Alkaline phosphatase activity in the osteoclasts induced by experimental tooth movement.

The localization of alkaline phosphatase (Alpase) activity in the osteoclasts was examined cytochemically. Alpase activity was located in the basolateral membrane in mature osteoclasts having ruffled borders and clear zones, and also in the basolateral membrane in the osteoclasts lacking a ruffled border or a clear zone on the bone surface. But in the preosteoclasts situated away from the bone surface the enzyme activity was noted in the whole plasma membrane. These results suggest that the localization of Alpase activity may be altered in relation to the changes in morphology associated with the functional activity of the osteoclasts.     

Varied cytochrome oxidase activities of the alveolar type I, type II and type III cells in rat lungs: quantitative cytochemistry

Cytochemically demonstrated cytochrome oxidase activities in mitochondria of rat pulmonary alveolar epithelial cells were quantitatively compared. As the standardized procedures, lungs were fixed for 10 min at 4 degrees C with 2% pure glutaraldehyde, and incubated for different times at 37 degrees C in a medium containing 1.0 mg/ml 3,3'-diaminobenzidine-4HCl, 1 mg/ml cytochrome c, 0.1 mg/ml catalase, 7% sucrose, and 0.1 M phosphate buffer, pH 7.4. Electron micrographs were then obtained, and the densities of the reaction deposits in the mitochondrial intermembrane-intracristal space were measured in an image analyzer, and were plotted in terms of time in minutes. The initial velocity (maximal rate) of the activity expressed as deposit accumulation rate (% area/60 min) filling the mitochondrial intermembrane-intracristal space of Type I, Type II and Type III cells was 40, 130 and 9.3, respectively. These results indicated that mitochondria are varied in the intensity of cytochrome oxidase activity among 3 types of pulmonary alveolar epithelial cells, and that the image-analyzing measurement of the accumulation rate of reaction product may be useful for quantitative analysis of enzyme activity, in particular, in the cells which are difficult to isolate from complex tissues.     

Quantitative cytochemical studies of cytochrome oxidase activity in rat dorsal root ganglion cells

The cytochrome oxidase activity of rat dorsal root ganglion cells was cytochemically and quantitatively measured. The 100 microns thick tissue slices fixed for 10 min were incubated in a DAB medium for 0, 30, 60, and 90 min at 37 degrees C, and electron-dense deposit areas within the mitochondrial intermembrane-intracristal spaces were measured with a computer-controlled image analyzer. The activity was expressed as deposit accumulation rate filling the mitochondrial space, (1 unit corresponds to the deposit filling 100% of the mitochondrial space per hour). Three different activities of the enzyme in either large pale cells or small dark cells of sensory neurons could be distinguished, based on the quantitative analysis, as large cells with intense (0.83 units), intermediate (0.38), and weak activity (0.22), and small cells with the same degrees of activity (1.04, 0.35, and 0.11, respectively). The results indicate that accumulation rate measurements of reaction product may be useful to quantitatively present the cytochrome oxidase reactivity of mitochondria, and that the degree of enzyme activity may contribute to the identification of functional differences in sensory neurons.     

Differentiation and function of osteoclasts cultured on bone and cartilage

We examined the differentiation and resorptive function of osteoclasts (OC) cultured on the slices of calcified bone, decalcified bone and hyaline cartilage, and found that OC differentiation depends on the co-cultured substratum, as well as osteoblast-derived factors. Bone marrow-derived macrophages (BMM) were formed from marrow cells of 5 week old ddY mice and cultured for 3 days on freeze-dried slices of calcified bone, decalcified bone or cartilage, all prepared from rabbit costal bone. BMM cultured on calcified bone slices exhibited tartrate-resistant acid phosphatase (TRAP) activity and were structurally characterized by multinucleation and ruffled border development. However, on decalcified bone slices, BMM seldom became multinucleated and exhibited weak TRAP activity. BMM cultured on cartilage slices were mononuclear, devoid of TRAP activity and structurally resembled mononuclear phagocytes. In SEM observations of co-cultured slices, resorption lacunae were formed only on calcified bone slices, and not on slices of decalcified bone and cartilage. Our results, therefore, indicated that BMM could differentiate into functional OC only on calcified bone slices, suggesting a key role of calcified components in the bone matrix for the terminal OC differentiation. Then, we cultured BMM on the same slices with yeast particles. In cultures with yeast particles, BMM exhibited intense TRAP activity, developed a ruffled border-like structure and formed resorption lacunae even on decalcified bone and cartilage slices. Vacuolar-type H+-ATPase was strongly expressed along the ruffled border membranes of these OC. Only the BMM that had not incorporated yeast particles developed a ruffled border, whereas the BMM that had incorporated yeast particles did not become multinucleated and lacked a ruffled border structure. Thus, our results further suggest that, even on uncalcified substrata, the terminal differentiation of BMM into functional OC is induced by an unidentified external stimulus, which may be contained in the cell membrane of yeast particles.     

Single- and Multiscale Laser Patterning of 3D Printed Biomedical Titanium Alloy: Toward an Enhanced Adhesion and Early Differentiation of Human Bone Marrow Stromal Cells

This study explores the enhancement of biocompatible titanium-based implants through surface functionalization for improved bone healing. Specifically, a near-beta type Ti-13Nb-13Zr alloy is 3D printed using laser powder bed fusion and subsequently textured using nanosecond (ns) and picosecond (ps) direct laser interference patterning (DLIP) to create single-scale and multi-scale surface textures. On these textures, the cell behavior, morphology, metabolic activity and osteogenic differentiation potential of human bone marrow stromal cells are assessed using fluorescence microscopy and MTS assays. Moreover, tissue non-specific alkaline phosphatase activity served as an early osteoblast production marker. Compared to untextured specimens, both types of textures exhibited higher metabolic activity and cell proliferation. Single-scale ns-DLIP textures encouraged cell extensions anchored in groove regions, while ps-DLIP textures with hierarchical structures promoted cell extensions attaching to nanostructures on sidewalls. The groove width and nanotopographies in groove areas facilitated cell spreading. Surface topography, roughness, and surface chemistry (surface energy, wettability) influenced cell adhesion, proliferation, and differentiation. A comprehensive evaluation of DLIP-generated surface textures, including their topography and chemical states, complements the factors affecting in vitro cell behavior. Overall, this research demonstrates the potential of surface-functionalized 3Dprinted titanium for a novel generation of biocompatible implants.     

酒精对心肌细胞超微结构及细胞色素C 氧化酶活性的影响

目的：通过给小鼠腹腔内注射不同浓度酒精,建立大量饮酒的动物模型,探讨酒精对心脏毒性作用的机理。方法：应用电镜技术观察心肌细胞的超微结构变化,以及酶细胞化学染色方法观察细胞色素C氧化酶(cytochrom C oxidase,Cox)活性的变化。结果：透射电镜下,实验各组心肌细胞肌原纤维排列紊乱,出现空泡,肌丝溶解,线粒体肿胀,嵴断裂甚至消失,细胞核形状不规则,部分染色质浓缩,边集于核膜下,呈斑块状;定位于线粒体内膜和嵴上的Cox活性减弱,电子密度降低。结论：酒精可以使心肌细胞超微结构发生异常改变,并且使线粒体上Cox活性减弱,从而导致心肌细胞结构和功能的改变。     

缺氧对大鼠心肌细胞色素氧化酶的影响——超微酶学图象分析

大鼠10只,分实验组(6只)和对照组(4只)。实验组大鼠(每次2只)置入密封玻璃容器内(容积5500ml),同时放入钠石灰100g,90分钟后杀死取左心室外侧壁作超微细胞色素氧化酶观察,并以正常大鼠作对照。对两组动物各50个线粒体的细胞色素氧化酶有关参数进行图象分析处理。结果表明,缺氧组动物心肌线粒体细胞色素氧化酶活性明显减弱。     

Ratio of polyamine synthesis and oxidation enzymes during cell proliferation and differentiation

Difluoromethylornithine (DFMO) was studied for its effect on the aminoxidase activity in the culture media during proliferation and differentiation of mouse fibroblasts. Single introduction of difluoromethylornithine into the culture medium increases the polyamine oxidase activity during 12 to 24 hours after the action. In the subsequent periods DFMO in 0.1 to 1 mM concentrations inhibits the proliferation and differentiation, but 0.01 mM concentration leads to their acceleration, which correlates with higher or lower aminoxidase activities respectively in the culture media.     

Multilayered Lipid Membrane Stacks for Biocatalysis Using Membrane Enzymes

Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single‐lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules, and greatly increase surface area and hence membrane protein concentration. Here, a supramolecular assembly of a multilayered lipid membrane system is reported in which poly‐l ‐lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo ₃ from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane‐bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. Using atomic force microscopy, CV, and fluorescence microscopy it is deduced that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and has significant potential for mimicry in biotechnology applications such as energy production or biosensing.     

A Novel Approach to Enhance Bone Regeneration by Controlling the Polarity of GaN/AlGaN Heterostructures

Rapid and effective bone regeneration is still a major challenge in the clinical treatment of various bone diseases. Although recently developed electroactive materials have demonstrated high bone regeneration potential, the instability of the electrical stimulation and the unclear effects of the charge polarity on osteogenic differentiation hinder their clinical applications. In this work, GaN/AlGaN materials with well-controlled polarity are used for the first time to induce endogenous electric stimulation and facilitate bone regeneration. By controlling the direction and magnitude of the piezoelectric and spontaneous polarization in the functional layer (GaN), charged GaN/AlGaN surfaces of opposite polarity, whose zeta potentials are within the range of the physiological potential, are obtained. Compared with N-polarity GaN/AlGaN (with a positively charged surface), Ga-polarity GaN/AlGaN (with a negatively charged surface) nanofilms show rapid and superior bone repair in vivo. In addition, the Ga-polarity GaN/AlGaN hetero-structures significantly promote the attachment, spreading, recruitment, and osteogenic differentiation of bone mesenchymal stem cells in vitro. Moreover, the bone morphogenetic protein-6 (BMP6) expression profile in the early stages of osteogenic differentiation reveals that BMP6 may be an electrically sensitive osteogenic protein. This work sheds light on the application of III-nitride materials in bone regeneration.     

Cytochemical energy-filtering transmission electron microscopy of mitochondrial free radical formation in paraquat cytotoxicity.

The generation of oxygen free radicals was investigated using cytochemistry and its energy-filtering transmission electron microscopy in reference to the toxic mediator for the herbicide paraquat. When isolated intact mitochondria from rat livers were incubated in a medium containing paraquat and NADH, a mitochondrial NADH-quinone oxidoreductase activity generated superoxide anions to cause the destruction of mitochondria which resulted in cell death. The superoxide anions were immediately converted into hydrogen peroxide, which then formed cerium perhydroxide deposits in the presence of cerium ions and precipitated on the outer surface of the mitochondrial outer membrane. This localization was also specifically identified by energy spectral imaging and image-electron energy loss spectral analyses. Precipitation reaction was scavenged by the addition of either cytochrome c or catalase and inhibited by dicoumarol (an inhibitor of NAD(P)H-quinone oxidoreductases). These cytochemical energy-filtering transmission electron microscopic results indicated that paraquat generated free radicals from the outer membrane of mitochondria.     

Micro/Nanometer‐Structured Scaffolds for Regeneration of Both Cartilage and Subchondral Bone

Treatment of osteochondral defects remains a great challenge in clinical practice because cartilage and subchondral bone possess significantly different physiological properties. In this study, the controlled surface micro/nanometer structure of bioactive scaffolds in a combination of biomaterial chemistry is harnessed to address this issue. Model bioactive biomaterials, bredigite (BRT) scaffolds, with controlled surface micro/nanostructure are successfully fabricated by combining 3D printing with a hydrothermal process. It is found that the growth of micro/nano–calcium phosphate crystals on the surface of BRT scaffolds notably enhances their compressive strength by healing the microcracks on the strut surface. The micro/nanostructured surface distinctly facilitates the spread and differentiation of chondrocytes by activating integrin αvb1 and α5b1 heterodimers, regulates cell morphology, and promotes osteogenic differentiation of rabbit bone marrow stromal cells (rBMSCs) through the synergetic effect of integrin α5b1 and RhoA, in which the microrod surface demonstrates the highest stimulatory effect on the differentiation of chondrocytes and rBMSCs. The in vivo study shows that the micro/nanostructured surface of the 3D printed scaffolds obviously promotes the regeneration of both cartilage and subchondral bone tissues. This study suggests that the construction of controlled micro/nanostructured surface in porous 3D scaffolds offers a smart strategy to induce bilineage bioactivities for osteochondral regeneration.     

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration).     

Surface Chemistry of Vitamin: Pyridoxal 5′‐Phosphate (Vitamin B6) as a Multifunctional Compound for Surface Functionalization

Vitamins are non‐toxic compounds that perform a variety of biological functions and also available in a large quantity. Other than the usage as food supplements, few attempts have been made to use them as functional materials. In this study, we report that vitamin B6, pyridoxal 5′‐phosphate (PLP), is a multi‐functional molecule for oxide surface chemistry. PLP‐immobilized surfaces exhibit superhydrophilicity and even hemophilicity, enhancing proliferation, migration, and differentiation of mammalian cells. Unlike existing molecules used so far in surface modification, PLP has an intrinsic chemical reactivity toward biomacromolecules due to the presence of the aldehyde group. In fact, RGD peptide is covalently tethered onto PLP surfaces directly in one step without any chemical activation. Furthermore, PLP‐functionalized implant device showed rapid bone healing. As vitamin B6 is a FDA approved molecule for human usage, the surface chemistry of vitamin B6 potentially allows a fast route for surface functionalized medical devices into clinic.     

Citrate‐Based Tannin‐Bridged Bone Composites for Lumbar Fusion

Conventional bone composites consistently fail to mimic the chemical composition and integrated organic/inorganic structure of natural bone, lacking sufficient mechanics as well as inherent osteoconductivity and osteoinductivity. Through a facile surface coating process, the strong adhesive, tannic acid (TA), is adhered to the surface of the natural bone component, hydroxyapatite (HA), with and without the immobilization of in situ formed silver nanoparticles. Residual functional groups available on the immobilized TA substituents are subsequently covalently linked to the citrate‐based biodegradable polymer, poly(octamethylene citrate) (POC), effectively bridging the organic and inorganic phases. Due to the synergistic effects of the tannin and citrate components, the obtained citrate‐based tannin‐bridged bone composites (CTBCs) exhibit vastly improved compression strengths up to 323.0 ± 21.3 MPa compared to 229.9 ± 15.6 MPa for POC‐HA, and possess tunable degradation profiles, enhanced biomineralization performance, favorable biocompatibility, increased cell adhesion and proliferation, as well as considerable antimicrobial activity. In vivo study of porous CTBCs using a lumbar fusion model further confirms CTBCs' osteoconductivity and osteoinductivity, promoting bone regeneration. CTBCs possess great potential for bone regeneration applications while the immobilized TA additionally preserves surface bioconjugation sites to further tailor the bioactivity of CTBCs.     

甲状腺显示胸腺样分化的癌超微病理结构观察

利用透射电子显微镜观察2例甲状腺显示胸腺样分化癌(carcinoma showing thymus-like differentiation,CASTLE)的超微病理结构特征,并在光镜下观察其组织学及免疫组织化学特点.电镜下肿瘤组织主要特点为癌细胞细胞核内以常染色质为主,核仁明显,胞质内含较多的线粒体及张力原纤维,细胞...     

Nucleoside Triphosphates as Promoters to Enhance Nanoceria Enzyme‐like Activity and for Single‐Nucleotide Polymorphism Typing

Nucleoside triphosphates (NTPs) can improve the oxidase‐like activity of nanoceria and the enhancement is correlated with the type of NTP. This effect is demonstrated to be as a result of the coupling of the oxidative reaction with the NTP hydrolysis reactions, as the nanoceria has both oxidase‐like and phosphatase‐like activities. The differences reflect the different dephosphorylation catalytic activities of nanoceria to the NTP used. Furthermore, based on the NTP‐promoted oxidase‐like activity of nanoceria and the differences among the different types of NTPs, series effective and high‐throughput colorimetric assays for single‐nucleotide polymorphism (SNP) typing are developed.     

Measure of enzymatic activity coincident with 2450 MHz microwave exposure.

Enzyme preparations were exposed to microwave radiation at 2450 MHz and enzymatic activity was simultaneously monitored spectrophotometrically with a crossed-beam exposure detection system. Enzymes studied were glucose 6-phosphate dehydrogenase from human red blood cells and yeast, adenylate kinase from rat liver mitochondria and rabbit muscle, and rat liver microsomal NADPH cytochrome c reductase. No difference was found between the specific activity at 25 degrees C of unirradiated controls and enzyme preparations irradiated at an absorbed dose rate of 42 W/kg.     

Stem Cell-Niche Engineering via Multifunctional Hydrogel Potentiates Stem Cell Therapies for Inflammatory Bone Loss

Effective therapies capable of simultaneously inhibiting inflammation and promoting bone healing remain to be developed for inflammatory bone disease. Stem cell therapies hold great promise for a variety of diseases, but their translation is hampered by low cell survival, rapid clearance, and limited functional integration of transplanted stem cells in target tissues. Herein, a multifunctional hydrogel-based stem cell niche engineering strategy is reported for the treatment of inflammatory bone loss. By rationally integrating different functional modules, an injectable hydrogel-based stem niche is engineered, which possesses temperature-triggered gelling performance, inflammation/oxidative stress-resolving activity, stem-cell binding and survival-enhancing capacity, and osteogenesis-promoting capability. Using ectomesenchymal stem cells (EMSCs), effectiveness of this functionally advanced synthetic stem cell niche is demonstrated in rats with periodontitis, a representative inflammatory bone loss disease. Synergistic effects of the multifunctional hydrogel and EMSCs are also confirmed, with respect to normalizing the pathological microenvironment and improving alveolar bone regeneration in the periodontal tissue. Mechanistically, inflammation/oxidative stress-resolving and osteogenic differentiation promoting capacities of the synthetic stem cell niche are mainly achieved by an incorporated nanotherapy via the GDF15/Atf3/c-Fos axis of the MAPK signaling pathway. Besides periodontitis, the newly engineered hydrogel-stem cell therapies are promising for the treatment of other inflammatory bone defects.     

Effects of substrate nanopatterning on human osteosarcoma cells (SaOs-2) behavior

Engineering of the cellular microenvironment has become an attractive strategy to guide cellular activities such as spreading, motility, proliferation and differentiation. From a technological perspective, the physical crosstalk between a cell and its surrounding represents a design parameter that may be modulated to achieve desired osseointegration in orthopedic and dental implants. In this study we use a surface engineering approach to tap into the interaction between the cell and its surroundings in order to modulate osteogenic adhesion-dependent differentiation and bone tissue formation.The effectiveness of this approach was studied by observing the in vitro cellular behavior of human osteoblastic cells (SaOs-2) seeded on silicon substrates with different nano-scale surface patterns.Our findings suggest that substrate nanopattern geometry can differentially control early cell differentiation decisions. Interestingly, rescue of the differentiation process by supplementation of growth medium with soluble differentiation factors did not appear to compensate for the differences in the early cellular fate decisions observed on the different patterns. Critically, combining surface topography and soluble differentiation factors appeared to modulate the speed of the differentiation process and its shut down at the end of terminal differentiation. Taken together, our findings suggest that cells recognize physical nano-scale topography as an instructive signal that is integrated to fine-tune the multimodal control and guidance of cell behavior and cell fate. This information may find utility in the design of orthopedic and dental implants or innovative bio-materials for regenerative medicine.