Weibull modulus and fracture strength of highly porous hydroxyapatite.

Porous hydroxyapatite (HA) is used in a variety of applications including biomedical materials such as engineered bone materials and microbe filters. Despite the utility of the Weibull modulus, m, as a gauge of the mechanical reliability of brittle solids, there have been very few studies of m for porous HA. A recent study of porous HA that included the current authors (Fan, X., Case, E.D., Ren, F., Shu, Y., Baumann, M.J., 2012a. Journal of the Mechanical Behavior of Biomedical Materials. 8, 21-36) showed increases in m for porosity, P, approaching PG, the porosity of the green (unfired) specimen. In this paper, 18 groups of highly porous HA specimens (12 groups fabricated in this study and 6 groups from Fan et al., 2012a) were analyzed with P values from 0.59 to 0.62, where PG=0.62. The partially sintered HA specimens were fractured in biaxial flexure using a ring-on-ring test fixture. The fracture strength decreased monotonically with decreasing sintering temperature, Tsinter, from 4.8MPa for specimens sintered at 1025°C-0.66MPa for specimens sintered at 350°C. However, the Weibull modulus remained surprisingly high, ranging from 6.6 to 15.5. In comparison, for HA specimens with intermediate values of P, from about 0.1-0.55, the Weibull modulus tended to be lower (ranging from about 4 to 11) than the highly porous specimens included in this study.

Part I: porosity dependence of the Weibull modulus for hydroxyapatite and other brittle materials.

Porous brittle materials are used as filters, catalyst supports, solid oxide fuel cells and biomedical materials. However the literature on the Weibull modulus, m, versus volume fraction porosity, P, is extremely limited despite the importance of m as a gauge of mechanical reliability. In Part I of this study, m is determined for 441 sintered hydroxyapatite (HA) specimens fractured in biaxial flexure for 0.08 ≤ P ≤ 0.62. In this study, we analyze a combined data set collected from the literature that represents work from a total of 17 different research groups (including the present authors), eight different materials and more than 1560 oxide and non-oxide specimens, the m versus P plot is "U-shaped" with a wide band of m values for P<0.1 (Region I) and P>0.55 (Region III), and a narrower band of m values in the intermediate porosity region of 0.1, and the Young's modulus E for the HA specimens tested in Part I along with literature data for other brittle materials. Both <σ(f)> and E are power law functions of the degree of densification, ϕ, where ϕ=1-P/P(G) and P(G) is the green (unfired) porosity.

Part II: fracture strength and elastic modulus as a function of porosity for hydroxyapatite and other brittle materials.

Part I of this paper discussed the Weibull modulus m, versus porosity P behavior of brittle materials, including HA. While the Weibull modulus m deals with the scatter in fracture strength data, this paper (Part II) focuses on two additional key mechanical properties of porous materials, namely the average fracture strength <σ(f)>, and Young's modulus E, for P in the interval from P≈ zero to P≈P(G) (the porosity of the unfired compacts). The <σ(f)> versus P data for HA from this study and the literature data for alumina, yttria stabilized zirconia (YSZ) and silicon nitride are described well by functions of ϕ, where ϕ=1-P/P(G)= the degree of densification. A similar function of ϕ applies to the versus P behavior of HA from this study and data from the literature for alumina, titanium and YSZ. All of the data analyzed in this study (Part II) are based on partially and fully sintered powder compacts (excluding green powder compacts), thus the <σ(f)>/σ(0) versus ϕ and /E(0) versus ϕ relationships may apply only to such specimens.

Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein.

BACKGROUND: Aggregation of the high-affinity IgE receptor (FcεRI) with the low-affinity IgG receptor (FcγRIIb) on basophils or mast cells has been shown to inhibit allergen-induced cell degranulation. Molecules cross-linking these two receptors might therefore be of interest for the treatment of allergic disorders. Here, we demonstrate the generation of a novel bispecific fusion protein efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of basophils to prevent pro-inflammatory mediator release. METHODS: Alternative binding molecules recognizing receptor-bound human IgE were selected from DARPin (designed ankyrin repeat protein) libraries. One of the selected DARPins was linked to the Fc-part of a human IgG(1) antibody for binding to FcγRIIb. RESULTS: The resulting anti-IgE DARPin-Fc fusion protein was not anaphylactogenic and inhibited allergen-induced basophil activation in whole blood assays. Both binding moieties of the fusion protein, namely the anti-IgE DARPin as well as the IgG(1) Fc-part, were required to achieve this inhibitory effect. Most importantly, inhibition was faster and more efficient than with Omalizumab, a humanized anti-IgE antibody currently used for the treatment of severe asthma. CONCLUSION: This novel anti-IgE DARPin-Fc fusion protein might represent a potential drug candidate for preventive or immediate treatment of allergic reactions.

Mn oxide coated catalytic membranes for a hybrid ozonation-membrane filtration: comparison of Ti, Fe and Mn oxide coated membranes for water quality.

In this study the performance of catalytic membranes in a hybrid ozonation-ceramic membrane filtration system was investigated. The catalytic membranes were produced by coating commercial ceramic ultrafiltration membranes with manganese or iron oxide nanoparticles using a layer-by-layer self-assembly technique. A commercial membrane with a titanium oxide filtration layer was also evaluated. The performance of the coated and uncoated membranes was evaluated using water from a borderline eutrophic lake. The permeate flux and removal of the organic matter was found to depend on the type of the metal oxide present on the membrane surface. The performance of the manganese oxide coated membrane was superior to that of the other membranes tested, showing the fastest recovery in permeate flux when ozone was applied and the greatest reduction in the total organic carbon (TOC) in the permeate. The removal of trihalomethanes (THMs) and haloacetic acids (HAAs) precursors using the membrane coated 20 times with manganese oxide nanoparticles was significantly better than that for the membranes coated with 30 or 40 times with manganese oxide nanoparticles or 40 times with iron oxide nanoparticles.

Biocompatibility and mechanical properties of diamond-like coatings on cobalt-chromium-molybdenum steel and titanium-aluminum-vanadium biomedical alloys.

Diamond-like carbon (DLC) films are favored for wear components because of diamond-like hardness, low friction, low wear, and high corrosion resistance (Schultz et al., Mat-wiss u Werkstofftech 2004;35:924-928; Lappalainen et al., J Biomed Mater Res B Appl Biomater 2003;66B:410-413; Tiainen, Diam Relat Mater 2001;10:153-160). Several studies have demonstrated their inertness, nontoxicity, and the biocompatibility, which has led to interest among manufacturers of surgical implants (Allen et al., J Biomed Mater Res B Appl Biomater 2001;58:319-328; Uzumaki et al., Diam Relat Mater 2006;15:982-988; Hauert, Diam Relat Mater 2003;12:583-589; Grill, Diam Relat Mater 2003;12:166-170). In this study, hydrogen-free amorphous, tetrahedrally bonded DLC films (ta-C) were deposited at low temperatures by physical vapor deposition on medical grade Co28Cr6Mo steel and the titanium alloy Ti6Al4V (Scheibe et al., Surf Coat Tech 1996;85:209-214). The mechanical performance of the ta-C was characterized by measuring its surface roughness, contact angle, adhesion, and wear behavior, whereas the biocompatibility was assessed by osteoblast (OB) attachment and cell viability via Live/Dead assay. There was no statistical difference found in the wettability as measured by contact angle measurements for the ta-C coated and the uncoated samples of either Co28Cr6Mo or Ti6Al4V. Rockwell C indentation and dynamic scratch testing on 2-10 µm thick ta-C films on Co28Cr6Mo substrates showed excellent adhesion with HF1 grade and up to 48 N for the critical load L(C2) during scratch testing. The ta-C coating reduced the wear from 3.5 × 10(-5) mm(3)/Nm for an uncoated control sample (uncoated Co28Cr6Mo against uncoated stainless steel) to 1.1 × 10(-7) mm(3)/Nm (coated Co28Cr6Mo against uncoated stainless steel) in reciprocating pin-on-disk testing. The lowest wear factor of 3.9 × 10(-10) mm(3)/Nm was measured using a ta-C coated steel ball running against a ta-C coated and polished Co28Cr6Mo disk. Student's t-test found that the ta-C coating had no statistically significant (p < 0.05) effect on OB attachment, when compared with the uncoated control samples. There was no significant difference (p < 0.05) in the Live/Dead assay results in cell death between the ta-C coated Co28Cr6Mo and Ti6Al4V samples and the uncoated controls. Therefore, these ta-C coatings show improved wear and corrosion (Dorner-Reisel et al., Diam Relat Mater 2003;11:823-827; Affato et al., J Biomed Mater Res B Appl Biomater 2000;53:221-226; Dorner-Reisel et al., Surf Coat Tech 2004;177-178:830-837; Kim et al., Diam Relat Mater 2004;14:35-41) performance and excellent in vitro cyto-compatibility, when compared with currently used uncoated Co28Cr6Mo and Ti6Al4V implant materials.

Confocal laser scanning microscopy as a tool for imaging cancellous bone.

Understanding the bimodal structure of cancellous bone is important for tissue engineering in order to more accurately fabricate scaffolds to promote bone ingrowth and vascularization in newly forming bone. In this study, confocal laser scanning microscopy (CLSM) was used to create detailed images of the bimodally porous intertrabecular space of defatted and deproteinized cancellous canine bone taken from the epiphysis of the humerus. The bimodal pore structure was imaged using both reflective and fluorescent modes in CLSM, resulting in four different, but complementary image types: (1) a Z-stack overlay, (2) a phi-Z scan, (3) a topographical map, and (4) a contour map. Submerging the bone in rhodamine B dye prior to fluorescent imaging enhanced the pore surface details, giving a more accurate pore size measurement. The average macropore diameter was found to be 260 +/- 97 microm while the average micropore diameter was 13 +/- 10 microm. When compared with common techniques, including microcomputed tomography, magnetic resonance imaging, scanning electron microscopy, and environmental scanning electron microscopy, for imaging cancellous bone, CLSM was found to be an effective tool, given its ability to nondestructively image the surface and near-surface pore structure.

The effects of combined ozonation and filtration on disinfection by-product formation.

The effects of combined ozonation and membrane filtration on the removal of the natural organic matter (NOM) and the formation of disinfection by-products (DBPs) were investigated. Ozonation/filtration resulted in a reduction of up to 50% in the dissolved organic carbon (DOC) concentration. Furthermore, humic substances were converted to non-humic substances, with changes in the humic and non-humic substance concentrations of up to -50% and +20%, respectively. Ozonation/filtration resulted in the formation of partially oxidized compounds from NOM that were less reactive with chlorine, decreasing the concentration of simulated distribution system total trihalomethanes (SDS TTHMs) and simulated distribution system halo acetic acids (SDS HAAs) by up to 80% and 65%, respectively. Reducing the molecular weight cut-off (MWCO) of the membranes resulted in reductions in the concentrations of SDS TTHMs and SDS HAAs. Using a membrane with a 5 kD MWCO, the minimum gaseous ozone concentration required to bring about effective NOM degradation and meet regulatory requirements for chlorinated DBPs was 2.5 g/m(3).

Hepatocyte growth factor (HGF) adsorption kinetics and enhancement of osteoblast differentiation on hydroxyapatite surfaces.

Hepatocyte growth factor (HGF) is a growth factor that promotes angiogenesis (tissue vascularization), cell motility, and cell differentiation, making it a potentially beneficial coating for bone implants. However, very little is known about maximizing HGF attachment to surfaces of tissue-engineered scaffolds. Here, we examine methods and kinetics of HGF adsorption onto a dense hydroxyapatite (HA) surface (used in bone implants) and determine the influence of HGF coating on osteoblast phenotype/differentiation. We demonstrate that incubating HA with HGF in solution (and not allowing the solution to dry) resulted in maximal surface adsorption that was not enhanced by extending incubation time beyond 2 days. Daily shaking of the coated HA surface did not remove adsorbed HGF. To further examine the effect of HA on osteoblast phenotype, MC3T3-E1 preosteoblasts were seeded onto HA or HGF-HA surfaces. Gene expression analyses indicate that HGF coating enhanced osteoblast differentiation as demonstrated by increased runx2 (a transcription factor important for osteoblast lineage and differentiation), alkaline phosphatase (marker of mid stage differentiation) and osteocalcin (marker of late stage differentiation) mRNA levels. Taken together, our results demonstrate that HGF can serve as an excellent bone implant coating based on its ability to readily adsorb to HA surfaces, maintain integrity over time, and enhance osteoblast differentiation.

Surface potential and osteoblast attraction to calcium phosphate compounds is affected by selected alkaline hydrolysis processing.

This study examines the link(s) between the suspension behavior of calcium deficient apatites (CDAs) and biphasic calcium phosphate (BCP), as measured by the zeta-potential, with respect to both whole bone and osteoblasts. CDA is fabricated by hydrolyzing an acidic CaP such as dicalcium diphosphate dihydrate (DCPD; CaHPO4.2H2O) and has a structure and composition close to bone apatite. Sintering CDA results in the formation of BCP ceramics consisting of mixtures of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), with the HA/beta-TCP weight ratio proportional to the Ca/P ratio of CDA. The choice of the base for the DCPD hydrolysis allows various ionic partial substitution of the formed CDA. Na for Ca partial substitution is of interest because of the resulting improvement in mechanical properties of the resulting BCP ceramics and NH4OH was used as a negative control. The zeta-potential was measured for these materials and the stability of the ceramic to bone interaction calculated. zeta-potential values decrease for CDA(NH4OH) versus CDA(NaOH) and increase for BCP(NH4OH) versus BCP(NaOH). While results of these analyses indicate that NH4OH and NaOH processed CDA and BCP will likely yield osteoblast attachment in vivo, differences in the zeta-potentials may explain varying degrees of cell attachment.

Electrostatic interactions as a predictor for osteoblast attachment to biomaterials.

The present study utilizes zeta (zeta)-potential analysis as an indicator of bonding of osteoblasts and whole bone to various biomaterials. Common metal alloys (316L stainless steel, CoCrMo, and Ti6Al4V) and bioceramics (hydroxyapatite and beta-tricalcium phosphate) used in orthopedic applications were suspended in particulate form in physiologic saline, both as-received and supplemented with bovine serum albumin (BSA). Metal alloys were also treated with NaOH washing to study the effect of such a surface treatment on the zeta-potential. The NaOH wash was found to increase the zeta-potential for CoCrMo and Ti6Al4V, but there was a decrease in the magnitude of the zeta-potential for 316L stainless steel. When the metal alloy powders were suspended in BSA-supplemented physiologic saline, the zeta-potential as a function of pH increased, thereby increasing the electronegativity gap and increasing the propensity for bonding between each of the metal alloys and bone. This increase is likely due to matrix proteins in the BSA, which adsorb onto the metal alloy surfaces, promoting bone growth. With the addition of BSA to each bioceramic system, a uniform decrease in zeta-potential was observed. However, the electronegativity gap remained large in each case, maintaining the anticipation of bonding. zeta-Potential analysis is an effective predictor of biomaterial attraction to osteoblasts and bone, providing a useful in vitro method for predicting such interactions.

Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts.

Hydroxyapatite describes both the natural mineral phase of bone as well as the widely used calcium-phosphate implant substitute. Given that hydroxyapatite is a major component of the in vivo surface with which osteoblasts interact, it is surprising that most studies examining the regulation of osteoblast growth and differentiation utilize plastic surfaces. Here we demonstrate that the phenotype of mouse MC3T3-E1 osteoblasts is significantly altered on hydroxyapatite compared with plastic surfaces. Specifically, alkaline phosphatase activity and messenger RNA levels, markers of early stages of osteoblast differentiation, are increased in osteoblasts cultured on hydroxyapatite. The precocious appearance of alkaline phosphatase activity on the hydroxyapatite surface suggests that osteoblast differentiation is activated earlier compared with plastic surfaces. Osteocalcin expression, a marker of late-stage differentiation, is also increased on hydroxyapatite and further demonstrates enhanced differentiation. Cell counts indicate that fewer osteoblasts are present on hydroxyapatite versus plastic surfaces 24 h after plating. Measurement of osteoblast attachment, apoptosis, and necrosis indicated no differences between surfaces. In contrast, the number of bromodeoxyuridine-incorporating cells was significantly decreased on hydroxyapatite compared with plastic surfaces. Taken together, our findings indicate that hydroxyapatite enhances osteoblast differentiation while also suppressing growth.