Phospholipase C signaling activated by parathyroid hormone mediates the rapid osteoclastogenesis in the fracture healing of orchiectomized mice.

BACKGROUND: The age-related osteoporosis is an increasing risk severely threatening the live quality of aged people. Human parathyroid hormone (hPTH) is applied to the therapy of osteoporosis successfully, however, the mechanism, especially the signaling pathway activated in the healing fracture by PTH is still unknown. METHODS: The once daily injections of hPTH(1-34) and GR (1-34) (the PLC deficient analog) into the orchiectomized male mice with bone fracture, were started at the second day after fracture and lasted for 4 weeks. To explore the role of phospholipase C signaling in the androgen-deficient fracture healing, the fracture healing were evaluated via radiography, micro-CT, biomechanics testing, serum biochemistry, bone marrow cell culture and gene expression quantification. RESULTS: After two weeks of fracture, both peptides significantly increased bone mineral density (BMD), bone mass content (BMC) and bone volume (BV/TV) in the healing area. However, compared to hPTH(1-34), GR(1-34) induced more woven bones, the higher BMC and BMD, as well as the less serum TRAP and osteoclasts. After four weeks of treatment, the effects of hPTH(1-34) on fracture healing showed no difference to those of GR(1-34). Consistently, GR(1-34) induced the similar osteogenesis but less osteoclastogenesis under the ex vivo condition immediately after administration compared to hPTH(1-34), which was verified by the weaker activation of RANKL, NFATC1, TRAP and Cathepsin K in GR(1-34) treatment. CONCLUSION: These results indicated that the PLC signaling activated by the intermittent injection of hPTH(1-34) leads to the bone resorption by rapidly activating the osteoclastogenesis in the fracture healing zone.

Characteristics of bone turnover markers in rapidly destructive coxopathy.

The purpose of this study was to clarify bone turnover marker levels in rapidly destructive coxopathy (RDC). Twenty patients with RDC (mean age, 72 ± 11 years; 3 men, 17 postmenopausal women), 111 with osteoarthritis (OA) (age, 60 ± 10 years; 15 men, 13 premenopausal women, 83 postmenopausal women), and 18 with osteonecrosis of femoral head (ON) (55 ± 14 years; 11 men, 3 premenopausal women, 4 postmenopausal women), and 100 patients with femoral neck fracture (FNF) (81 ± 10 years; 27 men, 73 postmenopausal women) were included. Serum tartrate-resistant acid phosphatase 5b (TRACP-5b), bone alkaline phosphatase (BAP), matrix metalloproteinase-3 (MMP-3) levels, and bone mineral density (BMD) of proximal femur and lumbar spine were investigated. TRACP-5b levels were significantly higher in RDC than in OA and ON, whereas BAP levels were higher in RDC than in OA (P < 0.05). MMP-3 levels were higher in RDC and ON than in OA (P < 0.05). TRACP-5b were higher in RDC than OA (P < 0.05) and FNF (P < 0.05) in performing propensity score matching; there were no differences in BMD between RDC and OA. TRACP-5b showed the largest area under the curve (AUC, 0.82) according to receiver operating characteristic (ROC) curve analysis for diagnosing RDC against OA and ON. AUCs of BAP and MMP-3 were 0.78 and 0.74. The respective sensitivities and specificities were 70.0 % and 85.3 % for TRACP-5b (cutoff, 623 mU/dl), 95.0 % and 57.1 % for BAP (13.8 U/l), and 70.0 % and 76.4 % for MMP-3 (52.7 ng/ml). The lack of differences in BMD suggested that high bone turnover marker levels may reflect osteoclast cell activation in RDC hips. Serum TRACP-5b and BAP could be RDC markers.

[Correlation between the content of transforming growth factor-beta 1, interleukin-1 beta and collagenase activity in proximal femur bone samples obtained from patients with femoral neck fracture]. / Wzajemne relacje pomiedzy zawartoscia transformujacego czynnika wzrostu-beta 1, interleukiny-1 beta i aktywnoscia kolagenazy w bioptatach kostnych blizszego konca kosci udowej u chorych po zlamaniu szyjki kosci udowej.

The aim of this study was to determine correlations between the content of TGF-beta1, IL-1beta and collagenase activity in bone samples of the femoral neck obtained from patients with femoral neck fracture. The material consisted of 42 samples of cancellous bone of femoral neck collected during hemiarthroplasty or total hip replacement procedure. 24 samples of cancellous bone from patients with osteoarthritis of the hip harvested during total hip replacement served as a control group. The content of TGF-beta1, IL-1beta was measured using ELISA and collagenase activity was measured with spectrofluorometry. In patients with fracture there was found a distinct inversely proportional correlation between the content of TGF-beta1 and IL-1beta. This correlation was not observed in the control group. In addition, a directly proportional relation between the content of TGF-beta1 and IL-1beta was discovered. These results confirm mutual correlations between examined cytokines in bone.

Allele frequency of the G-->T mutation of the col1A1 gene analyzed by an ARMS-PCR in osteoporotic subjects with femoral neck fractures.

The allele frequency of the G-->T mutation of COL1A1 gene (collagen type I alpha 1 gene, GenBank accession n. AF017178) was analyzed by a newARMS-PCR method in 240 osteoporotic subjects bearing a femoral neck fracture. The method is based on the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Normal and mutated alleles were detected by two PCRs, in which a common forward primer (positions 1307 to 1336 of the gene) and two reverse primers (positions 1566 to 1546), differing in the 3'-base (3'-C for the normal Sallele and 3'-A for the mutated sallele) are used. In the SS condition, amplification occurs only in one of the two PCRs, and in the ss condition only in the other. In the Ss condition both reactions give a product. This ARMS-PCR method avoids the use of any restriction enzyme, as described by Grant and colleagues in a previously published method based on a mismatched reverse primer which introduced a restriction site in the T-substituted (s) allele and in a method recently proposed by Vinkanharju and co-workers. In a survey for COL1A1 polymorphism in 240 osteoporotic subjects with femur fractures, here presented, a frequency of 80.6% was found for the G allele and 19.4% for the T allele. There were 66.7% dominant SS subjects, 27.9% Ss heterozygotes and 5.4% ss recessive homozygotes.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls.

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck's inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68-96 years). The density of eNOS+ cells (mm(-2)) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS+ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS+ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS+ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS+ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

Aromatase cytochrome P450 transcripts are detected in fractured human bone but not in normal skeletal tissue.

Peripheral conversion of gender steroid precursors has been implicated in playing a role in bone turnover in postmenopausal women. It has been reported that aromatase cytochrome P450 (P450arom) is present in primary bone and bone marrow (BM), and that P450arom mRNA has been identified in cultured BM and osteoblast-like cell lines. However, there are no reports that P450arom transcripts have been detected in skeletal tissue that has not been cultured. We therefore elected to test for the presence of P450arom mRNA in primary human bone and BM in normal and fractured necks of femora using the reverse transcription-polymerase chain reaction method. Although the RNA extracted from these tissues was of good quality as demonstrated by the expression of transcripts for interleukin-6, P450arom transcripts failed to be detected in normal primary cortical bone and fatty BM containing trabecular bone. However, P450arom transcripts were detected in the latter when they were cultured. Transcripts for P450arom were also detected in total RNA extracted from six fractured necks of femora and semiquantitative PCR demonstrated that P450arom mRNA was present in similar abundance in the same amount of RNA analyzed from buttock adipose tissue and fractured bone/BM. P450arom mRNA expression was also detected in cultured peripheral blood leukocytes, suggesting that this might be the source of the enzyme. In these cultures no correlation was detected between the expression of P450arom mRNA and cell proliferation. PCR failure was excluded in cases when P450arom transcripts failed to be detected in bone/BM by coamplifying RNA from human and rat brain mRNA, known to express P450arom mRNA, using primers that detect both P450arom mRNA from both species. These products were analyzed by Southern blot using oligonucleotide probes, which label either human or rat P450arom cDNA. The blots confirmed the absence of P450arom in nonfractured human bone and BM and preclude PCR failure. Our results indicate that P450arom mRNA is not detected in either normal human bone or BM, but can be induced in this microenvironment under pathological conditions. We propose that tissue grown in vitro is analogous to a wound and this explains why P450arom transcripts were detected in cultured normal skeletal tissue, whereas they failed to be detected in primary normal bone and BM.

Plasma aspirin esterase activity in elderly patients undergoing elective hip replacement and with fractured neck of femur.

Plasma aspirin esterase activity was measured in eight elderly patients undergoing elective hip replacement, and in 11 presenting with femoral-neck fracture. Elective hip surgery was associated with a marked and significant decline in esterase, from 330 +/- 35.9 (SE) (nmol salicylate/ml plasma/min) pre-operatively to 236 +/- 35.6 on the third post-operative day, and returning to normal (359 +/- 24.3) at recovery phase. Patients with fractured neck of femur showed a depressed esterase activity on admission (221 +/- 12.7), rising towards normal (290 +/- 15.4) on recovery. All of these changes were accompanied by a marked acute-phase response, both to injury and surgery. The data suggest that elderly patients experiencing injury or undergoing surgery may have significantly impaired drug metabolism, at least as far as this enzyme is concerned.

Vitamin D concentrations in women of postmenopausal age with fractures of the femoral neck.

Thirty-eight women of postmenopausal age, suffering from fractures of the femoral neck or vertebral bodies were studied in relation to differences in bone metabolism. The blood and urinary changes concerned in mineral and bone metabolism were recorded within 10 days of trauma, and in some femoral neck fractures, a histological study of the femoral heads removed in the course of prosthetic substitution was carried out. The patients with femoral neck fractures were older than those with vertebral fractures and had metabolic and histological findings suggestive of osteomalacia. A particularly significant difference between the two groups was the plasma level of 25 hydroxycholecalciferol, which was lower in femoral neck fractures, and the urinary excretion of calcium, which was also more reduced in femoral neck fractures. A deficit of vitamin D hepatic metabolite thus appears to be a risk factor for femoral neck fractures in old patients.

Is avoidance of sunlight a cause of fractures of the femoral neck in elderly Saudis?

A study of the frequency of admitted cases of femoral neck fractures to Riyadh Central Hospital, Riyadh, Saudi Arabia, showed admission of 95 patients over 40 years of age in a period of one year. The male to female ratio was 64% males to 36% females. Vitamin D nutritional status of patients with fractures of the neck of the femur expressed as 25-(OH)D3 was significantly lower (5.9 +/- 2.9 ng/ml) than of controls (9.7 +/- 4.7 ng/ml). Comparison of the housing types of both patients and controls showed that those living in traditional mud houses have significantly lower levels of 25-(OH)D3 than those occupying villas or flats. A third group of patients with low stores of vitamin D were exposed to natural ultraviolet light for a short period. This resulted in a significant increase in the levels of serum 25-(OH)D3 and a decrease in alkaline phosphatase activity. It is concluded that low levels of serum 25-(OH)D3 may play a role in the pathogenesis of femoral neck fractures in elderly Saudis and this may be due to minimal exposure to natural ultraviolet irradiation.

Changes in serum alkaline phosphatase after femoral fractures.

Osteomalacia may be a contributory factor in some patients in the development of fractures of the femoral neck and complicate the subsequent management. The level of serum alkaline phosphatase is often valuable in the diagnosis of metabolic bone disease but rises after any uncomplicated fracture, and since such a rise may limit the diagnostic usefulness of this measurement in detecting osteomalacia its extent was assessed in 106 patients. In the majority serum levels were normal on admission, rising after seven to nine days to reach a maximum within a month after fracture. Elevated levels on admission were found in patients with osteomalacia, liver damage or where there had been a delay of several weeks between injury and admission. In a small number of patients normal levels on admission subsequently reached very high values, usually in association with comminution or instability of the fracture. Elevated levels persisted for six to twelve weeks after fracture, the major influence upon the level at this time being the maximum value achieved rather than the presence of osteomalacia. If patients are to be screened for osteomalacia, the alkaline phosphatase must be measured within the first week after a fracture to avoid the distorting influences of the fracture itself.

[25-OH-vitamin D in the serum of patients with femoral-neck fractures]. / 25-OH-Vitamin D im Serum bei Patienten mit Schenkelhalsfrakturen.

In 10 women with femoral neck fracture, 10 with fracture(s) evolved from accidents (mainly traffic accidents) and 10 without disease (controls) in January and July, the 25-OH-vitamin D levels in serum were measured by a competitive protein binding assay. Patients with femoral neck fractures exhibited significantly (p less than 0.05) lower 25-OH-vitamin D levels than the other groups. Additionally, in patients with femoral neck fractures calcium x phosphate product in serum was subnormal (below 25). In Germany, prophylactic dietary supplementation of vitamin D seems feasible.