Osteogenic effects of bioactive glass on bone marrow stromal cells.

Bioactive glass (BG) is an effective synthetic bone graft material. BG granules of narrow size range (300-355 mum) have the ability to form new bone tissue inside excavations produced by in vivo resorption. Previously, we demonstrated that BG stimulates the differentiation of cultured osteoblast precursors if the glass surface was biomimetically modified by the formation of bone-like apatite and adsorption of serum proteins. We now report that modified BG can also increase the rate at which multipotential rat bone marrow stromal cells (rMSC) will undergo osteogenesis. BG promoted rMSC osteogenesis both when cells were plated in contact with BG and when cells were not directly in contact with the BG. Alkaline phosphatase activity, a marker of bone cell differentiation, was used as an indicator for osteogenesis. Alkaline phosphatase activity of rMSCs exposed to osteoinducers such as ascorbate, dexamethasone, and BMP-2 was enhanced in the presence of BG. The stimulatory effect of BG was more pronounced in rMSC cultures with low basal alkaline phosphatase activity than in those with higher activity. The enhanced differentiation of rMSCs was associated with both a change in rMSC morphology and altered chemical composition of the cell culture media. rMSCs cultured on BG in the presence of BMP or dexamethasone exhibited a more rounded osteoblast-like appearance as compared with cells grown on tissue culture plastic. In the presence of BG, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period, suggesting a continuous dissolution of surface-modified BG and resulting release of BG dissolution products. The data suggest that both surface- and solution-mediated events play a role in the osteogenic effect of BG.

Synthesis and evaluation of novel bioactive composite starch/bioactive glass microparticles.

The aim of the development of composite materials is to combine the most desired properties of two or more materials. In this work, the biodegradable character, good controlled-release properties, and natural origin of starch-based biomaterials are combined with the bioactive and bone-bonding properties of bioactive glass (BG). Novel, bioactive composite starch-BG microparticles were synthesized starting from a blend of starch and polylactic acid (50%/50% wt) with BG 45S5 powder using a simple emulsion method. Morphological and chemical characterization showed that these particles exhibited a spherical morphology with sizes up to 350 microm and that BG 45S5 was incorporated successfully into the composite particles. Upon immersion in a solution simulating body fluids, for periods up to 3 weeks, their bioactive nature was confirmed, as a calcium-phosphate layer resembling biological apatite was formed onto their surface. The short-term cytotoxicity of these materials was also tested by placing 24-h leachables of the materials extracted in culture medium in contact with a fibroblastic cell line (L929) up to 72 h. At this time period, two biochemical tests--MTT and total protein quantification--were performed. The results showed that these materials are not cytotoxic. These results constitute the basis of future encapsulation studies using bone-acting therapeutic agents such as bone morphogenetic proteins or other bone-relevant factors. The particles developed here may be very useful for applications in which controlled release, degradability, and bone-bonding ability are the main requirements.

The value of life and accident costing: a willingness-to-pay study amongst motorcyclists in Malaysia.

Motorcyclists constitute a large proportion of total road casualties in Asian countries Unfortunately, studies conducted for the purpose of evaluating the cost of traffic crashes, and cost-benefit analyses of safety interventions, are almost nonexistent in these countries. The loss-of-output approach to valuing life has been used for many years, yet this method has also long been criticised as it results in significant resource misallocation. This study attempts to overcome this problem by estimating the value of a statistical life among motorcyclists using the willingness-to-pay method that is commonly used in developed countries. The study recommends adopting a value of MYR1.1 million (almost five times the previous estimate) per statistical life for public policy analysis involving motorcycle safety.

Surface transformation of bioactive glass in bioreactors simulating microgravity conditions. Part I: experimental study.

Surface modified bioactive glass with surface properties akin to those of the bone mineral phase is an attractive candidate for use as a microcarrier material for 3-D growth of bone-like tissue in rotating wall vessel bioreactors (RWVs). The critical surface properties of this material are the result of reaction in solution. Because an RWV environment is completely different from conditions previously employed for bioactive glass testing, a detailed study of the surface reactions is warranted. Under properly chosen conditions, RWVs can also provide a simulated microgravity environment for the bioactive glass (BG) particles. In this sense, this study is also a report on the behavior of a bioactive material under microgravity conditions simulated on earth. A high aspect ratio vessel (HARV) and carefully selected experimental conditions enabled the simulation of microgravity in our laboratory. A complimentary numerical study was simultaneously conducted to ascertain the appropriateness of the experimental parameters (particle size, particle density, medium density, medium viscosity, and rotational speed) that ensure simulated microgravity conditions for the glass particles in the HARV. Physiological solutions (pH 7.4) with and without electrolytes, and also with serum proteins, were used to study the change in surface character resulting from simulated microgravity. Control tests at normal gravity, both static and dynamic, were also conducted. Solution and surface analyses revealed major effects of simulated microgravity. The rates of leaching of constituent ions (Si-, Ca-, and P-ions) were greatly increased in all solutions tested. The enhanced dissolution was followed by the enhanced formation of bone-like minerals at the BG surface. This enhancement is expected to affect adsorption of serum proteins and attachment molecules, which, in turn, may favorably affect bone cell adhesion and function. The findings of the study are important for the use of bioactive materials as microcarriers to generate and analyze 3-D bone-like tissue structures in bioreactors under microgravity conditions or otherwise.

Surface transformation of bioactive glass in bioreactors simulating microgravity conditions. Part II: numerical simulations.

The effects of simulated microgravity on the surface modification of bioactive glass (BG) in solution were studied using a numerical method. Models were developed for estimating the mass transfers of different chemical species from the surface of bioactive glass particles (microcarriers) suspended in the rotating liquid medium of a NASA-designed high aspect ratio vessel (HARV) bioreactor and on the bottom surface of a static vial. The concentration profiles resulting from chemical reactions and ionic transports were ascertained. Numerical results for the transport under simulated microgravity in the HARV and at normal gravity in the static vial were compared. These results were also compared with those of experiments to verify the enhancement of the reaction kinetics under simulated microgravity conditions. The experimental and numerical studies confirm that simulated microgravity conditions lead to the quick achievement of bioactive glass surface modification.

Silica sol-gel for the controlled release of antibiotics. II. The effect of synthesis parameters on the in vitro release kinetics of vanomycin.

Room temperature-processed silica sol-gel (xerogel) was investigated as a novel controlled release carrier of vancomycin for the treatment of osteomyelitis. Vancomycin-loaded xerogels were fabricated with varying water/alkoxysilane molar ratios and vancomycin concentrations. The goal of this study was to determine the effect of varying the aforementioned synthesis parameters on the daily in vitro release kinetics of vancomycin from the xerogel disks. A controlled, load-dependent, long-term release of vancomycin was observed for all of the molar ratios that were used in the study (4, 6, and 10). Variations in the water/alkoxysilane molar ratio affected the release process extensively. A cumulative release of about 90% of the original amount of vancomycin was found for molar ratios 6 and 10 by 21 and 14 days, respectively. Only about 30% was released from xerogels with a molar ratio of 4 after 21 days of immersion. A first-order release stage was followed by a steady release stage for xerogels with molar ratios of 6 and 10, whereas zero-order release was observed for xerogels with a molar ratio of 4. The findings of this study indicate that the release kinetics of vancomycin from xerogel can be tailored by varying the xerogel synthesis parameters.

Silica sol-gel for the controlled release of antibiotics. I. Synthesis, characterization, and in vitro release.

Room temperature processed silica sol-gel (xerogel) was investigated as a novel controlled release carrier of antibiotics (vancomycin). Xerogel characteristics, in vitro release properties, and bactericidal efficacy of the released antibiotic were determined. The xerogel/vancomycin composite showed a long-term sustained release (up to 6 weeks). In addition, bactericidal efficacy of released vancomycin was retained. The kinetics of release and the amount released were dose dependent. The initial, first-order release was followed by a near-zero-order release. The time to transition from the first- to zero-order release increased with vancomycin load (from 2 to 3 weeks with load increase from 2.2 to 11.1 mg/g). Regardless of the load, about 70% of the original vancomycin content was released by the transitional point, and the cumulative release after 6 weeks of immersion was about 90%. This study, combined with other reports documenting biocompatibility and controlled resorbability of the xerogel/drug composite in vivo, suggests that silica xerogel is a promising controlled release material for the treatment of bone infections.

Initial events at the bioactive glass surface in contact with protein-containing solutions.

Upon implantation, bioactive glass undergoes a series of reactions that leads to the formation of a calcium phosphate-rich layer. Most in vitro studies of the changes that occur on the surface of bioactive glass have employed the use of buffer solutions with compositions reflecting the ionic composition of interstitial fluid. Although these studies have documented the physical and chemical changes associated with bioactive glass immersed in aqueous media, they do not reveal the effect of serum proteins and cells that are present at the implantation site. In the present study, we document, using atomic force microscopy (AFM) and Rutherford backscattering spectrometry (RBS), significant differences in the reaction layer composition, thickness, morphology, and kinetics of formation arising from the presence of serum proteins. The data reveal that the uniform and rapid adsorption of serum proteins on the surface may serve to protect the surface from further direct interaction with the aqueous media, slowing down the transformation reactions. This finding is in agreement with previous studies that have shown that the presence of serum proteins significantly delays the formation of hydroxyapatite at the surface of bioactive glass. These data also support the hypothesis that initial reaction layers in vivo interact with cells in order to produce the tissue-bioactive glass interface typically observed on ex vivo specimens.

In vitro transformation of bioactive glass granules into Ca-P shells.

Bioactive glass (BG) granules of narrow size are excavated when implanted in mandibular bone of beagles. Bone tissue forms within these internally hollowed particles without a connection to the bone at the margins of the defect. In this study the internal excavation of BG granules was simulated by in vitro immersion experiments. Postimmersion solutions were analyzed for changes in Si, Ca, and P concentrations. Using scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis and Fourier Transform Infrared (FTIR) spectroscopy, granules were analyzed for compositional, morphologic, and structural changes resulting from immersion. Only when the solution was continuously replenished and only if this solution was composed of electrolyte- and protein-containing serum was excavation achieved. Without solution replenishment, that is, under so-called integral immersion conditions, the solution quickly became saturated in silicon, and the silicon no longer dissolved. When the glass was immersed in a solution with serum, a porous surface structure with fine precipitates was formed, in contrast to a dense surface reaction layer with closely packed globular precipitates that was formed in a solution without serum. The combined effect of continuous solution replenishment and the use of a solution containing serum proteins led to the formation of a surface reaction layer that did not impede continued corrosion. As such, all Si was released, and eventually a hollow Ca-P shell was formed. Thus this study supports the hypothesis that there is a physico-chemical mechanism of Si transport through the Ca-P-rich layer followed by Si dissolution. This mechanism may be operative in vivo and thereby may contribute to the observed in vivo excavation.

Sol-gel derived carrier for the controlled release of proteins.

Sol-gel derived porous silica carriers for the controlled release of proteins were synthesized using a room temperature process. The materials are intended to serve as both substrates for bone growth as well as to allow incorporated proteins such as growth factors to diffuse out and stimulate cell function and tissue healing. The data document that the in vitro release of trypsin inhibitor, a model protein of size similar to growth factors with documented effect in bone, was dose and time dependent during immersion up to nine weeks. The release pattern included an initially slow release, with further release occurring at a rate which is proportionate to the square root of time, an indicative of a diffusion-driven process.

Si-Ca-P xerogels and bone morphogenetic protein act synergistically on rat stromal marrow cell differentiation in vitro.

This study describes a novel bioactive xerogel glass as a carrier for bone morphogenetic protein (BMP) and the value of this carrier in terms of stimulating osteogenic activity of rat stromal marrow cells in vitro. These cells were seeded onto the surface of xerogel glass disks with BMP either incorporated in the glass, adsorbed to the surface of the glass, or added to the culture media and then compared to cells on glass with no added BMP or to cells on tissue culture plastic (TCP) with and without BMP. Cells were cultured for 6 and 10 days and examined for total DNA, alkaline phosphatase activity, and osteocalcin and total protein production. Stromal cell differentiation, as measured by alkaline phosphatase activity and osteocalcin synthesis was most increased when the BMP was incorporated or adsorbed onto the xerogel glass. Cells on xerogel glass without BMP were more differentiated than cells grown on plastic with BMP, thereby demonstrating the additive effect of a bioactive substrate and BMP on osteoblastic cell differentiation. These data indicate that xerogel glass effects differentiation of cells with osteogenic potential and that it can serve as a delivery vehicle for BMP.

Connectivity and control in the year 2000 and beyond.

By now, most executives are familiar with the famous Year 2000 problem--and many believe that their companies have the situation well in hand. After all, it seems to be such a trivial problem--computer software that interprets "oo" to be the year 1900 instead of the year 2000. And yet armies of computer professionals have been working on it--updating code in payroll systems, distribution systems, actuarial systems, sales-tracking systems, and the like. The problem is pervasive. Not only is it in your systems, it's in your suppliers' systems, your bankers' systems, and your customers' systems. It's embedded in chips that control elevators, automated teller machines, process-control equipment, and power grids. Already, a dried-food manufacturer destroyed millions of dollars of perfectly good product when a computer counted inventory marked with an expiration date of "oo" as nearly a hundred years old. And when managers of a sewage-control plant turned the clock to January I, 2000 on a computer system they thought had been fixed, raw sewage pumped directly into the harbor. It has become apparent that there will not be enough time to find and fix all of the problems by January I, 2000. And what good will it do if your computers work but they're connected with systems that don't? That is one of the questions Harvard Business School professor Richard Nolan asks in his introduction to HBR's Perspectives on the Year 2000 issue. How will you prepare your organization to respond when things start to go wrong? Fourteen commentators offer their ideas on how senior managers should think about connectivity and control in the year 2000 and beyond.

Effect of serum proteins and osteoblasts on the surface transformation of a calcium phosphate coating: a physicochemical and ultrastructural study.

Changes occurring at the surface of a calcium phosphate coating when in contact with osteoblasts versus those in acellular solutions were analyzed. The coating studied is one with a well-documented extensive effect on short-term bone growth stimulation. Precipitates associated with original crystals and organized in a weblike structure were observed after a 3-week culture with osteoblasts. The precipitates were identified as carbonated hydroxyapatite (c-HA). In contrast, no significant surface changes were detected after immersion in an acellular serum-containing solution. However, in an acellular serum-free solution simulating the ionic composition of plasma, precipitates, identified as c-HA, were abundantly formed. Dissolution of the original coating preceded precipitation. The data support the hypothesis that dissolution of synthetic calcium phosphate ceramics is an initial step in their transformation to a biologically equivalent apatite, and suggest that both solution-mediated (dissolution-precipitation) and cell-mediated mechanisms are involved in the surface transformation.

The effect of in vitro modeling conditions on the surface reactions of bioactive glass.

Using one parametric variation in solution composition, this paper documents that the surface reactions on bioactive glass (BG) 45S5 are exquisitely dependent upon the modeling conditions. The solutions used were 0.05 M tris hydroxymethyl aminomethane/HCl (tris buffer), tris buffer complemented with plasma electrolyte and/or serum, and serum. The reacted surfaces were analyzed using Fourier transform infrared (FTIR), scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDXA), and Rutherford backscattering spectroscopy (RBS). Post-immersion solutions were analyzed for changes in Ca and PO4 concentrations. After a short immersion (3 h), a crystalline, carbonated hydroxyapatite (c-HA) layer formed only in tris. Reaction surfaces of different structure, morphology, and composition were observed after various short and longer term immersions in all other solutions. They comprised two layers with the layer in contact with the bulk consisting mainly of Si; the outer layer, composed of Si, Ca, and P, was amorphous, and had a Ca/P ratio of about 1. Serum proteins adsorbed on the BG surfaces at the early stages of the solution-mediated BG reactions. Formation of a crystalline c-HA layer was delayed up to three or more days in solution with plasma ions. In the presence of serum, only amorphous surfaces composed of Si, Ca, and P were observed for any time up to seven days of immersion. The present data suggest that serum proteins adsorb in tandem with the occurrence of solution-mediated reactions leading to formation of a silica-gel. Amorphous Ca-P phases accumulate in the Si-rich matrix. Furthermore, the present data, in conjunction with the data published before, suggest that physicochemical and cell-mediated reactions occur in parallel to form the glass-tissue interfacial layer.

Calcium phosphate ceramic coatings as carriers of vancomycin.

Infection in the setting of total joint arthroplasty remains a challenging problem. Attention has turned to developing methods of local delivery of antibiotics for prophylaxis. Vancomycin loaded into calcium phosphate ceramic coatings on titanium alloy substrates is a clinically relevant concept in the setting of total joint arthroplasty. Drug loading was accomplished by immersion of ceramic-coated discs in vancomycin-containing simulated physiological solution; in some experiments drug loading by immersion was followed by lipid coating in egg phosphatidylcholine solutions. The kinetics of vancomycin release and the efficacy of drug inhibition of Staphylococcus aureus were determined in vitro in comparison to the release from currently used antibiotic-laden poly(methyl methacrylate) (PMMA). The loading by immersion provided effective release and inhibition at early time points (up to 24 h); however, the lipid-coated samples demonstrated significant release and effective bacterial inhibition up to 72 h. The two-step procedure, i.e. drug loading followed by lipid coating in order to slow antibiotic elution, is more effective than the conventional one-step loading. The study indicated that the osteoconductive calcium phosphate coatings have the potential to serve as drug carriers to prevent infection in the setting of total joint arthroplasty.

In vitro release kinetics of biologically active transforming growth factor-beta 1 from a novel porous glass carrier.

Sol-gel silica-based porous glass (xerogel) was used as a novel carrier material for recombinant human transforming growth factor-beta 1 (TGF-beta 1). Room temperature synthesis procedures included sol preparation, the addition of TGF-beta 1 solution to the sol, subsequent gelation and drying. After determination of optimal synthesis parameters, the material was assayed in vitro for its ability to release biologically active TGF-beta 1 in a controlled manner. Sustained release of TGF-beta 1 over a 7-day period was demonstrated. On the basis of published TGF-beta 1 potency, the amount released is capable of eliciting bone tissue reactivity. These findings suggest that this novel glass-growth factor composite may serve as an effective bone graft material for the repair of osseous defects.

Bone tissue reactions to an electrophoretically applied calcium phosphate coating.

Oral implants of a threaded design, calcium phosphate (CaP)-coated using an electrophoretic deposition technique, were compared to uncoated commercially pure (c.p.) titanium control in an animal study with 4 weeks and 6 months of follow-up, respectively. The 3D surface roughness of a CaP-coated implant was about three times greater than that of an uncoated control. Histomorphometric analyses of the direct bone-implant contact demonstrated a short-term advantage to the CaP-coated implants, whereas no significant difference to the uncoated titanium was found after 6 months. Comparison of the amount of bone inside or outside the threads showed similar values for test and control after 4 weeks. Significantly higher amounts of bone outside the uncoated c.p. titanium implants were measured after the long-term follow-up.

Effect of serum proteins on solution-induced surface transformations of bioactive ceramics.

The kinetics of immersion-induced surface transformation reactions of synthetic bone bioactive ceramics were studied in vitro in either protein-free or protein-containing simulated physiological solutions. Both solutions had an ion content similar to that of plasma. Synthetic ceramics used for the study included Ca-deficient hydroxyapatite (CDHA), stoichiometric HA either not well crystallized or well crystallized (s-HA nwc or s-HA wc), oxyhydroxyapatite (OHA), beta-tricalcium phosphate (beta-TCP), and porous coralline HA (I-HA) and calcium carbonate (CC). Only CDHA and nwc s-HA led to immediate precipitation in both protein-free and protein-containing solutions. In contrast, reactions of wc HA and I-HA showed lag times to onset of precipitation in the protein-free solution and a further delay in the presence of proteins. The reactions of nonapatitic ceramics whose lag times in the protein-free solution were longer than those of apatitic, were completely blocked in the presence of proteins within the duration of the experiment (up to 3 days). CDHA and nwc s-HA were the only ceramics that, in the presence of serum proteins, led to the formation of B-type carbonated apatite, typical for calcified tissue apatite.