Erik Fink Eriksen

Cancellous Bone Remodeling Occurs in Specialized Compartments Lined by Cells Expressing Osteoblastic Markers

We describe a sinus, referred to as a bone remodeling compartment (BRC), which is intimately associated with cancellous bone remodeling. The compartment is lined on its marrow side by flattened cells and on its osseous side by the remodeling bone surface, resembling a roof of flattened cells covering the bone surface. The flat marrow lining cells are in continuity with the bone lining cells at the margins of the BRC. We examined a large number of diagnostic bone biopsy specimens received during recent years in the department. Furthermore, 10 patients (8 women and 2 men, median age 56 [40–69] years) with the high turnover disease of primary hyperparathyroidism who were treated with parathyroidectomy and followed for 3 years were included in the histomorphometric study. Bone samples for the immuno‐enzyme staining were obtained from an amputated extremity of child. The total cancellous bone surface covered by BRC decreases by 50% (p < 0.05) following normalization of turnover and is paralleled by a similar 50% decrease in remodeling surface (p < 0.05). The entire eroded surface and two‐thirds of the osteoid surface are covered by a BRC. BRC‐covered uncompleted walls are 30% (p < 0.05) thinner than those without a BRC. This indicates that the BRC is invariably associated with the early phases of bone remodeling, that is, bone resorption, whereas it closes during the late part of bone formation. Immuno‐enzyme staining shows that the flat marrow lining cells are positive for alkaline phosphatase, osteocalcin, and osteonectin, suggesting that they are bone cells. The first step in cancellous bone remodeling is thought to be the lining cells digesting the unmineralized matrix membrane followed by their disappearance and the arrival of the bone multicellular unit (BMU). We suggest that the lining cell barrier persists during bone remodeling; that the old lining cells become the marrow lining cells, allowing bone resorption and bone formation to proceed under a common roof of lining cells; that, at the end of bone formation, new bone lining cells derived from the flattened osteoblasts replace the marrow lining cells thereby closing the BRC; and that the two layers of lining cells eventually becomes a single layer. The integrity of the osteocyte‐lining cell system is reestablished by the new generation of lining cells. The BRC most likely serves multiple purposes, including efficient exchange of matrix constituents and minerals, routing, monitoring, or modulating bone cell recruitment, and possibly the anatomical basis for the coupling of bone remodeling.

Polymorphisms in the osteoprotegerin gene are associated with osteoporotic fractures.

Osteoprotegerin (OPG) is a soluble receptor for RANKL and therefore a competitive inhibitor of osteoclast differentiation and activity. With this key role in the control of resorptive activity, we found that OPG is a candidate gene for genetic control of bone mass. We examined the promoter and the five exons with surrounding intron sequences of the OPG gene for polymorphisms in 50 normal patients and 50 patients with osteoporosis. We found 12 polymorphisms. Two sets of four and five polymorphisms, respectively, were in complete linkage. Subsequently, we examined the effect of the informative polymorphisms A163‐G (promoter), T245‐G (promoter), T950‐C (promoter), G1181‐C (exon 1), and A6890‐C (intron 4) on the prevalence of osteoporotic fractures, bone mass, and bone turnover in 268 osteoporotic patients and 327 normal controls. In A163‐G the variant allele G was more common among fracture patients: 34.0% versus 26.3% in normal controls (p < 0.05) and the odds ratio (OR) for a vertebral fracture, if an individual has the G allele, was 1.44 (1.00–2.08). In T245‐G the variant allele G was more common in osteoporotic patients: 12.4% versus 6.5% (p < 0.02) and the OR for vertebral fracture, if an individual has the G‐allele, was 2.00 (1.10–3.62). G1181‐C is located in the first exon and causes a shift in the third amino acid from lysine to asparagine. The CC genotype was less common among fracture patients: 26.3% versus 36.7% in the normal controls (p < 0.01). T950‐C and A6890‐C were not distributed differently among patients with osteoporosis and normal controls. None of the polymorphisms affected bone mineral density (BMD) or biochemical markers of bone turnover in the normal controls. In conclusion, we have examined the human OPG gene for polymorphisms and found 12. The rare alleles of the A163‐G and T245‐G were significantly more common among patients with vertebral fractures.

An Sp1 Binding Site Polymorphism in the COLIA1 Gene Predicts Osteoporotic Fractures in Both Men and Women

Genetic factors play an important role in the pathogenesis of osteoporosis, and recent studies have shown that a polymorphic Sp1 binding site in collagen type I α1 (COLIA1) gene is associated with bone mass and vertebral fractures in women from the U.K. Information on the predictive value of the COLIA1 Sp1 polymorphism in other populations is limited, however, and no studies have yet been performed in osteoporotic males. In view of this, we analyzed COLIA1 genotypes in relation to bone density and biochemical markers of bone turnover and the presence of osteoporotic fractures in a case‐control study of Danish men and women. COLIA1 genotype was determined by polymerase chain reaction analysis of genomic DNA extracted from peripheral blood samples and related to bone mass, biochemical markers of bone turnover, and the presence of fracture in a study of 375 osteoporotic vertebral fracture patients and normal controls. There was no significant effect of COLIA1 genotype on bone mass or biochemical markers when data from the control group (n = 195) and fracture group (n = 180) were analyzed separately. However, the genotype distribution was significantly different in the fracture cases compared with age‐matched controls (χ2 = 16.48, n = 249, p = 0.0003) due mainly to over‐representation of the ss genotype in the fracture patients (14.3% vs. 1.4%), equivalent to an odds ratio for vertebral fracture of 11.83 (95% confidence interval 2.64–52.97) in those with the ss genotype. Similar differences in genotype distribution between osteoporotic patients and controls were observed in both men (χ2 = 11.52, n = 95, p = 0.0032, OR = 2.04) and women (χ2 = 6.90, n = 154, p = 0.032, OR = 1.37). In keeping with the above, logistic regression analysis showed that the ss genotype was an independent predictor of osteoporotic fracture (p = 0.028). This study confirms that the COLIA1 Sp1 polymorphism is significantly associated with osteoporotic vertebral fractures. The association is seen in both men and women, and the effect on fracture risk appears to be partly independent of bone mineral density. Our results raise the possibility that genotyping at the Sp1 site could be of clinical value in identifying individuals at risk of osteoporotic fractures in both genders.

Bone morphogenetic protein-2 but not bone morphogenetic protein-4 and -6 stimulates chemotactic migration of human osteoblasts, human marrow osteoblasts, and U2-OS cells

Bone morphogenetic proteins (BMPs) have important functions for the differentiation of bone cells, but the exact role for bone remodeling and bone healing still needs to be defined. Migration of bone forming cells is an important physiological event both during bone healing and bone remodeling. The chemotatic properties of the bone morphogenetic protein family of growth factors have not been investigated. In this study the chemotactic effects of the bone morphogenetic proteins BMP-2, -4, and -6 have been quantitated toward human osteoblasts, human marrow stromal osteoblasts, and U2-OS human osteosarcoma cells. BMP-2 stimulated the migration of human stromal osteoblasts, human osteoblasts, and U2-OS cells with bell-shaped response curves in a dose-dependent manner with a 300% increase in cell migration at 1.0 ng/mL for human stromal osteoblasts and a 170-180% increase for human osteoblasts and U2-OS cells. At higher concentrations, migration decreased to background levels. BMP-4 and -6 did not show any effect on cellular migration. This study shows that BMP-2 can stimulate in vitro migration of human osteoblasts and human osteosarcoma cells. BMP-2 might play a role in the chemotactic recruitment of especially undifferentiated osteoblasts during bone remodeling and bone healing.

A TA repeat polymorphism in the estrogen receptor gene is associated with osteoporotic fractures but polymorphisms in the first exon and intron are not

Estrogen and the estrogen receptor (ER) play a central role in bone metabolism as illustrated by the loss of bone mass after menopause and the osteopenia in individuals with defect aromatase or ER. We therefore wanted to investigate the effect of polymorphisms in the ER‐α gene on bone mass, bone turnover, and the prevalence of osteoporotic fractures in a study of 160 women and 30 men with vertebral fractures and 124 women and 64 men who are normal. Three previously described polymorphisms, G261‐C in exon 1 and T‐C and A‐G in intron 1, in the ER gene were determined by restriction fragment length polymorphism (RFLP) using BstUI, Pvu II, and Xba I after polymerase chain reaction (PCR). A TA repeat polymorphism in the promoter region was examined by PCR and electrophoresis. The distribution of BstUI, Pvu II, and Xba I RFLPs was similar in the osteoporotic patients and the normal controls. No significant differences could be shown in bone mass or bone turnover between the genotypes. The mean number of TA repeats was lower in patients with osteoporotic fractures, 17.3 ± 2.8 versus 18.6 ± 2.8 in the normal controls (p < 0.01). This also was reflected in a significantly increased odds ratio of osteoporotic fractures in individuals with 11–18 repeats of 2.64 (95% CIs, 1.61‐4.34). Furthermore, bone mineral density (BMD) of the lumbar spine was lower in individuals with low mean number of repeats than in individuals with high mean number of repeats (0.790 ± 0.184 g/cm2 vs. 0.843 ± 0.191 g/cm2; p < 0.05). This difference also was found in BMD of the total hip. Using multiple linear regression, mean number of TA repeats was a predictor of lumbar spine BMD (p < 0.05) and a BMD‐independent predictor of fractures (p < 0.05). Mean number of TA repeats was not associated with levels of biochemical markers of bone turnover. All four polymorphisms were in linkage disequilibrium. A TA repeat polymorphism in the ER gene is associated with increased risk of osteoporotic fractures and a modest reduction in bone mass. Polymorphisms in the first exon and first intron of the ER gene are not associated with osteoporotic fractures, bone mass, or bone turnover.

Osteoporotic Fractures Are Associated with an 86‐Base Pair Repeat Polymorphism in the Interleukin‐1‐Receptor Antagonist Gene But Not with Polymorphisms in the Interleukin‐1β Gene

Interleukin‐1β (IL‐1β) is a potent stimulator of bone resorption, and has been implicated in the pathogenesis of high bone turnover and osteoporosis. IL‐1 receptor antagonist (IL‐1ra) is a competitive inhibitor of IL‐1β effects and the biological effects of IL‐1β are therefore proportional to the ratio IL‐1β/IL‐1ra. The coding regions of IL‐1β were examined for sequence variations by SSCP and sequencing after polymerase chain reaction (PCR) of genomic DNA. Three previously described polymorphisms (C−511‐T, G3877‐A and C3954‐T) in the IL‐1β gene were determined by restriction fragment length polymorphism (RFLP) using Ava I, Aci I, and Taq I after PCR. The 86‐base pair repeat polymorphism in IL‐1ra was examined by PCR and electrophoresis and the T11100‐C polymorphism in the IL‐1ra gene was examined by RFLP using MspA1I after PCR. All polymorphisms were related to bone mass, biochemical markers of bone turnover, and presence of fracture in a study including 389 osteoporotic patients with vertebral fractures and normal controls. Two normal women were heterozygous for a shift from cytosine to thymine (C3263‐T) in exon 4 of the IL‐1β gene. This substitution did not affect the amino acid sequence. We did not find other sequence variations in the IL‐1β gene apart from the already known polymorphisms. The distribution of C−511‐T, G3877‐A, and C3954‐T genotypes was similar in the osteoporotic and the normal controls. No significant differences could be shown in bone mass or bone turnover. In the IL‐1ra gene almost complete linkage was confirmed between the already known polymorphisms: G1731‐A, G1821‐A, A1868‐G, G1887‐C, T8006‐C, C8061‐T, 86 base pair variable number tandem repeat (VNTR), A9589‐T, and a new polymorphism: T1934‐C. The A1A1/A3 genotypes of the IL‐1ra VNTR polymorphism were significantly more frequent in osteoporotic patients (56.2%) compared with age‐matched normal controls (433%) (χ2 = 4.09; p = 0.043). The relative risk of osteoporotic fractures was increased to 1.68 (95% CI, 1.01–2.77) in individuals with A1A1/A3 genotypes. Bone mineral density (BMD) of the lumbar spine was reduced in individuals with A1A1/A3 genotypes (p = 0.014, analysis of variance [ANOVA]). The difference in bone mass between A1A1/A3 and A2A1/A2 tended to increase with increasing age. T11100‐C genotypes were distributed similarly in osteoporotic patients and normal controls and the polymorphism was without effect on bone mass and biochemical markers of bone turnover. In conclusion, an 86‐base pair repeat polymorphism in the IL‐1ra gene is associated with increased risk of osteoporotic fractures. Other polymorphisms in the IL‐1ra and the IL‐1β genes are not associated with osteoporotic fractures or alterations in bone mass or bone turnover.

Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms.

Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction‐mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication. After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different mechanisms and suggest that these two pathways may serve different purposes in coordinating osteoblast functions.

Polymorphisms in the transforming growth factor beta 1 gene and osteoporosis.

Transforming growth factor (TGF)-beta1 is the most abundant growth factor in human bone. It is produced by osteoblasts and inhibits osteoclast proliferation and activity and stimulates proliferation and differentiation of preosteoblasts. Several polymorphisms have been described in the TGF-beta1 gene. Previously, we and others have found associations between some of these polymorphisms and bone mass. We therefore wanted to examine if these polymorphisms are also predictors of osteoporotic fractures. The polymorphisms G(-1639)-A, C(-1348)-T, C(-765)insC, T(29)-C, G(74)-C, 713-8delC, C(788)-T, and T(816-20)-C were examined using RFLP and sequencing in 296 osteoporotic patients with vertebral fractures and 330 normal individuals. Bone mineral density (BMD) was examined at the lumbar spine and at the femoral neck by DXA. Genotype distributions were in H-W equilibrium. Linkage disequilibrium was found between the polymorphisms. The T(816-20)-C genotypes were distributed differently among osteoporotic patients and normal controls. The TT genotype was less common in individuals with osteoporotic fractures (chi(2) = 6.02, P < 0.05). BMD was higher in individuals with the TT-genotype (T(816-20)-C) at the lumbar spine, 0.960 +/- 0.173 g/cm(2) compared with individuals with the TC or CC genotypes: 0.849 +/- 0.181 g/cm(2) and 0.876 +/- 0.179 g/cm(2), respectively (P < 0.001, ANOVA). Similar differences between genotypes were found at the different hip regions as well as at the total hip. Individuals with the TT-genotype (C(-1348)-T) had higher bone mass at the femoral neck: 0.743 +/- 0.134 g/cm(2) compared with 0.703 +/- 0.119 g/cm(2) in individuals with TC or CC genotypes (P < 0.05). Individuals with the CC-genotype (T(29)-C) had higher bone mass at the femoral neck, 0.735 +/- 0.128 g/cm(2) compared with 0.703 +/- 0.120 g/cm(2) in individuals with TC or TT genotypes (P < 0.05) and at the total hip: 0.852 +/- 0.166 g/cm(2) vs. 0.818 +/- 0.149 g/cm(2), respectively (P < 0.05). None of the other polymorphisms were distributed differently in patients and controls and did not affect BMD. In conclusion, The TT genotype of the T(816-20)-C polymorphism is less common in patients with osteoporotic fractures and is associated with higher bone mass both at the lumbar spine and at the hip. The C(-1348)-T and T(29)-C polymorphisms were distributed similarly in osteoporotic patients and normal controls, however, the rare genotypes were associated with higher bone mass at the hip.

Long-term oral pamidronate treatment inhibits osteoclastic bone resorption and bone turnover without affecting osteoblastic function in multiple myeloma

Abstract: This study was performed as a cross‐sectional substudy to the Danish–Swedish Pamidronate Study, a randomized placebo‐controlled multicentre trial in multiple myeloma. The purpose was to evaluate the biological effects of long‐term treatment with oral pamidronate 300 mg daily on bone metabolism by using histomorphometry and analysis of cytokines and biochemical markers of bone turnover. Sixteen patients were included after median 27.5 months of protocolized treatment; 10 patients received active treatment and 6 patients placebo. When compared with placebo, pamidronate treatment was associated with: (a) marked decreased osteoclastic resorption rate (0.86±0.59 μm/d vs. 5.7±5.0 μm/d, p=0.002), and diminished activation frequency (0.20±0.18 yr−1 vs. 0.72±0.55 yr−1, p=0.014); (b) compensatory reduced volume referent bone formation rate (0.17±0.21 yr−1 vs. 0.71±0.54 yr−1, p=0.007), but unaltered mineral appositional rate; (c) neutral (–0.66±5.6 mm) vs. negative (–2.15±2.2 μm, p=0.013) bone balance per remodelling cycle; (d) higher trabecular bone volume (21.0±6.2% vs. 13.0±3.7%, p=0.01); (e) suppressed urinary excretion and serum levels of some of the biochemical markers of bone metabolism; and (f) significant reduction of circulating soluble interleukin‐6 receptor (IL‐6sR) (25.9±4.1 ng/ml vs. 32.1±6.6 ng/ml, p=0.04), and (g) a uniform tendency of lower serum and marrow plasma levels of IL‐6, IL‐1β, and TNFα. Thus oral pamidronate was absorbed in biologically active amounts, and reduced overall bone resorption and bone turnover without impairing osteoblastic bone formation. The observation that cytokine and cytokine receptor levels were reduced extends the possible and potential beneficial actions of bisphosphonates in multiple myeloma.

Comparison of five biochemical markers of bone resorption in multiple myeloma: elevated pre‐treatment levels of S‐ICTP and U‐Ntx are predictive for early progression of the bone disease during standard chemotherapy

Summary. Increased osteoclastic bone resorption is the major causal factor of bone disease in multiple myeloma. Recently, non‐invasive methods have been developed for the estimation of bone resorptive activity. To evaluate the biological sensitivity and clinical usefulness of five biochemical assays for measuring the C‐terminal telopeptide of collagen I (ICTP) in serum (β‐Crosslaps ELISA and ICTP radioimmunoassay) and urinary creatinine‐adjusted excretions of pyridinoline (PYR), deoxypyridinoline (DPD) and N‐terminal telopeptide of collagen I (Ntx), we performed a study of 34 consecutive newly diagnosed myeloma patients. Serum and morning‐fasting, second‐void urine samples were taken before the start of treatment. In total, 40 age‐ and sex‐adjusted healthy individuals served as controls. Results were expressed as Z‐scores. All test variables were highly significantly elevated in the patients (Pu2003<u20030·001). Serum (S)‐ICTP was elevated (Z‐scoreu2003>u20032) in most patients (85%) and showed significantly higher Z‐score values than the other markers. S‐ICTP remained more sensitive than the urinary assays when patients with impaired renal function were excluded from analysis. S‐ICTP and the urinary metabolites correlated significantly with skeletal morbidity. S‐β‐Crosslaps correlated with the bone morbidity only when patients with renal insufficiency were excluded from the analysis. High levels of S‐ICTP and urinary (U)‐Ntx correlated with an increased risk for early progression of bone lesions during standard melphalan–prednisolone treatment. U‐Ntx and S‐ICTP are sensitive tools for estimating the increased bone resorption in multiple myeloma and are clinically useful for identifying patients with increased risk of early progression of bone disease.