Ultrastructure of ceramic-bone interface using hydroxyapatite and beta-tricalcium phosphate ceramics and replacement mechanism of beta-tricalcium phosphate in bone

Hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) are useful for grafting and augmentation of bone tissue. Observation by transmission electron microscopy (TEM) was done to investigate the ultrastructures at the interfaces between the biomaterials and the adjacent tissue, and osteogenesis around the biomaterials in the present study. HA and beta-TCP ceramics were used in disk forms which had macropores and micropores, and were implanted between the parietal bone and the cranial periosteum of rats. Specimens were prepared for observation at 4 and 8 weeks postoperatively. The microscopic results indicated that an intervening layer was present on the surface of HA, whereas it was not present on the surface of beta-TCP. A characteristic fibrillar structure was observed in the intervening layer between HA and bone under decalcification by HCl. In beta-TCP, in reticular structures observed close to the bone tissue by optical microscopy, calcification and sparse collagen fibers were interspersed among the granules of beta-TCP. In addition, close to the interface between beta-TCP and bone, many osteocytes with numerous processes were present. Some processes were elongated towards the interface. These results revealed the difference in the ultrastructures of the interfaces between HA and beta-TCP, and the dissolution mechanism of beta-TCP in bone.     

Chemical analysis of silica doped hydroxyapatite biomaterials consolidated by a spark plasma sintering method

Silica (SiO(2)) and the silicate-based biomaterials play an important role due to their in vitro and in vivo biological response. The present study synthesized a novel nano-structured amorphous silica doped hydroxyapatite (HA) via an aqueous precipitation route. HA was prepared with 0, 1, 3 and 5 wt% silica, which are comparable to the measured silicon content of natural bone. After spray drying into micron sized powders, the silica doped HA (SiHA) powders were consolidated at 1000 degrees C with a dwell time of 3 min using a spark plasma sintering (SPS) technique. X-ray diffraction analysis showed a main apatite phase with minor secondary beta-tricalcium phosphate (beta-TCP) was observed in the as-consolidated SiHA compacts. Substitution of PO(4)(3-) by SiO(4)(4-) in the apatite structure resulting in a small increase in the lattice parameters in both a-axis and c-axis of the unit cell were identified by X-ray photoelectron spectrometer (XPS) analysis and Raman spectrometer investigation. The cell culture in vitro investigation demonstrated that the presence of silicon in the SPS consolidated compacts contributed to the relatively high cell proliferation ability when compared with phase pure HA.     

Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.     

Systematic fabrication of nano-carbonated hydroxyapatite/collagen composites for biomimetic bone grafts

A novel biomimetic self-assembly method was designed to create nano-carbonated hydroxyapatite/collagen (nCHAC) composites by means of incorporating various collagen and carbonate concentrations using solutions such as CaCl(2), H(3)PO(4), and Na(2)CO(3). At a given range of collagen and carbonate content, the nanosized inorganic phase of the newly synthesized material has a low degree of crystallinity which resembles that of natural bone. By manipulating the concentrations of collagen and carbonates, various morphologies of the nCHAC can be obtained. The crystal size of nCHAC is dependent on the concentration of carbonate and collagen present in the composites. For instance, higher collagen concentration results in smaller crystal nCHAC crystal size. Conversely, the higher the carbonate content, the smaller are the crystal size and the collagen fibril assembly. As the carbonate content increased, the plate-like crystals first became needle-like structures, subsequently short needle-like crystals and eventually became spherical particles. From this study, our method showcased the flexibility of fabricating various types of nCHAC composites which can be designed for different bone applications.     

Interaction of acidic poly-amino acids with octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of poly-L-aspartate (PASP) and poly-L-glutamate (PGLU). In the absence of the polyelectrolytes, the transformation of OCP into HA is complete in 48 h. Both poly-L-aspartate and poly-L-glutamate inhibit OCP hydrolysis. However, PGLU displays a greater inhibiting effect, as a result of the different extent of phase transformation obtained at the same polyelectrolyte concentration. The inhibition takes place through polyelectrolyte adsorption on the (100) face of OCP crystals, which prevents the splitting of OCP crystals along their c-axis and the transformation into the final very long, needle-like, apatitic crystals.     

Divalent Mn in calcium hydroxyapatite by pulse laser deposition

Pulse laser deposition (PLD) was used to deposit Mn containing calcium hydroxyapatite (HAMn). The PLD process ensures that the composition of the target and the deposited layer is the same. In some cases additional effort should be made to preserve some volatile components, namely OH. This was ensured by water steam supply. Calcium hydroxyapatite deposited by this method has the same properties as the target in respect to lattice parameters and valence state of Mn, which ensures the fixation between hard tissue and metal implants. This fact makes PLD grown HAMn layer covering implants to be improved for practical use.     

Carbonated hydroxyapatites precipitated in the presence of Ti

Hydroxyapatites (HA) were prepared by precipitation from an aqueous solution with Ti4+ (0-2500 microg/g) and with carbonate (0.8-4.0%) at pH 7.0. The uptake of Ti was found to be 75% of the original amounts. Stoichiometric ratios of Ca/P (1.67) were found for low carbonate samples. X-ray diffraction and IR spectroscopy have shown that samples have structural data characteristic for HA. Heat treatment and thermogravimetric analysis (20-900 degrees C) have shown carbonate decomposition enhanced by the presence of Ti and no transformation of the HA structure. It was also found that 0.2 mol of adsorbed and 0.6-0.8 mol of crystalline water are released from the samples during heating. Transmission electron microscopy revealed the formation of plate like crystals which increase in size with increase of carbonate content. Samples with high carbonate and high Ti content have irregular shape and are sensitive to electron beam irradiation as opposed to non-doped samples. Ti appears to have a destabilizing effect on HA. The incorporation of Ti in HA and the biological relevance of Ti in bone crystals is discussed.     

Crystallographical analysis of tooth enamel using milligram samples

An X-ray diffraction microanalytical method, in which sample is loaded onto a silver membrane filter, was applied to assess the crystal content in tooth enamel. Each enamel powder was first examined at room temperature, and then examined again at intervals after heating to 200, 400, 600, 800, and 1000 degrees C. The hydroxyapatite composition weight and crystal weight of the samples were derived from the standard calibration curves. The "crystal content ratio" was defined as the ratio of crystal weight to sample weight. The following results were obtained: (1) beta-tricalcium phosphate (beta-TCP) replaced the hydroxyapatite after heating at the high temperatures; (2) the "crystal content ratio" in the tooth enamel increased with the rise in temperature; and (3) the lattice parameters of the enamel apatite and the beta-TCP were changed by the heating. The X-ray diffraction technique has the potential to analyze the crystal content using milligram samples.     

Preparation and physicochemical properties of a novel hydroxyapatite/chitosan–silk fibroin composite

A novel hydroxyapatite/chitosan–silk fibroin (HA/CTS–SF) composite was prepared for bone repair and replacement by a coprecipitation method. It was revealed that the inorganic phase in the composite was carbonate-substituted HA with low crystallinity. The HA crystallites were found to be needle-like in shape with a typical size of 20–50 nm in length and around 10 nm in width. The composite exhibited a higher compressive strength than the precipitated HA without any organic source involved, which was closely related to the perfect incorporation of chitosan and SF macromolecules into the composite. The chemical interactions occurring between the mineral phase and the organic matrix were thought to improve the interfacial bonding and thus resulted in the enhanced mechanical property of the composite.     

In vitro studies of human and rat osteoclast activity on hydroxyapatite,beta-tricalcium phosphate,calcium carbonate

Investigations on the ceramic degradation caused by osteoclasts are designed to assess osteoclast-ceramic interactions and to determine which ceramics are more suitable for use as bone substitute. This study investigated the resorptive activity of osteoclasts on ceramics presenting different solubility rates. Osteoclasts isolated from new-born rat and from human giant cell tumour were cultured on different bioceramics: hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and calcium carbonate (calcite). Cytoskeletal was revealed by actin labelling and ceramic surfaces were observed by scanning electron microscopy (SEM). On all materials, the distribution of actin in typical ring was revealed. SEM examinations showed a clear difference in the shape and the depth of resorption lacunae on different ceramics. On pure HA, a superficial attack, clearly visible but very little extended. Numerous resorption lacunae, deep and well-delimited were observed on pure beta-TCP, but attacks less punctually were detected too. On pure calcite, an attack with form of spikes, very widespread but superficial was revealed. Degradation measurements revealed a significant increase of P release from the phosphocalcic ceramics and of Ca from all ceramics in the presence of osteoclasts. The both cell models found these characteristics, the rat osteoclasts were also an excellent model to study the ceramic resorption.     

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 +/- 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.     

Accumulation and interaction of calcium and manganese in Phytolacca americana

To further understand the hyperaccumulation of Mn, the present study investigated the accumulation of Ca and Mn and their interaction in Mn hyperaccumulator pokeweed (Phytolacca americana Linn.). Exogenous Ca was observed to have a distinctive impact on the Mn phytotoxicity and accumulation in pokeweed, but exogenous Mn had little influence on the accumulation of Ca. Both Ca and Mn accumulated in pokeweed were detected to be mainly in the form of oxalate. Investigation with SEM and TEM found there were two kinds of crystals in the leaves, Ca oxalate crystals and Mn-containing crystals. Further detection showed that there was no inclusion of Mn inside the Ca oxalate crystals, and that other elements, such as C, O and P, were present in the Mn-containing crystals. These results suggest that Ca oxalate crystals in pokeweed have no direct effect on the detoxification of Mn. In addition, the finding of element P and O in the Mn-containing crystals indicates that excess Mn could be deposited by phosphate, which could contribute to Mn accumulation and detoxification in pokeweed.     

Crystallization of bacteriophage MS2.

Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1% virus solution from 23 degrees C to 0 degrees C in the presence of poly(ethylene glycol) 6000. The crystals were colorless, needle-like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a two-dimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X-ray examination of the crystals confirmed their viral nature.     

In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape

Successful closure of bone defects in patients remains an active area of basic and clinical research. A novel and promising approach is the transplantation of human bone marrow stromal cells (BMSCs), which have been shown to possess a significant osteogenic potential. The extent and quality of bone formation by transplanted human BMSCs strongly depends on the carrier matrix with which cells are transplanted; to date, hydroxyapatite/tricalcium phosphate (HA/TCP) supports far more osteogenesis than any other matrix tested. In order to further improve the technique of BMSC transplantation, we studied whether commercially available HA/TCP particles, clinically approved as an osteoconductive material and commercially available as particles measuring 0.5-1.0 mm diameter, is an optimum matrix for promoting bone development by BMSCs. HA/TCP and HA particles of varying size were sieved into a variety of size ranges, from <0.044 mm to 1.0-2.0 mm. Transplants were formed by mixing 40 mg aliquots of particles with cultured passaged human BMSCs. They were placed in subcutaneous pockets in immunocompromised Bg-Nu-XID mice and harvested 4 or 10 weeks later. The transplants were examined histologically; the presence of bone within each transplant was evaluated using histomorphometry or blindly scored on a semiquantitative scale. Transplant morphology and the amount of new bone varied in a consistent fashion based on particle size and shape. Transplants incorporating HA/TCP particles of 0.1-0.25 mm size demonstrated the greatest bone formation at both 4 and 10 weeks; larger or smaller particles were associated with less extensive bone formation, while a size of 0.044 mm represented a threshold below which no bone formation could be observed. Flat-sided HA particles measuring 0.1-0.25 mm formed no bone. The differences in bone formation were not attributable to the differences in cell attachment among the groups. Instead, the size and spatial and structural organization of the particles within BMSC transplants appear to determine the extent of bone formation. These findings provide necessary information for the successful clinical application of BMSC transplantation techniques.     

Paracrystalline inclusions in various isolates of the blue-green bacteria Nostoc and Anabaena.

A number of different crystalline inclusions were observed in various isolates of Anabaena and Nostoc. Membrane-limited crystalline bodies were observed in 7 of 20 isolates of Anabaena and 19 of 29 isolates of Nostoc. These are spherical, single membrane-limited bodies from 0.6 to 0.1 micron in diameter. In most of the isolates they contained needle-like crystals 20 A in thickness and up to 80 nm in length. In 9 of the isolates the inclusions contained granular and fibrillar material. The number of bodies per cell varied in the different isolates from only a few, observed in many sections, up to 5 in a single section of A. subtropica (B1618). Crystalloids were observed in the cytoplasm of Anabaena sp. (1551), N. calcicola (B382), Nostoc sp. (588), and N. punctiforme (1629). In Anabaena sp. (1551) the roughly cuboidal inclusions 0.6 micron in diameter were composed of 100 A thick osmiophilic striations spaced to produce a 150 A periodicity. In Nostoc sp. (588) the elongate, 0.1 micron by 2.5 micron, crystalloids were composed of 100 A thick osmiophilic striations spaced to produce a 200 A periodicity. N. punctiforme (1629) and N. calciola (B382) contained intrathylakoidal crystalloids which consisted of short curved segments with 100 A thick osmiophilic striations producing a 200 A periodicity. Granular areas were observed in 2 isolates of Anabaena and 5 of Nostoc. These bodies found in various locations in the cells, were interpreted to be elongate structures 0.2 micron thick, 1.2 micron long and about 5 micron in depth. These inclusions were composed of 15 nm diameter granules which in some section planes appeared in rows spaced 20 nm apart. Spherical bodies up to 0.7 micron in diameter and of medium electron density were observed in 4 isolates of Anabaena and 2 of Nostoc. Convoluted inclusions were found in N. calcicola (B382) and Anabaena sp. (1551). These roughly spherical bodies up to 0.8 micron in diameter contain lighter swirled areas.     

Morphometry and ultrastructure of the squirrel monkey (Saimiri sciureus) vestibular nerve

Vestibular nerves of squirrel monkeys (Saimiri sciureus) embedded in plastics and epoxies were examined with light microscopy (LM) and transmission electron microscopy (TEM), and computerized measures were obtained and analyzed statistically. An average of 12,412 perikarya and 12,005 myelinated nerve fibers was obtained. Approximately 0.7% of the perikarya appeared unmyelinated under LM. About 500 unmyelinated fibers were counted. The cross-sectional area of 1,864 perikarya was 200-650 micron 2. The cross-sectional area of 1,346 nerve fibers was 3-11 micron 2 for the axoplasm and 11-12 micron 2 for the myelin sheath of the same fiber. Myelin thickness was directly proportional to the axoplasm cross-sectional area of the nerve fibers. The cross-sectional area of central axons and peripheral dendrites differed significantly (p less than 0.001). The initial segments of peripheral dendrites were usually smaller, but longer than the initial segments of the central axons. Both initial segments increased in diameter after the first node of Ranvier. Schmidt-Lantermann incisures were more abundant in thick and heavily myelinated fibers than in thin and lightly myelinated fibers. Larger perikarya usually had larger fibers and vice versa, within the first 100-200 micron from the first node of Ranvier. No major ultrastructural differences were found between myelinated and unmyelinated perikarya, except at the hillock region. The Nissl substance was preferentially located in the peripheral cytoplasm.     

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1–L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1–L2, (b) TCP/HA constructs group/L2–L3, and (c) autogenous bone graft group/L5–L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.     

The validity of the cholesterol nucleation assay.

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 micron filter or centrifuged at 37 degrees C for 2 h at 100,000 x g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 +/- 1.1 days to 1.8 +/- 0.2 days (mean +/- S.E., P less than 0.01, n = 5) when 10-100 micrograms/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 micron filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 +/- 0.7 days to 3.1 +/- 0.5 days (mean +/- S.E.) when 10 micrograms/ml cholesterol crystals were added before filtration (n = 10, P less than 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 micron filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 micron filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903-1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.     

Crystalline inclusions in the cytoplasm and nuclei of cells of acute myeloid leukaemia

In a survey by electron microscopy of peripheral blood and/or bone marrow from 230 adult patients with acute myeloid leukaemia, five were observed to contain crystalline inclusions in the cytoplasm of the leukaemic cells and a sixth contained crystals in the nuclei. In four cases, two of FAB type M2 and two of M4, the cytoplasmic crystals were hexagonal in section and 1-2 micron long. Two examples showed internal periodicities in the range 3.3-4.0 nm when the electronmicrographs were analysed by optical diffractometry. A single case of M1 contained smaller trapezoidal crystals with a 4.9nm periodicity. The sixth patient, with unusual cytological abnormalities and a rare t(3; 6) chromosomal translocation, contained six-sided crystals in the nuclei of some relatively undifferentiated cells. To the best of our knowledge such intranuclear crystals have not previously been reported in leukaemia. The relevance of the crystals to the leukaemic process is discussed.     

Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 micron 3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 micron 3, 0.3 micron in diameter, were resolved. Aquatic bacteria 0.05 micron 3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence. These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.