Hydroxy Diphosphonate Research Articles

99mTc-polyphosphonate labelling – Enhancement of a novel method for the quantification of osteogenic differentiation of MSCs in vitro

Bone tissue engineering is a fast-growing field in regenerative medicine. Consequently, there is a high demand for new, fast and reliable methods to track and quantify the osteogenic differentiation of cells. Recently, a novel method was published to non-destructively quantify the hydroxyapatite content of monolayer and 3-dimensional mesenchymal stem cell cultures using the ability of 99mTechnetium-methylene diphosphonate (MDP), a well-established tracer in clinical nuclear medicine, to bind to newly synthesized hydroxyapatite. In the present study, two other commonly used 99mTechnetium tracers, 2,3-dicarboxypropane-1,1-diphosphonate (DPD) and hydroxydiphosphonate (HDP), were evaluated to see if they could also be used for the same purpose. Furthermore, we investigated if labelling at various timepoints influenced the effectiveness of the labelling. The results were analysed using one-factor ANOVA followed by Bonferroni post-hoc testing. This revealed a highly significant difference between the three osteogenic groups at each timepoint compared to their corresponding negative controls. However, there was no statistically significant difference between the three different tracers (MDP, DPD, HDP) in the osteogenic groups. Therefore all three tracers are of similar value when quantifying the extracellular hydroxylapatite content in osteogenic stem cells cultures.

Assessment of relative uptake by mandibular condyles in a “normal” population

Hyperplasia of the mandibular condyle is self-limiting, but can lead to facial asymmetry, malocclusion, pain, and dysfunction of the temporomandibular joint (TMJ). Bone scintigraphy, particularly with single photon emission computed tomography (SPECT), is effective in assessing relative condylar uptake, but we know of no standardised methods or values. Our aim, therefore was to validate the values currently used to measure relative condylar uptake in our population. Between December 2015 and June 2018 44 patients had skull SPECT (15 male and 29 female patients, whose ages ranged from 4-33 years). They were having bone scans (hydroxydiphosphonate (HDP) −99MTc, 740 MBq ev) for unrelated reasons and had no known abnormalities of the head, facial asymmetry, or symptoms of the TMJ. Two research workers measured the relative uptake between the condyles using the summed transaxial images. The Hospital Ethics Committee approved the investigation. The maximum difference in condylar uptake was 8.33% with research worker 1 and 8.77% with research worker 2, and the mean (SD) differences were 3.03 (0.17) % and 3.29 (0.18) %, respectively. Data were tested for normality, and the t test and one-way ANOVA were used to assess the significance of differences. None was found in total counts either between sexes or age groups, and there were none between the total counts measured by the two research workers. We conclude that our results are within the published ranges, and the variation in condylar uptake was less than 5% in 37/44 patients, and in none was it 9% or more. When the results indicate less than 10%, but there is a high clinical suspicion of active hyperplasia, surgeons should use their clinical judgement to decide whether condylar surgery is required.

A review of paediatric administered radiopharmaceutical activities to determine compliance with prescription guidelines.

Attempts are underway to standardize paediatric administered activities, but equally important is knowing the actual activities administered to patients. In this work, paediatric administered activities are reviewed to determine compliance with the institution-prescribed guidelines. Paediatric administered activities for common studies at our institution, August 2011 to January 2017, have been analysed to determine their deviations from the set guideline tolerance of 10% from prescribed activities. The results for technetium-99m hydroxy diphosphonate (Tc-HDP), technetium-99m mercaptoacetyl triglycine (Tc-MAG3) and technetium-99m dimercaptosuccinic acid (Tc-DMSA), are presented here. Tc-MAG3 mean activities were close to the tolerance guideline at 10.3% SD. For Tc-HDP and Tc-DMSA, the prescribed guidelines were reviewed and reduced in May 2014 and September 2015, respectively. SDs for these studies over the two acquisition periods were different (8.9 and 6.6%, respectively, for Tc-HDP and 11.8 and 14.2%, respectively, for Tc-DMSA).The administered activities (dispensed minus residual activities) to patients depend on prescribed activities and dispensing and injecting techniques. Deviations from the prescribed activities are primarily because of issues related to residual activities, particularly with small activities prescribed in young patients. Small activities in small volumes make residual activities significant. The skill and experience of the nuclear medicine staff are essential in minimizing deviations from prescribed activities. It is important to measure residual to accurately determine the administered activities. If precautions are taken with dispensing and injecting techniques, it is possible to administer activities close to 10% of the prescribed activities. The regular review of the administered activities is essential to ensure that patients are not unnecessarily irradiated.

Increased technetium‐99 m hydroxy diphosphonate soft tissue uptake on bone scintigraphy in chronic kidney disease patients with secondary hyperparathyroidism: correlation with hyperphosphataemia

In bone scan patients with dialysis-treated chronic kidney disease (CKD) and hyperparathyroidism, soft tissue accumulation of technetium-99m hydroxy/methylene diphosphonate (Tc-99m-HDP/MDP) has been reported primarily in case reports and usually explained by hypercalcaemia and/or hyperphosphataemia. As human vascular smooth muscle cells produce hydroxyapatite during cell culture with increased phosphate levels and as Tc-99m-HDP/MDP primarily binds to hydroxyapatite, we hypothesized that soft tissue accumulation would be found in patients with hyperphosphataemia. We identified 63 CKD patients diagnosed with secondary hyperparathyroidism admitted for Tc-99m-HDP bone scan. Baseline characteristics and mean concentrations of biochemical markers (including P-calcium and P-phosphate) taken 0-3months prior to the bone scans were collected. Soft tissue uptake was detected on bone scans in 37 of 63 (59%) patients. Primary locations were in the heart (27/37=73%), muscles (12/37=32%), lung (9/37=24%) and gastrointestinal tract (6/37=16%), and 13 of 37 (35%) patients had simultaneous uptake in more than one location. Regarding biochemical markers, patients with soft tissue uptake only differed from patients without in terms of plasma phosphate levels (1·95±0·15 (n=37) versus 1·27±0·08 (n=26), P=0·0012). All patients with myocardial uptake (n=27) had a coronary arteriography-verified history of coronary artery disease (CAD), whereas CAD was only present in six of the 36 patients without myocardial uptake. In conclusion, dialysis-treated CKD patients with secondary hyperparathyroidism have a high incidence of soft tissue uptake, and this finding is strongly correlated with elevated phosphate, but not calcium values.

Extensive Peritoneal Uptake of Technetium-99m-Labelled Hydroxy Diphosphonate in a Patient Undergoing Hemodialysis

A 48-year-old African lady started peritoneal dialysis in 1994 after transplant failure. After several episodes of peritonitis and hemorrhagic ascites, hemodialysis was resumed in 2003 because of small bowel subocclusion. In 2006, an abdominal CT showed calcifications and dilatation of intestinal loops. Sclerosing peritonitis was diagnosed and she underwent enterolysis and resection of the peritoneal capsule. She remained asymptomatic until 2009 when she presented again with subocclusion. Because of generalized skeletal pain, a bone scan was performed, which showed Tc-99m hydroxy diphosphonate uptake on the peritoneum. The patient is currently on hemodialysis with parenteral nutrition.

Susceptibility of the collagenous matrix from bovine incisor roots to proteolysis after in vitro lesion formation.

The susceptibility of the organic matrix from permanent bovine incisor roots to proteolytic breakdown after in vitro lesion formation was investigated. Root surfaces were exposed to 0.1 M acetic acid, pH 4.0, to produce erosive lesions or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0, to produce subsurface lesions. After demineralization, the roots were treated with a bacterial collagenase. The quantity of enzyme-degradable collagen in the root tissue was found to be proportional to the calcium released during demineralization, until a plateau value was reached at calcium concentrations in solution of 3.3 mM at pH 4.0 and 2.7 mM at pH 5.0. The degradability of collagen was found to be substantially less in subsurface lesions than in erosive lesions. The presence of cementum-free areas did not affect the results. These findings suggest that the mineral component of the roots is composed of several fractions which differ in their solubility properties in weak acids.

Remineralization of Bovine Incisor Root Lesions in vitro: The Role of the Collagenous Matrix

This study deals with the role of collagen in the remineralization of bovine incisor root lesions. The degradability of the collagenous matrix after in vitro remineralization was also investigated. The root surfaces were exposed to 0.1 M acetic acid, pH 4.0, to produce erosive lesions or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0, to produce subsurface lesions. After demineralization, the roots were subjected to remineralization, with or without prior treatment with a bacterial collagenase to remove accessible collagen. Remineralization was carried out in solutions with a Ca/Pi molar ratio of 1.67, and [Ca]. [Pi] concentration products of 0.6, 1.35 or 2.4 mM2, with all solutions having an ionic strength of 0.15 M, and a pH of 7.0. After remineralization, the roots that were not treated with collagenase were incubated with the enzyme. The results indicate that remineralization of erosive lesions is surface-controlled and that of subsurface lesions diffusion-controlled. For both lesion types it was shown that the removal of collagen before remineralization did not affect the rate of mineral deposition in the root lesions. Nevertheless, remineralization in those lesions where accessible collagen had not been removed resulted in a significant reduction in the amount of degradable collagen. The observations of this study suggest that remineralization did not occur by nucleation of mineral on the organic matrix but rather by growth of residual crystals in the partially demineralized root tissue.

Degradation of bovine incisor root collagen in an in vitro caries model

The effects of pH, ionic strength and proteinases on the destruction of bovine incisor root collagen were studied. Experiments were done with powdered and intact root specimens. Completely demineralized root powder was subjected to solutions of varying pH and ionic strength: (a) 0.1 M acetic acid, pH 4.0, (b) 0.1 M acetic acid + 0.15 M KCl, pH 4.0, (c) 0.1 M Hepes, pH 7.0 or to (d) 0.1 M Hepes + 0.15 M KCl, pH 7.0 at 37 °C. The surfaces of intact root specimens were exposed to 0.1 M acetic acid, pH 4.0 (which resulted in erosive lesions) or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0 (which produced subsurface lesions) at 37 °C. After incubation, the extracts were analysed for soluble collagen and the insoluble matrices were treated with trypsin at 15 °C to determine the denatured collagen. To estimate sensitivity to non-specific proteinases, demineralized root powder was also treated with trypsin under physiological conditions of temperature, pH and ionic strength. The denaturation and subsequent solubilization of collagen material from the fibrils could be influenced by variations in pH and ionic strength but these effects were small when compared to proteolytic degradation under physiological conditions. This supports the hypothesis that, in root caries, destruction of exposed matrix collagen depends largely on the presence and activity of proteinases.

Release of Organic Matrix Components from Bovine Incisor Roots during in vitro Lesion Formation

The solubilization of organic matrix components during demineralization of powdered and intact root sections from adult bovine incisors was investigated. Root powder was demineralized with 0.1 mol/L acetic acid, pH 4.0, at 4 degrees C and 37 degrees C. Surfaces of intact root sections were subjected to 0.1 mol/L acetic acid, pH 4.0 (for production of erosive lesions), or to 0.1 mol/L lactic acid, 0.2 mmol/L methane hydroxy diphosphonate, pH 5.0 (for production of subsurface lesions at 37 degrees C). The solubilized organic material was analyzed for collagen, total noncollagenous protein (NCP), organic phosphate (Po), and proteoglycans (PGs), which were measured as chondroitin 4-sulfate (C-4-S). For root powder, a maximal release of NCPs and PGs was found only after neutralization of the extraction mixture. For both temperatures tested, the average amounts of liberated noncollagenous components (NCCs) were the same, i.e., 0.68 micrograms [NCP - Po], 0.11 microns Po, and 0.10 micrograms C-4-S per mumols released calcium. The amino acid composition of the NCP fraction revealed relatively high amounts of aspartic acid and serine. These findings indicate that the NCCs were easily liberated from the tissue, and that the NCP fraction consisted mainly of phosphoprotein. Demineralization of intact root sections resulted in average amounts of solubilized NCCs of 0.21 micrograms [NCP - Po], less than 0.01 micrograms Po, and less than 0.01 micrograms C-4-S per mumols released calcium, independent of incubation time and lesion type. The amino acid composition of all NCP fractions was virtually the same, high in glutamic acid, but lower in aspartic acid and serine when compared with the neutralized powder extracts. For both demineralization solutions, the amounts of solubilized collagen were 0.07 and 0.16 micrograms per micromol released calcium after three and 28 days of incubation, respectively. Our experiments indicate that phosphoprotein and proteoglycans may be released from root surfaces during the periods of neutral pH that follow acid demineralization.

Mineral Loss during Etching of Enamel Lesions

This study was aimed at determining the amount of mineral removed from incipient enamel lesions during acid etching. The rationale being that fissures which are scheduled for sealing will often have undetected demineralized regions. Lesions were formed in bovine enamel specimens using either an acidified gelatine gel or a lactate buffer containing methane hydroxy diphosphonate. Different parts of each lesion were acid-etched either for a 1- or for a 2-min period in 36% H3PO4 or served as control. Mineral content profiles were recorded on thin sections using computerized microradiography to determine the mineral loss resulting from acid etching. The results show that during etching a layer at the outer surface is removed completely. The thickness of this layer was greater for lesions when compared with sound enamel and depended on the lesion characteristics (porosity and fluoride content). The amount of mineral removed from the deeper layers of the lesion was very small; and in the innermost part of the lesions the mineral profiles of the control and acid-etched areas coincide. These observations indicate that the effect of etching is essentially restricted to a thin outer layer, irrespective of the porosity of the underlying tissue. Although the thickness of the etched-off layer was different between the two types of lesions, these findings were made for both types studied. In practical terms this implies that etching of enamel lesions does not cause an excessive mineral loss throughout the lesion.

Effect of polyvinyl phosphonates and ethane hydroxy diphosphonate on mineralization of ectopic bone

The effects of polymeric polyphosphonate and of ethane hydroxydiphosphonate on the development of ectopic bone were compared at different stages of bone development. The diphosphonate affected bone development at different stages, as reflected by alkaline phosphatase and aryl sulphatase activities, as well as by the creation of calcium ions by bone and on its readiness to undergo isotopic exchange. The polymeric polyphosphonate (mol. wt., 3500–8000), on the other hand, did not exhibit any of these effects, although it did inhibit the activity of the enzymes in vitro to an extent comparable with that of the diphosphonate. The results corroborate the assumption that the effects of polymeric polyphosphonates on hard tissues are confined to the extracellular space while the effects of diphosphonates on bone development are due to intracellular activity and not to sequestering of extracellular calcium.

Effects of ethane-1 hydroxy-1, 1-Diphosphonate (5 mg/kg/day dose) onquantitative bone histology in paget's disease of bone

The effects of ethane hydroxy diphosphonate (EHDP) on quantitative bone histology have been studied in 43 pagetic patients treated for 6 months at a dose of 5 mg/kg/day. To determine the therapeutic effects on pagetic bone and to appreciate the side effects in non-pagetic bone, the patients were biopsied either in pagetic or non-pagetic areas of the ilium before the beginning of the treatment and at 3, 6 and 9 months (i.e. 3 months post-therapy) after the inception of treatment. Tetracycline double labelling of bone was performed before biopsies in each patient to measure the calcification rate. The results demonstrate that 5 mg/kg/day EHDP given for 6 months has a marked antiosteoclastic effect which is prolonged after the cessation of treatment. Pagetic osteoclasts appear more sensitive to EHDP than normal osteoclasts. The new bone formed during treatment has a regular lamellar texture. At the same time, the high appositional rate of pagetic bone is reduced to a normal value. Marrow fibrosis is reduced along with the reduction in the osteoclast population. Thus, pagetic bone changes to bone normal in quantity, quality and metabolism. Dynamically, in nonpagetic areas, a transitory decrease of calcification rate Is noted at the end of the treatment, without morphological evidence of osteomalacia, except for 2 out of 43 cases. Histologically, EHDP given at a dose of 5 mg/kg/day for 6 months appears to be an effective and safe treatment for Paget's disease of bone.

Quantitative analysis of the distribution of 99mTc-labelled ethylene hydroxy diphosphonate in young rats.

The distribution of 99mTc-labelled ethylene 1-hydroxy-1,1-diphosphonate (EHDP) in young rats was studied 1, 3, 6 and 9 h after intraperitoneal injection. The concentration of 99mTc in bone was 8-10 times higher than that in the kidneys and 20-1000 times higher than those in other soft tissues. The concentration in bone remained constant throughout the observation time. In some of the animals sacrificed after 3, 6, or 9 h the uptake in the liver and spleen was higher than that found in the majority of the animals.

A comparison of phosphate bone-scanning agents in normal subjects and patients with malignant disease

None of the established bone-scanning compounds 85Sr, 87Srm nor 18F is entirely satisfactory and because of this the place of skeletal scanning is not clearly defined. 18F is the most effective of the available bone-scanning compounds but its 100 minute half-life makes distribution difficult and restricts its use in Britain to the Greater London Area. There has recently been considerable interest in certain compounds of phosphate which show great promise as bone scanning agents (Subramanian et al., 1972; Yano et al., 1973; Hosain, 1973; Citrin et al., 1974). The phosphate compounds polyphosphate, pyrophosphate and ethane hydroxy diphosphonate, are readily labelled with 99Tcm in the presence of the reducing agent stannous chloride. 99Tcm is one of the standard isotopes used in nuclear medicine. Its physical characteristics are ideal and it is produced from a simple generator on site. If Tc-labelled phosphates are shown to be effective and safe then bone scanning will be routinely available in hospitals with even the most limited isotope facilities. It is clearly important to know which of the currently available phosphate compounds, polyphosphate, pyrophosphate and ethane hydroxy diphosphonate (EHDP)* is the most effective agent. We have compared these three agents in 29 patients with known malignant disease of the skeleton to compare their relative efficiency in demonstrating bony metastases. We have also measured serial blood and whole-body levels in a number of normal subjects to establish which compound gives lowest blood activity and lowest whole-body radiation dose.

QUANTITATIVE BONE SCANNING: A METHOD FOR ASSESSING RESPONSE OF BONE METASTASES TO TREATMENT

Quantitative bone scanning using 99mTc-labelled ethane hydroxy diphosphonate and a gamma-camera / multi-channel analyser system provides a simple, non-invasive method of assessing the response of bone metastases to treatment.