A comparative analysis of strategies for isolation of matrix vesicles

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.     

Bifunctional Platinum(II) Complexes with Bisphosphonates Substituted Diamine Derivatives: Synthesis and In vitro Cytotoxicity

A series of N,N′‐dibisphosphonate‐containing 1,3‐propanediamine derivatives ( L1 – L6 ) and their corresponding dichloridoplatinum(II) complexes ( 1 – 6 ) have been synthesized and characterized by elemental analysis, ¹H‐NMR, ¹³C‐NMR, ³¹P‐NMR and HR‐MS spectra. The in vitro antitumor activities of compounds L1 – L6 and 1 – 6 were tested by WST‐8 assay with Cell Counting Kit‐8, indicating that platinum‐based complexes 1 – 6 showed higher cytotoxicity than corresponding ligands L1 – L6 against A549 and MG‐63, especially complex 2 which displayed comparable cytotoxicity to those of cisplatin and zoledronate after 48 h incubation. In addition, complexes 1 – 6 were more active in vitro on osteosarcoma cell line MG‐63 than normal osteoblast cell line hFOB 1.19. The structure‐activity relationship has been summarized based on the in vitro cytotoxicity of three series of platinum complexes from this and our previous studies. The in vitro bone affinity of platinum complexes was also tested by hydroxyapatite (HAP) chromatography in terms of capacity factor K′. Besides, in this paper, representative complex 2 , which has been proved to be a promising antitumor agent with high cytotoxicity and bone HAP binding property, was investigated for its mechanism of action producing cell death against MG‐63.     

Tunable hydroxyapatite wettability: Effect on adhesion of biological molecules

Surface wettability modifications of biocompatible materials and wettability patterning are attractive methods for directed biological cells immobilization for tissue engineering, drug delivery, gene transfer, etc. Hydroxyapatite is known as an implantable biomimetic material and a substrate for effective adhesion of biological cells of various origins. Here we report the use of a low-energy electron irradiation to achieve tunable wettability of the hydroxyapatite in a wide range of contact angles, from 10° to 100°, with accuracy of ±3°. The incident electrons generate electron/hole pairs resulting in significant variation of the surface potential of the hydroxyapatite semiconductor and give rise to pronounced wettability modification. Tailoring the gradually varied wettability state in the hydroxyapatite nanoceramics enabled the differential binding of biological materials with different surface properties, such as bovine serum albumin (BSA) and deoxyribonucleic acid (DNA).     

Evolution of sea urchin non-repetitive DNA

Summary New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin speciesStrongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, andLytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners,S. purpuratus, andS. franciscanus, which apparently diverged only 15 to 20 million years ago.     

High-resolution transmission electron microscopy of adult human peritubular dentine

Summary Single crystals from adult human peritubular dentine were studied by high-resolution transmission electron microscopy. Periodic fringe patterns were obtained from which the exact shape of the inorganic crystals were deduced. The crystals were found to have a mean length of 36.00±1.87 nm, a mean width of 25.57±1.37 nm, and a mean thickness of 9.76±0.69 nm. They consisted of platelets with a mean width-to-thickness ratio of 2.61, each being a flattened hexagonal prism of hydroxyapatite. Such conclusions are based upon a) the electron diffraction patterns that we obtained, and b) our comparison of the values of the periodic, equidistant fringes seen along different planes of sectioning with the corresponding theoretical values for hydroxyapatite.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

Spectral characterization of hydroxyapatite extracted from Black Sumatra and Fighting cock bone samples: A comparative analysis

At present, chicken business is occupying a major portion in the market and huge amount of bone wastes are dumped into the open places lead in environmental pollution. In this analysis, natural hydroxyapatite was extracted by thermal calcination process at different temperature ranges from 700 °C, 900 °C and 1100 °C and compared its spectral characteristics. The crystalline nature, functional groups and morphological characteristics of hydroxyapatite obtained from both bone samples were studied using XRD, FTIR and SEM analysis. The crystallite size, lattice parameters, specific surface area, volume and degree of crystallinity were measured using XRD data. The mean grain size of Black Sumatra and Fighting Cock bone hydroxyapatite was 62.67 nm and 31.34 nm respectively. The FTIR spectrum showed major peaks at 634.58 cm⁻¹ and 470.63 cm⁻¹, 1413.82 cm⁻¹ and 1460 cm⁻¹ indicates the presence of carbonate group and phosphate groups in both samples. The SEM micrograph confirmed the existence of maximum pores in matrix of fighting cock bone than Black Sumatra bone sample. Thus, the comparative analysis concluded that nano-sized hydroxyapetite obtained from bone wastes of fighting cock can be utilized as a low-cost biomaterial for the production of various implant coating materials and substitute for ceramics in bones and dentistry applications.     

三种不同表面活性剂对羟基磷灰石纳米颗粒的表面修饰的比较

目的:本研究旨在通过不同方法修饰羟基磷灰石纳米颗粒并检测其稳定性及分散性。方法:首先采用水合热合成法制备羟基磷灰石纳米颗粒,然后用透射电镜(TEM)和场发射扫描电镜(SEM)对其表面形态结构进行表征。我们首次用溴化十六烷三甲基铵(CTAB),PEG2000和人血清对羟基磷灰石纳米颗粒通过共价结合或表面吸附的方式进行表面嫁接,并利用透射电镜,傅里叶红外光谱(FT-IR)和X射线衍射(XRD)对新合成的这三种纳米羟基磷灰石复合物的形貌,结构和晶粒粒径进行表征。对这三种羟基磷灰石纳米颗粒悬浮液的时间沉降曲线进行分析。在分散性上通过检测这三种羟基磷灰石复合物悬浮液在不同pH值下的Zeta电位并绘制Zeta-pH曲线。结果:我们发现CTAB修饰的羟基磷灰石纳米颗悬浮液的悬浮稳定性最佳,其次是PEG2000,最后是人血清。在pH=7.0时,CTAB修饰的羟基磷灰石纳米颗粒的zeta电位值是25.68 m V,而PEG2000修饰的Zeta电位是4.32m V,人血清修饰的Zeta电位是-13.23m V。结论:CTAB表面修饰的羟基磷灰石纳米颗粒相对于其它两种表面活性剂复合物具有更好的分散性和悬浮稳定性,与DNA/RNA结合能力更强。本课题的结果给羟基磷灰石纳米颗粒载体的应用提供了一种新的选择,有望利用亲和力更高的基因载体实现基因治疗,具有广阔的应用前景。     

Role of matrix vesicles in biomineralization

Background

Matrix vesicles have been implicated in the mineralization of calcified cartilage, bone and dentin for more than 40 years. During this period, their exact role, if any in the nucleation of hydroxyapatite mineral, and its subsequent association with the collagen fibrils in the organic matrix has been debated and remains controversial.

Scope of Review

This review summarizes studies spanning the whole history of matrix vesicles, but emphasizes recent findings and several hypotheses which have been recently introduced to explain in greater detail how matrix vesicles function in biomineralization.

Major Conclusions

It is now generally accepted that matrix vesicles have some role(s) in mineralization; that they are the initial site of mineral formation; that MV bud from the plasma membrane of mineral forming cells, but that they take with them only a subset of the materials found in the parent membrane; that the three proteins, alkaline phosphatase, nucleotide pyrophosphatase phosphodiesterase and annexin V have important roles in the process and that matrix vesicles participate in regulating the concentration of PPi in the matrix. In contrast, many open questions remain to be answered.

General Significance

Understanding the role of matrix vesicles in biomineralization will increase our knowledge of this important process.     

Combined computational study of mechanical behaviour and drug delivery from a porous, hydroxyapatite-based bone graft

This paper presents a numerical model of a porous, hydroxyapatite-based bone graft also suitable as a drug delivery device. The graft was positioned in different sites and with different porosities inside a human femur model. The structural analyses were carried out to verify the graft mechanical strength, using the Tsai–Wu criterion, and the maximum porosity at which static failure does not occur. A local stress shielding risk was also calculated as the ratio between the bone stress in the intact condition and the stress after implantation of the graft. Drug release kinetics was calculated by means of the finite element method. High porosity grafts were found to fail in all implantation sites. Lower porosity grafts showed to have adequate strength if implanted in some positions, while provided insufficient resistance for other implantation sites. Drug release kinetics was found to be strongly dependent both on the porosity of the graft and the bone density near the bone-graft interface.