Biomimetic mineralization of dentin induced by agarose gel loaded with calcium phosphate

A novel biomimetic mineralization system was designed to induce a layer of hydroxyapatite on a demineralized dentin surface. This system was constructed as follows. A layer of 0.5% agarose gel containing 0.26M Na(2) HPO(4) was used to cover acid-etched dentin slices, followed by a layer of agarose gel without phosphate ions. Then a neutral 0.13M CaCl(2) solution was added onto the ion-free gel surface. The mineralization system (dentin-agarose gel containing phosphate ions-CaCl(2) solution) was kept in a water bath at 37°C, and the gel and CaCl(2) solution were replaced at various intervals. The results showed that the deposited hydroxyapatite crystals densely packed to each other, completely covered the dentin surface, and occluded the dentinal tubules after 10 days of biomimetic mineralization in vitro. Therefore, this method may provide the experimental basis for dentin remineralization and for a new method to treat dentin hypersensitivity and dental caries.     

成骨细胞力致钙响应和钙传递的研究进展

钙是骨系细胞内和细胞间的重要信号分子,同时也是细胞外矿物基质的主要组成部分。尽管100多年的科学实践已经证明力学刺激可以影响骨系细胞内的分子信号转导、细胞间的通讯和协同作用以及组织水平的骨矿化和骨吸收,但为了阐明力致骨重建的机制,仍需在力致钙响应和钙传递方面进行更加深入的研究。本文总结了近年来在成骨细胞的力致钙响应和钙传递方面的研究进展,包括:(1)研究细胞力致钙响应和传递的主要实验手段;(2)成骨细胞感受力学刺激并发生钙响应的钙来源和机制;(3)细胞间钙传递的路径;(4)成骨细胞内力致钙响应和细胞间钙传递的特征参数。最后对该领域未来可能的研究方向进行了展望。     

Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)-co-poly(?-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200–700 nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell–scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.     

The study on the degradation and mineralization mechanism of ion-doped calcium polyphosphate in vitro

Doping with different trace elements can significantly change the original degradability, mineralization, and biological properties of bone repair material. According to the fundamental research on prepared calcium polyphosphate (CPP) as a bone repair material by our group, this article began further exploration on the effect of doping different trace elements (K, Na, Mg, Zn, Sr) into CPP on its degradability and mineralization soaking in simulated body fluids. The results indicated that doped elements significantly changed the lattice parameters and cell volume of crystal, resulted in different types of crystal defect and surface charge distribution, and consequently changed the original degradability and mineralization of CPP. The conclusion is that doped ions with relatively smaller ionic radius and equivalent positive charge compared with Ca(2+) can greatly promote the degradability and mineralization of CPP, whereas doped ions with equivalent ionic radius and diverse positive charges compared with Ca(2+) provide less contribution on promoting the degradability and mineralization or even counteract.     

氟化钠致人成骨细胞凋亡相关基因分析

背景：目前氟致成骨细胞凋亡对线粒体凋亡通路研究不系统,缺乏系统连贯性,不能明确氟引起成骨细胞凋亡具体传导路径。目的：分析氟致成骨细胞凋亡的可能途径及分子特征。方法：以人成骨肉瘤细胞系Saos-2建立体外染氟模型,体外培养细胞后经不同质量浓度NaF(0,5,10,20,40,80 mg/L)处理。用流式细胞仪检测干预24 h后的线粒体膜电位;用PCR功能芯片检测84个与凋亡相关基因;对部分差异表达基因用免疫印记法予以验证。结果与结论：当氟化钠质量浓度为20,40,80 mg/L时,成骨细胞线粒体膜电位分别为27.0%,28.8%,38.6%      (P均< 0.05);PCR芯片检测发现13个基因表达上调,15个基因表达下调。免疫印记显示Bim、Caspase 9、Caspase 14、BCL2、BAX表达随氟化钠剂量增高而增强;Caspase 3在5 mg/L时表达减弱,10 mg/L以上表达逐渐增强。Caspase 7在各组的表达未见明显差异。Caspase 10表达随氟化钠剂量增高而减弱。结果提示,氟致成骨细胞凋亡可能是通过线粒体途径(包括内质网应激途径)及死亡受体途径。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

口腔科氢氧化钙制剂的临床应用评价

BACKGROUND: Calcium hydroxide has been used widely in clinical dental field, and its physical properties and bio-safety become more excellent along with the development of material science. OBJECTIVE: To summarize the physical properties, bio-safety and clinical application of calcium hydroxide materials. METHODS: A computer-based online research was performed in PubMed and CNKI databases using the keywords of “calcium hydroxide, calcium hydroxide preparation” in English and in Chinese, respectively, to retrieve relevant articles published from January 2010 to January 2016. RESULTS AND CONCLUSION: Calcium hydroxide preparation can be respectively divided into mono-component and bio-component, chemical curing and light curing, as well as power form and paste form according to its components, curing form and characteristics. Calcium hydroxide preparation has good physicochemical properties that can be used for dental root canal, root canal filling, direct pulp capping, indirect pulp capping, vital pulpotomy, and apexification. Clinical evaluation and experimental research have confirmed that the calcium hydroxide preparation has good biological safety and clinical effects; however, some urgent problems still need to be studied in depth.      

比较3种形态学方法观察成骨细胞矿化结节的应用价值

背景：矿化结节是成骨细胞分化成熟的标志,以往观察方法多采用茜素红等特殊染色法。 目的：比较茜素红染色-光镜、四环素荧光标记-激光扫描共聚焦显微镜、扫描电镜3种形态学观察成骨细胞矿化结节的方法,分析各自的特点及在骨病研究中的应用价值。 方法：将大鼠成骨细胞株UMR-106正常培养、每天换液,连续培养14 d,分别采用茜素红染色-光镜、四环素荧光标记-激光扫描共聚焦显微镜、扫描电镜观察矿化结节的形态结构,并利用扫描电镜结合能谱仪原位定量分析钙元素;此外,在培养中加入可抑制成骨细胞增殖、分化的肿瘤坏死因子α作为对照实验。 结果与结论：3种观察方法均可观察到正常成骨细胞矿化结节的形态结构。对于肿瘤坏死因子α抑制成骨细胞产生矿化结节的情况,经茜素红染色-光镜观察法,未见明显的矿化结节;而四环素荧光染色-激光扫描共聚焦显微镜观察法,可见清晰、稀少的矿化结节;扫描电镜观察法明显可见较少而细小的矿化结节,提示后二种方法灵敏度较高。此外,扫描电镜可将观察成骨细胞分泌钙质、形成矿化结节的亚细胞结构,与能谱元素分析结合,可实现矿化结节的定位与定量,在骨病研究中值得推广应用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Erosion process of calcium hydroxide cements in water.

Erosion process of calcium hydroxide cements was examined for 7 days by chemical analysis of eluates and observation of structural change of the eroded cements when the cements were immersed in water at 37 degrees C. The elution of salicylate from the set cements as well as that of Ca continued during the immersion time. In the early stage of the erosion, unreacted Ca(OH)2 was preferentially extracted from the cements in comparison with Ca-alkyl salicylate chelate of the cement matrix. The elution rate of N-ethyl o- and p-toluene sulphonamides, which were contained as plasticizer, was higher than those of Ca and salicylate. Insoluble inorganic filler remained at the surface of the cements after extraction of unreacted Ca(OH)2 and disintegration of the cement matrix. Types of salicylate ester seemed to affect the cement durability.     

Molecular basis of osteoclastogenesis induced by osteoblasts exposed to wear particles

In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al(2)O(3)) or 'ultra-high molecular weight polyethylene' (UHMWPE) particles for 24h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1 microg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al(2)O(3) HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al(2)O(3) wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.     

重组人骨形成蛋白-2对细胞成骨分化的作用

目的 进一步探讨rhBMP-2的促细胞成骨分化作用，以期找到合适的成骨分化标志作为rhBMP-2的定量活性测定指标。方法 首先表达制备rhBMP-2，用小鼠股部肌袋包埋法进行诱骨活性实验，然后检测rhBMP-2作用后的骨髓基质细胞（MSC）、NIH3T3和C2C12等3种细胞的碱性磷酸酶（ALP）和骨钙素（OC）、细胞总蛋白合成量以及细胞增殖的变化。结果 rhBMP-2具有良好的诱导骨形成的活性，可增加3种细胞的OC含量和蛋白合成量，对MSC的ALP活性变化影响明显，且可促进MSC的增殖，抑制NIH3T3细胞的生长。结论 rhBMP-2具有促进上述细胞向成骨细胞分化的作用；在一定剂量范围内，rhBMP-2的作用与细胞骨钙素合成量的增加呈线性正相关，故定量测定OC的含量基本可反映rhBMP-2的活性。     

Substrate geometry directs the in vitro mineralization of calcium phosphate ceramics

Repetitive concavities on the surface of bone implants have recently been demonstrated to foster bone formation when implanted at ectopic locations in vivo. The current study aimed to evaluate the effect of surface concavities on the surface mineralization of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics in vitro. Hemispherical concavities with different diameters were prepared at the surface of HA and β-TCP sintered disks: 1.8 mm (large concavity), 0.8 mm (medium concavity) and 0.4 mm (small concavity). HA and β-TCP disks were sintered at 1100 or 1200 °C and soaked in simulated body fluid for 28 days at 37 °C; the mineralization process was followed by scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction and calcium quantification analyses. The results showed that massive mineralization occurred exclusively at the surface of HA disks treated at 1200 °C and that nucleation of large aggregates of calcium phosphate started specifically inside small concavities instead of on the planar surface of the disks. Regarding the effect of concavity diameter size on surface mineralization, it was observed that small concavities induce 124- and 10-fold increased mineralization compared to concavities of large or medium size, respectively. The results of this study demonstrated that (i) in vitro surface mineralization of calcium phosphate ceramics with surface concavities starts preferentially within the concavities and not on the planar surface, and (ii) concavity size is an effective parameter to control the spatial position and extent of mineralization in vitro.     

Expression and ultrastructural immunolocalization of a major 66 kDa phosphoprotein synthesized by chicken osteoblasts during mineralization in vitro

Embryonic chicken osteoblasts cultured over a 30 day period were used as a model system for studying the expression of bone phosphoproteins during cellular differentiation and the possible role of these proteins in extracellular matrix mineralization. Accumulation of total phosphoprotein in the cultures, as determined by O-phosphoserine (Ser-P) and O-phosphothreonine (Thr-P) amino acid analysis, revealed a greater than 10-fold increase over the 30 day period. Total phosphoprotein synthesis, as assessed by (32P)-, (3H)-Ser-P, and (14C)-Thr-P protein labeling, showed the highest levels concurrent with initial mineral deposition within the matrix. The major phosphoprotein present in chicken bones and synthesized by the cultured osteoblasts had a molecular weight of approximately 66 kDa. This 66 kDa bone phosphoprotein (66 kDa BPP) was purified to homogeneity and was used for antibody production. Application of this antibody in Western blot analysis revealed that 66 kDa BPP was present only in protein extracts of mineralizing cultured osteoblasts and was absent in cultures of non-mineralizing chondrocytes, myoblasts, and tendon fibroblasts. The 66 kDa BPP in vitro accumulated continuously in the extracellular matrix in a manner that paralleled both phosphoprotein synthesis and total phospho-amino acid production. A comparison of the results obtained in vitro to those from developing embryonic tibiae in vivo demonstrated a similar qualitative and temporal expression of phosphoprotein and a continual accumulation of 66 kDa BPP in the matrix with advancing mineralization and developmental age. Ultrastructural immunocytochemistry using the 66 kDa BPP antibody and the protein A-gold technique revealed specific immunolabeling over electron-dense regions of mineralization in the cultures that appeared identical to the distribution of labeling observed in vivo (McKee et al.: Connect. Tissue Res., 21:21-29, 1989; Anat. Rec., 228:77-92, 1990). These results demonstrate that this major 66 kDa BPP was expressed concurrently with other differentiated osteoblast functions and suggests that it may play a role in the initiation or regulation of mineralization.     

正弦交变电磁场促进体外培养成骨细胞成熟矿化的时间效应

研究正弦交变电磁场(SEMFs)促进体外培养成骨细胞(OB)成熟矿化的时间效应。OB培养后随机分成7组,其中6组用50Hz、1.8mT SEMFs处理,处理时间为每天0.5、1.0、1.5、2.0、2.5和3.0h,1组0h(对照组)。倒置相差显微镜下观察各组细胞形态;48h后检测细胞增殖情况;第3、6、9、12d测定碱性磷酸酶(ALP)活性,第10d茜素红钙化结节染色。磁场处理8d后形成漩涡样分布的钙化节结;各磁场处理组均抑制细胞增殖;ALP活性从0.5～1.5h依次递增,从1.5～3.0h依次递减,1.5h组的ALP活性始终高于对照组(P<0.05),第9d时显著高于其他各磁场处理组(P<0.05);茜素红染色结果显示,1.0、1.5和2.0h组的钙化结节数明显多于对照组。50Hz、1.8mT SEMFs抑制体外培养OB的增殖,但对其分化成熟和矿化过程有显著促进作用,此作用与磁场处理时间有关,尤以1.5h促分化效果最为明显。     

A composite coating by electrolysis-induced collagen self-assembly and calcium phosphate mineralization

A composite coating that is composed of collagen protein and calcium phosphate minerals is considered to be bioactive and may enhance bone growth and fixation of metallic orthopedic implants. In this study, we have successfully developed a uniform collagen fibril/octacalcium phosphate composite coating on silicon substrate by electrolytic deposition (ELD). The coating deposition was done through applying a constant potential to the cathode in a three-electrode electrochemistry cell that contain a mild acidic (pH 4.8-5.3) aqueous solution of collagen molecules, calcium and phosphate ions. The coating process involved self-assembly of collagen fibrils and the deposition of calcium phosphate minerals as a result of cathode reaction and local pH increase. The two steps could be synchronized to form a bone-like composite at nanometer scale through proper adjustment of the solution and deposition parameters. Coating morphology, crystal structure and compositions were analyzed by optical and fluorescence microscopy, scanning and transmission electron microscopy, energy dispersive X-ray analysis, inductively coupled argon plasma optical emission spectrophotometry, and Fourier-transformed infrared spectroscopy. Under typical deposition conditions, the cathode (Si) surface formed a thin (100 nm) layer of calcium phosphate coating, on top of which a thick (approximately 100 microm) composite layer formed. The porous composite layer consists of a collagen fibril network on which clusters of octacalcium phosphate crystals nucleate and grow. By combining photolithography and ELD, we were also able to pattern the composite coating into regular arrays of squares. Preliminary results by nanoindentation tests showed that properly prepared composite coating may have higher elastic modulus and scratch resistance than monolithic porous calcium phosphate coating. The results not only provide a novel bioactive coating for biomedical implants, but also establish a new experimental protocol for studying biomineralization mechanisms of collagen based biological tissues.     

An explanation of the dose inhomogeneity caused by bolus in an electron portal

When an electron beam is incident upon a stepped surface, such as that which exists when a slab of bolus is present with it's square edge within the portal, the dose distribution is strikingly perturbed. An easily demonstrable explanation is given which utilizes the customary, flat-incident surface electron beam dose distribution.     

The reactivity of nano silica with calcium hydroxide

The reactivity of nano silica (SiO?) with calcium hydroxide (Ca(OH)?) was evaluated and characterized in this study. Ca(OH)? activated nano SiO? takes place through an exothermic process, which is mainly attributed to the breakdown of Si-O-Si bonds. Ca(2+) offsets the charge imbalance and bonds to Si-OH and Si-O(-) giving rise to calcium silicate hydrate (CSH) gel. Care has to be taken that the reactivity of nano SiO? with Ca(OH) ?significantly depends on the Q3 percentage in nano SiO ?. Q3 percentages significantly influence the reaction kinetic of nano SiO? . The higher Q3 percentage results in a higher reaction degree of nano SiO? with Ca(OH)? and shorter setting times of the pastes. The higher Q3 percentage results in a lower total reaction heat of nano SiO? with Ca(OH)?. It is suggested that the Q3 percentages of nano SiO? should be in excess of 30% to keep the satisfactory setting properties of the pastes for the application requirements of bone cement.     

The modulation of gene expression in osteoblasts by thrombin coated on biphasic calcium phosphate ceramic

For many years, fibrin sealants were associated with bone substitutes to promote bone healing. However, the osteoblastic response to fibrin sealant components remains poorly documented. In this study, MC3T3-E1 osteoblastic cells were cultured on biphasic calcium phosphate ceramic (MBCP) coated with Tissucol components (thrombin and fibrinogen). Analysis of osteoblastic differentiation markers by RT-PCR revealed that MBCP coated with Tissucol stimulated mRNA levels for osteocalcin and alkaline phosphatase (ALP). Of all the components of Tissucol, thrombin has been reported to affect osteoblastic behavior. Our results demonstrated that low thrombin concentrations (0.5-5 U/ml) stimulated mRNA levels for ALP, whereas high thrombin concentrations (50-100 U/ml) decreased mRNA levels for ALP and PTH/PTHrP receptor and also increased mRNA level for the osteoclastogenesis inhibitor OPG. As thrombin stimulated angiogenesis, we then wondered whether thrombin could influence the expression of angiogenic factors. Low thrombin concentrations were shown to up-regulate mRNA levels for VEGF-B and VEGF-R1, suggesting an autocrine/paracrine role for VEGF-B. Higher thrombin concentrations also up-regulated mRNA for VEGF-A and neuropilin-1. In conclusion, the association of MBCP with thrombin and fibrinogen appears to be a convenient scaffold for bone cell differentiation. Thrombin could also acts at the cellular level by increasing the angiogenic potential of osteoblasts as well as their responsiveness to thrombin and VEGF.     

Enzymatically induced mineralization of platelet-rich fibrin

Membranes of the autologous blood-derived biomaterial platelet-rich fibrin (PRF) were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, and subsequently incubated in calcium glycerophosphate (CaGP) solution to induce PRFs mineralization with calcium phosphate (CaP) to improve PRFs suitability as a material for bone replacement. Incorporated ALP retained its bioactivity and induced formation of CaP material within PRF membranes, as confirmed by SEM, EDS, FTIR, and von Kossa staining. The mass percentage attributable to CaP was quantified by lyophilization and measurement of the remaining mass fraction as well as by TGA. Cytocompatibility tests (LDH, MTT, and WST) with SAOS-2 cells showed that mineralized PRF did not release substances detrimental to cell vitality. Live/dead staining and SEM showed that mineralized PRF was colonized by cells. The results show that hydrogel biomaterials such as PRF can be mineralized through functionalization with ALP.     

The interaction between osteoclast-like cells and osteoblasts mediated by nanophase calcium phosphate-hybridized tendons

We developed a novel technique of hybridizing calcium phosphate (CaP) with bioorganic soft tissue using an alternating soaking process. By this technique, we hybridized CaP with a grafted tendon tissue to bond with a bone tunnel. Tendons were soaked in Ca and NaHPO(4) solutions alternately for 10 min. Needle-like CaP crystals 30-50 nm in length including low-crystalline apatite were deposited on and between collagen fibrils from the surface to 200 microm deep in the tendon. In light and transmission electron microscopic images, osteoclast-like cells and osteoblasts appeared on the implanted tendon and osteoid was observed on the tendon surface at 1 week postoperatively. At 2 weeks postoperatively, osteoclast-like cells resolved the tendon by forming Howship's lacuna-like spaces on the surfaces and osteoblasts formed osteoid in these spaces. Direct bonding between the implanted tendon and the newly formed bone was observed. At 3 weeks postoperatively, thick newly formed bone firmly bonded to tendon surface. From these results, we conclude that the tendons prepared by an accelerated CaP hybridization method efficiently enhance osteoclast-like cells and osteoblasts to bond the implanted tendons to newly formed bone.     

Uptake of calcium phosphate nanoshells by osteoblasts and their effect on growth and differentiation

The influence of calcium phosphate nanoshell materials on the uptake, viability, and mineralization of human fetal osteoblast cultures was evaluated. Proliferation rates and alkaline phosphatase activity of the cultures were unaffected by the addition of nanoshells to the growth media, but mineralization levels were enhanced by nearly 40%, in contrast to media prepared without nanoshells, or with other calcium phosphate nanomaterials. Nanoshells were internalized by macropinocytosis, and migrated toward the cell nucleus at a rate of 0.34 microm hr(-1). Dye-loaded nanoshells maintained high light emission intensity for over five days while inside the cells, where they could be used as intracellular markers for in vitro microscopic imaging. From these results, it appears that the CaP nanoshells could be developed into a safe sensor and delivery vehicle for osteoblast cell culture studies, whereas the carrier itself has intrinsic bioactivity and may itself upregulate the formation of new bone.