Zoledronic acid suppresses mineralization through direct cytotoxicity and osteoblast differentiation inhibition

J Oral Pathol Med (2012) 41 : 713–720 Background: Zoledronic acid (ZA) is prescribed to treat various metabolic bone diseases. Despite its efficacy in preventing bone loss, ZA has been linked to osteonecrosis of the jaw in several reports. However, a mechanism underlying this occurrence is still unclear. Objective: This study was to investigate causative roles of ZA on osseous cellular activities of pre‐osteoblastic cell line MC3T3‐E1 (MC3T3) and mesenchymal stem cell (MSC). Methods: Morphological analysis, RT‐PCR, annexin V/PI staining, together with mineralization, cell viability, and alkaline phosphatase (ALP) activity assays were performed. Results: Zoledronic acid treatment decreased bone nodule formation at all concentrations tested (0.01–100 μM). Cell morphologies of both cell types were altered from their normal appearances after the addition of ZA (≥5 μM), and cell viability was significantly inhibited at concentrations ≥0.1 μM for MC3T3 and at concentrations ≥10 μM for MSC. ZA (100 μM) induced apoptosis in MC3T3 and MSC. Furthermore, ALP activity from both cells was strongly reduced when exposed to ZA (≥1 μM for MC3T3 and ≥5 μM for MSC). ZA also down‐regulated Runx 2 and Col I mRNA expressions. Conclusion: With this in vitro study, ZA mediated defective bone mineralization by directly disrupting osteoblast/osteoprogenitor cellular activities at several levels, that is, cell proliferation, osteoblast differentiation, and osteoblast function of both pre‐osteoblastic cells and MSC.     

rhBMP-2和FGF-2对成骨细胞矿化及ENPP1、ANK、TNAP表达的影响

目的:研究重组人骨形态发生蛋白(rhBMP-2)和重组人碱性成纤维细胞生长因子(FGF-2)对成骨细胞(MC3T3-E1 Subclone 14)矿化及焦磷酸合成酶(ENPP1)、跨膜蛋白(ANK)和组织非特异性碱性磷酸酶(TNAP)表达的影响,探讨生长因子影响细胞矿化的机制。方法:将MC3T3-E1 Subclone 14细胞分成3组:成骨细胞诱导液(OS)成骨诱导培养组(对照组),OS与rhBMP-2培养组(rhBMP-2组),OS与FGF-2培养组(FGF-2组)。培养12 d后进行ALP活性检测及茜素红染色,实时荧光定量PCR检测矿化相关基因ENPP1、ANK和TNAP表达的差异。结果:rhBMP-2组ALP活性以及钙化结节明显高于对照组,ENPP1、ANK和TNAP均高表达;FGF-2组ALP活性以及钙化结节明显低于对照组,ENPP1和ANK呈高表达,TNAP低表达。结论:rhBMP-2和FGF-2通过调节ENPP1、ANK和TNAP的表达变化来影响骨的矿化。     

可注射型热触发仿天然骨材料的基础研究

目的：试图研制可注射型热敏仿天然骨材料,测定其理化特性,并将其初步应用于体外根尖孔破坏的离体牙模型上。方法：采用逆向蒸发法制备载碱性磷酸酶（alkalinephosphatase,ALP）的脂质体,考察其包封率、活性率及相变温度。将载ALP、载钙离子（Ca^24）和磷酸盐（Pi）3种脂质体与β-甘油磷酸盐（β-glycerophosphate,β-GP）混合制备矿化液,观测矿物质的形态、元素分析及红外光谱曲线;将I型胶原与以上矿化液混合制备的复合材料应用于根尖孔破坏的离体牙根尖周围,根管充填并统计分析。结果：载ALP的脂质体平均包封率为25．1％,平均活性率为16．5％,相变温度为37．3～40．1℃,峰值为39．2℃;与未加ALP者相比,所得矿化物形态和Ca／Pi比与天然骨中矿化物更接近,红外光谱曲线表明其为羟基磷灰石;体外模型显示该复合材料可在一定程度上支托根充材料,预防超充。     

重组人细胞外基质磷酸化糖蛋白对人牙髓细胞体外矿化的影响

目的:探讨细胞外基质磷酸化糖蛋白(Matrix extracellular phosphogl ycoprotein,MEPE)对人牙髓细胞(human dental pulpcells,HDPCs)在体外的矿化能力的影响,从而了解该蛋白在牙齿生长发育过程中的作用。方法:常规方法培养获得人牙髓细胞,实验组中使用的MEPEs的浓度为100μg/L。通过Vonkossa染色观察MEPE对人牙髓细胞矿化的影响。结果:MEPE蛋白能促进人牙髓细胞的矿化。结论:MEPE具有促进人牙髓细胞的矿化的能力,可能在牙齿生长发育过程中起着重要的作用。     

Bone formation after sinus augmentation with engineered bone

Objectives: The aim of the following investigation was to quantify the resorption rate of tissue-engineered bone grafts in the maxillary sinus using volume measurements. MATERIAL AND METHODS: Sinus floor augmentation using autologous bone grafts from the iliac crest (n=17, group 1) was compared with commercially produced transplants of human cells seeded on polyglycolid-polylactid (PLGA) scaffolds (Oral Bone) (n=14, group 2). RESULTS: The total resorption rate for autologous transplants 3 months post operation was 29%, while the tissue-engineered bone showed a resorption rate of 90%. The autologous bone had a bone density of up to 266-551 Hounsfield units (HU), while sufficient mineralization of tissue-engineered bone was found in only one case (152 HU). CONCLUSION: In this clinical study, the use of autologous cancellous bone grafts in sinus augmentation was more reliable than scaffolds containing cultured osteoblasts. Further tissue-engineered bone transplants should be examined to draw general conclusions about the use of tissue-engineered grafts compared with autologous bone grafts for maxillary sinus augmentation.     

Dentin structure in familial hypophosphatemic rickets: benefits of vitamin D and phosphate treatment

OBJECTIVE: To evaluate the outcome of 1-(OH) vitamin D and oral phosphate treatment on dentin structure in patients with familial hypophosphatemic rickets, and expression of SIBLINGs (a family of non-collagenous proteins involved in dentinogenesis) and osteocalcin. PATIENTS AND METHODS: Seven patients with familial hypophosphatemic rickets (age 3-16 years) were studied before or during treatment. Deciduous and permanent teeth were prepared for scanning electron microscopy (SEM) analysis and immunohistochemistry. RESULTS: Untreated or inadequately treated patients had necrotic teeth with impaired dentin mineralization including unmerged calcospherites and accumulation of non-collagenous proteins in wide interglobular spaces. Most of the primary incisors analyzed displayed fissures linking enamel subsurface to pulp horn. These elements may explain the bacterial penetration and dental abscesses despite the absence of carious lesions. Well-treated patients had healthy teeth with good dentin mineralization and little evidence of calcospherites. CONCLUSION: Treatment of hypophosphatemic children with 1-(OH) vitamin D and oral phosphate insures good dentin development and mineralization, and prevents clinical anomalies such as the dental necrosis classically associated with the disease. Starting treatment during early childhood and good adherence to the therapy are mandatory to observe these beneficial effects.     

Costly teeth? Gestation length in primates suggests that neonate dentition is not expensive to produce

Variation in the relationship between gestation length and body mass can arise because different types of tissue require varying amounts of energy to build, and not all species build such tissues in the same proportions. Given that a pregnant female has a finite amount of energy, trade-offs between investment in different tissues may occur. Here we examine if dental precocity accounts for variation in primate gestation length. If true, this could explain why folivorous species with precocial dentition have longer gestation lengths than predicted by neonatal brain and body mass. We compiled data on gestation length, neonate and adult female body and brain mass from the literature. We used published postcanine eruption schedules at 4 months of age and measured the total occlusal area as dental endowment to approximate dental precocity at birth. Species with embryonic delay in growth or altricial neonates were not considered because they represent grade shifts regarding gestation length. Consequently, our data were biased toward Simiiformes and Old World monkeys, specifically. We performed a phylogenetic generalized least squares regression (pGLS) of neonate brain mass in relation to neonate body mass, and a second pGLS with dental endowment as an additional predictor variable. Including dental endowment in the pGLS did not improve the model. Dental endowment did not systematically impact primate gestation length. Concordant with results from previous studies, this indicates that the energetically expensive period of tooth mineralization may occur postnatally. More data are required to examine if the results are typical across primates.     

透明质酸/壳聚糖复合支架的制备及其力学性能评价

目的　模拟天然骨组织的结构和成分,寻找适合骨组织工程的新型支架材料。 方法 以透明质酸、壳聚糖为基质材料,在微酸性环境中以一定配比与氯化钙和磷酸二氢钠混合,冷冻干燥得到多孔复合支架材料。然后在乙醇/水/尿素环境中分别陈化0、2、4、8、12和24 h,以生成产物钙磷盐前驱体转变为羟基磷灰石,最终制备出一种深度矿化的透明质酸/壳聚糖复合支架。并通过SEM、EDS等对支架进行表征,研究支架的形貌、成分及力学强度等性能。 结果 SEM观察显示,支架材料具有比较均匀的多孔结构,孔径大小为100~200 μm。EDS结果表明,复合支架在一次冻干之后形成的是磷酸氢钙（DCPD）,随着陈化时间的延长,DCPD逐渐向羟基磷灰石（HAP）转化。而压缩强度则表明经过原位矿化的支架力学性能显著提高。 结论 通过该法得到的透明质酸/壳聚糖复合支架可作为骨组织工程的新型支架材料。     

被动吸烟对鼠牙槽骨钙、磷含量及碱性磷酸酶活性的影响

目的 分析被动吸烟鼠牙槽骨钙、磷含量和碱性磷酸酶（AKP）的活性。方法 40只6周龄健康雄性Wistar鼠被随机等量分为实验组和对照组,每组再被随机等量分为15、30、45和60 d 4个亚组,烟熏法建立被动吸烟的大鼠模型,浓酸消化法提取牙槽骨内钙和磷,光谱分析法和改良Reddi法分别测定钙、磷含量和AKP的活性,行析因设计的方差分析。结果 与各对照组牙槽骨相比,被动吸烟鼠的钙、磷含量在15 d组略有升高,30 d组略有下降,差异无统计学意义（P〉0.05）,45、60 d组显著下降（P〈0.01）;AKP活性在15、30 d组略升高,45、60 d组略下降,差异无统计学意义（P〉0.05）。结论 本实验周期内,鼠牙槽骨钙、磷含量和AKP活性在被动吸烟初期略有升高,随被动吸烟时间的延长,钙、磷含量呈时间依赖性减少,AKP活性也呈时间依赖性减少的趋势。