Dynamic behavior of implants as a measure of osseointegration.

Research into the formation, destruction, and adaptation of bone around implants would benefit from a sensitive, nondestructive, noninvasive, and quantitative technique to assess the bone-implant interface. It is hypothesized that osseointegration can be quantified by sensing the mechanical impedance (or micromobility) of the implant when it is subjected to minute vibratory forces superimposed upon a quasi-static preload. To test this hypothesis, a total of 24 identical threaded, titanium root-form implants (10 x 3.75 mm, Osteo-Implant, New Castle, PA) were placed in the mandibles of 4 Walker hounds and allowed to heal submerged for 3 months. The implants were exposed and characterized for osseointegration using clinical observations, quantitative radiography, and a custom-designed impedance instrument. Subsequently, arbitrarily selected implants were ligated to induce bone loss and examined monthly over a 6-month study period. Following the terminal examination and euthanasia, quantitative histologic measurements were made of bone adjacent to the implant, including estimates of both crestal bone height and the percent bone (bone fraction). Linearized dynamic parameters (effective stiffness and effective damping) correlated well with radiographic and histologic measures of bony support (r2 values ranged from 0.70 to 0.89). Moreover, the presence of nonlinear stiffness was clearly associated with a bimodal "clinical impression" of osseointegration (P < .0003, 1-way analysis of variance). These results confirm that, in this animal model, mechanical impedance can be used as a measure of implant osseointegration.

Periodontal disease progression.

BACKGROUND: The objective of this investigation is to use noninvasive, state-of-the-art, diagnostic techniques to measure periodontal disease progression and model periodontal disease activity over time. In this investigation, digital subtraction radiography and an electronic controlled force periodontal probe capable of attachment level measurement were used to measure bone loss and attachment loss, respectively. The use of these nearly continuous measures of attachment and bone loss allowed detection of small amounts of disease activity and provided data to be used in modeling of the disease process over time. METHODS: Forty-four patients were studied for 18 months. Examinations used clinical attachment level measures at 1-month intervals and quantitative radiology at 6-month intervals. The sites were analyzed by regression for statistically significant changes. These data were used to determine sites of periodontal disease activity for testing various models of periodontal disease progression. RESULTS: Overall 22.8% of sites lost attachment, 5.4% gained, and 71.7% demonstrated no statistically significant change. The mean time to lose 1 mm of attachment was 8.4 +/- 0.6 months. In the first model tested a step-wise discriminant analysis was used to determine whether or not baseline measurements of plaque (PI), gingival inflammation (GI), attachment loss, and probing depth (PD) could be used to derive a satisfactory model for disease progression. Although the overall model was statistically significant with PI, PD, and GI contributing to the model (Wilks' lambda = 0.859, F = 5.71, P <0.0012), its predictive power was relatively weak. A considerably stronger significant model resulted when the rate of attachment loss over the first 6 months, baseline PI, and baseline GI were included (Wilks' lambda = 0.712, F = 14.17, P<0.00001). A significant model also resulted when bone loss during the first 6 months and baseline probing depth were included (Wilks' lambda = 0.438, F = 61.48, P<0.00001). When the last model was applied to each site, the sensitivity in predicting disease progression was 80.0% and the specificity in ruling out progressive disease was 93.9%. CONCLUSIONS: This study indicates that clinically significant progression of attachment loss in posterior tooth sites occurs as a frequent event in adult periodontitis. The modeling data also suggest that short-term (6 month) measures of periodontal disease progression greatly improve the ability to model attachment loss over a longer period in untreated periodontitis patients.

Assessment of osseointegration by nonlinear dynamic response.

Quantitative assessment of osseointegration remains a goal of researchers and clinicians alike. In this study, an instrument was designed for this purpose and tested in an animal model. Effective mechanical impedance, linearized for quasi-static force, was measured in 22 implants placed in the hind tibiae of 2 large hounds. The results demonstrate that in this animal long-bone model, the effective impedance of titanium root-form implants exhibits a degree of nonlinear behavior correlated with their state of osseointegration. This observation may be the basis for useful clinical instrumentation.

Production of very-high-amylose potato starch by inhibition of SBE A and B.

High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.

A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A.

Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.

Colonic bacterial activity and serum lipid risk factors for cardiovascular disease.

Antibiotics are being proposed for the treatment of cardiovascular disease. In the past, antibiotics were advocated for the control of hypercholesterolemia. We have therefore investigated the relation between colonic bacterial activity and serum lipids. In a four-phase randomized crossover study, we fed a different starch supplement during each 2-week phase to 24 healthy subjects. In two phases, supplements containing resistant starches were fed that reach the colon and are largely fermented by colonic bacteria. Fecal starch recovery therefore reflects the metabolic activity of colonic microflora. The control treatments were conventional starches. Blood lipid levels were obtained at the start and 4-day fecal collections at the end of each phase. Resistant starch supplements increased fecal starch excretion by 3.8 +/- 1.2 g/d more than conventional starches (P = .006). Mean starch excretion was related positively to pretreatment serum high-density lipoprotein (HDL) cholesterol (r = -.57, P = .003) and negatively to low-density lipoprotein (LDL) cholesterol (r = -.57, P = .004), apolipoprotein B:AI (r = -.56, P = .005), and fecal output of fusobacteria (r = -.73, P = .003) and bacteroides (r = -.72, P = .003). The ratio of fusobacteria to total anaerobes was also related to pretreatment LDL cholesterol (r = .56, P = .037). Differences in starch excretion between healthy subjects, as a measure of bacterial activity, accounted for 32% of the variation in pretreatment LDL cholesterol. The activity of colonic microflora therefore appears to influence serum lipid levels. Alterations of bacterial number and activity may provide an additional strategy to control serum lipid risk factors for cardiovascular disease.

Physiological effects of resistant starches on fecal bulk, short chain fatty acids, blood lipids and glycemic index.

OBJECTIVE: To assess the effects on fecal bulking, fecal short chain fatty acid (SCFA) production, blood lipids and glycemic indices of two different forms of resistant starch (RS2 and RS3) from a high-amylose cornstarch. METHODS: Twenty-four healthy subjects (12 men; 12 women) consumed four supplements taken for 2 weeks in random order separated by 2-week washout periods. The supplements were a low-fiber (control) and supplements providing an additional 30 g dietary fiber as wheat bran (high-fiber control) or the equivalent amount of resistant starch analyzed gravimetrically as dietary fiber from RS2 or RS3. Four-day fecal collections and 12-hour breath gas collections were obtained at the end of each period. Fasting blood was taken at the beginning and end of each period. Glycemic indices of supplements were also assessed. RESULTS: The wheat bran supplement increased fecal bulk 96+/-14 g/day compared with the low-fiber control (p<0.001) with the mean for both resistant starches also being greater (22+/-8 g/day) than the low-fiber control (p=0.013). On the resistant starch phases, the mean fecal butyrate:SCFA ratio, which has been suggested to have positive implications for colonic health, was significantly above the low-fiber control by 31+/-14% (p=0.035). Resistant starches did not alter serum lipids, urea or breath H2 or CH4. No significant differences in glycemic index were seen between the RS and control supplements. CONCLUSION: The potential physiological benefits of the resistant starches studied appear to relate to colonic health in terms of effects on fecal bulk and SCFA metabolism.

Comparison of lead bioavailability in F344 rats fed lead acetate, lead oxide, lead sulfide, or lead ore concentrate from Skagway, Alaska.

An animal model using rats was developed to initiate investigations on the bioavailability of different sources of environmental lead. Lead must be absorbed and transported to target organs like brain, liver, kidney, and bone, before susceptible cells can be harmed. The bioavailability and therefore the toxicity of lead are dependent upon the route of exposure, dose, chemical structure, solubility, particle size, matrix incorporation, and other physiological and physicochemical factors. In the present study male F344 rats were fed < or = 38 microns size particles of lead sulfide, lead oxide, lead acetate, and a lead ore concentrate from Skagway, Alaska, mixed into the diet at doses of 0, 10, 30, and 100 ppm as lead for 30 d. No mortality or overt symptoms of lead toxicity were observed during the course of the study. Maximum blood lead concentrations attained in the 100 ppm groups were approximately 80 micrograms/dl in rats fed lead acetate and lead oxide, and were approximately 10 micrograms/dl in those fed lead sulfide and lead ore concentrate. Maximum bone lead levels in rats fed soluble lead oxide and lead acetate were much higher (approximately 200 micrograms/g) than those seen in rats fed the less soluble lead sulfide and lead ore (approximately 10 micrograms); kidney lead concentrations were also about 10-fold greater in rats fed the more soluble compared to the less soluble lead compounds. However, strong correlations between dose and tissue lead concentrations were observed in rats fed each of the four different lead compounds. Kidney lesions graded as minimal occurred in 7/10 rats fed 30 ppm and in 10/10 rats fed 100 ppm lead acetate, but not at lower doses or from other lead compounds. Similarly, urinary aminolevulinic acid excretion, a biomarker for lead toxicity, was increased in rats fed 100 ppm lead acetate or lead oxide, but was unaffected at lower doses or by the less soluble lead compounds. Although the histological and biochemical responses to lead toxicity were restricted to the more soluble lead compounds in this study, lead from Skagway lead ore concentrate and lead sulfide was also bioavailable, and accumulated in proportion to dose in vulnerable target organs such as bone and kidney. Longer-term studies with different mining materials are being conducted to determine if tissue lead continues to increase, and whether the levels attained are toxic. Data from such studies can be used to compare the toxicity and bioavailability of lead from different sources in the environment.

Planning interactive implant treatment with 3-D computed tomography.

Treatment planning for dental implants presents special challenges to the dentist. The authors review the conventional radiographic and non-radiographic methods for the assessment of the implant site. A new dental interactive treatment planning program under development at the University of Alabama School of Dentistry Department of Periodontics is described.

A periodontal probe with automated cemento--enamel junction detection-design and clinical trials.

An automated periodontal probe has been developed to measure pocket depth and attachment loss in a single measurement under controlled force conditions. A natural anatomic landmark, the cemento-enamel junction (CEJ), is used as a reference for attachment level measurements. The CEJ is detected automatically by immediate digital postprocessing of simultaneous measurements of probe tip acceleration and displacement during probing. Clinical trials in the beagle dog model for naturally occurring periodontitis have shown that the automated probe, when used with 35 g probing force, has a repeatability of 0.16 mm and a bias of 0.09 mm. These results indicate that the automated periodontal probe is capable of attachment level measurements with a high degree of repeatability and validity.

Synthesis and possible applications of biotin-linked copper clusters.

The trifunctional aziridine XAMA-7 (CAS 57116-45-7) has been used to form crosslinks between a deep red-violet copper cluster of the type Cu(I)8Cu(II)6pen12Cl5- (pen=penicillamine) and molecules with biological activity such as d-biotin and proteins. A complex containing biotin, bovine serum albumin and the copper cluster displayed activity toward affinity columns of avidin on Agarose, and the red-violet pigment was immobilized on the gel. This interaction was completely blocked in gels which had been pretreated with d-biotin carboxylic acid. The free and biologically active versions of the cluster have some potential for biomedical applications. For example, the short-lived positron emitter 64Cu (suitable for positron tomography) may be carried in the cluster's structure. The cluster is paramagnetic, but it is a relatively weak effector of water proton spin-lattice relaxation. Other members of this structural group of inorganic compounds may have better magnetic properties, and the crosslinking reaction with aziridines appears to be generally applicable to the group.

Design and applications of biomimetic anthraquinone dyes. Purification of calf intestinal alkaline phosphatase with immobilised terminal ring analogues of C.I. reactive blue 2.

A 330-fold one-step purification of alkaline phosphatase from a crude calf intestinal extract has been achieved using specific elution with inorganic phosphate (5 mM) from a purpose designed adsorbent comprising a terminal ring phosphonate analogue of C.I. Reactive Blue 2 coupled to Sepharose CL-6B-200. The resulting alkaline phosphatase preparation displayed a specific activity in excess of 1000 U/mg and was of equivalent purity to commercial "high purity" preparations as deduced by sodium dodecyl sulphate polyacrylamide gel electrophoresis and specific activity comparisons.

Bacterial metabolism of alpha-pinene: pathway from alpha-pinene oxide to acyclic metabolites in Nocardia sp. strain P18.3.

Over 20 gram-positive bacteria were isolated by elective culture with (+/-)-alpha-pinene as the sole carbon source. One of these strains, Nocardia sp. strain P18.3, was selected for detailed study. alpha-Pinene-grown cells oxidized, without lag, alpha-pinene, alpha-pinene oxide (epoxide), and the cis and trans isomers of 2-methyl-5-isopropylhexa-2,5-dienal. No other tested terpene was oxidized at a significant rate. alpha-Pinene was not metabolized by cell extracts in the presence or absence of NADH or NADPH. Cell extracts catalyzed a rapid decyclization of alpha-pinene oxide, in the absence of added cofactors, with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. Further oxidation of the aldehyde to the corresponding acid occurred in the presence of NAD. Both activities were induced by growth with alpha-pinene. A rapid, nonenzymic transformation of the cis aldehyde into the trans isomer occurred in glycine buffer. The trans isomer was also a substrate for the NAD-linked aldehyde dehydrogenase. The distribution of the alpha-pinene oxide lyase in alpha-pinene-utilizing Pseudomonas spp. was also investigated and was compatible with the two alternative ring-cleavage sequences that have been proposed on the basis of accumulated metabolites.

Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3.

alpha-Pinene oxide is an intermediate in the degradation of alpha-pinene by Nocardia sp. strain P18.3 and some Pseudomonas strains. The epoxide is cleaved by a lyase which catalyzes a concerted reaction in which both rings of the bicyclic structure are cleaved with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. The enzyme has been purified to homogeneity from Nocardia sp. strain P18.3. It was induced by growth with alpha-pinene and constituted 6 to 7% of the soluble protein of cell extracts. The apparent molecular weight of the native enzyme was 50,000 by ultracentrifugal analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave two dissimilar subunits with apparent molecular weights of 17,000 and 22,000. The enzyme was devoid of prosthetic groups, had no cofactor requirement, and had a broad pH activity range, a Km for alpha-pinene oxide of 9 microM, and a turnover number of 15,000. Inhibitors included sulfhydryl reactive compounds, terpene epoxides, and pinane derivatives with substituent groups at carbon 3. A mechanism for the concerted reaction has been proposed in which decyclization is initiated by donation of a proton from the catalytic center to the oxygen of the epoxide with consequent destabilization. In vitro the enzyme was inactivated during catalysis, and a reactive cationic intermediate may be responsible for this phenomenon. The enzyme should be classified as a lyase EC 4.99.-.-.

The rCBF response to Diamox in normal subjects and cerebrovascular disease patients.

Age-related norms for the regional cerebral blood flow (rCBF) response to Diamox (acetazolamide) were based on studies of 55 normal subjects at rest and on studies of 33 of these 55 normal subjects following an intravenous injection of Diamox (22 mg/kg). After the Diamox injection, rCBF increased at all locations measured in all subjects. On average, rCBF increased 1.7 times. The following were found for rCBF in both resting and Diamox-treated subjects: 1) rCBF decreased significantly with increasing age; 2) slope and intercept for the regression of rCBF on age were largest for frontal detectors, intermediate for parietal detectors, and smallest for occipital detectors; 3) rCBF hyperfrontality was most noticeable in younger subjects; 4) in subjects of any age, 95% confidence intervals for rCBF were relatively large (expected value +/- 30%) and lower 95% confidence intervals for Diamox rCBF tended to overlap the upper 95% confidence intervals for resting rCBF; and 5) side-to-side percentage difference in rCBF did not have a significant regression on age and tended to be less than 10% to 20%. Diamox did not have an important effect on blood pressure, pulse rate, or respiratory rate. The normative data for the rCBF response to Diamox was used in evaluating 20 patients with cerebrovascular disease. Forty percent of these patients, all of whom exhibited angiographic evidence of potentially hemodynamically significant lesions, had normal rCBF at rest and after Diamox injection. Twenty percent had normal resting flows with abnormal Diamox-activated flows. Asymmetry in rCBF was the most sensitive indicator of a potential abnormality in cerebral perfusion. Thirty percent of the abnormal studies showed only significant asymmetry. It is suggested that rCBF studies at rest and after Diamox treatment, with age-related norms, may be useful in the management of patients with cerebrovascular disease.

Implication of histidine at the active site of exo-beta-(1-3)-D-glucanase from Basidiomycete sp. QM 806.

The enzyme, exo-beta-(1-3)-D-glucanase, (EC 3.2.1-) obtained from a culture filtrate of Basidiomycete sp. QM 806, has been obtained in a highly purified form and preliminary investigations on its mechanism of action have been reported (Peterson, D. R., and Kirkwood, S. (1975) Carbohydr. Res. 41, 273-283). Studies reported in this paper, have provided strong evidence for the role of histidine in the catalytic site of this carbohydrase. Chemical modifications of the amino acid residues in the enzyme with diazotized 5-amino-1H-tetrazole or tetranitromethane caused irreversible loss of enzyme activity which varied according to the time of exposure to, or concentration of the inhibitor. Prior incubation of the enzyme with a substrate considerably reduced the extent of this inhibition. Amino acid analysis of the enzyme treated in these ways clearly indicated that the substrate protected histidine residues from chemical modification by the diazotized 5-amino-1H-tetrazole. Chemical modification of both histidine and tyrosine residues were effected by incubating the enzyme with the inhibitors described above. Although evidence is presented to suggest that tyrosine is not directly involved in the active site of the enzyme (the catalytic site or the binding site), the role of this residue in the maintenance of the enzyme conformation is discussed. Enzyme assays carried out either in aqueous or deuterated buffer systems provided further evidence which is consistent with the proposed enzyme mechanism.

A new periodontal probe with automated cemento-enamel junction detection.

A new automated periodontal probe has been developed which measures attachment level relative to the cemento-enamel junction in a single measurement. The probe tip automatically enters the periodontal pocket and retracts under controlled force. As the probe tip transverses the cemento-enamel junction, the electronics detect an alteration in the acceleration of the probe tip. Thus, the location of the cemento-enamel junction is determined automatically. The repeatability and accuracy of the probe was assessed in vitro. Measurements of attachment level were repeatable to 0.03 mm, and the probe had a net accuracy of 0.04 mm.