Eriksen Ef

Chemotaxis of human osteoblasts

The in vitro chemotactic response of human osteoblasts was investigated towards the following growth factors: TGF‐β, PDGFs, FGFs and IGFs. Human osteoblasts grown from trabecular bone after enzymatic digestion were studied. TGF‐β stimulated the migration of human osteoblasts in a dose‐dependent manner with a four‐fold increase in migrated cells at 100 pg/ml, which was the optimum concentration. PDGF‐BB also stimulated migration four‐fold in a dose‐dependent manner with a maximum response at 10 ng/ml. PDGF‐AA, IGF‐I and IGF‐II stimulated migration two‐fold at 100 ng/ml. The results show that TGF‐P and PDGF‐BB are important regulators of human osteoblast migration, but other growth factors IGF‐I, IGF‐II and PDGF‐AA may also stimulate osteoblast migration. Our results additionally suggest that TGF‐P and PDGF‐BB may participate in the recruitment of osteoblasts during bone remodeling since both TGF‐P and PDGF‐BB are found in bone matrix and could be released during osteoclastic bone resorption. They furthermore support a possible use of TGF‐P and PDGF‐BB in growth factor‐induced osteogenesis.

Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol.

BACKGROUNDnTransforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-beta1 and calcitriol.nnnDESIGNnIn vitro study employing two models of normal human osteoblasts: human bone marrow stromal cells [hMS/(OB)] containing osteoprogenitor cells and trabecular bone osteoblasts (hOB), which are mature osteoblasts. A reverse-transcriptase-polymerase-chain-reaction assay was employed to measure steady state mRNA levels of TGF-beta(s) isoforms and receptors. Effects of short-term treatment of TGF-beta1 on osteoblast proliferation and differentiation markers were assessed. The effect of cotreatment of calcitriol (10-8 M) and TGF-beta1 on osteoblast differentiation was also determined.nnnRESULTSnBoth hMS(OB) and hOB cells expressed mRNA transcripts of TGF-beta1, TGF-beta2, TGF-beta 3, TGF-beta type I and type II receptors. TGF-beta 1 stimulated osteoblast proliferation in hMS(OB) and in hOB cultures. In hOB cultures, TGF-beta1 stimulated AP production and cotreatment with calcitriol induced a synergistic increase in AP levels to 250 +/- 61% of calcitriol-treated controls. Effects of TGF-beta1 and calcitriol were less pronounced in hMS(OB) cultures. TGF-beta1 inhibited collagen type I production in hMS(OB) cells and these effects were abolished in presence of calcitriol. In presence of calcitriol, TGF-beta1 increased collagen type I production in hOB cells. In both hOB and hMS(OB) cultures, TGF-beta1 inhibited osteocalcin production.nnnCONCLUSIONSnTGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol, it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production, other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo.

Telomere shortening during aging of human osteoblasts in vitro and leukocytes in vivo: lack of excessive telomere loss in osteoporotic patients

We have compared the telomere length, as assessed by Southern analysis, of telomere restriction fragments (TRFs) generated by RsaI/HinfI digestion of genomic DNA in: (i) in vitro cultured human trabecular osteoblasts undergoing cellular aging; and (ii) peripheral blood leukocytes (PBL) obtained from three groups of women: young (aged 20-26 years, n = 15), elderly (aged 48-85 years, n = 15) and osteoporotic (aged 52-81 years, n = 14). The mean TRF length in human osteoblasts undergoing aging in vitro decreased from an average of 9.32 kilobasepairs (kb) in middle-aged cells to an average of 7.80 kb in old cells. The rate of TRF shortening was about 100 bp per population doubling, which is similar to what has been reported for other cell types, such as human fibroblasts. Furthermore, there was a 30% decline in the total amount of telomeric DNA in senescent osteoblasts as compared with young cells. In the case of PBL, TRF length in the DNA extracted from young women was slightly longer (6.76 +/- 0.64 kb) than that from a group of elderly women (6.42 +/- 0.71 kb). A comparison of TRFs in the DNA extracted from the PBL from osteoporotic patients and from age-matched controls did not show any significant differences (6.47 +/- 0.94 versus 6.42 +/- 0.71 kb, respectively). Therefore, using TRF length as a marker for cellular aging in vitro and in vivo, our data comparing TRFs from osteoporotic patients and age-matched controls do not support the notion of the occurrence of a generalized premature cellular aging in osteoporotic patients.

Treatment with 1,25‐dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25‐dihydroxyvitamin D3 (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long‐term subculturing. In order to study in vitro age‐related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast‐specific genes in early‐passage (<u200950% lifespan completed) with those of late‐passage cells (>u200990% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late‐passage cells compared to early‐passage cells, suggesting an in vitro age‐related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age‐related changes. Furthermore, calcitriol (10−8M) treatment of early‐passage osteoblastic cells inhibited their proliferation by 57u2009±u20091% and stimulated steady state mRNA levels of AP (1.7u2009±u20090.1‐fold) and OC (1.8u2009±u20090.2‐fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7u2009±u20090.2‐fold) and OC (3.0u2009±u20090.3‐fold) in late‐passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age‐related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment. J. Cell. Physiol. 186:298–306, 2001.

Growth hormone, insulin-like growth factors and bone remodelling

Growth hormone (GH) plays a major role in postnatal longitudinal bone growth owing to its ability to stimulate precursor cells in the epiphyseal cartilage [1,2]. These effects are mediated both directly via GH receptors in the cell membrane and indirectly via increased synthesis of insulin-like growth factors (IGF-I and -II). Apart from the effects on skeletal size, these hormones also affect the mature skeleton via effects on bone remodelling in adults [3–5]. This review will limit its scope to the description of the effects of these growthpromoting peptides on adult bone remodelling.

Typing of Pasteurella multocida from haemorrhagic septicaemia in Danish fallow deer (Dama dama)

Isolates of Pasteurella multocida ssp. multocida (n = 31) from a Danish population of fallow deer which succumbed to haemorrhagic septicaemia during 1992–1993 and isolates from the palatine tonsils of apparently healthy fallow deer from the same area (n=6) were typed and compared with P. multocida from other sources. Plasmids were not observed in the fallow deer strains and one unique pattern was observed by ribotyping using HindIII and by pulsed‐field gel eletrophoresis using SalI as restriction endonuclease. All Danish fallow deer isolates belonged to serotype B:3,4. On restriction endonuclease analysis using HhaI as restriction endonuclease, all had a profile identical to that of a fallow deer isolate from the United Kingdom: profile 0033 of Wilson et al. On restriction endonuclease analysis using HpaII as restriction endonuclease, the Danish fallow deer isolates had a unique profile, designated 0062, which differed slightly from that of a fallow deer isolate from the United Kingdom. P. multocida from other animal species were genotypically different from the fallow deer isolates. It is concluded that a specific clone of P. multocida was responsible for the outbreak of haemorrhagic septicaemia among Danish fallow deer. A carrier rate of 27% was demonstrated among apparently normal animals from the same population.

Ultrastructure of human osteoblasts and associated matrix in culture

The ultrastructure, as visualized by transmission electron microscopy, of cells obtained from human bone explants and subsequently cultured is described along with the electron microscopic appearance of the associated intercellular matrix. The cells were characterized as osteoblasts on the basis of immunohistochemical, enzymatic, and functional criteria. Although the osteoblasts could be cultured in standard culture media and always appeared singly, not forming syncytia, the cultures were eventually confluent and formed multilayers. The cells were fusiform or cuboidal with diameters ranging between 10–15 μm. The cytoplasm was characterized by numerous large mitochondria, and especially by a very prominent RER. The intercellular matrix was woven with collagen fibres surrounding large numbers of matrix vesicles. In areas with matrix vesicles, evidence for osteoblast activity, i.e. mineralization related to matrix vesicles, could be observed after incubation with β‐glycerophosphate. In conclusion, we provide evidence that human osteoblasts cultured in vitro synthesize collagen and produce a matrix with vesicles capable of initiating mineralization processes.

Solid-phase radioimmunoassay of immunoglobulins G, A and M: applicability in analysis of sucrose gradients

A simple and sensitive solid-phase radioimmunoassay for the detection of immunoglobulins G, A and M in sucrose gradients is described. The solid-phase consisted of immunoglobulins adsorbed to polystyrene tubes. Using buffers without detergent and 125I-labeled sheep anti-rabbit IgG as radioligand, the assay was able to detect 0.8 ng per tube in the IgG assay and 1.6 ng per tube in the IgA and IgM assays. Standard curves with antigen dissolved in 10% and 32% sucrose were superimposable and did not deviate from standard curves with antigen dissolved in buffer without sucrose. Using these techniques on ultracentrifugation samples from patients with systemic lupus erythematosus, Schönlein-Henoch nephritis and IgA glorulonephritis it was possible to detect both immunoglobulin fragments and immunoglobulin aggregates at the same time without prior dialysis of the samples.

The Use of Bone Biopsies in the Diagnosis of Male Osteoporosis

Publisher Summary The incidence of fractures is higher in men than women from adolescence through mid life. This difference is ascribed to a difference in high energy fractures between the two genders. After 50 years, however, the incidence of fractures increases with aging in men as well as women, but the age-adjusted incidence of both hip and vertebral fractures in men is about half of that in women. Another important difference pertaining to osteoporosis pathogenesis between males and females is the higher prevalence of secondary osteoporosis in men. While around 20% of osteoporotic women exhibit other diseases causing bone loss and fracture, the prevalence of secondary osteoporosis in men has been cited to be 40–50% in most studies. This chapter aims to review how bone biopsies have helped us understand the differences in bone remodeling between males and females that are responsible for the different bone loss patterns in the two sexes; review the role of bone biopsies in the elucidation of possible secondary causes in male osteoporosis; and finally review possible new information on bone quality, which can be gained from studies of bone biopsies.