可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损

背景：自体髂骨移植一直被认为是骨缺损修复的“金标准”,但其来源有限。 目的：验证应用可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损的效果。 方法：80只雄性SD大鼠建立双侧颅骨缺损模型,随机分为3组：骨修复材料+骨碎补总黄酮组采用可注射骨修复材料结合骨碎补总黄酮灌胃修复大鼠颅骨缺损;骨修复材料+去离子水组采用可注射骨修复材料结合去离子水灌胃修复大鼠颅骨缺损;羟基磷灰石+去离子水组采用羟基磷灰石结合去离子水灌胃修复大鼠颅骨缺损,1次/d,持续8周。于建模后2,4,8周取颅骨标本进行苏木精-伊红染色和Masson染色组织学观察。 结果与结论：羟基磷灰石组新骨形成和材料降解速度较慢;可注射骨修复材料组新骨形成和材料降解较羟基磷灰石组快,利于血管及纤维组织长入;骨碎补总黄酮灌胃可以促进血管及纤维组织长入材料,促进成骨。与羟基磷灰石相比,可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损,可促进新骨形成,缩短骨缺损修复时间。     

大鼠骨质疏松伴颅骨极量缺损模型的建立北大核心

背景:骨质疏松伴骨缺损疾病逐渐成为一个重要的社会健康问题。建立动物模型是研究该疾病的基础,且目前还没有关于建立该疾病动物模型的报道。目的:建立骨质疏松伴颅骨极量缺损SD大鼠模型。方法:将20只雌性SD大鼠随机分为2组,实验组行双侧卵巢切除,12周后再造8 mm颅骨极量缺损;对照组只摘除双侧卵巢周围同等质量的脂肪组织,12周后再造8 mm颅骨极量缺损。颅骨极量缺损后12周,检测离体股骨近端、胫骨远端三维骨矿含量、骨密度及血清碱性磷酸酶水平,并进行颅骨组织切片病理观察。结果与结论:两组大鼠颅骨缺损区均未完全愈合,并且骨修复效果不明显,对照组形成新生骨量较实验组多;实验组股骨近端、胫骨远端的三维骨矿含量和骨密度均较对照组显著降低(P<0.05),血清碱性磷酸酶水平较对照组明显升高(P<0.05);与对照组比较,实验组颅骨骨小梁明显变细变小,骨髓腔明显增大,皮质骨显著变薄,新生骨基质形成明显减少。结果表表明,骨质疏松伴颅骨极量缺损SD大鼠模型建立成功。     

辛伐他汀复合Bio-Oss修复兔下颌骨缺损

背景：研究发现辛伐他汀具有促进新骨形成的作用,但其成骨机制及成骨效果目前仍然存在争议。 目的：对比观察Bio-Oss/辛伐他汀复合材料与单纯Bio-Oss修复材料修复兔下颌骨骨缺损区的成骨效果。 方法：12只新西兰大白兔下颌骨双侧制备缺损,随机将一侧采用辛伐他汀复合Bio-Oss修复缺损区;另一侧采用单纯Bio-Oss修复缺损区。两组均覆盖Bio-Gide胶原膜。植骨后4,8,12周分别处死各组兔子4只,通过大体观察,X射线及口腔锥形束CT影像学观察,组织学切片观察,定性定量对比分析植骨区牙槽骨形成情况。 结果与结论：植骨后4,8,12周新骨形成逐渐增多,随着高阻射的Bio-Oss骨粉逐渐降解,在各时间点密度值测量结果辛伐他汀复合Bio-Oss组均显著低于单纯Bio-Oss组(P < 0.05)。新生骨百分比测量结果辛伐他汀复合Bio-Oss均显著高于单纯Bio-Oss组(P < 0.05)。提示辛伐他汀具有促进Bio-Oss骨粉吸收的效果,在骨缺损修复中具有促进新骨生成的作用。     

rhBMP-2/D,L-PLA泡沫复合人工骨修复骨缺损的实验研究

用层压技术研制的聚乳酸 ( D,L- PLA)泡沫材料与人基因重组骨形成蛋白 - 2 ( rh BMP- 2 )复合 ,形成新型复合人工骨 ,并通过兔颅骨缺损修复实验验证复合人工骨的骨缺损修复能力。结果表明 ,研制的复合人工骨具有较强的骨缺损修复能力     

镁锶合金修复兔桡骨骨缺损的实验研究

目的研究Mg-Sr合金对兔桡骨骨缺损的修复效果。方法将30只新西兰大耳白兔随机分为Mg-Sr合金组和空白对照组两组,每组15只。手术制备双侧桡骨中段1.0cm骨缺损模型,镁锶合金组缺损部位植入Mg-Sr合金;空白对照组不植入任何材料作为对照。两组分别于术后4、8和12周各处死5只动物取材,行X线片、组织学检查及Micro-CT,观察各组对骨缺损的修复效果。结果 Mg-Sr合金组骨缺损得到了修复,空白对照组仅有少量新骨形成,但骨缺损未得到修复。结论 Mg-Sr合金对兔桡骨骨缺损有很好的修复效果。     

组织工程化骨修复犬牙槽骨缺损的组织学观察

目的观察组织工程化骨修复犬牙槽骨缺损过程中的新骨形成及其矿化程度。方法全麻及无菌条件下抽取犬胸骨骨髓,体外诱导培养犬自体骨髓间充质干细胞。取成年杂种犬8只,随机分成实验组和对照组,每组4只。实验组将已分化的自体成骨细胞与Bio-Oss骨胶原复合修复犬牙槽骨缺损:对照组牙槽骨缺损处仅植入Bio-Oss骨胶原。每组分别于术后4周,8周各处死2只动物,标本常规切片后行HE和改良Masson三色染色,光学显微镜下观察各组标本的组织学表现。结果实验组4周时即可见骨缺损修复区骨胶原内蓝色的新骨形成,随着时间的推移,形成新骨逐渐矿化成为红色的成熟骨组织,8周时骨缺损修复区可见大片新骨呈岛状或条索状排列,新生牙槽骨骨化效果明显优于对照组,Masson染色为红色。结论组织工程化骨促进犬牙槽骨缺损组织的修复。     

异体骨基质联合重组人骨形成蛋白2与红骨髓修复兔骨缺损

目的：采用NaOH消蚀脱细胞制备异体骨基质(BMM)，观察其与重组人骨形成蛋白2(rhBMP)2及自体红骨髓(ARM)联合移植的成骨效果，为该材料应用提供实验依据。方法：用6％NaOH消蚀兔皮质骨。将支架、rhBMP2及ARM组合分组，移植修复兔桡骨节段性骨缺损，定期X线、组织学及生物力学检测，观察成骨效果。结果：(1)支架几乎未见细胞成分，骨基质一胶原纤维及骨盐结构无改变，无明显排异反应。(2)BMM／rhBMP2／ARM共同移植，修复效果优于其它组，应用ARM组有早期修复现象。结论：采用NaOH消蚀法制作的骨基质支架，可用于骨组织工程；ARM促进早期成骨，与rhBMP2联合应用，可有效修复骨缺损。     

纳米晶胶原基骨复合骨髓单个核细胞修复下颌骨缺损

背景：纳米晶胶原基骨复合骨髓单个核细胞可促进各种干细胞生长,诱导新骨形成和成血管化,促进最终成骨。 目的：探讨骨髓单个核细胞复合纳米晶胶原基骨支架材料修复兔下颌骨缺损的可行性。 方法：选择健康新西兰大白兔27只,制备新西兰大白兔双侧下颌骨人工制备骨缺损模型,分为3组,实验组骨缺损处植入自体骨髓单个核细胞复合纳米晶胶原基骨支架材料,对照组骨缺损处植入纳米晶胶原基骨支架材料,空白组骨缺损处不植入任何材料。术后4,8,12周制备组织标本,行大体观察、影像学分析、苏木       精-伊红染色、扫描电镜检测。 结果与结论：影像学检查及组织学染色显示,实验组骨缺损处愈合程度、成骨速度及质量明显优于其他组;扫描电镜显示实验组材料与骨接触紧密,组织相容性好,无炎症刺激反应;分析牙CT数据及新骨形成检测结果表明,实验组骨修复情况优于其他组(P < 0.05)。表明骨髓单个核细胞复合纳米晶胶原基骨支架材料具有骨诱导和骨形成作用,可用于修复颌骨缺损。     

组织工程骨-软骨复合组织修复兔膝关节部分缺损的实验研*

目的制备具备关节解剖形态的组织工程骨软骨,行原位移植修复兔关节大面积缺损,探讨组织再生方式及特点,为进一步研究提供研究基础。方法以兔耳原代软骨细胞和第三代成骨诱导的骨髓间充质干细胞为种子细胞,制备具有解剖形态的明胶-硫酸软骨素-透明质酸钠-明胶-陶瓷化骨软骨支架。分为复合细胞组﹙A﹚,无细胞组﹙B﹚和不修复组﹙C﹚。进行体内原位移植,术后不同时间进行HE染色,免疫组化染色及PCR检测。结果复合细胞组与其他两组相比,该组伴随支架吸收同时可以形成大量细胞外基质。而随着体内植入时间延长,不同组别间则出现了明显区别,从HE及免疫组化染色结果观察复合细胞组越来越具备正常骨-软骨组织学表现,而无细胞支架组则表现为纤维化改变。结论修复大面积的骨-软骨组织缺损,需要采用支架复合细胞的方式,单纯采用支架其修复效果是不可靠的。     

柚皮苷-壳聚糖/羟基磷灰石复合支架修复大鼠颅骨缺损

背景:骨碎补的有效成分柚皮苷具有补肝肾强筋骨的传统功效,能增加骨痂厚度,提高骨折愈合质量。目的:探究载中药骨碎补有效成分柚皮苷壳聚糖/羟基磷灰石复合支架的骨传导和骨诱导性能。方法:将一定钙磷比的羟基磷灰石前体液与含柚皮苷的壳聚糖溶液在碱性条件下原位结晶、冷冻干燥,获得柚皮苷-壳聚糖/羟基磷灰石多孔支架。将15只成年SD大鼠随机分成空白组(n=5)、对照组(n=5)和实验组(n=5),建立直径5 mm颅骨骨缺损模型,空白组未填充生物材料,对照组填充壳聚糖/羟基磷灰石支架,实验组填充柚皮苷-壳聚糖/羟基磷灰石复合支架。术后4周取材,CT扫描观察颅骨修复情况,苏木精-伊红染色观察颅骨修复的形态学,骨形态发生蛋白2和血管内皮生长因子免疫组化染色后观察缺损区域局部成骨活性因子的表达。结果与结论:(1)CT扫描显示,空白组大鼠颅骨未见明显骨生成,仅在缺损边缘可见少量新生骨;对照组于缺损孔隙可见新生骨形成,新生骨较少;实验组骨缺损修复良好,新生骨组织与缺损孔隙周围颅骨密度相似,大面积新生骨广泛填充了缺损孔隙。(2)苏木精-伊红染色显示,空白组缺损区填充以稀薄的疏松网状纤维组织,可见大量炎性反应病灶,...     

Difference in soft tissue response between immediate and delayed delivery suggests a new mechanism for recombinant human bone morphogenetic protein 2 action in large segmental bone defects

The ability of recombinant human bone morphogenetic protein 2 on absorbable collagen sponge (rhBMP2/ACS) to regenerate bone in segmental defect has been well characterized. However, clinical results of rhBMP2/ACS constructs in secondary reconstruction of large mandibular and craniofacial defects have not been consistent. We hypothesized that rhBMP2 delivery triggers an endogenous response in the soft tissues surrounding the defect, in the form of expression of BMP2 and vascular endothelial growth factor (VEGF). Such osteogenic response will occur only after immediate, as opposed to delayed, rhBMP2 delivery, suggesting a new explanation to the difference in bone regeneration between the two settings. A 35-mm segmental bone and periosteum defect was created on one side of the mandible in 16 dogs divided in three groups. Group 1 (Gp1, n=6) ACS was loaded with 8 mL of rhBMP2 (0.2 mg/mL). In Gp2 (n=5) the same dose of rhBMP2/ACS was delivered into the defect 4 weeks after surgery. In Gp3 (control; n=5) the defect was reconstructed using ACS loaded with 8 mL of buffer only (devoid of rhBMP2). Tissues were collected after 12 weeks of reconstruction in all groups. Direct measurement of physical dimensions of regenerates and bone morphometry was performed to evaluate bone regeneration. The mRNA expression of both BMP2 and VEGF in the soft tissue surrounding the defect was evaluated using real-time quantitative PCR. Both BMP2 and VEGF proteins were quantified in immunostained sections. Immunoflurescence colocalization of BMP2 and acetylated low density lipoprotein (AcLDL) was done to detect the source of BMP2. Immediate delivery yielded better bone regeneration. Both BMP2 and VEGF mRNA expression was upregulated only in Gp1 (+7.3, p=0.001; +1.53, p=0.001, respectively). BMP2 protein was significantly higher in the immediate reconstruction group; however, VEGF protein was undetected in the examined sections. Immediate delivery of rhBMP2 seemed to induce endogenous release of BMP2 from the surrounding soft tissues, an effect that was lacking in delayed delivery and may explain the variability of clinical results associated with BMP2 use. Colocalization of BMP2 and endothelial cells (ECs) suggested that ECs could be the source of endogenous BMP2.     

表面修饰羟基磷灰石修复骨缺损骨形态发生蛋白-2的表达

目的了解精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石（hydroxyapatite,HA）修复节段性骨缺损局部骨形态发生蛋白-2（bone morphogenefic protein-2，BMP-）的表达。方法以骨髓基质干细胞（marrow stromal cels，MSCs）复合Arg-Gly-Asp（RGD）多肽表面修饰的HA或单纯材料培养制备组织工程骨，选择60只新西兰白兔。制作15mm长的桡骨节段性骨缺损模型，根据植入不同的材料分为A、B、C、D组。A组：骨缺损区植入MSCs复合RGD多肽表面修饰的HA培养制备的组织工程骨；B组：骨缺损区植入MSCs复合HA培养制备的组织工程骨；C组：骨缺损区植入RGD多肽表面修饰的HA；D组：骨缺损区植入HA。术后4周取材，行修复区局部BMP-2免疫组化分析。结果术后4周各组骨缺损区均有新骨生成，修复区局部BMP-2表达水平依次为：A〉B〉C〉D（P〈0．05）。结论RGD多肽表面修饰对以HA为支架材料组织工程骨的修复作用有明显优化作用。     

可吸收骨蜡的生物安全性的动物实验研究*

目的研制一种以共聚物Pluronic为基础的可吸收骨蜡,通过动物实验证实其无毒性。方法将固态的Pluronic F88和固态的Pluronic P85按质量比混合、加热、溶解、分装、高压灭菌、冷却,制得可吸收骨蜡;小白鼠12只随机分成实验组和对照组,将可吸收骨蜡样品浸提液注入到实验组小白鼠腹腔内,观察两组动物的一般情况;54只健康SD大白鼠分为可吸收骨蜡组、爱惜康骨蜡组和空白组,每组18只。将可吸收骨蜡植入到大鼠的颅骨缺损处,术后2w、4w、8w测定血常规、血肝肾功。切取植入区的颅顶骨,取出肝、肾,制作组织切片。大体观察颅骨植入区组织有无炎症反应情况,在显微镜下观察肝、肾形态学变化,镜下观察对植入物周围进行定性评价其炎症反应。结果与对照组相比,可吸收骨蜡浸提液组小白鼠术后24h、48h、72h内观察,小鼠呼吸节律匀称,无呼吸困难、腹泻、等腹部刺激症,体重未见明显减轻,无死亡;术后2w、4w时,可吸收骨蜡组与爱惜康骨蜡组血常规作组间单因素方差分析,具有统计学意义(<0.05);术后2w、4w、8w时血生化指标ALT、AST各组组间均有具有统计学意义(<0.05);UREA、CR、UA指标无统计学意义(>0.05)。术后2w在显微镜下观察肝、肾组织结构无明显变化,颅顶骨骨缺损处少量炎性细胞,术后4w、8w镜下观察肝、肾、骨缺损处均无明显变化,无炎性细胞。结论可吸收骨蜡是一种安全、无毒性、可用于骨创面止血的材料。     

大鼠股骨缺损模型中BBP增强BMP-2的骨诱导作用*

目的验证在大鼠节段性骨缺损模型中骨形态发生蛋白结合肽（BMP Binding Peptide,BBP）对于重组人骨形态发生蛋白-2（recombinent human bone morphogenetic protein-2,rhBMP-2）骨诱导作用的影响。方法 70只缺损大鼠分别分成7组,每组不同剂量的rhBMP-2＋/-1000 gBBP,4w和8w后分别摄片,动物8w后处死,股骨样本分别手工评估,采用uCT测量骨容积,随后分别采用组织学和生物力学分析。结果高剂量（10 g）rhBMP-2组术后8w可见骨愈合,骨缺损处骨完全覆盖和桥接,但低剂量（5 g和2 g）rhBMP-2组术后8w骨愈合欠佳。与单独应用rhBMP-2相比,使用低剂量的rhBMP-2复合一定量的BBP可以取得更满意的骨形成量。BBP增强rhBMP-2的骨形成活性发生于4~8w时,而在术后早期并无明显作用。单纯应用BBP仅可见骨缺损处局部的钙化,未见骨愈合。结论 BBP能显著增强rhBMP-2的骨形成活性,这种增强作用需要一定时间来产生效果;其活性发生于术后4~8w时,在术后早期并无明显作用。而且BBP本身并没有骨诱导潜力,仅仅能增强rhBMP-2的骨形成活性。BBP起到缓释作用,与rhBMP-2紧密结合后,让rhBMP-2缓慢而持久的释放。     

非诺贝特对高甘油三酯血症并骨质疏松大鼠骨代谢的影响

目的探讨非诺贝特对去卵巢骨质疏松并高甘油三酯（TG）血症大鼠股骨中细胞核因子κB受体活化因子配体/护骨素（RANKL/OPG）mRNA表达的影响。方法将40只3月龄雌性SD大鼠,用果糖饲养复制高TG模型,大鼠共分为去卵巢＋果糖组、去卵巢＋果糖＋非诺贝特（FF）组、去卵巢＋普食组、假手术＋果糖组。12周后取股骨检测细胞核因子κB受体活化因子配体（RANKL）mRNA、护骨素（OPG）mRNA表达水平。结果去卵巢＋果糖组的TG水平高于去卵巢＋普食组（P〈0.01）,也高于去卵巢＋果糖＋FF组（P﹤0.01）;去卵巢＋果糖组RANKL mRNA/OPG mRNA水平,均高于去卵巢＋果糖＋FF组、去卵巢＋普食组和假手术＋果糖组（P〈0.01或〈0.05）。结论非诺贝特可通过降低TG而保持RANKLmRNA/OPGmRNA的平衡。     

In Situ Gene Expression Analysis During BMP2-induced Ectopic Bone Formation in Mice Shows Simultaneous Endochondral and Intramembranous Ossification

We examined the molecular progression of ectopic bone development upon application of recombinant human bone morphogenetic protein-2 (rhBMP2), using a commercial collagen type I carrier, in the hind quarter muscles of mice. We performed a gene expression study using mRNA in situ hybridisation to compare embryonic cartilage and bone formation with BMP2-induced ectopic bone formation. As bone growth can be induced postnatally or in adult animals, we examined the expression of molecules regulating embryonic bone development. We found that the mRNAs of the same molecules, such as Indian hedgehog (IHH), parathyroid hormone (PTH)/PTH-related peptide receptor (PPR) and BMPs, that regulate embryonic cartilage and bone development, are expressed during BMP-induced ectopic bone formation, suggesting parallels in the mechanisms controlling these processes. Our studies support by molecular means the previous findings in rats that BMP2-induced ectopic bone formation in mice undergoes bone development involving both modes, endochondral and intramembranous ossification, simultaneously at different sites of the implant.     

Adenovirus BMP2-induced osteogenesis in combination with collagen carriers

Adenovirus BMP2 gene therapy has potential of a robust endogenous BMP2 production, while circumventing many of the problems currently associated with recombinant BMP2. The study objective was to determine and compare the ability of adenovirus BMP2 ex vivo gene therapy in combination with three types of collagen carriers to release BMP2 in vitro and to induce heterotopic bone formation in vivo. Human CD45-negative bone marrow cells were ex vivo transduced with a chimeric Ad5F35BMP2. The bioactivity of BMP2 produced by the transduced cells without a carrier, or in combination with three types of collagen carriers (injectable gel, microporous sponge, collagen-mineral composite) was measured and compared to rhBMP2. The heterotopic osteoinductivity assay was performed in immunocompromised NOD/SCID mice. A statistically significant decrease in the amount of rhBMP2 and adenoviral BMP2 released in vitro from the collagen-mineral composite carrier was noted (21% and 12%, respectively), whereas the amounts of rhBMP2 and adenoviral BMP2 released from the gel or sponge carriers were comparable. In vivo, 14 days post-implantation, no bone was formed consistently in groups with the empty Ad5F35HM4 control vector. New bone formation was evident radiographically and histologically in all groups with the Ad5F35BMP2-transduced cells irrespective of the presence or absence of a carrier. The presence of a carrier resulted in osteogenesis limited to the implantation site, and was most pronounced for solid (sponge, composite) carriers. The physical characteristics of the carrier determined the new bone spatial distribution at the site. Solid carriers reduced the clearance of AD5F35-transduced cells by the host immune cells. Adenoviral ex vivo BMP2 gene therapy in combination with collagen carriers with distinct physical characteristics offers the prospects of adjusting this approach to optimally match the specific therapeutic requirements.     

Characterization of human ethmoid sinus mucosa derived mesenchymal stem cells (hESMSCs) and the application of hESMSCs cell sheets in bone regeneration

Mesenchymal stem cells (MSCs) have been extensively applied in the field of tissue regeneration. MSCs derived from various tissues exhibit different characteristics. In this study, a cluster of cells were isolated from human ethmoid sinus mucosa membrane and termed as hESMSCs. hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation. In particular, hESMSCs displayed strong osteogenic differentiation potential, and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Next, hESMSCs were prepared into a cell sheet and combined with a PSeD scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats, which showed excellent reparative effects. Additionally, ELISA assays revealed that secreted cytokines, such as BMP-2, BMP-4 and bFGF, were higher in the hESMSCs conditioned medium, and immunohistochemistry validated that hESMSCs cell sheet promoted the expression of BMP signaling downstream genes in newly formed bone. In conclusion, hESMSCs were demonstrated to be a class of mesenchymal stem cells that possessed high self-renewal capacity along with strong osteogenic potential, and the cell sheet of hESMSCs could remarkably promote new bone regeneration, indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone regeneration.     

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.