Hydroxylamine as an oxygen nucleophile. Structure and reactivity of ammonia oxide.
Ammonia oxide is revealed as a stable molecule in a crystal structure and as a likely reactive species in many reactions of hydroxylamine.
Journal Article↗
Biomedical subjects
John E Davies
Publications and source records attributed to John E Davies.
Platelet releasate increases the proliferation and migration of bone marrow-derived cells cultured under osteogenic conditions.
Concentrated platelets and their products are currently being used as a clinical tool to accelerate endosseous wound healing. However, there is little understanding regarding the actions of platelets and platelet-released products on osteogenic cells. We show, herein, that releasate from thrombin-activated platelets increases the migration and proliferation of osteogenic cultures of bone marrow cells. Using a scratch wound assay, we demonstrated that platelet releasate (PR) stimulated up to a 2.4 +/- 0.5-fold increase in wound closure in serum-free medium, relative to a control containing thrombin. In the presence of serum, the addition of PR resulted in a 1.45 +/- 0.13-fold increase in scratch closure. To isolate cell migration from the effects of cell proliferation, cell monolayers were pre-incubated with 5, 10 and 20 microg/ml of Mitomycin C (MMC), which is a potent inhibitor of cell proliferation. This resulted in a large decrease in the leading front of scratch closure, which indicates that PR stimulated cell mitogenesis. However, irrespective of MMC pre-treatment, PR stimulated a motogenic response. These results provide evidence of possible mechanisms by which platelets could influence bone regeneration.
Alkylating Agents↗
Soluble factor cross-talk between human bone marrow-derived hematopoietic and mesenchymal cells enhances in vitro CFU-F and CFU-O growth and reveals heterogeneity in the mesenchymal progenitor cell compartment.
The homeostatic adult bone marrow (BM) is a complex tissue wherein physical and biochemical interactions serve to maintain a balance between the hematopoietic and nonhematopoietic compartments. To focus on soluble factor interactions occurring between mesenchymal and hematopoietic cells, a serum-free adhesion-independent culture system was developed that allows manipulation of the growth of both mesenchymal and hematopoietic human BM-derived progenitors and the balance between these compartments. Factorial experiments demonstrated a role for stem cell factor (SCF) and interleukin 3 (IL-3) in the concomitant growth of hematopoietic (CD45+) and nonhematopoietic (CD45-) cells, as well as their derivatives. Kinetic tracking of IL-3alpha receptor (CD123) and SCF receptor (CD117) expression on a sorted CD45- cell population revealed the emergence of CD45-CD123+ cells capable of osteogenesis. Of the total fibroblast colony-forming units (CFU-Fs) and osteoblast colony-forming units (CFU-O), approximately 24% of CFU-Fs and about 22% of CFU-Os were recovered from this population. Cell-sorting experiments demonstrated that the CD45+ cell population secreted soluble factors that positively affect the survival and proliferation of CFU-Fs and CFU-Os generated from the CD45- cells. Together, our results provide insight into the intercellular cytokine network between hematopoietic and mesenchymal cells and provide a strategy to mutually culture both mesenchymal and hematopoietic cells in a defined scalable bioprocess.
Cell Differentiation↗
Platelet interactions with calcium-phosphate-coated surfaces.
Many studies have shown that calcium-phosphate (CaP)-coated endosseous implants exhibit more peri-implant bone formation and bone contact at early healing times than uncoated implants. Since the rate of healing is influenced by blood/implant interactions and possibly the degree of blood platelet activation, the aim of this study was to determine whether the topography, microtopography, or the presence of calcium (Ca) and phosphate (PO(4)) ions in the implant surface plays a predominant role in platelet activation. We define the threshold between topography and microtopography as the limit of the scale range of platelets themselves; thus, a microtopographic surface is defined by one which exhibits features 3mum. With the help of four international collaborating laboratories, we prepared 11 titanium and CaP-modified titanium surfaces each with different (micro)topographies and interrogated these surfaces with both platelet adhesion (lactate dehydrogenase activity) and platelet activation (microparticle formation and P-selectin expression) assays. Our results show that: calcium (Ca)- and phosphate (PO(4))-containing surfaces of increasing surface microtopographical complexity exhibit increasing platelet activation; surfaces with similar surface microtopographies show similar levels of platelet activation regardless of the presence of Ca and PO(4) in the surface; and that surface microtopography is responsible for platelet activation rather than the presence of Ca and PO(4) in the surface.
Blood Platelets↗
Thrombin mediated migration of osteogenic cells.
Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.
Actins↗
Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors.
We describe the isolation of a nonhematopoietic (CD45-, CD34-, SH2+, SH3+, Thy-1+, CD44+) human umbilical cord perivascular (HUCPV) cell population. Each HUCPV cell harvest (2-5 x 10(6), depending on the length of cord available) gave rise to a morphologically homogeneous fibroblastic cell population, which expressed alpha-actin, desmin, vimentin, and 3G5 (a pericyte marker) in culture. We determined the colony-forming unit-fibro-blast (CFU-F) frequency of primary HUCPV cells to be 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 10(10) HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. The frequency of this CFU-osteogenic subpopulation at P1 was 2.6/10(5) CFU-F, which increased to 7.5/10(5) CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/10(4) and 1.3/10(4) CFU-F, respectively, for P1 and P2. CFU-O were not seen at P0 in either osteogenic or non-osteogenic culture conditions, but P0 HUCPV cells did contain a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC-/-). This population increased to 95% following passage and cryopreservation (P5). We conclude that, due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC-/- phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies.
Cell Differentiation↗
Bone marrow genesis after subcutaneous delivery of rat osteogenic cell-seeded biodegradable scaffolds into nude mice.
This study describes the generation of an active hematopoietic marrow within the confines of a biodegradable, macroporous polyester scaffold, seeded with rat osteogenic cells, after subcutaneous implantation in nude mice. A macroporous, poly(DL-lactide-co-glycolide) polymer scaffold, into which resorbable calcium phosphate particles were incorporated, was seeded with rat bone marrow-derived cells. Scanning electron microscopy of the cell-seeded scaffold demonstrated confluent cell colonization. Scaffolds seeded with cells were implanted under the dorsum of immunocompromised mice for 5 weeks. Histological analysis revealed bone formation along the scaffold pores creating bony cavities within which a host-derived, hematopoietic marrow was observed which included hematopoietic precursors, megakaryocytes, fat cells, and numerous marrow sinusoids. In those areas where bone was not elaborated on the scaffold surface, no marrow genesis was observed and the scaffold interstices were filled with fibrous tissue. These results demonstrate the utility of this biodegradable scaffold in delivery of a phenotypically functional cell population for bone tissue and bone marrow engineering applications. Moreover, the recapitulation of hematopoietic marrow tissue within the engineered bony cavities also provides a new experimental environment with which to further investigate the interactions of hematopoietic and nonhematopoietic compartments of the marrow microenvironment.
Animals↗
Preparation and characterization of a highly macroporous biodegradable composite tissue engineering scaffold.
A unique composite scaffold for bone-tissue engineering applications has been prepared by combining biodegradable poly(lactide-co-glycolide) (PLGA) with bioresorbable calcium phosphate (CaP) cement particles through the process of particle fusion and phase separation/particle leaching. The scaffold is characterized by a highly interconnected macroporosity, with macropores of 0.8-1.8 mm and porosities ranging from 81% to 91%, and improved mechanical properties with respect to the polymer alone, producing excellent dimensional stability. The scaffold properties were controlled by adjusting the processing parameters, including PLGA molar mass and concentration, CaP/PLGA ratio, and porogen size. The differences in mechanical properties between dry, wet/room temperature, and wet/37 degrees C testing conditions, of which the latter are more relevant for materials to be employed in a biological milieu, were investigated. Thus, a scaffold made from PLGA IV 1.13, PLGA concentration 12.5%, and CaP/PLGA ratio 2:1 exhibited significantly different compressive strengths of 0.16 MPa and 0.04 MPa when tested under dry and wet/37 degrees C conditions, respectively. .
Absorbable Implants↗
Fibrin-filled scaffolds for bone-tissue engineering: An in vivo study.
Recently, fibrin sealants that typically contain supra physiological concentrations of fibrinogen and thrombin have been investigated as matrices to facilitate the delivery of cells within biodegradable scaffolds for tissue engineering applications. It is well known from in vitro experiments that the thrombin concentration present during fibrin polymerization influences the structural properties of fibrin, and these can affect cell invasion. This study was conducted to determine whether the structural properties of fibrin can affect bony wound healing in vivo. Drill hole defects were created in the distal femurs of 20 rats. Four experimental groups were used: nontreated defects, scaffolds alone, and scaffolds filled with fibrin polymerized with either a low thrombin concentration [fibrin(low T)] or a high thrombin concentration [fibrin(high T)]. The area of bone formed at 2, 5, and 11 days after implantation was determined histomorphometrically. After 5 days, scaffolds filled with fibrin(high T) were infiltrated with less bone than empty scaffolds (p < 0.05), but no statistical difference was found between the empty scaffolds and the scaffolds filled with fibrin(low T). After 11 days, both fibrin-filled scaffolds significantly delayed bony wound healing (p < 0.004). Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the two fibrin formulations showed no difference in gamma-gamma crosslink formation. This work demonstrates that fibrin sealants in their present state are not ideal for enhancing bone-tissue invasion into scaffolds, and that the structural properties of fibrin matrices may be an important design parameter for maximizing host tissue invasion during wound healing.
Animals↗
Solution-processible conjugated electrophosphorescent polymers.
We report the synthesis and photophysical study of a series of solution-processible phosphorescent iridium complexes. These comprise bis-cyclometalated iridium units [Ir(ppy)(2)(acac)] or [Ir(btp)(2)(acac)] where ppy is 2-phenylpyridinato, btp is 2-(2'-benzo[b]thienyl)pyridinato, and acac is acetylacetonate. The iridium units are covalently attached to and in conjugation with oligo(9,9-dioctylfluorenyl-2,7-diyl) [(FO)(n)] to form complexes [Ir(ppy-(FO)(n))(2)(acac)] or [Ir(btp-(FO)(n))(2)(acac)], where the number of fluorene units, n, is 1, 2, 3, approximately 10, approximately 20, approximately 30, or approximately 40. All the complexes exhibit emission from a mixed triplet state in both photoluminescence and electroluminescence, with efficient quenching of the fluorene singlet emission. Short-chain complexes, 11-13, [Ir(ppy-(FO)(n)-FH)(2)(acac)] where n = 0, 1, or 2, show green light emission, red-shifted through the FO attachment by about 70 meV, but for longer chains there is quenching because of the lower energy triplet state associated with polyfluorene. In contrast, polymer complexes 18-21 [Ir(btp-(FO)(n))(2)(acac)] where n is 5-40 have better triplet energy level matching and can be used to provide efficient red phosphorescent polymer light-emitting diodes, with a red shift due to the fluorene attachment of about 50 meV. We contrast this small (50-70 meV) and short-range modification of the triplet energies through extended conjugation, with the much more substantial evolution of the pi-pi* singlet transitions, which saturate at about n = 10. These covalently bound materials show improvements in efficiency over simple blends and will form the basis of future investigations into energy-transfer processes occurring in light-emitting diodes.
Journal Article↗
Investigation of conjugate addition/intramolecular nitrone dipolar cycloadditions and their use in the synthesis of dendrobatid alkaloid precursors.
The sequential intramolecular conjugate addition of the oxime 13 followed by intramolecular dipolar cycloaddition of the intermediate nitrone 14 affords a mixture of the isoxazolidines 15, 16 and 17. The tricyclic 6,5,5-adduct 15 is believed to be the product of kinetic control and can be equilibrated with the epimeric tricyclic 6,5,5-isoxazolidine 17 through a beta-elimination/conjugate addition process. Conditions have been developed for the two-step conversion of the ketone 12 under thermodynamic control into the racemic tricyclic 6,6,5-adduct 16 which is the core precursor of all the known histrionicotoxin alkaloids.
Alkaloids↗
A concise synthesis of the octalactins.
The total synthesis of octalactins A and B has been achieved in 15 steps (longest linear sequence) and 10% overall yield from commercially available materials. Key steps include the Paterson-Aldol reaction for the rapid assembly of the carbonate 46, methylenation of 46 and subsequent Claisen rearrangement of the corresponding alkenyl-substituted cyclic ketene acetal to provide the core unsaturated medium-ring lactone 47, and the use of enzyme-mediated acetate deprotection in the presence of a medium-ring lactone.
Antineoplastic Agents↗
Lemierre's syndrome: how a sore throat can end in disaster.
Lemierre's syndrome is characterised by a history of recent oropharyngeal infection, clinical or radiological evidence of internal jugular vein thrombophlebitis and isolation of an anaerobic pathogen. We present a case report and review the literature.
Bacteremia↗
A tricycloheptane product in cationic rearrangements.
The bicycloheptene 6 rearranged in acid to give the tricycloheptane 9, as shown by an X-ray crystal structure determination of the p-nitrobenzoate 10 derived from it. Earlier results in the literature had already indicated that this isomer was the thermodynamic sink. This apparently crowded structure, with its three contiguous quaternary centres, is, nevertheless, lower in energy than other accessible but less crowded structures, because of electronic stabilisation of the more substituted cyclopropane ring conjugated to the ester group.
Journal Article↗
Bone formation on two-dimensional poly(DL-lactide-co-glycolide) (PLGA) films and three-dimensional PLGA tissue engineering scaffolds in vitro.
For some bone tissue engineering strategies, direct contact of newly synthesized bone with a scaffold is important for structural continuity and stability at the scaffold/bone interface. Thus, as the polymer degrades, the support function of the scaffold could be adopted by the developing bone structure. This study was designed to determine whether poly(DL-lactide-co-glycolide) with a comonomer ratio of 75:25 supports bone apposition in vitro. Osteogenic cells derived from rat bone marrow cells were cultured for 2 weeks on polymeric two-dimensional films and three-dimensional tissue engineering scaffolds. Bacteriological grade polystyrene and tissue culture polystyrene dishes served as negative and positive controls for interfacial bone deposition, respectively. The surfaces of the prepared substrates were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle, scanning electron microscopy, and atomic force microscopy. After cell culture, the elaborated matrix was examined using scanning electron microscopy, transmission electron microscopy, and energy dispersive spectroscopy. The results show that poly(DL-lactide-co-glycolide) supports appositional bone growth on both two-dimensional films and three-dimensional scaffolds, including the formation of a mineralized cement line matrix. Furthermore, surface topographical features are not required for the adherence of the cement line matrix to the polymer.
Animals↗
Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independent survival and expansion.
OVERVIEW: We show the existence of adult human mesenchymal progenitor cells (hMPCs) that can proliferate, in a cytokine-dependent manner, as individual cells in stirred suspension cultures (SSC) while maintaining their ability to form functional differentiated mesenchymal cell types. MATERIALS AND METHODS: Ficolled human bone marrow (BM)-derived cells were grown in SSC (and adherent controls) in the presence and absence of exogenously added cytokines. Phenotypic, gene expression, and functional assays for hematopoietic and nonhematopoietic cell populations were used to kinetically track cell production. Limiting-dilution analysis was used to relate culture-produced cells to input cell populations. RESULTS: Cytokine cocktail influenced total and progenitor cell expansion, as well as the types of cells generated upon plating. Flow cytometric analysis of CD117, CD123, and CD45 expression showed that cytokine supplementation influenced SSC output. The concomitant growth of CD45(+) and CD45(-) cells in the cultures that exhibited the greatest hMPC expansions suggests that the growth of these cells may benefit from interactions with hematopoietic cells. Functional assays demonstrated that the SSC-derived cells (input CFU-O number: 1990+/-377) grown in the presence of SCF+IL-3 resulted, after 21 days, in the generation of a significantly greater number (p<0.05) of bone progenitors (33,700+/-8763 CFU-O) than similarly initiated adherent cultures (214+/-75 CFU-O). RT-PCR analysis confirmed that the SSC-derived cells grown in osteogenic conditions express bone-specific genes (Cbfa1/Runx2, bone sialoprotein, and osteocalcin). CONCLUSIONS: Our approach not only provides an alternative strategy to expand adult BM-derived nonhematopoietic progenitor cell numbers in a scalable and controllable bioprocess, but also questions established biological paradigms concerning the properties of connective-tissue stem and progenitor cells.
Adult↗
The hemolytic and cytotoxic properties of a zeolite-containing root filling material in vitro.
OBJECTIVE: This in vitro study characterized the hemolysis and cytotoxicity of ZUT, an experimental glass ionomer cement (GIC) sealer with an added antimicrobial-containing zeolite (0.2% Zeomic w/w). STUDY DESIGN: ZUT, Ketac-Cem (GIC component of ZUT), Ketac-Endo, and two AH 26 sealer formulations were tested at various times after mixing. Hemolysis produced by standardized specimens was determined spectrophotometrically (n = 6/material). Cytotoxicity was assessed by using a Millipore Filter test with a HeLa cell monolayer (n = 10/material). Tests were repeated, and results were analyzed with a one-way analysis of variance (alpha = .05). RESULTS: Disks of AH 26 containing silver produced the most hemolysis of all test groups (P < .0001). Compared to controls, GICs and AH 26 formulations were noncytotoxic at 1 and 6 hours after mixing, respectively (P > .05). Addition of Zeomic did not increase the cytotoxic and hemolytic activity of Ketac-Cem (P > .05). CONCLUSION: Overall results suggest ZUT is less cytotoxic than AH 26 and possesses characteristics similar to the other GIC formulations tested.
Analysis of Variance↗
Effect of platelet releasate on bone cell migration and recruitment in vitro.
The use of platelets and platelet products has become increasingly popular clinically as a means of accelerating endosseous wound healing. It is likely that growth factors released by activated platelets at the site of injury play a role in bone regeneration by stimulating the migration and proliferation of bone cells. In this study, a novel in vitro assay was developed to study the effects of platelet releasate (PR) collected from activated platelet concentrate on rat bone marrow-derived cells. Cultures of primary rat bone marrow cells were overlaid with a fibrin matrix, and the number of cells migrating within the three-dimensional matrix and the leading front of migration were quantified. The addition of PR to the top of the fibrin gels at different time points caused a 25% increase in the leading front of migration and a 3.5-fold increase in the number of migrating cells. Platelet releasate was also shown to have a mitogenic effect on bone cells in proliferation studies. Comparison between migration and proliferation data indicated that PR stimulates the initial recruitment of bone marrow cells to migration. This assay further allowed the determination that rat bone marrow cells are capable of exerting contractile forces on fibrin matrices and that matrix contraction is directly related to the migratory activity of cells. The results provide a potential mechanism to explain why biologically active platelet-derived factors enhance endosseous wound healing.
Animals↗