The effect of three-dimensional demineralized bone matrix on in vitro cumulus-free oocyte maturation

The physiological role of cumulus cell surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. Collagen-based demineralized bone matrix (DBM) is a valuable biomaterial for the three-dimensional (3-D) cell culture. The present study was designed to determine whether in vitro maturation (IVM) of cumulus-free oocytes in mice could be improved by using the 3-D DBM co-culture system. The results indicated that the denuded oocytes cultured in 3-D DBM co-culture system with cumulus cells showed close similarity of cortical granules (CGs) distribution pattern, had more normal maturation-promoting factor (MPF) level and zona pellucida (ZP) hardening level to the in vivo matured oocytes, and the best preimplantation development after being activated by in vitro fertilization (IVF) or parthenogenetic activation. Thus, 3-D DBM collagen scaffold could serve as a tool for fundamental in vitro studies of cells or tissues under the environment that closely assembles the in vivo conditions.     

The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins

Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.     

The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer

The vacuolar-ATPase (v-ATPase) is a proton transporter found on many intracellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cancer cells may contribute to their invasive properties in vitro. Its relevance to human cancer tissues remains unclear. We investigated whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic cancer, and its effect on matrix metalloproteinase (MMP) activation in vitro. The intensity of v-ATPase staining increased significantly across the range of pancreatic histology from normal ducts to pancreatic intraepithelial neoplasms (PanIN), and finally pancreatic ductal adenocarcinoma (PDAC). Low-grade PanIN lesions displayed polarized staining confined to the basal aspect of the cell in the majority (86%) of fields examined. High-grade PanIN lesions and PDAC showed intense and diffuse v-ATPase localization. In pancreatic cancer cells, PM-associated v-ATPase colocalized with cortactin, a component of the leading edge that helps direct MMP release. Blockade of the v-ATPase with concanamycin or short-hairpin RNA targeting the V?E subunit reduced MMP-9 activity; this effect was greatest in cells with prominent PM-associated v-ATPase. In cells with detectable MMP-2 activities, however, treatment with concanamycin markedly increased MMP-2's most activated forms. V-ATPase blockade inhibited functional migration and invasion in those cells with predominantly MMP-9 activity. These results indicate that human PDAC specimens show loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. Thus, v-ATPase selectively modulates specific MMPs that may be linked to an invasive cancer phenotype.     

维甲酸对胰腺癌细胞生长的抑制作用及其机制研究

目的：观察两种维甲酸对人胰腺癌细胞生长的抑制作用及其可能机制。方法：应用ＭＴＴ以法，流式细胞仪，裸鼠肾包膜下移植瘤抑制试验和透射电镜技术进行检测。结果；ＲＡ抑制胰腺癌细胞生长，６ｄ组抑制５０Ａ％细胞生长的药物浓度全反式ＲＡ和１３－顺式为１０－３０μｍｏｌ／Ｌ和３０－５０μｍｏｌ／Ｌ。     

The matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase profile in colorectal polyp cancers

Aims:  The matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system has a major role in tumour invasion and metastasis. Roles in pathways involved in early tumour development are also being identified for this system, and the aim of this study was to define the expression profile of the major MMPs and TIMPs in colorectal polyp cancers.
Methods and results:  The expression and cellular localization of individual MMPs and TIMPs was determined in colorectal polyp cancers by immunohistochemistry. All the MMPs and TIMPs showed immunoreactivity in carcinomatous epithelium. MMP1 ( P  < 0.001), MMP2 ( P  = 0.003), MMP3 ( P  = 0.004), TIMP1 ( P  = 0.01) and TIMP2 ( P  < 0.001) showed significant increases in immunoreactivity in carcinomatous epithelium compared with adenomatous epithelium. MMP7 showed immunoreactivity in carcinomatous epithelium, but showed no immunoreactivity in either normal epithelium or adenomatous epithelium. MMP and TIMP expression was limited in normal epithelium to MMP1, MMP2 and TIMP3.
Conclusions:  This study defines the expression profile of MMPs and TIMPs in colorectal polyp cancers and shows that the increased expression of MMPs and TIMPs occurs at an early stage of colorectal neoplasia. It provides evidence to support the hypothesis that these molecules have a key involvement in the early stages of tumour development.     

Removal of dentin matrix proteoglycans by trypsin digestion and its effect on dentin bonding

The aim of this study was to investigate the effect of trypsin digestion on removal of chondroitin sulfate proteoglycans (CS-PGs) in demineralized dentin, and subsequent dentin bonding. Bovine dentin fragments were demineralized, treated with or without trypsin, stained with cupromeronic blue, and observed under transmission electron microscopy. Demineralized sections with or without trypsin digestion were also subjected to immunohistochemical analysis with anti-chondroitin-4-sulfate (C4S) monoclonal antibody, 2-B-6. The presence of galactosamine and glucosamine in the trypsin digest was confirmed by amino acid analysis. Bond strength testing was performed on trypsin treated and control specimens where samples were either kept moist or dried and re-wet, then bonded. Bond strength significantly decreased after trypsin treatment (p < 0.05). TEM, immunohistochemical, and amino acid analyses demonstrated that trypsin digestion efficiently removed C4S-PGs from demineralized dentin matrix. This study indicates that the detrimental effects observed on dentin bonding by trypsinization may be due in part to the removal/cleavage of the C4S-PGs, and further underscore the importance of C4S-PGs on dentin bonding.     

Net expansion of dried demineralized dentin matrix produced by monomer/alcohol saturation and solvent evaporation

The purpose of this work was to determine if nonaqueous methacrylate monomer/alcohol mixtures could expand dried collapsed demineralized dentin matrix. Thin disks (ca. 200 microm) of human dentin were demineralized and placed in wells beneath contact probes of linear variable differential transformers. The probes were placed on water-saturated expanded matrices to record the shrinkage associated with drying. Monomer mixtures containing hydroxyethyl methacrylate, 2,2-bis[4-(2-hydroxy-3 methacryloyloxy)propoxyphenyl] propane, or triethyleneglycol dimethacrylate were mixed with methanol or ethanol at alcohol/monomer mass fraction % of 90/10, 70/30, 50/50, or 30/70. They were randomly applied to the dried matrices to determine the rate and magnitude of expansion; then shrinkage was recorded during evaporation of the alcohols. The results indicated that matrix expansion was positively correlated with the Hoy's solubility parameters for hydrogen bonding forces (delta(h)) of the monomer/solvent mixtures (p < 0.001). Expansions were more rapid with methanol-containing than with ethanol-containing monomer mixtures. For the test solutions, triethyleneglycol dimethacrylate-containing mixtures produced the slowest rate of matrix expansion and hydroxyethyl methacrylate-containing mixtures the most rapid expansion. When the solvents were evaporated, the matrix shrank in proportion to the solvent content and the delta(h) of the monomer-solvent mixtures. The results indicate that expansion of dried, collapsed dentin matrices requires that the delta(h) of the mixtures be larger than 17 (J/cm(3))(1/2). The greater the delta(h) of the monomer solutions, the greater the rate and extent of expansion.     

The effect of crosslinking heparin to demineralized bone matrix on mechanical strength and specific binding to human bone morphogenetic protein-2

Demineralized bone matrix (DBM) is a collagen-based scaffold, but its low mechanical strength and limited BMP-2 binding ability restrict its application in bone repair. It is known that heparin could be immobilized onto scaffolds to enhance their binding of growth factors with the heparin-binding domain. Here, we crosslinked heparin to DBM to increase its BMP-2 binding ability. To our surprise, the mechanical strength of DBM was also dramatically increased. The compression modulus of heparin crosslinked DBM (HC-DBM) have improved (seven-fold increased) under wet condition, which would allow the scaffolds to keep specific shapes in vivo. As expected, HC-DBM showed specific binding ability to BMP-2. Additional studies showed the bound BMP-2 exerted its function to induce cell differentiation on the scaffold. Subcutaneous implantation of HC-DBM carrying BMP-2 showed higher alkaline phosphatase (ALP) activity (2 weeks), more calcium deposition (4 and 8 weeks) and more bone formation than that of control groups. It is concluded that HC-DBM has increased mechanical intensity as well as specific BMP-2 binding ability; HC-DBM/BMP-2 enhances the osteogenesis and therefore could be an effective medical device for bone repair.     

人纳米脱钙骨基质复合物的理化性质和安全性

背景：脱钙骨基质和骨形态发生蛋白已被证实具有良好的骨诱导性,但有关纳米脱钙骨基质的研究较少,其理化性质和生物安全性尚不明确。 目的：在前期实验制备人纳米脱钙骨基质的基础上加载重组人骨形态发生蛋白2,分析人纳米脱钙骨基质复合重组人骨形态发生蛋白2的理化性质及生物安全性。 方法：采用改良Urist法制备人脱钙骨基质,并进行纳米化处理,再将骨形态发生蛋白2与其按特定比例混合,冻干塑型行以下实验：①热源实验：将材料浸提液经耳静脉注入兔体内。②毒性实验：将材料浸提液与生理盐水分别经尾静脉注入小白鼠体内。③植入实验：在兔两侧后肢肌肉内分别植入实验材料和β-磷酸三钙。 结果与结论：冻干塑型后,纳米人脱钙骨基质材料表面致密,孔隙直径100-400 μm,孔隙分布欠均匀,孔隙率小于30%,以碳、氧和氮为主要元素组成。人纳米脱钙骨基质复合重组人骨形态发生蛋白2材料无热源效应,注射后未见兔体温有明显波动。急性全身毒性实验结果表明人纳米脱钙骨基质复合重组人骨形态发生蛋白2材料符合国家相关规定,注射后未见小鼠出现明显毒性反应。人纳米脱钙骨基质复合重组人骨形态发生蛋白2材料植入兔体内的炎症反应明显轻于β-磷酸三钙植入后的反应。结果表明人纳米脱钙骨基质复合重组人骨形态发生蛋白2是一种无毒、组织相容性好、生物利用度高、炎症反应轻的纳米同种异体骨移植替代物。     

The effect of aging on crack-growth resistance and toughening mechanisms in human dentin

Crack-growth experiments in human dentin have been performed in situ in an environmental scanning electron microscope to measure, for the first time, the crack-growth resistance curve (R-curve) for clinically relevant (<250 microm) crack extensions and to simultaneously identify the salient toughening mechanisms. "Young" dentin from donors 19-30 years in age and "aged" dentin from donors 40-70 years in age were evaluated. The "young" group had 0-4% of its tubules filled with apatite; the "aged" group was subdivided into "opaque" with 12-32% filled tubules and "transparent" with 65-100% filled tubules. Although crack-initiation toughnesses were similar, the crack-growth resistance of "young" dentin was higher by about 40% compared to "aged" dentin. Mechanistically, this behavior is interpreted in terms of three phenomena: (i) gross crack deflection of the growing crack, (ii) microcracks which initiated at unfilled tubules in the high stress region in the vicinity of a propagating crack (no microcracks formed at filled tubules), and (iii) crack propagation which followed a local trajectory through unfilled tubules and deflected around filled tubules. The higher toughness of the "young" dentin was related to enhanced microcracking (at unfilled tubules) ahead of the growing crack, which (i) shields the crack by activating multiple crack tips and by reducing the local stress intensity through crack deflection and (ii) leads to the formation of crack bridges from "uncracked ligaments" due to the incomplete coalescence of these microcracks with the main crack tip. With age, the role of these toughening mechanisms was diminished primarily to the lower fraction of unfilled, and hence microcracked, tubules.     

All-trans-retinoic acid suppresses matrix metalloproteinase activity and increases collagen synthesis in diabetic human skin in organ culture

Diabetes increases susceptibility to chronic skin ulceration. The etiology of chronic wound formation in diabetic individuals is multifactoral but may be accelerated by changes in the structure and function of the skin secondary to impaired fibroblast proliferation, decreased collagen synthesis, and increased matrix metalloproteinase (MMP) expression. This study explored the effects of all-trans-retinoic acid (RA) on cellular and biochemical features of diabetic human skin in organ culture. Two-mm skin biopsies from hip or ankle were obtained from diabetic subjects and incubated for 9 days in the absence or presence of 2 micro mol/L RA. Hip skin from non-diabetic individuals served as control. Following organ culture incubation, untreated and RA-treated tissue was examined histologically after staining with hematoxylin and eosin. In parallel, organ culture-conditioned medium collected on days 5 and 7 was assayed for levels of active and total MMP-1 (interstitial collagenase) and MMP-9 (gelatinase B). The same organ culture fluids were assayed for the presence of soluble collagen. In comparison with skin from non-diabetic individuals, diabetic skin demonstrated no major differences in overall epidermal thickness or collagen production (both were increased in RA-treated tissue as compared to non-RA-treated tissue). In contrast, levels of MMP-9 (active forms) were elevated in organ culture fluid from diabetic skin as compared to non-diabetic control skin. In the presence of RA, active forms of both MMP-1 and MMP-9 were reduced. Together, these data suggest that RA has the capacity to improve structure and function of diabetic skin, and that a major effect is on reduction of collagen-degrading MMPs.     

Changes in stiffness of demineralized dentin following application of collagen crosslinkers

It is thought that increasing the strength of the dentin matrix using crosslinking agents may improve both the strength and the durability of resin-dentin bonds. The purpose of this study was to evaluate the effect of two collagen crosslinking agents (glutaraldehyde, GD and grape seed extract, GSE) on the modulus of elasticity of demineralized dentin. Sound molar fragments were fully demineralized and divided into five groups according to the type and concentration of crosslinking agents: 2.5% GD; 5% GD, 25% GD; 0.65% GSE; 6.5% GSE. Specimens were immersed in their respective solution and tested at baseline, 10 min, 30 min, 1 h, 2 h, 4 h. The elastic modulus of dentin was significantly affected by the treatment (p < 0.01) and exposure time (p < 0.01). There was a statistically significant interaction between the two factors evaluated (treatment vs. time p < 0.01). Mean baselines values varied between 4.8 and 6.2 MPa in water; after 4 h of treatment the values increased between 34.9 and 242.5 MPa, that were treatment time and agent dependent. The use of these collagen crosslinkers to increase the stiffness of demineralized dentin, was both concentration and time dependent.     

Quantitative structure-activity relationship studies on matrix metalloproteinase inhibitors: hydroxamic acid analogs

A quantitative structure-activity relationship study has been conducted on two different series of acyclic hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The results suggest that in a few cases, the hydrophobic property of the molecules is the major governing factor. However, in some cases, the polarizability of the molecules is shown to be dominant. The two enzymes, MMP-9 and MMP-13, are shown to behave in a similar fashion with any group of inhibitors.     

Comparison of matrix metalloproteinase expression in normal and cirrhotic human liver

 To study the extend of ongoing tissue remodelling in end-stage cirrhosis, the expression of different matrix metalloproteinases [interstitial collagenase (MMP-1), M_r 72000 gelatinase (MMP-2), stromelysin-1 (MMP-3) and stromelysin-3 (MMP-11)] and of TIMP-1 was studied in 13 cirrhotic livers explanted at transplantation. The results were compared with those obtained in normal liver. Western blot, northern blot, ELISA, RT-PCR and zymogram analysis were used. Proenzymes of stromelysin-1 and -3, interstitial collagenase and M_r 72000 gelatinase were positive in normal liver, while activated enzymes were not detectable in western blot analysis. In cirrhosis proenzyme levels of the studied MMPs were reduced to a mean of 60–70%, but mRNA expression and gelatin-degrading activity increased. TIMP-1 expression was detectable on mRNA level and by ELISA in normal liver and also increased in cirrhosis. Our results show that mRNA expression of certain matrix metalloproteinases is increased in end-stage liver cirrhosis, while the amount of proenzyme is decreased, indicating enhanced MMP proenzyme turnover. These data suggest that besides increased TIMP-1 activity, altered MMP expression may also play a part in fibroproliferation in liver disease. Received: 6 June 1997 / Accepted: 1 September 1997     

可塑形脱矿骨基质/透明质酸骨泥的制备与细胞相容性

背景：应用预制成形的材料修复不规则骨缺损时,常出现材料与骨缺损形状不匹配的问题,制备可任意塑形骨泥对促进不规则骨缺损的愈合具有重要意义。 目的：构建可塑形脱钙骨基质/透明质酸骨泥,筛选最佳复合比例并评价其细胞相容性。 方法：选取合格供体皮质骨制备脱钙骨基质。分别配置浓度为0.75%,1.5%,3%,4.5%的透明质酸溶液并检测其黏度,以450 mg脱钙骨基质与上述各浓度透明质酸溶液1 mL混合,制备不同比例的脱钙骨基质/透明质酸骨泥。37 ℃条件下将骨泥浸泡在PBS平衡液中,观察其离散时间,筛选最佳复合比例的脱钙骨基质/透明质酸骨泥。应用脱钙骨基质/透明质酸骨泥浸提液培养L-929小鼠成纤维细胞,1,4,7 d采用CCK-8法检测骨泥的细胞毒性。 结果与结论：随透明质酸浓度的增加其溶液黏度增大,脱钙骨基质/透明质酸骨泥的离散时间延长,3%浓度的透明质酸与450 mg脱钙骨基质复合制备的骨泥具有良好可塑形能力,离散时间为8 h,为最佳复合比例。脱钙骨基质/透明质酸骨泥浸提液培养L-929小鼠成纤维细胞1,4,7 d的细胞增殖率分别为93.72%,101.65%,97.68%,细胞毒性级别为0或1级。表明脱钙骨基质/透明质酸复合质量比为15∶1制备的骨泥具有良好的可塑形能力及抗离散能力,细胞毒性轻微。     

Influence of dentinal polyelectrolytes on wet demineralized dentin,a bonding substrate

The objective of this study was to show the influence of dissolved dentinal polyelectrolytes on the characteristics of dentin (bonding substrate) demineralized by citric acid in the absence or presence of ferric chloride. The demineralizing agent was an aqueous mixture of 0, 1, 3, or 10% ferric chloride in 10% citric acid (10-0, 10-1, 10-3, 10-10, respectively). The hypothesis was that the concentration of dissolved dentinal noncollagenous substances, mainly polyelectrolytes soluble in water, must be decreased by their aggregation with ferric ions, which changes the characteristics of demineralized dentin, the rates of demineralization, and dehydration. Cervical bovine dentin was prepared in 3 x 2 x 2-mm blocks, each weighing 20.0 +/- 0.5 mg. The rate of demineralization was investigated by measuring the weight loss resulting from demineralization by immersion in 10 mL of conditioner at 2-h intervals. The dehydration rate of wet demineralized dentin was determined using two methods: (1) weight loss in a desiccator under 263 Pa pressure and (2) differential scanning calorimetry (DSC). Twenty, 12, 8, and 4 h were required to complete demineralization of the blocks with the 10-0, 10-1, 10-3, and 10-10 solutions, respectively. The 10-10 wet demineralized dentin showed the highest rate of dehydration, followed in descending order by the 10-3, 10-1, and 10-0 specimens. Ferric chloride in dentin conditioners provided both a higher rate of dentin demineralization and a higher dehydration rate of wet demineralized dentin. These results suggest that in the presence of ferric chloride, a decreasing amount of dissolved polyelectrolytes aggregated with ferric ions in the substrates may increase the permeability of dentin to water and citric acid. Improvement of monomer permeability is essential to the preparation of good hybridized dentin, providing a more stable and reliable bonding and also protecting the dentin and pulp from infection. A further study of bonding substrates is required in order to understand the role of hybridized dentin in improved dental treatment.     

The central inhibitory effect of interleukin-1 on gastric acid secretion

The effect of interleukin-1 (IL-1) on gastric secretory functions was examined in pylorus-ligated conscious rats. Intracisternal (i.c.) injection of IL-1 beta (1-100 ng) induced dose-related, long-lasting inhibition of gastric acid output, which was due to the reductions of both the amount and the acid concentration of the gastric juice. A much higher dose of IL-1 alpha was required to achieve identical effects on gastric acid secretion when it was given by intravenous routes. The i.c. injection of IL-1 alpha also had an inhibition of gastric secretion. This inhibitory effect of i.c. applied IL-1 beta on gastric acid secretion was completely abolished in indomethacin-pretreated animals but not in reserpine-pretreated ones. These results suggest that IL-1 may have an inhibitory action on the regulation of gastric secretory functions by its central action which is dependent on the eicosanoid metabolism.     

The effect of gradual mild acid hydrolysis on serologic activities of glycoproteins from human erythrocytes.

M and N blood group glycoproteins from O group erythrocytes were hydrolyzed with acid under various conditions that produced a gradual release of sialic acid. The preparations desialized to different degree were tested for M, N, NVg and AHp activities. M and N blood group activity was decreased upon release of sialic acid, and AHp activity was exposed "de novo" and increased upon desialization. NVg activity always increased during the initial period of acid hydrolysis, but prolonged heating in acid solution caused subsequent partial inactivation of glycoprotein. This decrease in NHp activity was not related either to the extent of desialization, or to fragmentation of the glycoprotein molecule.     

Mineralization processes in demineralized bone matrix grafts in human maxillary sinus floor elevations.

For reconstruction of the severely resorbed lateral maxilla for dental implant placement, one of the successful procedures is to elevate the maxillary sinus floor by implanting demineralized bone matrix (DBM). We studied bone formation in DBM grafts in the lateral maxilla in humans by means of histology and histomorphometry. Six months after grafting, at the time of dental implantation biopsies were taken from the grafted areas of seven patients. All biopsies contained mineralized matrix (MM) in the grafted area. At close inspection, three types of mineralization were found. First, lamellar biomineralization was seen in and near the maxillary host bone. Second, remineralization was observed in some particles that probably had not been completely demineralized. In the area connecting the graft and host bone, where woven bone was formed against DBM particles, a third mechanism was detected. In this case many dotlike foci of remineralization appeared close to the bone-DBM interface. The remineralized DBM and woven bone were both subsequently remodeled. Bone formation was most active in the area adjoining the maxillary host bone. We conclude that in human sinus floor elevation, allogenic DBM increases mineralized tissue volume by osteoconduction that is supported by the remineralization processes. Osteoinduction by this material seems questionable.     

The inhibitory effect of immunoglobulin E on monoamine uptake in human platelets

The uptake of the monoamine radiolabelled tracer 125I-meta-iodo-benzyl-guanidine (MIBG) was studied in vitro in platelets from 15 healthy volunteers and five allergic patients whose serum IgE concentration was 37 +/- 33 and 650 +/- 200 IU/ml, respectively. The MIBG uptake was determined by means of a gamma counter after incubation in a buffer. Binding of IgE to platelets significantly reduced MIBG uptake both in healthy subjects and in allergic patients. This reduction was significantly related to the action of IgE as it did not occur after binding of IgG. Moreover, MIBG uptake plotted versus increasing concentration of IgE (ranging from 10 to 3000 IU/ml) was fitted by a classical sigmoidal curve which was not significantly different between healthy subjects and allergic patients. Lastly, the effect of IgE on MIBG uptake was due to the binding of the sole IgE as it was not modified by subsequent addition of anti-IgE. It is concluded that the binding of the sole IgE to its specific receptor on platelets alters the transport of monoamines.