Osteoblast Density Research Articles

Age-Related Changes in Bone Morphology Are Accelerated in Group VIA Phospholipase A 2 (iPLA 2β)-Null Mice

Phospholipases A(2) (PLA(2)) hydrolyze the sn-2 fatty acid substituent, such as arachidonic acid, from phospholipids, and arachidonate metabolites are recognized mediators of bone modeling. We have previously generated knockout (KO) mice lacking the group VIA PLA(2) (iPLA(2)beta), which participates in a variety of signaling events; iPLA(2)beta mRNA is expressed in bones of wild-type (WT) but not KO mice. Cortical bone size, trabecular bone volume, bone mineralizing surfaces, and bone strength are similar in WT and KO mice at 3 months and decline with age in both groups, but the decreases are more pronounced in KO mice. The lower bone mass phenotype observed in KO mice is not associated with an increase in osteoclast abundance/activity or a decrease in osteoblast density, but is accompanied by an increase in bone marrow fat. Relative to WT mice, undifferentiated bone marrow stromal cells (BMSCs) from KO mice express higher levels of PPAR-gamma and lower levels of Runx2 mRNA, and this correlates with increased adipogenesis and decreased osteogenesis in BMSCs from these mice. In summary, our studies indicate that age-related losses in bone mass and strength are accelerated in iPLA(2)beta-null mice. Because adipocytes and osteoblasts share a common mesenchymal stem cell origin, our findings suggest that absence of iPLA(2)beta causes abnormalities in osteoblast function and BMSC differentiation and identify a previously unrecognized role of iPLA(2)beta in bone formation.

Ultrathin gradient films using thiol‐ene polymerizations

AbstractThe application of surface‐attached, thiol‐ene polymer films for controlling material properties in a gradient fashion across a surface was investigated. Thiol‐ene films were attached to the surface by first depositing a thiol‐terminated self‐assembled monolayer and performing a thiol‐ene photopolymerization reaction on the surface. Property gradients were created either by creating and modifying a gradient in the surface thiol density in the SAM or by changing the polymerization conditions or both. Film thickness was modified across the substrate by changing either the density of the anchoring thiol functional groups or by changing the reaction conditions such as exposure time. Thicker films (1–11 nm) were obtained by polymerizing acrylate polymer brushes from the surface with varying exposure time (0–60 s). The two factors, that is, the surface thiol density and the exposure time, were combined in orthogonal directions to obtain thiol‐ene films with a two‐dimensional thickness gradient with the maximum thickness being 4 nm. Finally, a thiol‐acrylate Michael type addition reaction was used to modify the surface thiol density gradient with the cell‐adhesive ligand, Arg‐Gly‐Asp‐Ser (RGDS), which subsequently yielded a gradient in osteoblast density on the surface. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 7027–7039, 2006

Porous hydroxyapatite–tricalcium phosphate bioceramics

Porous bioceramics hydroxyapatite–tricalcium phosphate (HA–TCP), aimed to be applied in clinic, was fabricated by powder metallurgy and evaluated using both in vitro and in vivo models. Porous HA–TCP was supposed as a partially biodegradable material and designed as a scaffold for bone reconstruction or regeneration. The material processing was proposed and the physical properties as well as the microstructure feature were characterised here. Biological postulation of the relationship between seeding density, proliferation and viability of human osteoblasts cultured on the porous HA–TCP was quantitatively measured. Bone reconstruction was investigated both in vitro and in vivo by use of these biodegradable scaffolds with pore sizes ranged from 200 to 400 μm in diameter. The degradable bioceramic supported cellular proliferation seeded on the scaffold and showed normal differentiated function in vitro. Suitable pore size of the porous bioceramic was required if promotion of bone reconstruction was desired. Clinical trials showed that the bioceramics were successfully applied for bone reconstruction and regeneration and could be partially degraded in human body in 18 months. This approach suggests the feasibility of using porous HA–TCP bioceramics for the transplantation of autogenous osteoblasts to regenerate bone tissue.

Better osteoblast adhesion on nanoparticulate selenium— A promising orthopedic implant material

Apart from problems such as poor osseointegration, stress shielding, and wear debris-associated bone cell death, a major concern of metallic orthopedic implants is that they slowly corrode under in vivo environments. It is possible that continuous tissue exposure to metallic corrosion products limits orthopedic implant efficacy; this is especially true for patients receiving implants due to bone cancer. To date, there is no metallic orthopedic implant available in the market that specifically deals with the prevention and/or recurring cancer that may happen in these patients. The objective of this study was to deal with these problems in an integrated way by introducing a new biomaterial to the orthopedic community with anticancer chemistry: selenium (Se). In this study, six types of Se compacts were tested for bone cell (osteoblast) adhesion under in vitro conditions. Two types of cylindrical compacts were made with conventional Se metal particles in the micron (6.539 +/- 1.364-microm diameter) and submicron (0.963 +/- 0.139-microm diameter) range. These two types of compacts were chemically etched with different concentrations of NaOH to create two additional types of Se particles in each category: conventional size particles with nanosurface roughness and nanometer particles (0.204- to 0.264-microm diameter). Results showed for the first time, enhanced osteoblast adhesion on particulate surfaces of the compacts made from conventional Se compared with reference nonparticulate wrought titanium sheets. More importantly, this study provided the first evidence that osteoblast density was further increased on the surfaces of the Se compacts with nanometer particles. These initial findings indicate that there may be a promising future for nanoparticulate Se as an anticancer biocompatible orthopedic material.

Morphological, stereological and radiological changes in pinealectomized chicken cervical vertebrae

Considerable evidence supports the hypothesis that melatonin plays an important role in osteogenesis. We carried out an experiment to investigate histological and radiological changes in the cervical vertebrae of pinealectomized chickens. Thirty new-hatched chicks were divided into two groups: pinealectomized group (n=15) and nonpinealectomized control (n=15). Surgical pinealectomies were performed in Hybro broiler chickens at the age of 3 days. At 8 wk, one animal from each group was examined using computed tomography scanner to obtain density histograms of four consecutive vertebrae between the 4th and 7th cervical vertebral segments of chickens. At the end of the study, all animals were killed for subsequent measurement of bone mineral density (BMD) and macroscopic, radiographic and histopathological evaluation of specimens. We found that the profile of the density histogram in nonpinealectomized control group showed a spike profile for the vertebral body, indicating an increase in the amount of higher density tissues in this region, while a plateau-shaped profile was obtained for the vertebral body in pinealectomy group, indicating the presence of heterogenous bone tissue. Accordingly, the mean value of BMD in pinealectomy group was significantly lower at the vertebral body in chickens compared with control subjects (P<0.001). At the end of the experiment, almost all of the chickens in pinealectomy group developed a scoliotic curvature and the mean weight and length of the cervical vertebral bodies of the pinealectomized chickens were significantly lower than those of control group (P<0.001). Although the numerical density of osteocytes and osteoblasts in pinealectomy group was significantly higher than that from the control group, total number of osteocytes but not osteoblasts in cervical vetrebrae from pinealectomized animals was significantly lower than that from nonpinealectomized control animals (P<0.05). In conclusion, the results of the current study demonstrated for the first time pinealectomy-induced histomorphometrical changes in chicken vertebral column using stereological methods, suggesting that pineal gland/melatonin may have an osteoinductive effect on bone formation, but further studies are needed to elucidate the relationship of our findings with some disorders such as postmenopausal and/or senile osteoporosis.

Increased viable osteoblast density in the presence of nanophase compared to conventional alumina and titania particles

In the present in vitro study, osteoblast (bone-forming cells) viability and cell density were investigated when cultured in the presence of nanophase compared to conventional (i.e. micron) alumina and titania particles at various concentrations (from 10,000 to 100 μg/ml of cell culture media) for up to 6 h. Results confirmed previous studies of the detrimental influences of all ceramic particulates on osteoblast viability and cell densities. For the first time, however, results provided evidence of increased apoptotic cell death when cultured in the presence of conventional compared to nanophase alumina and titania particles. Moreover, since material characterization studies revealed that the only difference between respective ceramic particles was nanometer- and conventional-dimensions (specifically, phase and chemical properties were similar between respective nanophase and conventional alumina as well as titania particles), these results indicated that osteoblast viability and densities were influenced solely by particle size. Such nanometer particulate wear debris may result from friction between articulating components of orthopedic implants composed of novel nanophase ceramic materials. Results of a less detrimental effect of nanometer—as compared to conventional-dimensioned particles on the functions of osteoblasts provide additional evidence that nanophase ceramics may become the next generation of bone prosthetic materials with increased efficacy and, thus, deserve further testing.

OECD report outlines water management improvements

Bone transport (BT) for segmentary bone defects is a well-known technique as it enables correction with new bone formation, which is similar to the previous bone. Despite the high number of experimental studies of distraction osteogenesis in bone lengthening, the types of ossification and histological changes that occur in the regenerate of the bone transport process remain controversial.The aim of this study is to provide the complete evolution of tissues and the types of ossification in the regenerate during the different phases of bone formation after BT until the end of the remodelling period.A histological study was performed using ten adult sheep that were submitted to BT. The types of ossification as well as the evolution of different tissues in the regenerate were determined using histomorphometry and inmunohistochemical studies. The evolution of trabeculae thickness, osteoblast and osteoclast densities, relationship between collagen types and changes in vascularization were also studied.Ossification was primarily intramembranous, with some focus of endochondral ossification in isolated animals. The cell counts showed a progression of cellular activity from the periphery to the centre, presenting the same progression as the growth of bone trabeculae, whose trabeculae thickness was quadrupled at the end of remodelling. Inmunohistochemical studies confirmed the prevalence of type I collagen and the ratio of the Type I/Type II collagen ratio was found to be 2.48. The percentages of the vascularized areas were proximally higher than distally in all animals, but distal zone obtained higher rates than the central region.Bone transport regenerate exhibits a centripetal ossification model and a mixed pattern with predominance of intramembranous over endochondral ossification. The data obtained resemble partially to those found in models of bone lengthening applied to large animals. This study provides a detailed structural characterization of the newly formed tissue, which may help to explain the development of the regenerate of bone transport in humans. It will also serve for future mechanobiological models that may aid research on the effect of loading or distractor stiffness in clinical results.

Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering

Poly(ethylene glycol) (PEG) hydrogels were investigated as encapsulation matrices for osteoblasts to assess their applicability in promoting bone tissue engineering. Non-adhesive hydrogels were modified with adhesive Arg-Gly-Asp (RGD) peptide sequences to facilitate the adhesion, spreading, and, consequently, cytoskeletal organization of rat calvarial osteoblasts. When attached to hydrogel surfaces, the density and area of osteoblasts attached were dramatically different between modified and unmodified hydrogels. A concentration dependence of RGD groups was observed, with increased osteoblast attachment and spreading with higher RGD concentrations, and cytoskeleton organization was seen with only the highest peptide density. A majority of the osteoblasts survived the photoencapsulation process when gels were formed with 10% macromer, but a decrease in osteoblast viability of ∼25% and 38% was seen after 1 day of in vitro culture when the macromer concentration was increased to 20 and 30 wt%, respectively. There was no statistical difference in cell viability when peptides were added to the network. Finally, mineral deposits were seen in all hydrogels after 4 weeks of in vitro culture, but a significant increase in mineralization was observed upon introduction of adhesive peptides throughout the network.

Expression and activity of NAD(P)H:quinone oxidoreductase (NMO1) in human osteoblastic cells

NAD(P)H:quinone oxidoreductase (NMO1; EC 1.6.99.2), also called DT-diaphorase, is involved in the reduction of coenzyme Q, an important cellular lipophilic antioxidant that can function as an intermediate electron carrier in plasma membrane-associated electron transport regulating cell growth. We examined the ability of normal human trabecular osteoblastic cells to express NAD(P)H:quinone oxidoreductase (NMO1) and studied its modulation during cell proliferation and growth arrest. We found that confluent primary human trabecular osteoblastic cells derived from healthy individuals constitutively express NMO1 activity, measured using 2-6 dichlorophenol indophenol (DCI) or menadione (vitamin K3) as substrate, and NADPH or NADH as electron donor. We also found that NMO1 activity was related to osteoblastic cell growth. NMO1 activity increased with osteoblastic cell density. Confluent growth-arrested cells expressed an eightfold higher NMO1 specific activity than proliferative human osteoblastic cells. Reverse-transcription polymerase chain reaction analysis showed that NMO1 mRNA levels did not differ in growth-arrested confluent cell and growing cells, suggesting that the increased NMO1 activity with cell density was due to posttranslational events. Harvesting and replating the cells at low density resulted in a 93.4% loss of NMO1 enzymatic activity. Removal of serum from high-density growth-arrested cells resulted in a 48.5% decrease in NMO1 activity. NMO1 activity does not appear to be related to induction of osteoblast differentiation because treatment with the differentiating agent 1,25(OH) 2 vitamin D 3 had no effect on NMO1 activity. The finding that human osteoblastic cells express NMO1 constitutively and that NMO1 activity increases with density-dependent growth inhibition suggest a role for NAD(P)H:quinone oxidoreductase in the control of growth arrest in normal human osteoblastic cells.

The role of osteoblast density and endogenous interleukin-6 production in osteoclast formation from the hemopoietic stem cell line FDCP-MIX C2GM in coculture with primary osteoblasts.

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C2GM was shown to be strongly dependent on osteoblast density. In cocultures of C2GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 x 10(4) cells/cm2), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 x 10(4) cells/cm2). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear.

Etidronate inhibits the thyroid hormone-induced bone loss in rats assessed by bone mineral density and messenger ribonucleic acid markers of osteoblast and osteoclast function

TSH-suppressive doses of thyroid hormone are associated with bone loss. We have previously reported that L-T4 decreases femoral, but not vertebral bone mineral density (BMD) in rats. As bisphosphonates are able to decrease bone resorption, especially in high bone turnover states, we investigated the potential effects of etidronate disodium (EHDP) on L-T4-induced bone loss in the rat model by assessing BMD and gene expression of osteoblast (osteocalcin, osteopontin, type I collagen, and alkaline phosphatase), osteoclast (tartrate-resistant acid phosphatase), and cell growth (histone) markers in the skeleton. L-T4 administered for 20 days decreased BMD in the femur, but had no effect on the lumbar spine. EHDP alone had no effect on femoral or vertebral BMD, but did prevent the L-T4-induced bone loss in the femur. L-T4 increased mRNA levels of alkaline phosphatase, tartrate-resistant acid phosphatase, and histone H4 in the femur, but not in the vertebrae. EHDP, which alone had no effect on gene expression in the femur or vertebrae, inhibited the effect of L-T4 on mRNA markers in the femur. The results demonstrate that EHDP can prevent the L-T4-induced decrease in femoral BMD in rats that is associated with the prevention of changes in mRNA markers of osteoclast and osteoblast function. EHDP and other bisphosphonate compounds may be useful in the prevention of thyroid hormone-induced bone loss in humans.

Etidronate inhibits the thyroid hormone-induced bone loss in rats assessed by bone mineral density and messenger ribonucleic acid markers of osteoblast and osteoclast function.

TSH-suppressive doses of thyroid hormone are associated with bone loss. We have previously reported that L-T4 decreases femoral, but not vertebral bone mineral density (BMD) in rats. As bisphosphonates are able to decrease bone resorption, especially in high bone turnover states, we investigated the potential effects of etidronate disodium (EHDP) on L-T4-induced bone loss in the rat model by assessing BMD and gene expression of osteoblast (osteocalcin, osteopontin, type I collagen, and alkaline phosphatase), osteoclast (tartrate-resistant acid phosphatase), and cell growth (histone) markers in the skeleton. L-T4 administered for 20 days decreased BMD in the femur, but had no effect on the lumbar spine. EHDP alone had no effect on femoral or vertebral BMD, but did prevent the L-T4-induced bone loss in the femur. L-T4 increased mRNA levels of alkaline phosphatase, tartrate-resistant acid phosphatase, and histone H4 in the femur, but not in the vertebrae. EHDP, which alone had no effect on gene expression in the femur or vertebrae, inhibited the effect of L-T4 on mRNA markers in the femur. The results demonstrate that EHDP can prevent the L-T4-induced decrease in femoral BMD in rats that is associated with the prevention of changes in mRNA markers of osteoclast and osteoblast function. EHDP and other bisphosphonate compounds may be useful in the prevention of thyroid hormone-induced bone loss in humans.

Histomorphometric study on osteocyte-osteoblasts relationships

The formation of new bone involves both the deposition of bone matrix, and the formation of a network of cells embedded within the bone matrix, called osteocytes. Osteocytes derive from bone-synthesising cells (osteoblasts) that become buried in bone matrix during bone deposition. The generation of osteocytes is a complex process that remains incompletely understood. Whilst osteoblast burial determines the density of osteocytes, the expanding network of osteocytes regulates in turn osteoblast activity and osteoblast burial. In this paper, a spatiotemporal continuous model is proposed to investigate the osteoblast-to-osteocyte transition. The aims of the model are (i) to link dynamic properties of osteocyte generation with properties of the osteocyte network imprinted in bone, and (ii) to investigate Marotti׳s hypothesis that osteocytes prompt the burial of osteoblasts when they become covered with sufficient bone matrix. Osteocyte density is assumed in the model to be generated at the moving bone surface by a combination of osteoblast density, matrix secretory rate, rate of entrapment, and curvature of the bone substrate, but is found to be determined solely by the ratio of the instantaneous burial rate and matrix secretory rate. Osteocyte density does not explicitly depend on osteoblast density nor curvature. Osteocyte apoptosis is also included to distinguish between the density of osteocyte lacuna and the density of live osteocytes. Experimental measurements of osteocyte lacuna densities are used to estimate the rate of burial of osteoblasts in bone matrix. These results suggest that: (i) burial rate decreases during osteonal infilling, and (ii) the control of osteoblast burial by osteocytes is likely to emanate as a collective signal from a large group of osteocytes, rather than from the osteocytes closest to the bone deposition front.

Effects of donor age on osteogenic cells of rat bone marrow in vitro

The effect of donor age on the production of bone-like tissue and expression of cellular alkaline phosphatase was examined in cultures of cells obtained from rat bone marrow. Stromal cells were obtained from the bone marrow of young (5–6 weeks) and old (18 months) rats and cultured in vitro. After 28 days in first subculture, the following were quantitied: (1) the total number of mineralised nodules and the size distribution of nodules and (2) the density of osteoblasts and osteocytes associated with nodules in histological sections. The doubling times of the cultures and the numbers of cells in cultures which expressed alkaline phosphatase activity were determiend in seperate experiments. Cells from young cultures produced three times more bone-like nodules than old cultures, although no differences were seen in the size distribution of nodules, or on osteoblast and osteocyte density. Doubling times for both groups were similar. The numbers of alkaline phosphatase (AP) positive cells was reduced by half in old cultures. These data show that this model may be useful for the study of the mechanisms of ageing on osteogenesis, and demonstrate a reduced osteogenic capacity in old cultures. The results suggest that this effect may be due to a reduction in the generation of cells of osteoblast lineage during ageing.

Multiple immunoreactive forms of osteocalcin in uremic serum.

Circulating osteocalcin, which normally reflects the rate of bone formation, is elevated in uremia. In 18 patients receiving maintenance hemodialysis, serum osteocalcin levels were directly related to the bone formation rate (r = 0.88, P less than 0.001), osteoblastic osteoid surface density (r = 0.65, P less than 0.01), and osteoclastic resorptive surface density (r = 0.75, P less than 0.001). Multiple regression analysis showed that osteocalcin levels remained positively correlated with osteoclastic resorption when the bone formation rate was held constant (P less than 0.01). The intimation that the coupling of bone formation and resorption could not explain the relationship between osteocalcin and resorption led us to determine whether fragments of this abundant matrix protein are released by bone resorption and retained in uremia. Sera from dialysis patients with renal osteodystrophy were fractionated by sequential gel filtration and HPLC, and assayed for immunoreactive osteocalcin. When normal serum was analyzed, a single sharp peak was found. In pooled sera from patients with high osteoclastic resorptive surfaces identified by histomorphometry, we found five additional immunoreactive peaks, while three additional peaks were detected in sera from patients with lower osteoclastic surfaces. Bio-Gel P-10 chromatography showed that these multiple peaks were of lower molecular weight than intact osteocalcin. We suggest that the liberation of bone matrix by osteoclasts contributes to the circulating osteocalcin immunoreactivity in uremia.