Effect on osseointegration of two implant macro-designs:A histomorphometric analysis of bicortically installed implants in different topographic sites of rabbit's tibiae.

BACKGROUND: To evaluate the effect of two different implant macro-designs on the sequential osseointegration at bicortically installed implants in the rabbit tibia. A further aim is to compare the osseointegration at different topographic zones. MATERIAL AND METHODS: 27 New Zealand rabbits were implemented. Two implants, one for each macro-design (Ticare Inhex® or Ticare Quattro®, Mozo-Grau, Valladolid, Spain), were randomly implanted in the diaphysis or metaphysis of each tibia. The flaps were sutured to allow a submerged healing. The animals were sacrificed after 2, 4 or 8 weeks. Ground sections were prepared and analyzed. RESULTS: No statistically significant differences were found between the two groups for newly formed bone in contact with the implant surface, being about 16%, 19% and 33% in both groups, after 2, 4, and 8 weeks of healing. Bone apposition was slightly higher in the diaphysis, reaching values of 36.4% in the diaphysis, and 29.3% in the metaphysis at 8 weeks of healing. It was observed that the implant position showed a statistical significance regarding BIC values at 4 and 8 weeks (p<0.05). Multivariate analysis fails to detect statistical significant differences for the interaction between implant designs and topographic site. Ticare Quattro® design had a slight better BIC values at diaphysis sites across healing stages, but without reaching a statistical significance. CONCLUSIONS: The both implant macro-designs provided similar degrees of osseointegration. Bone morphometry and density may affect bone apposition onto the implant surface. The apposition rates were slightly better in diaphysis compared to metaphysis.

Complement proteins regulating macrophage polarisation on biomaterials.

One of the events occurring when a biomaterial is implanted in an host is the protein deposition onto its surface, which might regulate cell responses. When a biomaterial displays a compromised biocompatibility, distinct complement pathways can be activated to produce a foreign body reaction. In this article, we have designed different types of biomaterial surfaces to study the inflammation process. Here, we used different concentrations of (3-glycidoxypropyl)-trimethoxysilane (GPTMS), an organically-modified alkoxysilane as a precursor for the synthesis of various types of sol-gel materials functionalizing coatings for titanium implants to regulate biological responses. Our results showed that greater GPTMS surface concentrations induced greater secretion of TNF-α and IL-10 on RAW 264.7 macrophages. When implanted into rabbit tibia, osseointegration decreased with higher GPTMS concentrations. Interestingly, higher deposition of complement-related proteins C-reactive protein (CRP) and ficolin-2 (FCN2), two main activators of distinct complement pathways, was observed. Taking all together, inflammatory potential increase seems to be GPTMS concentration-dependent. Our results show that a greater adsorption of complement proteins can condition macrophage polarization.

Electrothermal atomic absorption spectrometric diagnosis of familial hypercholesterolemia.

We have developed a new nonradioactive assay to identify human low-density lipoprotein receptor defects. It is based on the incubation of cultured cells with colloidal gold-LDL conjugates and quantitation of the gold associated with the cells by electrothermal atomic absorption spectrometry. After an oxidative treatment with nitric and hydrochloric acids, the biological matrix interferes neither with the gold recovery nor with the gold measurements, which are linear, at least from 0.15 to 3 ng of gold. When cells expressing a functional LDL receptor are incubated with increasing amounts of colloidal-gold LDL conjugates, the obtained saturation curve parallels that described when [125I]LDL is used as ligand. Moreover, this new assay allows us to clearly distinguish among fibroblasts from normal subjects or from heterozygous or homozygous patients of familial hypercholesterolemia, a very common autosomal disease. The assay is easy to perform, is sensitive, and avoids the use of radioactive compounds. Therefore, it could be successfully employed in the clinical diagnosis of this disease. Furthermore, since the methodology developed here can be applied to quantify the association of other gold-conjugated ligands to cells, it could have a widespread use in a variety of clinical and basic research studies.

Mutational analysis of sickle haemoglobin (Hb) gelation.

The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants.

A rapid procedure suitable to assess quantitatively the endocytosis of colloidal gold and its conjugates in cultured cells.

We measured the endocytic uptake of low-density lipoproteins (LDLs) conjugated to colloidal gold in cultured cells, either by counting gold particles on electron micrographs or by inductively coupled plasma (ICP) mass spectrometry (MS). Both procedures are comparable but the latter requires a considerably shorter time and allows analysis of a much larger sample. In addition, ICP MS, compared to alternative radioactive or fluorescent procedures, offers the major advantage of using the same probe to quantify the endocytic uptake and to follow it by electron microscopy. Therefore, ICP MS analysis provides an easy, rapid, and sensitive quantification of endocytosis that complements the electron microscopic studies.

Nucleation and polymerization of sickle hemoglobin with Leu beta 88 substituted by Ala.

We have measured the solubility, and the rates of homogeneous and heterogeneous nucleation on sickle hemoglobin (HbS beta 6 Glu-->Val) additionally modified by site-directed mutagenesis to possess Ala rather than Leu at beta 88, which forms part of the receptor site for beta 6 Val in the sickle polymer. The solubility of the hemoglobin is increased at all temperatures, and is about 29 g/dl at 25 degrees C. Polymerization kinetics, induced by laser photolysis and observed by light-scattering intensity, showed exponential growth with rates about 300 times slower than experiments done on similar concentrations of HbS. When polymerization is carried out in small volumes, the time of measurable light-scattering signal to reach one-tenth of its final value (denoted as the tenth time) showed stochastic fluctuations, as is seen in pure HbS. Homogeneous nucleation rates were measured by observing distributions of tenth times and these rates were slowed by the mutation by almost 1000-fold relative to pure HbS. The kinetics, including the exponential progress curves and shape of the tenth time distributions, are well described by the double nucleation mechanism for polymerization. Analysis of the homogeneous nucleation rates leads to the surprising conclusion that the mutation has scarcely changed the energy of the intermolecular contacts despite the increase in solubility of the double mutant. This conclusion is supported by the stereochemistry of the modified contact site, in which the amount of exposed hydrophobic surface appears to be unchanged by the mutation. The increased solubility must therefore result from decreased motional freedom of molecules within the polymer, which could arise from tighter packing into the enlarged receptor pocket. This points up the ability of kinetic analysis to reveal important thermodynamic properties of assembly, and underlines the importance of the vibrational degrees of freedom in setting the final equilibrium constant. Chemical modifications to restrict vibrations and enhance the cost of polymerization may prove useful in constructing compounds to act as inhibitors of sickle cell gelation.

Use of inductively coupled plasma-mass spectrometry for the quantitation of the binding and uptake of colloidal gold-low-density lipoprotein conjugates by cultured cells.

The binding and endocytic uptake of low-density lipoprotein (LDL) particles by cells, transiently or permanently transfected with the human LDL receptor cDNA, was investigated, under different situations, using colloidal gold-LDL conjugates. The amount of gold associated with the various cells, which bind and internalize LDL to different extents, was estimated by inductively coupled plasma-MS. In all cases, the existing differences in LDL binding and uptake were clearly detectable with this procedure. We conclude, therefore, that inductively coupled plasma-MS provides an appropriate assay system for the rapid quantitation of these processes. This procedure also recognizes differences in LDL receptor expression in human lymphocytes and, therefore, it could be of value for the differential diagnosis of LDL receptor defects in familial hypercholesterolemia in various cell types. In addition, this easily performed methodology can also be applied to a variety of other problems requiring quantitation of colloidal gold associated with cells.

Solubility of sickle hemoglobin measured by a kinetic micromethod.

We have developed a photolytic method to determine the concentration of reactive hemes in a solution in the presence of a trace amount of CO. By measurement of the bimolecular rate of CO binding, and by calibration of the rate constant under equivalent conditions, the concentration of the reactive hemes can be determined. In a solution of sickle hemoglobin, the molecules in the gel contribute negligibly to the recombination rate, allowing the concentration of the molecules in the solution phase to be determined. To optimize signal to noise, modulated excitation methods were employed, although the method could also be used with pulse techniques and suitable signal averaging. Because the optical method employs a microspectrophotometer, only a few microliters of concentrated Hb solution is required to reproduce the entire temperature dependence of the solubility previously determined by centrifugation using milliliter quantities of solutions of the same concentration. This should be especially useful for studies of site-directed mutants, and we present results obtained on one such HbS in which Leu 88 beta has been replaced by Ala. The free energy difference in the polymerization of the Leu 88 beta double mutant is consistent with known differences in the amino acid hydrophobicities. The calibration required for these experiments also provides an excellent determination of the activation energy for binding the first CO to deoxy Hb.

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.

Biochemical and functional properties of recombinant human sickle hemoglobin expressed in yeast.

Previous studies had indicated that recombinant and natural human sickle hemoglobin had similar chemical properties (Martin de Llano, J. J., Schneewind, O., Stetler, G., and Manning, J. M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 918-922). In the present study, additional biochemical and physiological characterization of some primary through quaternary structural features of recombinant sickle hemoglobin are described. The molecular weight of the purified recombinant sickle hemoglobin was identical to natural sickle hemoglobin as determined by mass spectrometry, thus excluding extensive post-translational modification in the yeast system. Carboxypeptidases A and B together catalyzed the release of COOH-terminal amino acids at the same rate for recombinant and natural hemoglobin S, consistent with identity in their primary and secondary structures in this region of the molecule. The tryptic peptide maps of natural and recombinant hemoglobins were practically indistinguishable, indicating the same internal protein sequences for recombinant and natural hemoglobins. As a probe of the secondary structure of recombinant sickle Hb, the reactivity of the SH group of Cys-93(beta) was investigated for the glutathione sickle hemoglobin adduct, which has significant anti-gelling and anti-sickling properties. The position of glutathione at Cys-93(beta) was established by direct mass spectrometric analysis of enzyme digests; reduction of this derivative to the unmodified chains was also observed by mass spectrometry and by isoelectric focusing. The oxygen equilibrium curves of recombinant and natural sickle hemoglobin at high protein concentration were superimposable with identical Hill coefficients of 3.3. The response of recombinant sickle hemoglobin to chloride with respect to a lowered oxygen affinity was identical to that of natural sickle hemoglobin. The gelation properties of recombinant and natural sickle hemoglobins were identical at the high hemoglobin concentrations that occur in the red cell. Therefore, the yeast expression system synthesizes a completely functional recombinant sickle hemoglobin with the same biochemical and physiological properties as natural sickle hemoglobin with respect to features characteristic of its primary through quaternary structures.

Recombinant human sickle hemoglobin expressed in yeast.

Sickle hemoglobin has been expressed in the yeast Saccharomyces cerevisiae after site-directed mutagenesis of a plasmid containing normal human alpha- and beta-globin genes. Cassette mutagenesis of this plasmid was achieved by inserting a DNA fragment containing the beta-globin gene in the replicative form of M13mp18 to make a point mutation and then reconstituting the original plasmid containing the mutated beta-globin gene. Pure recombinant hemoglobin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel electrophoresis, isoelectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and functional properties (P50, cooperativity, and response to 2,3-bisphosphoglycerate). In yeast and in mammalian cells, cotranslational processing yields the same N-terminal valine residues of hemoglobin alpha- and beta-chains, but in bacterial expression systems the N terminus is extended by an additional amino acid because the initiator methionine residue is retained. Since the N-terminal valine residues of both chains of hemoglobin S participate in important physiological functions, such as oxygen affinity, interaction with anions, and the Bohr coefficient, the yeast expression system is preferable to the bacterial system for recombinant DNA studies. Hence, mutagenesis employing this expression system should permit definitive assignments of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention. The engineering of this system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.

Increased electrophoretic mobility of sodium sulfite-treated jack bean urease.

Sodium sulfite is a widely used activity-protective agent for the storage of urease. However, this reagent produces a 10% increase in the anodic electrophoretic mobility of native urease. Changes in the hydrodynamic properties of the enzyme are not involved in that modification. The observed change is related to an increased negative charge of the protein molecule in the presence of sodium sulfite. The results are discussed in terms of sulfitolysis of the single disulfide bond in the urease monomer. It is remarkable that the modification occurs at neutral pH. Our results show that removing sodium sulfite and reversing its effect by treatment with 2-mercaptoethanol are required prior to any study involving native urease.

Selective silver staining of urease activity in polyacrylamide gels.

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.