Tetracycline Labeling Research Articles

Congenital insensitivity to pain: Fracturing without apparent skeletal pathobiology caused by an autosomal dominant, second mutation in SCN11A encoding voltage-gated sodium channel 1.9

Congenital insensitivity to pain (CIP) comprises the rare heritable disorders without peripheral neuropathy that feature inability to feel pain. Fracturing and joint destruction are common complications, but lack detailed studies of mineral and skeletal homeostasis and bone histology. In 2013, discovery of a heterozygous gain-of-function mutation in SCN11A encoding voltage-gated sodium channel 1.9 (Nav1.9) established a distinctive CIP in three unrelated patients who suffered multiple painless fractures, self-inflicted mutilation, chronic diarrhea, and hyperhidrosis.Here, we studied a mother and two children with CIP by physical examination, biochemical testing, radiological imaging including DXA, iliac crest histology, and mutation analysis.She suffered fractures primarily of her lower extremities beginning at age two years, and had Charcot deformity of both ankles and joint hypermobility. Nerve conduction velocity together with electromyography were normal. Her children had recurrent major fractures beginning in early childhood, joint hypermobility, and chronic diarrhea. She had an excoriated external nare, and both children had hypertrophic scars from scratching. Skin collagen studies were normal. Radiographs revealed fractures and deformities. However, lumbar spine and total hip BMD Z-scores, biochemical parameters of mineral and skeletal homeostasis, and iliac crest histology of the mother (after in vivo tetracycline labeling) were normal. Genomic DNA from the children revealed a unique heterozygous missense mutation in exon 23 (c.3904C>T, p.Leu1302Phe) of SCN11A that is absent in SNP databases and alters an evolutionarily conserved amino acid.This autosomal dominant CIP reflects the second gain-of-function mutation of SCN11A. Perhaps joint hypermobility is an unreported feature. How mutation of Nav1.9 causes fracturing remains unexplained. Lack of injury awareness is typically offered as the reason, and was supported by our unremarkable biochemical, radiological, and histological findings indicating no skeletal pathobiology. However, low-trauma fracturing in these patients suggests an uncharacterized defect in bone quality.

Flow perfusion culture of MC3T3-E1 osteogenic cells on gradient calcium polyphosphate scaffolds with different pore sizes.

Calcium polyphosphate is a biodegradable bone substitute. It remains a challenge to prepare porous calcium polyphosphate with desired gradient porous structures. In this study, a modified one-step gravity sintering method was used to prepare calcium polyphosphate scaffolds with desired-gradient-pore-size distribution. The differences of porous structure, mechanical strength, and degradation rate between gradient and homogenous calcium polyphosphate scaffolds were evaluated by micro-computed tomography, scanning electron microscopy, and mechanical testing. Preosteoblastic MC3T3-E1 cells were seeded onto gradient and homogenous calcium polyphosphate scaffolds and cultured in a flow perfusion bioreactor. The distribution, proliferation, and differentiation of the MC3T3-E1 cells were compared to that of homogenous calcium polyphosphate scaffolds. Though no significant difference of cell proliferation was found between the gradient and the homogenous calcium polyphosphate scaffolds, a much higher cell differentiation and mineralization were observed in the gradient calcium polyphosphate scaffolds than that of the homogenous calcium polyphosphate scaffolds, as manifested by increased alkaline phosphatase activity (p < 0.05). The improved distribution and differentiation of cultured cells within gradient scaffolds were further supported by both (18)F-fluorine micro-positron emission tomography scanning and in vitro tetracycline labeling. We conclude that the calcium polyphosphate scaffold with gradient pore sizes enhances osteogenic cell differentiation as well as mineralization. The in vivo performance of gradient calcium polyphosphate scaffolds warrants further investigation in animal bone defect models.

Polypyrrole coating on poly-(lactide/glycolide)-β-tricalcium phosphate screws enhances new bone formation in rabbits

Polypyrrole (PPy) has gained interest as an implant material due to its multifunctional properties and its high compatibility with several cell and tissue types. For the first time, the biocompatibility and osteointegration of PPy coating, incorporated with chondroitin sulfate (CS), were studied in vivo by implanting PPy-coated bioabsorbable bone fixation composite screws of poly-(lactide/glycolide) copolymer (PLGA) and β-tricalcium phosphate (TCP) into New Zealand white rabbits. Uncoated bioabsorbable polymer composite screws and commercially available stainless steel cortical screws were used as reference implants. The rabbits were euthanized 12 and 26 weeks after the implantation. The systemic effects were evaluated from food and water consumption, body weight, body temperature, clinical signs, blood samples, internal organ weights, and histological examination. Local effects were studied from bone tissue and surrounding soft tissue histology. New bone formation was evaluated by micro-computed tomography, tetracycline labeling and torsion tests. Torsion tests were performed in order to capture the peak value of the torsion force during the course of the screw’s loosening.The coated screws induced significantly more bone formation than the uncoated screws. In addition, none of the implants induced any systemic or local toxicity. The results suggest that PPy is biocompatible with bone tissue and is a potential coating for enhancing osteointegration in orthopedic implants.

Are Biochemical Markers of Bone Turnover Representative of Bone Histomorphometry in 370 Postmenopausal Women?

The levels of bone formation and resorption can be assessed at the tissue level by bone histomorphometry on transiliac bone biopsies. Systemic biochemical markers of bone turnover reflect the overall bone formation and resorption at the level of the entire skeleton but cannot discriminate the different skeletal compartments. Our aim was to investigate the correlations between the serum biochemical markers of formation and resorption with histomorphometric parameters. We performed post hoc analysis of a previous clinical study. Patients were selected from the general population. A total of 371 untreated postmenopausal osteoporotic women aged 50 to 84 years with a lumbar T-score ≤ -2.5 SD or ≤ -1 SD with at least one osteoporotic fracture. Transiliac bone biopsies were obtained after a double tetracycline labeling, and blood samples were collected. The static and dynamic parameters of formation and bone resorption were measured by histomorphometry. Serum biochemical markers of formation (bone alkaline phosphatase [ALP]; procollagen type I N-terminal propeptide [PINP]) and resorption (C-terminal crosslinking telopeptide of collagen type 1 [sCTX]) were assessed. The mean values of biochemical markers were: bone ALP, 15.0 ± 5.2 ng/mL; PINP, 56.2 ± 21.9 μg/mL; and sCTX, 0.58 ± 0.26 ng/mL. Bone ALP and PINP were significantly correlated with both the static and dynamic parameters of formation (0.21 ≤ r' ≤ 0.36; 0.01 ≥ P ≥ .0001). sCTX was significantly correlated with all resorption parameters (0.18 ≤ r' ≤ 0.24; 0.02 ≥ P ≥ .0001). Bone turnover markers were significantly but modestly associated with bone turnover parameters measured in iliac cancellous bone. The iliac crest bone may not represent perfectly the whole bone turnover.

Perivascular Stem Cells at the Tip of Mouse Incisors Regulate Tissue Regeneration

Cells with in vitro properties similar to those of bone marrow stromal stem cells are present in tooth pulp as quiescent cells that are mobilized by damage. These dental pulp stem cells (DPSCs) respond to damage by stimulating proliferation and differentiation into odontoblast-like cells that form dentine to repair the damage. In continuously growing mouse incisors, tissue at the incisor tips is continuously being damaged by the shearing action between the upper and lower teeth acting to self-sharpen the tips. We investigated mouse incisor tips as a model for the role of DPSCs in a continuous natural repair/regeneration process. We show that the pulp at the incisor tip is composed of a disorganized mass of mineralized tissue produced by odontoblast-like cells. These cells become embedded into the mineralized tissue that is rapidly formed and then lost during feeding. Tetracycline labeling not only revealed the expected incorporation into newly synthesized dentine formation of the incisor but also a zone covering the pulp cavity at the tips of the incisors that is mineralized very rapidly. This tissue was dentine-like but had a significantly lower mineral content than dentine as determined by Raman spectroscopy. The mineral was more crystalline than dentine, indicative of small, defect-free mineral particles. To identify the origin of cells responsible for deposition of this mineralized tissue, we genetically labeled perivascular cells by crossing NG2(ERT2) Cre and Nestin Cre mice with reporter mice. A large number of pericyte-derived cells were visible in the pulp of incisor tips with some having elongated, odontoblast-like shapes. These results show that in mouse incisors, rapid, continuous mineralization occurs at the tip to seal off the pulp tissue from the external environment. The mineral is formed by perivascular-derived cells that differentiate into cells expressing dentin sialo-phosphoprotein (DSPP) and produce a dentine-like material in a process that functions as continuous natural tissue regeneration.

Calcium silicate cement-induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two-photon fluorescence microscopy.

Two-photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate-based restorative material (Biodentine(TM) ) and a glass ionomer cement (GIC:​Fuji​IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate-free tetracycline, and tetracycline-free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8-week storage at 37 °C, dentine samples were imaged using two-photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine(TM) in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra- and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement-free or PBS-free media, or with GIC. It could therefore be concluded that Biodentine(TM) induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate-rich media, which was selectively detectable using the tetracycline labelling.

1,25-Dihydroxyvitamin D3 prevents bone loss of the secondary spongiosa in arthritic rats by an increase of bone formation and mineralization and inhibition of bone resorption.

BackgroundActive vitamin D metabolites have been shown to have protective effects in experimental arthritis especially when used as preventive treatment. However, because the direct effects of 1,25-dihydroxyvitamin D3 (1,25(OH) 2D3) on bone formation and resorption are very complex, the net effect of 1,25(OH)2D3 on histomorphometric parameters of bone turnover and mineralisation should be investigated. Therefore, we examined the influence of 1,25(OH)2D3 therapy on arthritis-induced alterations of periarticular and axial bone as well as disease activity, inflammation and joint destruction in antigen-induced arthritis (AIA) of the rat.MethodsAIA was induced in 20 eight-week-old female Wistar rats. 10 rats without arthritis were used as healthy controls. AIA rats received 1,25(OH)2D3 (0.2 μg/kg/day, i.p., n = 10) or vehicle (n = 10) at regular intervals for 28 consecutive days beginning 3 days before arthritis induction. Bone structure of the secondary spongiosa of the periarticular and axial bone was analyzed using histomorphometry. Parameters of mineralization were investigated using tetracycline labelling. Clinical disease activity, inflammation and joint destruction were measured by joint swelling and histological investigation, respectively.ResultsAIA led to significant periarticular bone loss. 1,25(OH)2D3 treatment resulted in a highly significant increase in trabecular bone volume and bone formation rate in comparison to both vehicle-treated AIA and healthy controls at periarticular (p < 0.01 and p < 0.001, respectively) and axial bone (p < 0.001 and p < 0.001, respectively). In addition, bone resorption was reduced by 1,25(OH)2D3 at the axial bone (p < 0.05 vs. vehicle-treated AIA). Joint swelling as well as histological signs of inflammation and joint destruction were not influenced by 1,25(OH)2D3.ConclusionsThe results of the study indicate a marked osteoanabolic effect of 1,25(OH)2D3 presumably due to a substantial increase in mineralization. Thus, 1,25(OH)2D3 may be an effective osteoanabolic treatment principle to antagonize the inflammation-associated suppression of bone formation in rheumatoid arthritis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-345) contains supplementary material, which is available to authorized users.

Effects of Eucommia ulmoides extract on longitudinal bone growth rate in adolescent female rats.

Eucommia ulmoides is one of the popular tonic herbs for the treatment of low back pain and bone fracture and is used in Korean medicine to reinforce muscles and bones. This study was performed to investigate the effects of E. ulmoides extract on longitudinal bone growth rate, growth plate height, and the expressions of bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) in adolescent female rats. In two groups, we administered a twice-daily dosage of E. ulmoides extract (at 30 and 100 mg/kg, respectively) per os over 4 days, and in a control group, we administered vehicle only under the same conditions. Longitudinal bone growth rate in newly synthesized bone was observed using tetracycline labeling. Chondrocyte proliferation in the growth plate was observed using cresyl violet dye. In addition, we analyzed the expressions of BMP-2 and IGF-1 using immunohistochemistry. Eucommia ulmoides extract significantly increased longitudinal bone growth rate and growth plate height in adolescent female rats. In the immunohistochemical study, E. ulmoides markedly increased BMP-2 and IGF-1 expressions in the proliferative and hypertrophic zones. In conclusion, E. ulmoides increased longitudinal bone growth rate by promoting chondrogenesis in the growth plate and the levels of BMP-2 and IGF-1. Eucommia ulmoides could be helpful for increasing bone growth in children who have growth retardation.

Bony adaptation after expansion with light-to-moderate continuous forces

The purpose of this study was to evaluate the biologic response of dentoalveolar bone to archwire expansion with light-to-moderate continuous forces. With a split-mouth experimental design, the maxillary right second premolars of 7 adult male dogs were expanded for 9 weeks using passive self-ligating brackets (Damon Q; Ormco, Orange, Calif) and 2 sequential archwires (0.016 × 0.022-in copper-nickel-titanium alloy, followed by 0.019 × 0.025-in copper-nickel-titanium alloy). Intraoral and radiographic measurements were made to evaluate tooth movements and tipping associated with expansion; archwire forces were measured using a force gauge. Microcomputed tomography was used to compare buccal bone height, total tooth height, total root height, and buccal bone thickness. Bone formation was evaluated histologically using tetracycline and calcein fluorescent labels and hematoxylin and eosin stains. Buccal expansion was produced by forces between 73 and 178 g. Compared with the control side, which showed no tooth movement, the experimental second premolars were expanded by 3.5 ± 0.9 mm and tipped by 15.8°. Buccal bone thickness was significantly thinner (about 0.2 mm) in the coronal aspects and significantly thicker (about 0.9 mm) in the apical aspects over the mesial roots. The tipping and expansion significantly (P <0.05) reduced buccal bone height (ie, caused dehiscences) at the mesial (about 2.9 mm) and distal (about 1.2 mm) roots. Bony apposition occurred on the trailing edges of tooth movement and on the leading edges of the second premolar apices. The axial microcomputed tomography slices indicated, and the bone histomorphometry and histology demonstrated, newly laid-down bone on the periosteal side of the buccal cortical surfaces. Ordered osteoblast aggregation was also evident on the periosteal surfaces of buccal bone, just cervical to the apparent center of rotation of the tooth. Tooth and root heights showed no significant differences between the experimental and control second premolars. Buccal expansion with light-to-moderate continuous forces produced 3.5 mm of tooth movement, uncontrolled tipping, and bone dehiscence, but no root resorption. Bone formation on the periosteal surfaces of cortical bone indicates that apposition is possible on the leading edge of tooth movements.

Effects of Up to 5 Years of Denosumab Treatment on Bone Histology and Histomorphometry: The FREEDOM Study Extension

Denosumab reduced bone resorption, increased bone mineral density (BMD), and decreased new vertebral, hip, and nonvertebral fracture risk in postmenopausal women with osteoporosis in the FREEDOM trial. Consistent with its mechanism of action, transiliac crest bone biopsies from subjects treated with denosumab for 1 to 3 years demonstrated reduced bone turnover that was reversible upon treatment cessation. Long-term denosumab treatment for up to 6 years in the FREEDOM extension provides sustained bone turnover reduction and continued low fracture incidence. Here, we evaluate 5 years of denosumab treatment on bone remodeling at the tissue level. Transiliac crest bone biopsies were obtained from 41 subjects (13 cross-over and 28 long-term from the FREEDOM placebo and denosumab groups, respectively) at year 2 of the FREEDOM extension, representing up to 5 years of denosumab treatment. Demographics for this subset were comparable to the overall extension cohort. The mean (SD) duration from the last denosumab dose to the first dose of tetracycline was 5.7 (0.5) months. Qualitative bone histology assessed in all biopsy samples was unremarkable, showing normally mineralized lamellar bone. Structural indices, including trabecular bone volume, number, and surface, were similar between cross-over and long-term groups. Bone resorption was decreased as reflected by eroded surface in cross-over and long-term subjects. A total of 11 of 13 (85%) cross-over subjects and 20 of 28 (71%) long-term subjects had specimens with double or single tetracycline label in trabecular and/or cortical compartments; specimens from 5 cross-over subjects and 10 long-term subjects were evaluable for dynamic trabecular bone parameters. Dynamic remodeling indices were low for both groups and consistent with reduced bone turnover with denosumab. In conclusion, denosumab treatment through 5 years resulted in normal bone quality with reduced bone turnover. These observations are consistent with its mechanism of action and associated with continued BMD increases and low fracture incidence.

Effects of teriparatide on cortical histomorphometric variables in postmenopausal women with or without prior alendronate treatment

Cortical bone, the dominant component of the human skeleton by volume, plays a key role in protecting bones from fracture. We analyzed the cortical bone effects of teriparatide treatment in postmenopausal women with osteoporosis who had previously received long-term alendronate (ALN) therapy or were treatment naïve (TN). Tetracycline-labeled paired iliac crest biopsies obtained from 29 ALN-pretreated and 16 TN women were evaluated for dynamic histomorphometric parameters of bone formation at the periosteal, endocortical and intracortical bone compartments, before and after 24months of teriparatide treatment. At baseline, the frequency of specimens without any endocortical and periosteal tetracycline labeling, and the percentage of quiescent osteons, was higher in the ALN than the TN group. Endocortical and periosteal mineralizing surface (MS/BS%), periosteal bone formation rate (BFR/BS), mineral apposition rate (MAR) and the number of intracortical forming osteons were significantly lower in the ALN-pretreated patients than in the TN group. Following teriparatide treatment, the frequency of endocortical and periosteal unlabeled biopsies decreased; in the ALN-pretreated group the percentage of quiescent osteons decreased and, in contrast, forming and resorbing osteons were increased. Teriparatide treatment resulted in significant increases of MAR in the endocortical, and MS/BS% in the periosteal compartment in the ALN-pretreated group. Most indices of bone formation remained lower in the ALN-pretreated group compared with the TN group at study end. Endocortical wall width was increased in both ALN-pretreated and TN groups. Cortical porosity and cortical thickness were significantly increased in the ALN-pretreated group after teriparatide treatment. Our results suggest that 24months of teriparatide treatment increases cortical bone formation and cortical turnover in patients who were either TN or had previous ALN therapy.

18F-fluoride Positron Emission Tomography Measurements of Regional Bone Formation in Hemodialysis Patients with Suspected Adynamic Bone Disease

18F-fluoride positron emission tomography (18F-PET) allows the assessment of regional bone formation and could have a role in the diagnosis of adynamic bone disease (ABD) in patients with chronic kidney disease (CKD). The purpose of this study was to examine bone formation at multiple sites of the skeleton in hemodialysis patients (CKD5D) and assess the correlation with bone biopsy. Seven CKD5D patients with suspected ABD and 12 osteoporotic postmenopausal women underwent an 18F-PET scan, and bone plasma clearance, K i, was measured at ten skeletal regions of interest (ROI). Fifteen subjects had a transiliac bone biopsy following double tetracycline labeling. Two CKD5D patients had ABD confirmed by biopsy. There was significant heterogeneity in K i between skeletal sites, ranging from 0.008 at the forearm to 0.028 mL/min/mL at the spine in the CKD5D group. There were no significant differences in K i between the two study groups or between the two subjects with ABD and the other CKD5D subjects at any skeletal ROI. Five biopsies from the CKD5D patients had single tetracycline labels only, including the two with ABD. Using an imputed value of 0.3 μm/day for mineral apposition rate (MAR) for biopsies with single labels, no significant correlations were observed between lumbar spine K i corrected for BMAD (K i/BMAD) and bone formation rate (BFR/BS), or MAR. When biopsies with single labels were excluded, a significant correlation was observed between K i/BMAD and MAR (r = 0.81, p = 0.008) but not BFR/BS. Further studies are required to establish the sensitivity of 18F-PET as a diagnostic tool for identifying CKD patients with ABD.

OP0042 Increased modeling-based bone formation and decreased bone resorption on endocortical surfaces in male cynomolgus monkeys treated with sclerostin antibody

BackgroundBone formation on previously quiescent surfaces occurs through a process known as bone modeling, which differs from the resorption-coupled formation that occurs during bone remodeling. The robust anabolic effects of...

OP0251 Bone Histology and Histomorphometry: Effects of 5 Years of Denosumab in the Freedom Study Extension

BackgroundDenosumab (DMAb) reduces bone turnover in transiliac crest bone biopsies, which is reversible on treatment cessation (Reid JBMR 2010; Brown JBMR 2011). Long-term DMAb treatment sustains reduction in bone turnover...

The Significance of Calcified Fibrocartilage on the Cortical Endplate of the Translational Sheep Spine Model

To gain an understanding of the vertebral cortical endplate and factors that may affect the ability to achieve skeletal attachment to intervertebral implants and fusion, this study aimed to characterize the hypermineralized tissue on the cortical endplate of the vertebral body on a commonly used animal model. Skeletally mature sheep were injected with tetracycline prior to euthanasia and the C2-C3, T5-T6, and L2-L3 spinal motion segments were excised and prepared. Vertebral tissues were imaged using backscatter electron (BSE) imaging, histology, and tetracycline labeling was used to assess bone remodeling within different tissue layers. It was determined that the hypermineralized tissue layer was calcified fibrocartilage (CFC). No tetracycline labels were identified in the CFC layer, in contrast to single and double labels that were present in the underlying bone, indicating the CFC present on the cortical endplate was not being actively remodeled. The average thickness of the CFC layer was 146.3 ± 70.53 µm in the cervical region, 98.2 ± 40.29 µm in the thoracic region, and 150.89 ± 69.25 µm in the lumbar region. This difference in thickness may be attributed to the regional biomechanical properties of the spine. Results from this investigation indicate the presence of a nonremodeling tissue on the cortical endplate of the vertebral body in sheep spines, which attaches the intervertebral disc to the vertebrae. This tissue, if not removed, would likely prevent successful bony attachment to an intervertebral device in spinal fusion studies and total disc replacement surgeries.

Миграции грызунов в зоне влияния Восточно-Уральского радиоактивного следа (радиобиологический аспект)

We investigated migrations of mouse-like rodents in the Eastern Urals Radioactive Trace (EURT) zone using the method of small mammal group marking by tetracycline (additionally to the data of radionuclide self-marking). The originality of small mammals' population in the EURT zone is defined by a configuration of radioactively polluted area and features of animals' migrations. The tetracycline label is detected in the dentine of upper incisors fluorescing in UV light. A high migration activity of rodents is observed both in the EURT zone and adjacent areas. When migrating, rodents used the certain ways of moving. Our new data allowed us to conclude that the EURT zone, as well as any other areas, is inhabited by a population with a constantly changing set of individuals, i.e. a flowing population. It is the convincing evidence of the absence of any isolation. Migrations of small mammals in the narrow and extended EURT zone (1) considerably decrease the probability that certain adaptive changes may be fixed and inherited in a series of generations; (2) are the base of transmission of radio-induced effects in adjacent areas. The fact of a flowing population should be taken into account in a wide spectrum of investigations when analyzing the remote consequences of chronic radiation influence.

Case of femoral diaphyseal stress fracture after long-term risedronate administration diagnosed by iliac bone biopsy

Bisphosphonate excessively inhibits bone resorption and results in pathological fracture of the femur or ilium. The subject of this study was administered risedronate for 7 years; we suspected an easy fracture of the femoral diaphysis. In this study, we report the results of this patient’s bone biopsy and bone morphometric analysis. A 76-year-old female patient presented with right femoral pain. Bone mineral density of the anteroposterior surface of the 2nd to 4th lumbar vertebrae (L2–L4) was decreased and levels of bone turnover markers were high. Therefore, we initiated treatment with risedronate. As she continued the medication, urinary levels of cross-linked N-terminal telopeptides of type I collagen and alkaline phosphatase (bone-type isozyme) were found to be within the normal ranges. After 7 years of administration, the patient experienced pain when she put weight on the right femur and right femoral pain while walking. Plain radiographic examination revealed polypoid stress fracture-like lesions on the right femoral diaphysis and on the slightly distal-lateral cortical bone. Similar lesions were observed on magnetic resonance imaging and bone scintigraphy. We suspected severely suppressed bone turnover. Bone biopsy was obtained after labeling with tetracycline, and bone morphometric analysis was performed. On microscopic examination, slight double tetracycline labeling was observed. The trabeculae were narrow, and the numbers of osteoblasts and osteoclasts were decreased. Further, rates of bone calcification and bone formation were slow. Hence, we diagnosed fracture as a result of low turnover osteopathy. Risedronate was withdrawn, and Vitamin D3 was administered to improve the bone turnover. At 6 months, abnormal signals on magnetic resonance imaging had decreased and her pain while walking or undergoing the stress test disappeared as well. Thus, long-term administration of bisphosphonates may lead to easy fracture, although bone turnover markers were observed to be within the normal range. During bisphosphonate administration, physicians need to monitor closely and treat their patients for any pain experienced in the femoral region while walking or undergoing a stress test.

Double and quadruple tetracycline labeling of bone: Impact of the label itself

To the Editor: Quantitative histomorphometry of bone is a well-recognized technique with selected applications in clinical patient evaluation and in clinical research and drug development that allows assessment of the safety of drugs and their mechanism of action. Recently, two publications have examined issues in modern histomorphometry and have updated the ASBMR Histomorphometry Nomenclature Committee's recommendations on nomenclature, symbols, and units.1, 2 Here we draw attention to an issue originally described by Parfitt and colleagues3 which has implications for the conclusions that might be drawn from dynamic assessment of bone formation. In most histomorphometry applications, the bone is labeled with a tetracycline that binds to the mineralizing surface of bone. This has become a standard method that allows calculation of the rate of bone formation in all four bone compartments (cancellous, endocortical, intracortical, and periosteal bone). In most clinical situations, two separate labels of the same form of tetracycline (usually tetracycline HCl, oxytetracycline, or demethylchlortetracycline) are administered, each separated from the other by a period of 12 or to 14 days, and the biopsy is performed about 1 week after completion of the second label. For some applications, a different label may be used for the second of the two labels; the difference in the color of the two labels under UV light allows unambiguous assignment of single labels to the first or second labeling period. Several years ago we described a quadruple-labeling technique4 that allows short-term longitudinal data in a single biopsy with the first two tetracycline labels given in the usual format, but separated by several weeks from the second set of labels. In order to distinguish the first and second sets of labels under UV light, we used the same tetracycline for the first set of two labels and a different one for the second set. Using that technique, with demethylchlortetracycline as the first pair of labels and tetracycline HCl for the second set, we demonstrated the early effects of teriparatide on bone formation at the cellular level. With that sequence, we found that in cancellous bone of subjects on no osteoporosis medication, the second set of labels was almost always shorter than the first set, and consequently the proportion of the surface covered by label, the mineralized surface (MS/BS), calculated as double + 50% of the single-labeled surface, was less with the second set of labels (2.94 ± 1.13% demethylchlortetracycline versus 2.14 ± 0.83% tetracycline HCl), a difference that did not achieve statistical significance (n = 9). A similar trend was seen at the endocortical junction. Given the crescent-shaped anatomy of many cancellous bone packets, we might have expected that, if anything, the second set of labels should be longer, even in untreated control subjects. The effects of teriparatide were clearly evident, with a marked increase in MS/BS (that exceeded by several-fold any expected differences in label length due to the different forms of tetracycline). We now report in a separate experiment, in which tetracycline HCl was given as the initial set of labels and demethylchlortetracycline as the final set, in cancellous bone of untreated subjects, that the second labels are now longer than the first set (2.82 ± 0.76 tetracycline HCl versus 3.76 ± 0.65 demethylchlortetracycline; p < 0.05, n = 14). This strongly supports the original data from Parfitt and colleagues3 which suggested that the demethylchlortetracycline label was always longer than the oxytetracycline label, irrespective of the order in which the labels were administered. We believe that our data confirms the recommendation3 that if two different tetracyclines are being administered the differences in label length need to be taken into consideration. We suggest that for consistency across studies demethylchlortetracycline always be used for the initial set of labels followed by tetracycline HCl for the second set, and that a correction factor be applied using control data from the same laboratory, to correct for the differences in label length that are likely due to just differential binding to mineralizing bone. This work was supported by NIAMS Grant number: 5RO1AR56651-2. Robert Lindsay* , Hua Zhou , Felicia Cosman* , Jeri Nieves* §, David Dempster ¶, * Helen Hayes Hospital, Clinical Research Center, West Haverstraw, NY, USA, Department of Medicine, Columbia University, New York, NY, USA, Helen Hayes Hospital, Regional Bone Center, West Haverstraw, NY, USA, § Mailman School of Public Health, Columbia University, New York, NY, USA, ¶ Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

Occurrence of new bone-like tissue formation in uremic tumoral calcinosis

A 55-year-old woman who had been on hemodialysis for 5years was admitted for evaluation of a hard mass in the right hip region. Her serum calcium (Ca)–phosphate (P) product was elevated. Radiographs showed periarticular calcified masses in the soft tissues around both hips and shoulders, which were characteristic of uremic tumoral calcinosis (UTC). Biopsy specimens were obtained from both right hip mass and the right iliac crest. Histological examination of hip mass revealed bone-like tissue with marrow, as well as calcified material. The bone-like tissue was categorized as heterotopic ossification (HO), because it had been formed inside soft tissue where bone-like tissue does not normally exist. Histological analysis of HO showed the formation of cancellous bone-like tissue. Woven mineralized bone-like tissue was predominant over lamellar bone-like tissue. High bone turnover combined with osteitis fibrosa-like lesion was diagnosed because of an increase of the fibrous volume, as well as clear double tetracycline labeling. Near a site of HO, numerous ALP- and Runx2-positive cuboidal osteoblast-like cells and TRAP- and cathepsin K-positive multinucleated osteoclast-like cells were noted. Histomorphometric analysis of the right iliac crest revealed osteitis fibrosa. This is the first report of HO in a patient with UTC. After parathyroidectomy, the patient's Ca–P imbalance was corrected and UTC subsided. Although the mechanism by which new bone-like tissue formation arises in the soft tissues has not yet been determined, secondary hyperparathyroidism may have contributed to the progression of UTC in this patient.

Three-Dimensional Analysis of ABM/ P-15 With Sodium Hyaluronate Carrier in Sinus Augmentation

and tissue harvesting, microCT scans were performed on each of the specimens in order to provide objective measurements in evaluating the quality of the newly regenerated bone. Following imaging, specimens were fixed for 48 hours in a 10% neutral-buffered solution, sequentially dehydrated in 70%, 95%, and 100% ethanol, and then infiltrated and embedded in methylmethacrylate. Embedded specimens were sectioned to 25-um thickness and examined under ultraviolet light to evaluate new bone formation as marked by tetracycline labeling.