Reactions and Surface Transformations of a Bone-Bioactive Material in a Simulated Microgravity Environment

A comprehensive program to investigate the expeditious in vitro formation of three-dimensional bone-like tissue is currently underway at the University of Pennsylvania. The study reported here forms a part of that program. Three-dimensional bone-like tissue structures may be grown under the simulated microgravity conditions of NASA designed Rotating Wall Bioreactor Vessels (RWV's). Such tissue growth will have wide clinical applications. In addition, an understanding of the fundamental changes that occur to bone cells under simulated microgravity would yield important information that will help in preventing or minimizing astronaut bone loss, a major health issue with travel or stay in space over long periods of time. The growth of three-dimensional bone-like tissue structures in RWV's is facilitated by the use of microcarriers which provide structural support. If the microcarrier material additionally promotes bone cell growth, then it is particularly advantageous to employ such microcarriers. We have found that reactive, bone-bioactive glass (BBG) is an attractive candidate for use as microcarrier material. Specifically, it has been found that BBG containing Ca- and P- oxides upregulates osteoprogenitor cells to osteoblasts. This effect on cells is preceded by BBG reactions in solution which result in the formation of a Ca-P surface layer. This surface further transforms to a bone-like mineral (i.e., carbonated crystalline hydroxyapatite (c-HA)). At normal gravity, time-dependent, immersion-induced BBG reactions and transformations are greatly affected both by variations in the composition of the milieu in which the glass is immersed and on the immersion conditions. However, the nature of BBG reactions and phase transformations under the simulated microgravity conditions of RWV's are unknown, and must be understood in order to successfully use BBG as microcarrier material in RWV'S. In this paper, we report some of our recent findings in this regard using experimental and numerical methods. BBG composition 45S5, the most reactive among known bone-bioactive glasses, was chosen for the study. BBG 45S5 behavior in physiological solutions was tested in simulated microgravity and compared with that at normal gravity. On the basis of our numerical study, we have chosen the BBG granule size to be in the range 40-70 microns, and a RWV rotational speed of 10 rpm. Our numerical study has shown that these parameters enable the microcarrier to remain suspended in the medium without experiencing collisions with the wall of the vessel. Immersion-induced changes in the solution composition and the material surface were analyzed after immersion

Solution mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSC) on PLGA by promote the formation of a calcium phosphate rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, we tested whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSC, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell derived factors which promoted distant MSC differentiation. Thus PLGABG composites demonstrates significant potential as a bone replacement material

Effect of surface activated poly(dimethylsiloxane) on fibronectin adsorption and cell function

Cell function on biomaterials may depend on surface chemistry and concentration (as well as conformation) of protein molecules. To understand the interplay between these two effects, fibronectin (Fn) was physi-sorbed on a smooth, activated poly(dimethylsiloxane), films spun cast on silicon wafers. Contact angle goniometry, ellipsometry, Atomic force microscopy and Rutherford backscattering spectrometry were used to characterize the nanoscale roughness and thickness of the films. The films were activated by exposure to 30 min ultraviolet ozone radiation. Water contact angle measurements indicated higher hydrophobicity (\u3e 100o) prior to surface activation. Tapping mode AFM scans showed that the activation process produced a rougher substrate (Ra \u3e 0.50 nm). Fibronectin surface coverage after incubating PDMS in 2.5µg/mL of Fn was significantly higher than on non-activated surface, possibly due to favorable hydrophobic interactions between PDMS and Fn. To investigate the effect of surface activation on MC3T3-E1 osteoblast-like cells, cell spreading on PDMS and activated PDMS (30 min) coated with 2.5 µg/mL Fn was studied. Cells plated on the activated Fn-coated PDMS, for 15 min, in DMEM (with serum) showed higher cell attachment. Cell spreading after 72 h plating was clearly favored on the hydrophilic substrates as well. The increase in cell area is attributed to favorable conformational changes in absorbed Fn molecules on these substrates

Adhesion of MC3T3-E1 cells to RGD peptides of different flanking residues: Detachment strength and correlation with long-term cellular function

We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (τ50) of RGD peptides of different flanking residues was found: RGDSPK ≻ RGDSVVYGLR ≈ RGDS ≻ RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily α√-integrin-mediated. In contrast, the τ50 value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6-7 times higher and involved both α5β1 and ανβ3 integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides

Bioactivity in silica/poly(γ-glutamic acid) sol–gel hybrids through calcium chelation

Bioactive glasses and inorganic/organic hybrids have great potential as biomedical implant materials. Sol–gel hybrids with interpenetrating networks of silica and biodegradable polymers can combine the bioactive properties of a glass with the toughness of a polymer. However, traditional calcium sources such as calcium nitrate and calcium chloride are unsuitable for hybrids. In this study calcium was incorporated by chelation to the polymer component. The calcium salt form of poly(γ-glutamic acid) (γCaPGA) was synthesized for use as both a calcium source and as the biodegradable toughening component of the hybrids. Hybrids of 40 wt.% γCaPGA were successfully formed and had fine scale integration of Ca and Si ions, according to secondary ion mass spectrometry imaging, indicating a homogeneous distribution of organic and inorganic components. 29Si magic angle spinning nuclear magnetic resonance data demonstrated that the network connectivity was unaltered with changing polymer molecular weight, as there was no perturbation to the overall Si speciation and silica network formation. Upon immersion in simulated body fluid a hydroxycarbonate apatite surface layer formed on the hybrids within 1 week. The polymer molecular weight (Mw 30–120 kDa) affected the mechanical properties of the resulting hybrids, but all hybrids had large strains to failure, >26%, and compressive strengths, in excess of 300 MPa. The large strain to failure values showed that γCaPGA hybrids exhibited non-brittle behaviour whilst also incorporating calcium. Thus calcium incorporation by chelation to the polymer component is justified as a novel approach in hybrids for biomedical materials

The inhibition of \u3cem\u3eStaphylococcus epidermidis\u3c/em\u3e biofilm formation by vancomycinmodified titanium alloy and implications for the treatment of periprosthetic infection

Peri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections

Zoonosis emergence linked to agricultural intensification and environmental change

A systematic review was conducted by a multidisciplinary team to analyze qualitatively best available scientific evidence on the effect of agricultural intensification and environmental changes on the risk of zoonoses for which there are epidemiological interactions between wildlife and livestock. The study found several examples in which agricultural intensification and/or environmental change were associated with an increased risk of zoonotic disease emergence, driven by the impact of an expanding human population and changing human behavior on the environment. We conclude that the rate of future zoonotic disease emergence or reemergence will be closely linked to the evolution of the agriculture–environment nexus. However, available research inadequately addresses the complexity and interrelatedness of environmental, biological, economic, and social dimensions of zoonotic pathogen emergence, which significantly limits our ability to predict, prevent, and respond to zoonotic disease emergence