Bisphosphonate treatment of type I diabetic mice prevents early bone loss but accentuates suppression of bone formation.

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Previous studies demonstrate that T1-diabetes decreases osteoblast activity and viability. Bisphosphonate therapy, commonly used to treat osteoporosis, is demonstrated to inhibit osteoclast activity as well as osteoblast apoptosis. Therefore, we examined the effect of weekly alendronate treatments on T1-diabetes induced osteoblast apoptosis and bone loss. Bone TUNEL assays identified that alendronate therapy prevents the diabetes-induced osteoblast death observed during early stages of diabetes development. Consistent with this, alendronate treatment for 40 days was able to prevent diabetes-induced trabecular bone loss. Alendronate was also able to reduce marrow adiposity in both control diabetic mice compared to untreated mice. Mechanical testing indicated that 40 days of alendronate treatment increased bone stiffness but decreased the work required for fracture in T1-diabetic and alendronate treated mice. Of concern at this later time point, bone formation rate and osteoblast markers, which were already decreased in diabetic mice, were further suppressed in alendronate-treated diabetic mice. Taken together, our results suggest that short-term alendronate treatment can prevent T1-diabetes-induced bone loss in mice, possibly in part by inhibiting diabetes onset associated osteoblast death, while longer treatment enhanced bone density but at the cost of further suppressing bone formation in diabetic mice.

Surface microcracks signal osteoblasts to regulate alignment and bone formation.

Microcracks are present in bone and can result from fatigue damage due to repeated, cyclically applied stresses. From a mechanical point, microcracks can dissipate strain energy at the advancing tip of a crack to improve overall bone toughness. Physiologically, microcracks are thought to trigger bone remodeling. Here, we examine the effect of microcracks specifically on osteoblasts, which are bone-forming cells, by comparing cell responses on microcracked versus non-microcracked hydroxyapatite (HA) specimens. Osteoblast attachment was found to be greater on microcracked HA specimens (p<0.05). More importantly, we identified the preferential alignment of osteoblasts in the direction of the microcracks on HA. Cells also displayed a preferential attachment that was 75 to 90 µm away from the microcrack indent. After 21 days of culture, osteoblast maturation was notably enhanced on the HA with microcracks, as indicated by increased alkaline phosphatase activity and gene expression. Furthermore, examination of bone deposition by confocal laser scanning microscopy indicated preferential mineralization at microcrack indentation sites. Dissolution studies indicate that the microcracks increase calcium release, which could contribute to osteoblast responses. Our findings suggest that microcracks signal osteoblast attachment and bone formation/healing.

Interactions of aqueous NOM with nanoscale TiO2: implications for ceramic membrane filtration-ozonation hybrid process.

The combined effect of pH and calcium on the interactions of nonozonated and ozonated natural organic matter (NOM) with nanoscale TiO2 was investigated. The approach included characterization of TiO2 nanoparticles and NOM, extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) modeling of NOM-TiO2 and NOM-NOM interactions, batch study on the NOM adsorption onto TiO2 surface, and bench-scale study on the treatment of NOM-containing feed waters using a hybrid process that combines ozonation and ultrafiltration with a 5 kDa ceramic (TiO2 surface) membrane. It was demonstrated that depending on pH and TiO2 loading, the adsorption of NOM species is controlled by either the availability of divalent cations or by preozonation of NOM. XDLVO surface energy analysis predicts NOM adsorption onto TiO2 in the ozone-controlled regime but not in the calcium-controlled regime. In both regimes, short-range NOM-NOM and NOM-TiO2 interactions were governed by acid-base and van der Waals forces, whereas the role of electrostatic forces was relatively insignificant. Ozonation increased the surface energy of NOM, contributing to the hydrophilic repulsion component of the NOM-NOM and NOM-TiO2 interactions. In the calcium-controlled regime, neither NOM-TiO2 nor NOM-NOM interaction controlled adsorption. Non-XDLVO interactions such as intermolecular bridging by calcium were hypothesized to be responsible for the observed adsorption behavior. Adsorption data proved to be highly predictive of the permeate flux performance.

MC3T3-E1 osteoblast attachment and proliferation on porous hydroxyapatite scaffolds fabricated with nanophase powder.

Porous bone tissue engineering scaffolds were fabricated using both nano hydroxyapatite (nano HA) powder (20 nm average particle size) and micro HA powder (10 microm average particle size), resulting in sintered scaffolds of 59 vol% porosity and 8.6 +/- 1.9 microm average grain size and 72 vol% porosity and 588 +/- 55nm average grain size, respectively. Scanning electron microscopy was used to measure both the grain size and pore size. MC3T3-E1 osteoblast (OB) attachment and proliferation on both nano HA and micro HA porous scaffolds were quantified. As expected, OB cell number was greater on nano HA scaffolds compared with similarly processed micro HA scaffolds 5 days after seeding, while OB attachment did not appear greater on the nano HA scaffolds (p < 0.05).

Fabrication of catalytic membranes for the treatment of drinking water using combined ozonation and ultrafiltration.

The removal of disinfection byproducts and their precursors was investigated using a combined ozonation-ultrafiltration system. A commercial membrane was coated 20 or 40 times with iron oxide nanoparticles (4-6 nm in diameter). With this membrane, the concentration of dissolved organic carbon was reduced by >85% and the concentrations of simulated distribution system total trihalomethanes and simulated distribution system halo acetic acids decreased by up to 90% and 85%, respectively. When the coated membrane was used, the concentrations of aldehydes, ketones, and ketoacids in the permeate were reduced by >50% as compared to that obtained with the uncoated membranes. Hydroxyl or other radicals produced at the iron oxide coated membrane surface as a result of ozone decomposition are believed to have enhanced the degradation of the natural organic matter, thereby reducing the concentration of disinfection byproducts. While increasing the number of times the membrane was coated from 20 to 40 did not significantly reduce the concentrations of most of the parameters measured, it did result in a significant decrease in the concentrations of ozonation byproducts. Increasing the sintering temperature from 500 to 900 degrees C also resulted in an improvement in the removal of the ozonation byproducts.

Effects of ozonation on the permeate flux of nanocrystalline ceramic membranes.

Titania membranes, with a molecular weight cut-off of 15 kD were used in an ozonation/membrane system that was fed with water from Lake Lansing, which had been pre-filtered through a 0.45 microm glass fiber filter. The application of ozone gas prior to filtration resulted in significant decreases in membrane fouling. The effects of ozonation could not be explained by physical scouring of the filter cake. Decrease in the pH resulted in a concomitant increase in the dissolved ozone concentration in the feed water and in an improvement in permeate flux recovery. Increasing the ozone concentration beyond a threshold value had no beneficial effect on permeate flux recovery. Ozone decomposition, resulting in the formation of OH or other radicals at the membrane surface, is thought to result in the decomposition of organic foulants at the membrane surface and reduce the extent of membrane fouling.

Adsorption of serum fetuin to hydroxylapatite does not contribute to osteoblast phenotype modifications.

Osteoblasts exhibit enhanced differentiation and altered gene profiles when cultured on hydroxyapatite (HA) compared to plastic surfaces. To begin determining mechanisms for this response, we used proteomics to identify proteins predominantly found in osteoblasts on HA but not plastic surfaces. Two-dimensional gel electrophoresis and Western analyses indicate that fetuin is abundant in extracts from HA, but not plastic surfaces. Incubation of HA and plastic surfaces with cell culture medium (containing 10% serum) under cell-free conditions shows that fetuin is predominantly derived from the culture medium serum and readily adsorbs to the HA surface. However, we did detect low levels of fetuin B mRNA in osteoblasts. Serum albumin, actin-beta, apolipoprotein-AI, and vimentin also adsorbed to HA. To determine the role of fetuin in the HA-induced osteoblast phenotype changes, osteoblasts were seeded onto fetuin-coated or uncoated HA under serum-free conditions. Osteoblast morphology was similar on both HA surfaces, suggesting that HA alone (without adsorbed serum proteins) is sufficient for cell attachment and spreading. Similarly, genes previously reported to be modulated by HA (glvr-1, DMP-1, osteoglycin, and proliferin 3) were modulated even in the absence of fetuin or other serum proteins. These data show that HA surface can be enriched selectively with fetuin from serum; however, neither fetuin or other serum proteins are required to mediate HA-induced osteoblast attachment, spreading, or changes in expression of genes examined. This finding suggests that factors intrinsic to HA are required for the response.

Biaxial flexure testing of calcium phosphate bioceramics for use in tissue engineering.

This study analyzes data from 206 CaP specimens (68 HA, 70 BCP, and 68 beta-TCP) fractured via biaxial flexure testing. Specimens were divided into four groups: (a) Group I, dry; (b) Group II, wet (day 0, immersion time approximately 5-10 s); (c) Group III, after immersion in media for 21 days (day 21); and (d) Group IV, after culturing osteoblasts (OBs) on the surface for 21 days (day 21 with cells). X-ray diffraction verified the presence of minor second phases in HA and beta-TCP while BCP was a biphasic mixture of HA and beta-TCP with minor phases present. The statistical significance (p < 0.05) of differences in the measured biaxial flexure fracture strength, S, between groups was assessed via one-way ANOVA with Tukey's test. Also, a two-parameter Weibull analysis assessed the mechanical reliability of each group. Osteoblasts increase the biaxial flexure fracture strength in a statistically significant way compared to both the HA discs in Groups II and III. Scanning electron microscope examination revealed grain boundary grooving on the sintered surfaces and with thermal expansion anisotropy, likely leads to the observed rapid strength decline upon exposure to media found in Groups II, III and IV.

Osteoblasts respond to hydroxyapatite surfaces with immediate changes in gene expression.

Bone mineral contains hydroxyapatite (HA). This is the surface that mature osteoblasts and osteocytes interact with. Synthetic HA is widely used in orthopedic surgeries as an implant or implant coating. The bone-like HA surfaces increase implant union and bone formation; however, the mechanisms accounting for this effect on osteoblasts are not known. In this study, we compared gene expression profiles of osteoblasts responding to HA or plastic surfaces for 24 h. Expression profiles were also compared between HA discs processed with gravity-sieved compared with combined gravity and air-jet-sieved HA powders. The latter, composed of smaller HA particles, exhibits an increase in grain boundary surface area. Discs made with either HA powder similarly up-regulated osteoblast expression of 10 genes (including proliferin 3, Glvr-1, DMP-1, and tenascin C) and down-regulated 15 genes (such as osteoglycin) by more than 2-fold compared with plastic surfaces. The overall changes are indicative of an immediate (24-h) response to the HA surface and a trend toward osteoblast differentiation. In addition, subsets of modulated genes exist that are unique to each HA subtype. Taken together, we identified HA responsive genes evident within 24 h of surface contact, indicating a critical role for extracellular mineral surfaces in the regulation of osteoblast gene expression and phenotype.