密度、股骨骨密度、腰椎骨密度及血清OC、TRACP 5b,胫骨微组织结构和股骨生物力学性能显著差异有统计学意义(0.01P0.05)。与模型组比较,阳性对照组大鼠全身与股骨骨密度、血清OC、TRACP 5b和股骨生物力学性能极显著差异有统计学意义(P0.01)。药物处理组与阳性对照相比无统计差异。结论 25 mg/kg的异补骨脂素能增加SD大鼠骨密度,改善骨组织微结构,有效提升骨生物力学性能,促进骨形成,降低骨质疏松骨折发生的风险。     

异紫杉脂素治疗对去卵巢大鼠骨量流失和骨密度降低保护作用的机制研究

目的探索异紫杉脂素(isotaxiresinol,IXO)对去卵巢大鼠骨密度和骨量的影响,并探讨其作用机制。方法将大鼠随机分为以下各组:假手术组(Sham)、去卵巢大鼠组(OVX)、去卵巢大鼠+异紫杉脂素组(IXO)。其中IXO组大鼠给予异紫杉脂素100 mg/(kg·d)治疗12周。Micro-CT和HE切片观察骨组织变化。通过ELISA检查用于分析骨代谢指标,进行蛋白质印迹分析以评估PI3K、Akt和RUNX-2的蛋白表达。结果 12周时,OVX组大鼠骨密度和骨量较Sham组显著降低。而IXO能显著改善去卵巢大鼠的骨密度和股骨干骺端骨小梁微观结构。去卵巢大鼠在IXO给药治疗后可显著降低P1NP和β-CTX水平(P0.05);用IXO治疗可上调去卵巢大鼠的PI3K、Akt和RUNX-2蛋白表达。结论本研究提示异紫杉脂素可以通过激活PI3K/Akt信号通路对去卵巢大鼠骨量流失和骨密度降低起到保护作用。     

黄芪多糖治疗对去卵巢诱导骨质疏松大鼠骨密度、骨量和骨代谢的影响

目的探索黄芪多糖对去卵巢大鼠骨量和骨代谢以及BMP-2/Smads信号通路的影响。方法30只雌性SD大鼠进行去卵巢手术或假手术。正常饲养12周后被分为黄芪多糖组(ASNT组)、对照组(CON组)和去卵巢组(OVX组),其中黄芪多糖组每天给予400 mg/kg黄芪多糖治疗。通过骨密度、骨代谢指标、Micro-CT以及WB检测评估ASNT对骨质疏松症大鼠骨量、骨代谢以及BMP-2/Smads信号通路的影响。结果经过12周治疗,ASNT组大鼠骨密度较OVX组显著增加(P<0.05);ASNT治疗12周可以降低血清ALP和OC水平,增加血钙含量,增加股骨骨密度。经过12周治疗,ASNT组大鼠骨体积分数(BV/TV)、表面积体积分数(BS/TV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)较OVX组显著增加;而骨小梁间隙(Tb.Sp)较OVX组显著降低,差异比较有统计学意义(P<0.05)。WB结果表明OVX大鼠骨组织的BMP-2/Smsds信号通路受到抑制;ASNT可以通过上调BMP-2、p-Smad1和p-Smad5的表达显著激活BMP-2/Smsds信号通路传导。结论研究表明ASNT对去卵巢大鼠骨密度和骨量具有保护作用,可能和激活BMP-2/Smads信号通路有关。     

补肾壮骨方对去卵巢大鼠骨结构和骨代谢作用的研究

目的研究补肾壮骨方对骨质疏松大鼠骨结构和骨代谢的影响及其可能的作用机制。方法用SD雌性大鼠复制去卵巢骨质疏松模型,然后分别灌服补肾壮骨方水提液高剂量(1.4 g/kg)和低剂量(0.7 g/kg)12周。用生化法测定血清钙(S-Ca)、血清磷(S-P)、尿钙(U-Ca/Cr),尿磷(U-P/Cr)以及血清中高密度脂蛋白(high-density lipoprotein,HDL)、低密度脂蛋白(low-density lipoprotein,LDL)、总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)的含量;Elisa法测定I型胶原氨基端前肽(procollagen I N-terminal propeptide,PINP)、I型胶原羧基端肽(collagen I carboxyl terminal peptide,CTX-I)、尿脱氧吡啶啉(deoxypyridine,DPD)、骨钙素(osteocalcin,OCN)的含量;用放免法测定血清碱性磷酸酶(alkaline phosphatase,ALP)的活性;用双能X线(DEXA)法、Viva CT和万能试验机测定骨密度、骨微结构和骨生物力学特性;用Western blot法测定骨组织蛋白酶(cathepsin)K表达。结果补肾壮骨方水提液能升高血清中的钙、磷、HDL-C和PINP的含量,降低血清中TC、TG、LDL、ALP、OCN和CTX-I及尿液中的钙、磷、DPD的含量。同时,补肾壮骨方水提液可以升高去卵巢大鼠股骨的骨密度,改善骨微结构,增加骨强度,降低胫骨Cathepsin K的表达水平。结论补肾壮骨方水提液能够抑制去卵巢大鼠的骨量丢失和骨强度下降,改善去卵巢大鼠的骨代谢,其作用机制可能与抑制Cathepsin K的表达有关。     

红景天苷激活PI3K/AKT通路改善绝经后骨质疏松症实验研究

目的探讨红景天苷激活PI3K/AKT通路改善绝经后骨质疏松症的作用。方法建立绝经后骨质疏松症大鼠模型,随机分为模型组、LY294002(PI3K/Akt通路抑制剂)组、红景天苷组、红景天苷+LY294002组,每组12只,另取12只设为假手术组。分组处理后,通过骨科生物力学测试仪测定大鼠股骨生物力学指标弹性模量、最大载荷、屈服载荷;测定大鼠股骨骨矿盐含量;运用苏木精-伊红(HE)染色检测各组大鼠骨组织病理变化;通过酶联免疫吸附法(ELISA)检测血清白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;运用蛋白免疫印迹法检测脑组织PI3K/Akt通路相关蛋白表达情况。结果与假手术组相比,模型组大鼠骨组织呈现骨小梁稀疏断裂、数目明显减少,有较多空隙、不能连接成网等病理损伤,血清IL-6及TNF-α水平均显著升高(P0.05),股骨弹性模量、最大载荷、屈服载荷、骨矿盐含量、骨组织p-PI3K/PI3K、p-Akt/Akt水平均显著降低(P0.05);与模型组相比,红景天苷组大鼠骨组织病理损伤减轻,血清IL-6及TNF-α水平降低(P0.05),股骨弹性模量、最大载荷、屈服载荷、骨矿盐含量、骨组织p-PI3K/PI3K、p-Akt/Akt水平升高(P0.05); LY294002组大鼠骨组织病理损伤加重,血清IL-6及TNF-α水平升高(P0.05),股骨弹性模量、最大载荷、屈服载荷、骨矿盐含量、骨组织p-PI3K/PI3K、p-Akt/Akt水平降低(P0.05)。与LY294002组相比,红景天苷+LY294002组大鼠骨组织病理损伤减轻,血清IL-6及TNF-α水平降低(P0.05),股骨弹性模量、最大载荷、屈服载荷、骨矿盐含量、骨组织p-PI3K/PI3K、p-Akt/Akt水平升高(P0.05)。与红景天苷组相比,红景天苷+LY294002组大鼠骨组织病理损伤加重,血清IL-6及TNF-α水平升高(P0.05),股骨弹性模量、最大载荷、屈服载荷、骨矿盐含量、骨组织p-PI3K/PI3K、p-Akt/Akt水平降低(P0.05)。结论红景天苷可能通过激活PI3K/Akt通路改善绝经后骨质疏松症。     

补肾固本方对大鼠骨质疏松模型中PI3K/AKT/mTOR信号通路表达的调控研究

目的观察补肾固本方对去卵巢骨质疏松(osteoporosis,OP)大鼠骨组织中PI3K/AKT/mTOR信号通路的影响,探讨补肾固本方干预OP的作用机制。方法将100只SD大鼠随机分为5组:正常组(C组)、模型组(M组)、补肾固本方组(B组)、补肾固本方+PI3k受体特异性阻断剂LY294002组(B+L组)、PI3k受体特异性阻断剂LY294002组(L组),除正常组外,其余4组建立去卵巢大鼠OP模型。药物连续干预12周后,采用ELISA方法检测血清中E2、BGP、ALP水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测各组股骨组织中LC3、Beclin1、caspase-9 mRNA的表达情况,蛋白质印迹法(Western blot)检测各组股骨组织中PI3K、AKT、mTOR蛋白的表达。结果补肾固本方可显著增高OP大鼠血清E2水平,降低BGP、ALP水平,显著上调LC3、Beclin1表达水平,降低caspase-9、PI3K、p-AKT、mTOR的表达;而上述变化能够被PI3k受体特异性阻断剂LY294002所阻断,且其差异具有统计学意义(P0.05)。结论补肾固本方减少去卵巢后大鼠OP的发生,其机制可能与抑制PI3K/AKT/mTOR信号通路及其下游基因蛋白表达有关。     

骨形态发生蛋白-2/胶原/掺锶羟基磷灰石材料修复颅骨缺损

目的 制备骨形态发生蛋白-2( BMP-2)/胶原/掺锶羟基磷灰石材料并探讨其修复大鼠颅骨缺损的可行性和有效性.方法 扫描电镜观察Ⅰ型胶原制备单纯胶原、胶原/羟基磷灰石、胶原/掺锶羟基磷灰石、BMP-2/胶原/掺锶羟基磷灰石4组骨修复材料表面结构.用BMP-2/胶原/掺锶羟基磷灰石材料浸提液进行细胞毒性试验和体外溶血试验评价其生物相容性.在大鼠头颅制备颅骨极限骨缺损模型,分别植入4种骨修复材料.术后12周CT扫描观察骨缺损修复影像学.苏木素-伊红(HE)和Masson染色观察骨缺损组织学变化,并在骨缺损及其周围新生骨部位行骨桥蛋白( OPN)和β-连环蛋白(β-catenin)免疫组织化学染色.结果 在扫描电镜下观察发现单纯的胶原材料为交织样物质结构,胶原/羟基磷灰石材料为交织晶体板状结构胶原/掺锶羟基磷灰石和BMP-2/胶原/掺锶羟基磷灰石材料晶体结构为单晶体交织状.BMP-2/胶原/掺锶羟基磷灰石材料浸提液对细胞相对增殖率(RGR)无显著影响(P＞0.05),材料的细胞毒性为1级.骨缺损CT扫描平均CT值分别为(98.5±10.2)、(208.4±19.5)、(418.4±27.1)、(476.8±30.5)hu,BMP-2/胶原/掺锶羟基磷灰石材料缺损部位CT值最高.HE和Masson染色见BMP-2/胶原/掺锶羟基磷灰石组骨质愈合完全,原骨缺损处多为红色成熟骨.胶原/掺锶羟基磷灰石组植入区内蓝色的新生骨较多.胶原/羟基磷灰石材料组,植入区在植入材料边缘新生骨形成,界限仍然清晰.单纯胶原组骨质未愈合,骨缺损处为淡蓝色条索状结构,中间未见骨形成.对比其他3组,BMP-2/胶原/掺锶羟基磷灰石组存在大量棕色的OPN和β-catenin染色阳性新生骨组织,差异有统计学意义(P＜0.05).结论 BMP-2/胶原/掺锶羟基磷灰石材料促进骨修复能力强于单纯胶原、胶原/羟基磷灰石、胶原/掺锶羟基磷灰石材料.     

A novel nano-hydroxyapatite/synthetic polymer/bone morphogenetic protein-2 composite for efficient bone regeneration

BACKGROUNDEfficient bone regeneration using recombinant human bone morphogenetic protein-2 (BMP-2) is needed to reduce side effects caused by high-dose BMP-2 use. The composite material of polylactic acid–polyethene glycol (PLA-PEG) for sustained release and an osteogenic nano-hydroxyapatite (nHAp) can contribute to efficient bone regeneration by BMP-2.STUDY DESIGNAn experimental in vitro and in vivo study.PURPOSEThe objective of this study is to investigate the effectiveness of a novel composite material of PLA-PEG and nHAp as a carrier for BMP-2.METHODSThe release kinetics of BMP-2 from the composites was investigated by ELISA. Thirty-six male Sprague–Dawley rats underwent posterolateral spinal fusion on L4–L5 with three different doses of BMP-2 (0 µg [control], 3 µg [low dose], and 10 µg [high dose]). Weekly µCT results and histology and a manual palpation test at 8 weeks postoperatively were used for assessment of the spinal fusion.RESULTSELISA demonstrated the sustained release of BMP-2 until day 21. µCT and manual palpation test demonstrated a solid fusion in 91.6% (11/12) of specimens in both the low- and high-dose groups. N mice in the control group attained bony fusion (0%, 0/9). nHAp was resorbed between 2 and 4 weeks postoperatively, and regenerated fusion mass at 8 weeks postoperatively consisted of only newly formed bone.CONCLUSIONSThe nHAp/PLA-PEG composite enabled efficient bone regeneration with low-dose BMP-2. The sustained release of BMP-2 by PLA-PEG and the osteogenic and biodegradable scaffold of nHAp might contribute to efficient bone regeneration.CLINICAL SIGNIFICANCEThis novel composite material has potential in clinical applications (spinal fusion, large bone defect and non-union) by enabling efficient bone formation by BMP-2.     

Vascularized bone marrow transplantation: A new surgical approach using isolated femoral bone/bone marrow

BACKGROUND: Orthotopic composite tissue (limb) transplantation in rats is a unique model for vascularized bone marrow transplantation because bone marrow cells and bone marrow stroma are transplanted by microsurgical means, thus creating immediate bone marrow space and engraftment. However, it contains a skin component and other musculoskeletal tissues that complicate issues related to tolerance induction. MATERIALS AND METHODS: To study only aspects of vascularized bone marrow transplantation, we created a new isolated vascularized bone marrow transplant model in rats. The common iliac (or femoral) artery and vein were microsurgically anastomosed to the recipient abdominal aorta and inferior vena cava in an end-to-side fashion, respectively. Syngeneic male Lewis (RT1(1), n = 20) and allogeneic male BN (RT1(n), n = 10) donors were transplanted to female Lewis recipients. To establish rejection criteria, we examined histopathology and used the polymerase chain reaction (PCR) to assess microchimerism of donor male bone marrow cells in the peripheral blood of female recipients using rat Y chromosome (sex-determining region Y)-specific primers. RESULTS: All recipients were healthy and remained stable without major complications for up to 300 days posttransplant. Morphologically, syngeneic male Lewis bone marrow showed a near-normal appearance. Allogeneic male BN bone marrow was clearly rejected. Male bone marrow cells were detected by PCR in the peripheral blood of all syngeneic recipients, but not in allogeneic blood specimens. CONCLUSIONS: A new surgical approach to bone marrow transplantation was established. This consisted of the vascularized femoral bone/bone marrow transplant. Further analyses regarding the ability of vascularized femoral bone marrow transplants to induce systemic transplantation tolerance in adult rats will provide insights into not only various issues of immunology but also the potential clinical application of vascularized bone marrow transplantation.     

Composite vascularized skin/bone transplantation models for bone marrow-based tolerance studies

There is an ongoing need to understand the mechanisms of bone marrow-based allograft tolerance. This is important in clarifying the diverse variables influencing the ultimate outcome of the solid organ and composite tissue transplants. To establish bone marrow transplantation as a routine clinical application, further experimental studies should be conducted to overcome the obstacles related to the bone marrow transplantation. These obstacles include graft versus host disease, immunocompetence, and toxicity of the conditioning regimens. For these purposes, novel experimental models are needed.In an attempt to provide a reliable research tool for bone marrow-based tolerance induction studies, we introduced different experimental models of modified vascularized skin/bone marrow (VSBM) transplantation technique for tolerance induction, monitoring, and maintenance studies.In this skin/bone transplantation model, the technical feasibility of concurrent or consecutive transplantation of the combination of bilateral vascularized skin, vascularized bone marrow, or vascularized skin/bone marrow transplants was investigated.Isograft transplantations were performed between genetically identical Lewis (LEW, RT1) rats. Five different experimental designs in 5 groups of 5 animals each were studied. Group I: Bilateral vascularized skin (VS) transplantation; group II: bilateral vascularized skin/bone transplantation; group III: vascularized skin transplantation on one side and vascularized skin/bone transplantation on the contralateral side; group IV: vascularized bone transplantation on one side and vascularized skin/bone transplantation on the contralateral side; group V: vascularized bone transplantation on one side and vascularized skin transplantation on the contralateral side.Successful transplantations were performed in all groups. The survival of the isograft transplants was evaluated clinically and histologically. All skin flaps remained pink and pliable and grew new hair. The viability of the compact bone, bone marrow and skin at 100 days posttransplant was confirmed by histologic evaluation, and bone marrow revealed active hematopoiesis.Bilateral skin/bone transplantation model may serve as an experimental tool to study new strategies in tolerance induction by altering the amount of the immunogenic load in the form of skin transplant and bone marrow delivery in the vascularized form, allowing for expedited engraftment of stem and progenitor cells.     

Preparation and degradation characteristic study of bone repair composite of DL—polylactic acid／hydroxyapatite／decalcifying bone matrix

Objective:To explore the preparative method and study the degradation characteristics of bone repair composite of DL-polyactic acid(PDLLA) /hydroxyapatite(HA)/decalcifying bone matrix(DBM) in vitro.Methods:An emulsion blend method was developed to prepare the composite of PDLLA/HA/DBM in weight ratio of PDLLA:HA:DBM=1.2-2:1.5:1.The dynamic changes of weight,biomechanical property and pH value of PDLLA/HA/DBM and PDLLA in phosphate buffered saline(PBS,pH7.4)were studied respectively through degradation tests in vitro.Results:Without being heated,PDLLA,HA and DBM could be synthesized with the emulsion blend method as bone composite of PDLLA/HA/DBM,wich had both osteoconductive and osteoinductive effects.The diameter of the aperture was 100-400μm and the gap rate was 71.3%.During degradation,the pH value of PDLLA solution decreased lightly within 2 weeks,but decreased obviously at the end of 4 weeks and the value was 4.0 .While the pH value of PDLLA/HA/DBM kept quite steady and was 6.4 at the end of 12 weeks.The weight of PDLLA changed little within 4 weeks,then changed obviously and was 50% of its initial weight at the end of 12 weeks.While the weight of PDLLA/HA/DBM changed little within 5 weeks ,then changed obviously and was 60% of the initial weight at the end of 12 weeks.The initial biomechanical strength of PDLLA was 1.33 MPa, decreased little within 3 weeks, then changed obviously and kept at 0.11 MPa at the end of 12 weeks.The initial biomechanical strength of PDLLA/HA/DBM was 1.7 MPa, decreased little within 4 weeks, then changed obviously and kept at 0.21 MPa at the end of 12 weeks.Conclusions:The emulsion blend method is a new method to prepare bone repair materials.As a new bone repair material,PDLLA/HA/DBM is more suitable for regeneration and cell implantation,and the environment during its degradation is advantageous to the growth of bone cells.     

Perspectives on bone densitometry: Past/present/future

Osteoporosis is defined as a decrease in bone mass and structural changes in bone leading to an increased fracture incidence. Therefore, early diagnosis in terms of prophylaxis and treatment are of great interest. Over the past several decades there has been considerable progress in the development and application of non-invasive methods of bone mass measurements or bone densitometry. This article reviews basic methodology and developments in bone densitometry including the early approaches like radiogrammetry, photo densitometry, neutron activation analysis and compton scattering techniques, present methods like single and dual x-ray absorptiometry and quantitative computed tomography, and recent developments like quantitative ultrasound, magnetic resonance techniques and structural analysis. The widespread interest in bone densitometry will initiate further improvement of established methods and development of new applications.     

OPG/RANKL/RANK系统与转移性骨肿瘤

OPG、RANKL、RANK是破骨细胞分化、成熟过程中最重要的分子,OPG/RANKL/RANK系统在肿瘤骨转移过程中具有重要作用.肿瘤细胞可通过扰乱OPG/RANKL/RANK系统原有的平衡状态使破骨细胞异常激活,导致肿瘤骨转移部位溶骨性破坏.尽管成骨性破坏并非前列腺癌细胞直接干扰OPG/RANKL/RANK系统的结果,但局部破骨细胞激活导致的骨质破坏仍是肿瘤转移并形成成骨性破坏的必要条件.OPG可与TRAIL结合而抑制其对肿瘤细胞及肿瘤血管内皮细胞的凋亡诱导活性,促进肿瘤细胞生长及转移.通过检测和干预OPG/RANKL/RANK系统,可早期发现并有效控制肿瘤骨转移.     

Amorphous/crystalline interrelationships in bone mineral

Amorphous calcium phosphate was found to be a major mineral component of skeletal tissue by X-ray diffraction techniques. This non-crystalline bone mineral phase can be converted into crystalline apatitein vitro upon exposure to water for a prolonged period of time. The amorphous calcium phosphate content of whole rat femur, tibia-fibula and calvarium decreases as the crystalline apatite content of these bones increases with advancing age. In addition, hypophysectomized whole rat femora contain more amorphous bone mineral than do normal controls. No significant differences in amorphous/crystalline bone mineral composition exist between control and denervated whole rat femora during disuse osteoporosis. However, rachitic chick bone contains more amorphous calcium phosphate than does normal bone tissue, regardless of the manner in which the disease state is reached. Moreover, alterations in chick bone lipid and mucopolysaccharide content coincide with corresponding alterations in the amorphous/crystalline mineral content of the tissue. It is suggested that amorphous calcium phosphate is the first mineral deposited during the calcification process and that the amorphous bone mineral fraction can act as a metabolically active, metastable precursor of crystalline bone apatite. It is further suggested that the amorphous calcium phosphate fraction of bone mineral may also exist in a stabilized form.

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Zusammenfassung Das amorphe Calciumphosphat wurde mittels Röntgenstrahlendiffraktionsmethoden als eine der wichtigsten Komponenten des Knochengewebes erkannt. Dieser nicht kristallisierte Teil des Knochenminerals kann in vitro durch Wassereinwirkung während längerer Zeit in kristallines Apatit umgewandelt werden. Bei Ratten nimmt der Gehalt an amorphem Calciumphosphat der ganzen Femora, Tibia, Fibula und Calvaria mit zunehmendem Alter ab, während der Gehalt an kristallinem Apatit steigt. Dazu enthalten die Femora von hypophysektomierten Ratten mehr amorphes Knochenmineral als die von normalen Tieren. Es besteht kein signifikanter Unterschied im relativen Gehalt von amorphem und kristallinem Knochenmineral zwischen Kontroll- und denervierten Rattenfemora bei Immobilisationsosteoporose. Jedoch enthalten Knochen von rachitischen Hühnern, ungeachtet auf welche Art die Krankheit hervorgerufen wurde, mehr amorphes Calciumphosphat als normales Knochengewebe. Ferner stimmen die Veränderungen des Lipids- und Mucopolysaccharidgehaltes der Hühnerknochen mit entsprechenden Veränderungen im Gehalt an amorphem und kristallinem Mineral des Gewebes überein. Es wird vorgeschlagen, daß amorphes Calciumphosphat das erste Mineral ist, welches während des Verkalkungsprozesses abgelagert wird, und daß dieser Anteil an amorphem Knochenmineral als ein metabolisch aktiver, metastabiler Vorgänger des kristallinen Knochenapatites wirken kann. Dieser amorphe Calciumphosphatanteil des Knochenminerals könnte auch in einer stabilen Form vorkommen.

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Résumé L'analyse de diffraction par rayons X a montré que le phosphate de calcium amorphe est un des principaux constituants du squelette. Cette phase solide, non cristallisée de l'os peut se transformer en apatite cristalline in vitro après avoir été en contact avec de l'eau pendant une période suffisamment longue. Chez le rat, la teneur en phosphate de calcium amorphe des fémurs, tibias, péronés et des calottes crâniennes décroît, alors que la teneur en apatite cristalline de ces os croît, avec l'âge. De plus. les fémurs de rats hypophysectomisés contiennent une plus grande proportion de minéral osseux sous forme amorphe que ceux de témoins normaux. En ce qui concerne la proportion de minéral amorphe et cristallin dans l'ostéoporose d'immobilisation, on n'observe pas de différence significative entre des fémurs dénervés et des os témoins. Cependant, l'os de poulets rachitiques contient plus de phosphate de calcium amorphe que le tissu osseux de poulets sains, quelle que soit la manière dont on a provoqué le rachitisme. De plus, des modification de la teneur de l'os de poulet en lipides et en mucopolysaccharides correspondent à des modifications du rapport entre minéral amorphe et cristallin de l'os. Nous suggérons que le phosphate de calcium amorphe est la première forme minérale déposée au cours de la calcification et que la fraction amorphe est un précurseur métaboliquement actif et métastable de l'apatite cristalline: Il semble d'autre part que le phosphate de calcium amorphe peut également exister sous une forme stable.

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This work was supported in part by PHS Grant DE-10945 from the National Institute of Dental Research, U.S.A. Publication No. 24 from the Laboratory of Ultrastructural Biochemistry.     

Demineralized bone matrix and hydroxyapatite/tri-calcium phosphate mixture for bone healing in rats

Purpose: Hydroxyapatite/tri-calcium phosphate (HA/TCP) mixture is an osteoconductive material used as a bone graft substitute, and demineralised bone matrix (DBM) is an osteoinductive material. A combination of DBM and HA/TCP mixture would probably create a composite with both osteoconductive and osteoinductive properties. The purpose of this study was to determine the effect of the combination of DBM and HA/TCP mixture on healing of rat radius segmental defects. Methods: Twenty-four adult male Wistar rats were used. Bilateral radial defects were created in each animal. Radial defects were implanted with DBM, HA/TCP mixture and a combination of both substances. Control defects were left unfilled. Ten weeks after implantation, the animals were sacrificed, and the radii were evaluated by radiograhic and histopathological studies. Results: The use of DBM alone demonstrated improved healing on radiographic and histological studies compared to other groups and the control group. There were no differences between the other two groups and the control group. Conclusion: The DBM group showed the best healing response. Combined use of DBM and HA/TCP mixture did not improve bone healing, and the osteoinductive properties of DBM were inhibited by HA/TCP mixture.