Expression of the osteogenic phenotype in porous hydroxyapatite implanted extraskeletally in baboons

A porous hydroxyapatite was used as a morphogenetic matrix to study early tissue formation preceding the morphogenesis of bone in extraskeletal sites of the baboon (Papio ursinus). Porous hydroxyapatites, obtained by hydrothermal conversion of the calcium carbonate exoskeleton of coral, were implanted extraskeletally in 16 baboons. Specimens were harvested at days 30, 60 and 90, and processed to obtain decalcified sections for histomorphometry, and undecalcified sections for enzyme histochemical demonstration of alkaline phosphatase, immunohistochemical demonstration of laminin and type I collagen, and for comparative histologic analysis. At day 30, the tissue that invaded the porous spaces showed mesenchymal condensations at the hydroxyapatite interface, and prominent vascular penetration. Collagen type I staining was localized within mesenchymal condensations. Bone had not formed in any specimen harvested at day 30. At days 30 and 60, alkaline phosphatase staining was initially localized in the invading vasculature, and subsequently found in cellular condensations prior to their transformation into bone, and in capillaries close to cellular condensations. Laminin staining was localized around invading capillaries adjacent to and within mesenchymal condensations, and in capillaries in direct contact with the hydroxyapatite. Bone had formed by day 60; cartilage, however, was never observed. By day 90, bone formation within the porous spaces was often extensive. Goldner's trichrome stain and fluorescence microscopy of tetracycline-labeled specimens demonstrated nascent mineralization within condensations during initial bone morphogenesis. Coating the hydroxyapatite with collagen type I prepared from baboon bone did not increase the amount of bone formation. In this hydroxyapatite-induced osteogenesis model in primates, vascular invasion and bone differentiation appear to be accompanied by a specific temporal sequence of alkaline phosphatase expression. The differentiation of osteogenic cells in direct apposition to the hydroxyapatite suggests that this substratum may act as a solid state matrix for adsorption and controlled release of endogenously-produced bone morphogenetic proteins. The porous hydroxyapatite, as used in this bioassay in primates, may be an appropriate delivery system for bone morphogenetic proteins for the controlled initiation of therapeutic osteogenesis.     

Sustained response to intravenous alendronate in postmenopausal osteoporosis

We studied the effects of alendronate (amino-hydroxybutylidene bisphosphonate) on biochemical indices of bone turnover and on lumbar spinal bone mineral density in 15 postmenopausal women with vertebral osteoporosis. Alendronate 7.5 mg daily was administered intravenously as a slow infusion for four consecutive days. Treatment was associated with a significant decrease in serum calcium (p < 0.01), fasting urinary calcium excretion (p < 0.01) and hydroxyproline excretion within several days followed a later decrease in serum alkaline phosphatase activity that showed a significant reduction at two months after treatment (p < 0.05). Serum calcium reverted to pretreatment values by the second week after infusion, but the decrease in alkaline phosphatase, urinary calcium, and hydroxyproline excretion persisted to six months after infusion. There was a 3% mean increase in lumbar bone mineral density at six months (p < 0.01). A transient lymphopenia or leucopenia was noted in eight patients and a short-lived fever in six. No other side effects were observed. This study demonstrates that shortterm exposure to high intravenous doses of alendronate induces suppression of bone resorption in osteoporosis that persists for at least 6 months after infusion. We conclude that a short exposure to high intravenous doses induces sustained effects on bone turnover in much the same manner as that observed in Paget's disease of bone.     

Angiographic demonstration of vertebral osteoid osteoma

A case of osteoid osteoma in the left lamina of the T-8 vertebra is reported. The roentgenograms of the thoracic spine showed a hyperostotic round mass with associated scoliotic changes and the myelograms revealed complete obstruction of the spinal subarachnoid space. By selective spinal angiography a peculiar ring-like vascular stain was visualized in the area of hyperostosis which may be characteristic of an osteoid osteoma.     

Osteoporosis after spinal cord injury

Immobilisation secondary to spinal cord injury (SCI) is associated with marked and rapid atrophy of trabecular bone. The purpose of this study was to evaluate bone mineral density (BMD) in both the upper and lower extremities following SCI sustained for various lengths of time and to correlate the BMD to the level of the lesion, time from injury, spasticity and serum calcium, phosphorus and alkaline phosphatase (ALP) levels. A study was undertaken in 41 SCI patients with a mean age of 35.8 +/- 12.7 years. A significant difference in BMD between upper and lower extremities of the paraplegics were found. BMD of upper and lower extremities were similar in tetraplegies. The BMD values were significantly different when the upper extremity scores of paraplegics and tetraplegics were compared but BMD scores of the lower extremities were similar in the two groups. The decrease in BMD was less in the spastic patients when compared to the flaccid group. There was a positive correlation between time from injury and the degree of BMD deficit in the paralysed areas. In the whole group of patients a significant positive correlation was found between the duration of SCI and serum ALP levels.     

Monitoring of bone turnover biological, preanalytical and technical criteria in the assessment of biochemical markers

A review is given summarizing the present knowledge of bone turnover markers with special emphasis on biological, preanalytical and technical criteria in the proper judgement of efficacy and limitations of the methods employed. The marker substances may be either measures of bone formation or bone resorption. Markers of bone formation are bone alkaline phosphatase, osteocalcin and the carboxyl-terminal propeptide of procollagen type I. Bone alkaline phosphatase has proved to be superior to total alkaline phosphatase activity with respect to diagnostic sensitivity and specificity. Immunochemical techniques for measuring bone alkaline phosphatase show a cross-reactivity of 14-20% with liver alkaline phosphatase. However, this does not compromise the clinical usefulness of these assays except for patients with severe liver diseases. Osteocalcin is strictly bone-specific but shows numerous disadvantages with respect to apparent instability and discordant results as obtained by different methods; however, in certain diagnostic situations (corticosteroid-induced osteopenia, absence of destroyed bone architecture) osteocalcin may serve as a sensitive bone turnover marker. The carboxyl-terminal propeptide of procollagen type I generally shows low discriminating power in the diagnosis of bone diseases. The urinary excretion of pyridinium "cross-links' has been carefully evaluated so far with respect to analytical performance and clinical usefulness. This marker may be a substitute for 4-hydroxyproline measurements as the method of choice for assessment of bone resorption. There are other degradation products from the telopeptide regions of bone-derived collagen type I which are excreted into the urine (N-telopeptides, CrossLapsTM); these analytes are promising tools in the assessment of bone resorption but require further evaluation, in particular with respect to their extraskeletal clearance and putative origin outside bone. Moreover, their clinical usefulness may vary depending on the patient group examined. In contrast, the serum concentration of the cross-linked telopeptide region of collagen type I seems to lack both diagnostic specificity and sensitivity in the majority of patient groups. Tartrate-resistant acid phosphatase (as determined by the presently available methods) cannot be recommended as a routine tool for assessment of bone resorption.     

Changes in skeletal metabolism in pubertal girls can be revealed by biochemical parameters of calcium metabolism and bone turnover

Biochemical changes related to skeletal turnover in puberty were investigated in a sample of 67 girls aged 8-14 years. The following biochemical parameters were measured in serum: total calcium, phosphate, magnesium, total alkaline phosphatase, osteocalcin, and calcium and hydroxyproline in the second morning urine. Thirty-five premenarchal girls (8-11 years) had significantly lower serum calcium, and higher alkaline phosphatase and phosphate than those menstruating regularly (N = 32, 12-14 years). A statistically significant negative correlation of serum parameters and age was found for phosphate and alkaline phosphatase in all subjects, and for calcium and magnesium only in the premenarchal girls. These results indicated the more intensive processes of skeletal metabolism occurring in prepubertal age and early puberty to reflect in basic biochemical parameters of calcium and bone metabolism. Analysis of correlation between biochemical parameters showed alkaline phosphatase and phosphate to correlate positively with hydroxyproline excretion and negatively with urinary calcium in all subjects. In the subjects after menarche, osteocalcin correlated with alkaline phosphatase and phosphate. Thus, biochemical parameters indirectly reflected physiologic changes occurring with bone turnover in puberty. Variations in bone turnover during puberty, including a more pronounced bone formation during prepubertal or early stages, can be indirectly observed through biochemical parameters related to calcium and bone metabolism. Investigations of skeletal growth and puberty would benefit from specific markers of bone remodeling and "basic" biochemical parameters, as it might disclose subtle metabolic relationships.     

Mechanics of Drag Embedment Anchors in a Soft Seabed

This paper presents an analysis of the load capacity and trajectory of a drag embedment anchor in a soft seabed. Anchor capacity relationships are developed for an idealized anchor comprising a rectangular fluke and a cylindrical shank. Geometric variables considered for the anchor include fluke length, fluke thickness, shank length, angle between fluke and shank, and shank thickness. Parametric studies are presented investigating the effect of these variables on anchor capacity and performance. A method of anchor trajectory prediction during drag embedment is developed by considering anchor behavior in conjunction with the mechanics of the anchor line. The anchor trajectory simulations indicate that an equilibrium condition rapidly develops during embedment in which the rate of anchor rotation is identical to the rate of change in the anchor line uplift angle at the shackle point. At the equilibrium state, the anchor load capacity normalized by soil strength remains constant and the anchor is in a state of incipient rotation. The anchor line angle at the seabed also influences anchor trajectory. This angle varies throughout embedment according to the mechanics of the anchor line in both the seabed and the water column.     

Restriction fragment length polymorphism of genes of the alpha 2(XI) collagen, bone morphogenetic protein-2, alkaline phosphatase, and tumor necrosis factor-alpha among patients with ossification of posterior longitudinal ligament and controls from the Japanese population

STUDY DESIGN: The present study analyzed the restriction fragment length polymorphism patterns of alpha 2(XI) collagen, bone morphogenetic protein-2, alkaline phosphatase, and tumor necrosis factor-alpha genes in patients with ossification of the posterior longitudinal ligament. This study investigates the genetic polymorphism of bone-induced factors in patients with ossification of the posterior longitudinal ligament and compares it with healthy control subjects. OBJECTIVES: To clarify the genetic markers linked to ossification of the posterior longitudinal ligament. SUMMARY OF BACKGROUND DATA: Ossification of the posterior longitudinal ligament is a genetic disease associated with abnormal calcium metabolism involving the posterior longitudinal ligament. Previous genetic studies have not identified the pathologic mechanism of ossification of the posterior longitudinal ligament. Histopathologic studies of ossification of the posterior longitudinal ligament and the animal model, the spinal hyperostotic mouse, have revealed an increase in Type XI collagen and bone morphogenetic protein-2 expression. METHODS: Eighteen Japanese patients with ossification of the posterior longitudinal ligament and 51 healthy, unrelated control subjects were investigated for the restriction fragment length polymorphism patterns of COL11A2, bone morphogenetic protein-2, alkaline phosphatase, and tumor necrosis factor-alpha, genes with various restriction endonucleases. RESULTS: The gene frequencies of COL11A2 obtained with BamHl (10.0 kb fragment) and HindIII (19.0 kb fragment) observed in patients with ossification of the posterior longitudinal ligament were higher compared with control subjects (0.43 and 0.14, respectively). These differences were statistically significant (BamHl P = 0.018; Hindlll P = 0.046). Two new restriction fragment length polymorphism patterns were detected of the bone morphogenetic protein-2 gene with Mspl and Taql and one already known restriction fragment length polymorphism pattern of the tumor necrosis factor-alpha gene with Ncol. However, they were not significantly different from the control subjects. CONCLUSIONS: Seven restriction fragment length polymorphisms of COL11A2 gene were identified. Two of them (BamHl, 10.0/10.0 kb genotype; HindIII, 19.0/19.0 kb genotype) were significantly different in patients with ossification of the posterior longitudinal ligament.     

Molecular weights of three mouse milk caseins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and kappa-like characteristics of a fourth casein

Caseins of mouse milk are phosphoproteins which precipitate at pH 4.6, stain blue with "Stains-all," and stain red with "Stains-all" following alkaline phosphatase digestion. Four caseins were separated electrophoretically in sodium dodecyl sulfate-polyacrylamide gels varying from 8.5 to 15% acrylamide. Molecular weights for three of these proteins were 43,200, 27,700, and 25,900. The molecular weights determined for bovine alphas1 and beta caseins by this method were similar to those previously obtained by other methods. A fourth mouse casein contained carbohydrate, phosphorus, and sialic acid. This protein was rennin-sensitive and behaved anomalously on sodium dodecyl sulfate polyacrylamide gels, as did bovine kappa-casein. Because of similarities with bovine kappa-casein, it was designated with "kappa-casein" of mouse milk.     

Chronic osteomyelitis of the spine managed with a free flap of latissimus dorsi. A case report

STUDY DESIGN: A patient with intractable spinal osteomyelitis who underwent surgery 12 times with persistent exposed bone is presented. OBJECTIVES: To demonstrate the effectiveness of free-flap grafting for managing difficult spinal osteomyelitis wounds. SUMMARY OF BACKGROUND DATA: Conventional procedures can usually achieve wound closure, but they may not work for advanced cases. METHODS: A free latissimus dorsi flap was transferred for reconstruction. The muscle component was used to obliterate the dead space and cover the exposed bone, and the skin component was used for tension-free closure of the wound. RESULTS: The wound healed dramatically. There was no recurrence of infection at 2-year follow-up evaluation. CONCLUSIONS: For an intractable spinal osteomyelitis wound, a free flap should be considered, although the surgery is difficult. Technical precautions in performing this operation are given.     

An investigation into the immunochemical properties of the isoenzymes of human alkaline phosphatase

A study was carried out to investigate the immunochemical properties of the alkaline phosphatases present in liver, placenta, intestine, and kidney. Butanol extracts of the tissues were examined by antigen-antibody crossed electrophoresis, and the immunoprecipitates formed by the enzymes were identified by use of a specific stain for alkaline phosphatase. The results obtained revealed three immuno-chemical types of alkaline phosphatase, designated L, P, and PI respectively. The distribution of these enzymes among the tissues examined was as follows: type L was the only enzyme detected in liver extract and type P the only one detected in placental extract. The extracts of intestine and kidney both contained two isoenzymes, one of which was type L and the other type PI.     

Concomitant pneumococcal appendicitis, peritonitis, and meningitis

A 9-year-old boy developed pneumococcal meningitis and peritonitis following appendectomy. Subsequent pathologic examination showed Gram-positive diplococci in the appendix. Cultures of the peritoneal fluid, blood, and spinal fluid showed Diplococcus pneumoniae. The experience illustrates the danger of assuming that all pneumococcus peritonitis is the primary variety and the advisability of routine Gram stain of the peritoneal fluid at operation in order to select the appropriate antibiotic.     

Effects of gadolinium on proliferation, differentiation and calcification of primary mouse osteoblasts in vitro

To evaluate the effects of Gd on proliferation, differentiation and mineralization function of primary osteoblasts (OBs) in vitro, we tested cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell differentiation by alkaline phosphatase (ALP) activity assay, synthesis of type ? collagen, and oil red O and alizarin red S (ARS) stain assays. The results indicated that effects of Gd on the proliferation, osteogenic differentiation, mineralization function and adipocytic transdifferentiation of primary OBs de-pended on concentration and incubation time, but were not dose-dependent. It was suggested that the effect of Gd on bone metabolism was complicated, and concentration and culture time were key factors for switching the biological effects of Gd from damage to protection.     

A simple combination of Rf value and melting-point determination for the identification of barbiturates

Patients (219) with prostatic adenocarcinoma were classified on the basis of whether or not their bone scans were positive for metastasis. Acid and alkaline phosphatase determinations and clinical evaluations for bone metastases were reviewed. Of those with proved metastases, 43% had no bone pain, 39% had normal acid phosphatase levels, 23% normal alkaline phosphatase levels, 19% normal levels of both enzymes, and 15% normal enzyme levels without bone pain. Twenty-four per cent of the patients with normal enzyme levels and clinically unsuspected bone metastases had bone scans which proved positive for metastasis; 62% of these had normal radiographs.     

Installation of Torpedo Anchors: Numerical Modeling

Torpedo anchors are used as foundations for mooring deep-water offshore facilities, including risers and floating structures. They are cone-tipped cylindrical steel pipes ballasted with concrete and scrap metal and penetrate the seabed by the kinetic energy they acquire during free fall through the water. A mooring line is usually connected at the top of the anchor. The design of such anchors involves estimation of the embedment depth as well as short-term and long-term pullout capacities. This paper describes the development of a computational procedure that leads to prediction of torpedo-anchor embedment depth. The procedure relies on a computational fluid dynamics (CFD) model for evaluation of the resisting forces on the anchor. In the model, the soil is represented as a viscous fluid and the procedure is applied to axially symmetric penetration of the seabed. The CFD approach provides estimates of not only the embedment depth but the pressure and shear distributions on the soil-anchor interface and in the soil.     

Estrogen protection against bone resorbing effects of parathyroid hormone infusion. Assessment by use of biochemical markers

OBJECTIVE: Because parathyroid hormone (PTH) stimulates bone resorption, resistance to its actions might help maintain bone mass. We tested the hypothesis that the effects of estrogen on bone are accomplished in part by decreasing the sensitivity of the skeleton to the resorbing effects of PTH. STUDY DESIGN: Comparison of response to PTH infusion in untreated and estrogen-treated postmenopausal women with osteoporosis. INTERVENTION: (1-34) human PTH, 0.55 U/(kg.h), was infused intravenously over 20 hours. SETTING: The inpatient clinical research unit of a referral hospital. PATIENTS: Women with primary postmenopausal osteoporosis who were untreated (n = 15) or treated with estrogen (n = 17). MAIN OUTCOME MEASURES: Skeletal turnover indices including hydroxyproline, deoxypyridinoline, pyridinoline, tartrate-resistant acid phosphatase, alkaline phosphatase, bone Gla protein, and insulin-like growth factor-1. RESULTS: All basal indices were higher in untreated than in estrogen-treated women, but statistical differences were seen only for deoxypyridinoline and pyridinoline. During the 20-hour infusion, hydroxyproline/creatinine increased 0.023 mumol/mumol in untreated women but only 0.010 mumol/mumol in estrogen-treated women (P < 0.05). Corresponding changes for deoxypyridinoline/creatinine were 14.6 mumol/mumol and 3.5 mumol/mumol (P = 0.06). Tartrate-resistant acid phosphatase and pyridinoline increased only in untreated group. A circadian rhythm in circulating bone Gla protein was seen in both groups without clear PTH-induced effects or differences between groups. Alkaline phosphatase levels decreased and insulin-like growth factor-1 levels increased in both groups with no distinction between untreated and estrogen-treated women [corrected]. CONCLUSION: The estrogenized postmenopausal osteoporotic skeleton is less sensitive to the bone resorbing effects of acutely administered PTH. There are no differential effects on bone formation.     

Experimental Testing of Pile-to-Cap Connections for Embedded Pipe Piles

For bridges supported by piles, acceptable system performance under seismic loading depends on effective pile-to-cap connections. A fixed pile-to-cap connection is often desirable to help control deflections during lateral loading when soft soils are present. While reinforcement bar cages that extend from the pile into the cap are effective in providing a fixed pile-to-cap connection, it is more economical to rely on pile embedment to provide fixity and moment resistance. This study investigated embedded pile-to-cap connections for concrete-filled pipe piles. Four full-scale specimens, each consisting of a cap with two piles, were investigated in the field under cyclic loading. The specimens had minimal reinforcement and varying amounts of pile embedment. Results show that the moment resistance of pile-to-cap connections can be significantly greater than what is typically calculated based on the flexural reinforcement and embedment bearing. Excess moment capacity may be explained by friction between the pile and the cap at the connection. This friction mechanism is described and discussed in the context of experimental results from other studies.     

Similarities in the phenotypic expression of pericytes and bone cells

Bovine brain microvessel pericytes, bone cells, and fibroblasts were grown in tissue culture in 3%, 21%, or 60% oxygen for 7 weeks. Alkaline phosphatase activity was highest in bone cells and pericytes grown in 3% oxygen, with the activity higher in the former than the latter. Alkaline phosphatase activity was very low in fibroblasts at every oxygen concentration. Osteocalcin concentration was higher in bone cells than in pericytes, was not detected in fibroblasts, and in bone cells and pericytes the concentration was highest in 21% oxygen. Other bovine brain microvessel pericytes were grown in 3% or 21% oxygen for 3 to 24 days in the presence or absence of bone morphogenetic protein 2 and in the presence or absence of parathyroid hormone. At Day 3 of culture, alkaline phosphatase activity was highest in 21% oxygen in the presence of bone morphogenetic protein 2. By Day 17 of culture, alkaline phosphatase activity was highest in 3% oxygen whether bone morphogenetic protein was present or not. Cyclic adenosine monophosphate production in pericytes in response to parathyroid hormone stimulation was very modest when compared with that of bone cells, and this response was not found to be significantly altered by bone morphogenetic protein 2, duration of culture, or the oxygen concentration during incubation. These findings show that the microvessel pericyte is capable of exhibiting several oxygen dependent, phenotypic characteristics ascribed to osteoblasts.     

Epidemiological study of osteoporosis

An epidemiological investigation of osteoporosis was carried out in a community in which fishing is the primary industry, and correlations were investigated between the bone mass and various risk factors for osteoporosis and biochemical findings. In 852, who could be examined directly, of the 3541 males and females aged 40 years or above living in Nansei Town, Watarai District, Mie Prefecture, the age, sex, height, body weight, years after menopause, married or unmarried, area of residence, daily activity, and intakes of milk, fish, and alcohol were studied by direct oral inquiry, and the serum calcium, inorganic phosphorus, alkaline phosphatase, and total protein were measured. The subjects were divided into a decreased bone mass group who showed grade I or more advanced loss in bone mass and a normal bone mass group who showed no or only slight loss in bone mass on the basis of microdensitometry (MD). Eighty-two subjects (9.6%) were classified as the decreased bone mass group. Items that showed a close correlation with sigma GS/D, which is an index of bone density, were age, years after menopause, serum alkaline phosphatase level, height, and body weight in females, age and serum alkaline phosphatase level in males. In the decreased bone mass group, 79 of the 82 subjects were females. Significant differences were observed between the decreased bone mass group and the normal bone mass group in age, years after menopause, serum alkaline phosphatase level, height, and body weight. From these results, a high age, being a female, low height, and a low body weight were found to be factors correlated with osteoporosis. Also, the bone mass was correlated with the serum alkaline phosphatase level and years after menopause.     

NASBA technology: isothermal RNA amplification in qualitative and quantitative diagnostics

The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. approximately 15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10-20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation.